Focus Issue Editorial: Biotic StressJin, Hailing; Mitchum, Melissa; Panstruga, Ralph; Stone, Julie
doi: 10.1104/pp.19.00330pmid: 30940733
Devastating diseases caused by pathogens and pests have threatened crop production, human health, and the stability of global economies. Extensive efforts to more fully understand the mechanisms of plant immune responses and pathogen virulence have yielded new insights that may aid in maintaining or enhancing yields and nutritional quality, as plant pathogen ranges fluctuate in response to climate change and consumer preferences also evolve. Progress in our understanding has been both rapid and significant, warranting this “Focus Issue on Biotic Stress,” which includes four diverse Update Reviews on recent advances in the field, 12 primary Research Articles covering various research areas on plant host interactions with pathogens and pests, and one Breakthrough Technologies. Regulation of phytohormone biosynthesis and signaling is a key component of plant–biotic stress interactions. Defense suppression and developmental reprogramming via alterations to phytohormone biosynthesis and signaling pathways are critical mechanisms used by pests and pathogens to thrive and cause disease. In their Update, Gheysen and Mitchum (2019) describe how plant-parasitic cyst and root-knot nematodes alter defense and developmental programs by manipulating plant hormone pathways to establish specialized feeding sites within host roots. The authors also highlight how these nematodes have evolved to secrete a variety of plant peptide hormones that mimic endogenous plant-growth–regulating peptides and hijack developmental programs to establish effective feeding sites. At invasion sites, plants attempt to defend themselves by reinforcing the cell wall against insect and pathogen attack, producing defensive compounds such as callose. In maize (Zea mays), benzoxazinoids are known to enhance callose deposition and promote resistance to phloem feeding sap-sucking aphids. In this issue, Varsani et al. (2019) demonstrate that maize inbred line Mp708 utilizes a newly elucidated defense mechanism via 12-oxo-phytodienoic acid, independent of the jasmonic acid pathway, as a key regulator of enhanced callose deposition to limit colonization by corn leaf aphid (Rhopalosiphum maidis). Using the Arabidopsis (Arabidopsis thaliana)–two-spotted spider mite (Tetranychus urticae) plant herbivore model system, Santamaría et al. (2019) identified a role for the Toll-interleukin receptor–lectin domain protein Phloem Protein2 (PP2)-A5 in resistance against two-spotted spider mites. PP2-A5–overexpressing plants showed enhanced resistance to mite herbivory, whereas loss-of-function mutants exhibited much more extensive leaf damage. Metabolite profiling revealed that altered hormonal signaling, rather than changes in indole glucosinolates, underpin PP2-A5–mediated resistance to provide new insight into the plant–spider mite interaction. Ali et al. (2019) describe the identification and characterization of a RING-type E3 ubiquitin ligase, JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1)-ASSOCIATED UBIQUITIN LIGASE1 (JUL1), which ubiquitinates JASMONATE-ASSOCIATED VQ-MOTIF GENE1 (JAV1/VQ22), a repressor of jasmonate-mediated defense responses. The JAV1/JUL1 functional module was discovered to act as a specific coordinator of plant defense responses against the generalist herbivore Spodoptera litura. Nutrient acquisition from host cells is a key necessity for plant-attacking pests and microbial intruders. Whereas insects and nematodes can use physical devices, such as stylets, to gain access to nutrient-rich plant phloem sap or to establish specialized feeding sites, the challenging barrier afforded by plant cell walls is harder to address for microbes. In the case of biotrophic and hemibiotrophic pathogens, dedicated structures such as haustoria or infection hyphae facilitate the establishment of an interface to plant host cells. In their Update, Judelson and Ah-Fong (2019) describe how mutual exchange of biomolecules at the oomycete-plant interface impact disease outcomes. The authors not only elaborate on the task of the interface as a trading place for nutrients, but also highlight its role as a battleground enabling plant defense and microbial counterdefense. Carbohydrates are key nutrients absorbed by pathogenic microbes and required to maintain infection. In an elegant study using a fluorescence resonance energy transfer-based biosensor to trace Glc distribution in fungal cells, Sosso et al. (2019) studied carbohydrate partitioning in the interaction between maize and its biotrophic fungal pathogen, Ustilago maydis, causing smut disease. The authors discovered comprehensive alterations in carbohydrate allocation during infection, which happens alongside changes in expression levels of maize genes encoding sugar transporters of the SWEET family. Additionally, they found a steep Glc gradient in fungal hyphae, with highest Glc levels in the hyphal tips. Whereas sugar transporters thus seem essential for carbohydrate delivery to microbial intruders, they rather surprisingly can also play a role in conferring disease resistance. The wheat (Triticum aestivum) Lr67 gene encodes a high-affinity Glc-proton symporter that transports Glc from the apoplastic space to the cytosol. A particular mutation in the gene, resulting in a single amino acid substitution within a transmembrane domain, leads to dominantly inherited resistance to multiple fungal pathogens (rusts and powdery mildew). Milne et al. (2019) have now demonstrated that the orthologous barley transporter, HvSTP13, has similar transport characteristics to wheat Lr67. The authors further revealed that the multipathogen resistance phenotype could be recapitulated in barley (Hordeum vulgare) by transgenic expression of the resistance-conferring allele of wheat Lr67, demonstrating conservation of the underlying resistance mechanism between grasses. Communication between plants and microbes is essential during pathogen infection. Rybak and Robatzek (2019) provided an excellent overview in their “Update” on the important roles of extracellular vesicles in secretion and exchange of protein, RNA, lipid, and metabolites in interacting hosts and microbes. Both microbial- and plant-derived extracellular vesicles, their contents and functions in pathogenesis and plant immune responses, were discussed. Soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins are key components of vesicle trafficking in plants and eukaryotic pathogens. Cao et al. (2019) identified a rice Qa-Soluble n-ethylmaleimide-sensitive factor attachment protein receptor protein, syntaxin121, which accumulates at fungal penetration sites and contributes to rice resistance against Magnaporthe oryzae infection. This study reinforces the importance of vesicle trafficking for host resistance against fungal pathogens. Han et al. (2019) characterized an extracellular chitinase, MoChi1 from the rice blast fungus M. oryzae, which suppresses chitin-induced reactive oxygen species responses. They identified an interacting protein of MoChi1 from rice, a jacalin-related Mannose-Binding Lectin (OsMBL1). OsMBL1 is induced by M. oryzae infection and positively regulates rice immune responses. Isolation of apoplast extracts is a critical step for secretion and vesicle trafficking studies, which is especially challenging in monocots. Gentzel et al. (2019) developed a simple method to collect apoplast contents in maize, and established an easy protocol to measure apoplast hydration. They demonstrate applicability of the procedure by the efficient recovery of a bacterial maize pathogen (Pantoea stewartii ssp stewartii) residing in the apoplastic space. Cytoplasmic sensor proteins composed of an amino-terminal coiled-coil or Toll-interleukin receptor domain, a central nucleotide-binding domain, and carboxyl-terminal Leucine-rich repeats represent the canonical versions of plant NLR-type resistance proteins. These typically confer isolate-specific resistance against a given pathogen by direct or indirect perception of a pathogen effector protein or directly act on a host target protein(s). This type of immunity is usually referred to as effector-triggered immunity (ETI). A recently emerged variation of this canonical scheme is the integration of other protein domains (integrated domains, IDs) into plant NLR proteins. These IDs are presumed to mimic authentic host targets of pathogen effectors to serve as decoys to attract effector proteins and trigger a boosted immune response. In an Update article, Grund et al. (2019) provide a nice overview about the current state-of-the-art regarding NLRs with IDs. The authors describe their distribution, illustrate experimentally validated examples, and discuss potential mechanisms for effector recognition by NLRs with IDs. During ETI, oxidants generate a largely unexplored shift in intracellular redox potential. In their Breakthrough Technology article, McConnell et al. (2019) report a novel method, comprising an enrichment procedure coupled to mass spectrometry-based quantification, to study the plant redoxome (i.e. proteome-wide oxidative modifications of proteins caused by oxidants) during ETI. The authors demonstrate the usefulness of the new technology by comparing the redoxome of wild-type and oligopeptidase mutant plants defective in ETI. Dracatos et al. (2019) also took advantage of a comparatively new method, “MutChromSeq,” to clone a leaf rust resistance gene (Rph1) in a grass species (barley) with a large and highly complex genome. The procedure rests on sequencing of flow-sorted chromosomes from multiple individual mutants and comparative analysis to identify candidate genes. By this approach, the authors recognized a single candidate gene for Rph1, which encodes an NLR protein and represents the first leaf rust resistance gene cloned from cultivated barley. Notably, Rph1 is closely related to the Arabidopsis antibacterial resistance gene RPM1. MAPK cascades are important signaling components in plant immunity. Li et al. (2019) identified a group D MAPK gene from rice (Oryza sativa), OsMAPK20-5, which negatively regulates ethylene and nitric oxide pathways. Silencing of OsMAPK20-5 confers broad resistance to the two most destructive pests of rice, adult brown planthopper (Nilaparvata lugens) and white-backed planthopper (Sogatella furcifera). Epigenetic regulation has been recently recognized as an important molecular mechanism for gene regulation during plant–pathogen interactions. Chromatin modification was implicated in regulating the production of volatile organic compounds (VOCs) and secondary metabolites in filamentous fungi. Estrada-Rivera et al. (2019) have elucidated the function of a histone deacetylase HDA-2 in chromatin modification of Trichoderma atroviride, a beneficial fungus that promotes plant growth. HAD-2 regulates not only the levels of VOCs, but also the profiles of the VOCs, impacting plant growth. Nuclear lamina is a key structure that provides docking sites for chromatin, critical for gene regulation and maintaining proper nuclear function and morphology. Choi et al. (2019) identified a family of candidate nuclear lamina proteins in Arabidopsis, Crowded Nuclei (CRWN), which impact plant development and defense responses. Loss-of-function of CRWN proteins induces salicylic acid biosynthesis, expression of immune-responsive genes, spontaneous defense responses, and cell death. CRWN genes are essential, because knocking out all four family members is lethal. These Updates and Research Articles elucidate how interactions between plants and pathogens/pests lead to beneficial or detrimental outcomes. We look forward to future advancements based on our understanding of plant biotic stress responses aimed at developing innovative and eco-friendly control strategies for plant protection. ACKNOWLEDGMENTS We thank all of the authors and reviewers that collectively contributed to this Focus Issue. LITERATURE CITED Ali MRM , Uemura T, Ramadan A, Adachi K, Nemoto K, Nozawa A, Hoshino R, Abe H, Sawasaki T, Arimuraa G ( 2019 ) The ring-type E3 ubiquitin ligase JUL1 targets the VQ-motif protein JAV1 to coordinate jasmonate signaling . Plant Physiol 179 : 1273 – 1284 Google Scholar Crossref Search ADS PubMed WorldCat Cao W-L , Yu Y, Li M-Y, Luo J, Wang R-S, Tang H-J, Huang J, Wang J-F, Zhang H-S, Bao Y-M ( 2019 ) OsSYP121 accumulates at fungal penetration sites and mediates host resistance to rice blast . Plant Physiol 179 : 1330 – 1342 Google Scholar Crossref Search ADS PubMed WorldCat Choi J , Strickler S, Richards EJ ( 2019 ) Loss of CRWN nuclear proteins induces cell death and salicylic acid defense signaling . Plant Physiol 179 : 1315 – 1329 Google Scholar Crossref Search ADS PubMed WorldCat Dracatos PM , Barto¡ J, Elmansour H, Singh D, Karafiátová M, Zhang P, Steuernagel B, Svačina R, Cobbin JAC, Clark B, et al. ( 2019 ) The coiled-coil NLR Rph1, confers leaf rust resistance in barley cultivar Sudan . Plant Physiol 179 : 1362 – 1372 Google Scholar Crossref Search ADS PubMed WorldCat Estrada-Rivera M , Rebolledo-Prudencio OG, Pérez-Robles DA, del Carmen Rocha-Medina M, del Carmen González-López M, Casas-Flores S ( 2019 ) Trichoderma histone deacetylase HDA-2 modulates multiple responses in Arabidopsis . Plant Physiol 179 : 1343 – 1361 Google Scholar Crossref Search ADS PubMed WorldCat Gentzel I , Giese L, Zhao W, Alonso AP, Mackey D ( 2019 ) A simple method for measuring apoplast hydration and collecting apoplast contents . Plant Physiol 179 : 1265 – 1272 Google Scholar Crossref Search ADS PubMed WorldCat Gheysen G , Mitchum MG ( 2019 ) Phytoparasitic nematode control of plant hormone pathways . Plant Physiol 179 : 1212 – 1226 Google Scholar Crossref Search ADS PubMed WorldCat Grund E , Tremousaygue D, Deslandes L ( 2019 ) Plant NLRs with integrated domains: unity makes strength . Plant Physiol 179 : 1227 – 1235 Google Scholar Crossref Search ADS PubMed WorldCat Han HY , Song L, Peng C, Liu X, Liu L, Zhang Y, Wang W, Zhou J, Wang S, Ebbole D, et al. ( 2019 ) A Magnaporthe chitinase interacts with a rice jacalin-related lectin to promote host colonization . Plant Physiol 179 : 1416 – 1430 Google Scholar Crossref Search ADS PubMed WorldCat Judelson HS , Ah-Fong AMV ( 2019 ) Exchanges at the plant-oomycete interface that influence disease . Plant Physiol 179 : 1198 – 1211 Google Scholar Crossref Search ADS PubMed WorldCat Li J , Liu X, Wang Q, Huangfu J, Schuman MC, Lou Y ( 2019 ) A group D MAPK protects plants from autotoxicity by suppressing herbivore-induced defense signaling . Plant Physiol 179 : 1386 – 1401 Google Scholar Crossref Search ADS PubMed WorldCat McConnell EW , Berg P, Westlake TJ, Wilson KM, Popescu GV, Hicks LM, Popescu SC ( 2019 ) Proteome-wide analysis of cysteine reactivity during effector-triggered immunity . Plant Physiol 179 : 1248 – 1264 Google Scholar Crossref Search ADS PubMed WorldCat Milne RJ , Dibley KE, Schnippenkoetter W, Mascher M, Lui ACW, Wang L, Lo C, Ashton AR, Ryan PR, Lagudah ES ( 2019 ) The wheat Lr67 gene from the sugar transport protein 13 family confers multipathogen resistance in barley . Plant Physiol 179 : 1285 – 1297 Google Scholar Crossref Search ADS PubMed WorldCat Rybak K , Robatzek S ( 2019 ) Functions of extracellular vesicles in immunity and virulence . Plant Physiol 179 : 1236 – 1247 Google Scholar Crossref Search ADS PubMed WorldCat Santamaría ME , Martínez M, Arnaiz A, Rioja C, Burow M, Grbic V, Díaz V ( 2019 ) An Arabidopsis TIR-lectin two-domain protein confers defense properties against Tetranychus urticae. Plant Physiol 179 : 1298 – 1314 Google Scholar Crossref Search ADS PubMed WorldCat Sosso D , van der Linde K, Bezrutczyk M, Schuler D, Schneider K, Kämper J, Walbot V ( 2019 ) Sugar partitioning between Ustilago maydis and its host Zea mays L during infection . Plant Physiol 179 : 1373 – 1385 Google Scholar Crossref Search ADS PubMed WorldCat Varsani S , Grover S, Zhou S, Koch KG, Huang P-C, Kolomiets MV, Williams WP, Heng-Moss T, Sarath G, Luthe DS, et al. ( 2019 ) 12-oxo-phytodienoic acid acts as a regulator of maize defense against corn leaf aphid . Plant Physiol 179 : 1402 – 1415 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 This work was supported by the US National Institutes of Health (grant no. R01GM093008 to H.J.), the National Science Foundation (grant no. IOS1557812 to H.J.), and the US Department of Agriculture-National Institute for Food and Agriculture (grant no. 2019-70016-29067 to H.J.); the National Science Foundation (grant no. IOS1456047 to M.M.), the US Department of Agriculture-National Institute for Food and Agriculture (grant no. 2015-67013-23511 to M.M.), the North Central Soybean Research Program (to M.M.), and the Missouri Soybean Merchandising Council (to M.M.); the Deutsche Forschungsgemeinschaft-funded Priority Programme SPP1819 (“Rapid Evolutionary Adaptation: Potential and Constraints”; grant no. PA 861/14-1 to R.P.) and the French Agence Nationale de la Recherche-Deutsche Forschungsgemeinschaft co-funded project “X-KINGDOM-MIF” (grant no. PA 861/15-1 to R.P.); and North Central Multistate Research projects (to J.S.). 2 Author for contact: [email protected]. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00330 © 2019 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
The Immune Redoxome: Effector-Triggered Immunity Switches Cysteine Oxidation ProfilesMhamdi, Amna
doi: 10.1104/pp.19.00207pmid: 30940734
The changing climate impacts the interactions between plants and their surrounding environment, including pathogen invaders (Mhamdi and Noctor, 2016). Plant immune responses are often associated with increased production of reactive oxygen species (ROS), also known as the oxidative burst (Lamb and Dixon, 1997), leading to wide-ranging changes in leaf primary and secondary metabolism (Chaouch et al., 2012). The impact of pathogen-triggered ROS bursts on protein oxidation states is still only partly characterized, but cysteine (Cys) residues on sensitive proteins can undergo a panoply of reversible oxidations, such as sulfenylation, nitrosylation, and glutathionylation. The resulting redox modifications can affect multiple protein functions, such as catalytic activity, reactivity toward surrounding cell components, and subcellular localization. One well-studied case is the redox regulation of NONEXPRESSER OF PR GENES1 (NPR1), which is required for salicylic acid-induced PATHOGENESIS-RELATED gene expression and resistance against pathogens. Reduction of NPR1 tetramers to monomers is associated with relocalization of the protein from the cytosol to the nucleus (Mou et al., 2003), but such reductive changes may be preceded or accompanied by oxidative responses to ROS bursts. In general, how metabolic changes initiated by thiol oxidation shifts function in host defense is largely unknown. Recent evidence suggests that oxidative stress proteomes might be strongly influenced by the type of trigger and target particular metabolic processes in specific subcellular compartments (Waszczak et al., 2014; Akter et al., 2015; De Smet et al., 2019). However, defining the specific signatures that are relevant to plant defense strategies is a challenging task. In this issue of Plant Physiology, McConnell et al. (2019) explore the effects of effector-triggered immunity on sensitive protein Cys residues in plant tissues. The authors profile the redoxome in the Arabidopsis (Arabidopsis thaliana) Columbia-0 wild type as well as mutant lines defective in the two major salicylic acid-binding thimet oligopeptidases TOP1 and TOP2, which alter effector-triggered immunity (Moreau et al., 2013). This study reveals that changes in the thiol oxidation state during the immune response occur in a dynamic, wave-like pattern, with increased oxidation at 8 h after bacterial challenge and a subsequent decrease at 12 h. Moreover, the amplitude of the wave is lower in top1 top2 relative to Columbia-0, with fewer oxidized proteins and fewer oxidized Cys residues per protein. Furthermore, McConnell et al. (2019) show that Cys residues sharing similar oxidation characteristics belong to proteins acting in similar pathways and that immune responses target specific metabolic pathways such as proteasome degradation, glycolysis, and amino acid biosynthesis. This is in agreement with recent findings that oxidative stress triggered switches in chloroplastic sulfenome targets, in particular, amino acid metabolism (De Smet et al., 2019). However, it remains unclear whether these Cys residues are required for signal perception and propagation in any given biological context or whether they are simply a consequence of an upstream oxidation event. Hence, the next logical but challenging step will be to validate the biological and physiological relevance of specific oxidation events at Cys residues and reveal how they might be associated with stress resilience. Analysis of the overlap between immunity-triggered shifts in Cys oxidation and published data sets for protein oxidation triggered by intracellular oxidative stress revealed surprisingly little overlap. This may point to a specific apoplastic ROS burst signature. This specific effect is intriguing but difficult to prove within the context of available data, knowing that most of these data sets have been obtained using an array of technologies and biological materials. Another possible issue is the broad variety of oxidative modifications that Cys thiols can undergo, some of which might have been differentially enriched in some experiments compared with others. In this context, bias can be introduced if specific or inadequate enrichment occurs during the experimental procedure, artificially exposing buried thiols that are otherwise not sensitive to oxidation. Potential overlap between various redoxomes may also be minimized by recent advancements in mass spectrometry that have allowed profiling of Cys thiol oxidation using different detection methods based on multistep protocols to allow subpopulations of oxidized Cys residues to be targeted (Liu et al., 2014; Waszczak et al., 2014; Akter et al., 2015; Slade et al., 2015). A future challenge in this field is to develop breakthrough protocols that allow site-specific and quantitative assessment of redoxomes combined with a potential analysis of other posttranslational modifications on neighboring residues (e.g. phosphorylation). Finally, given the apparent complexity of the protein redox network, it will be important to produce a comprehensive picture of temporal and spatial changes that will give insights into the flow of redox signals between subcellular compartments and from the primary site of infection to adjacent cells. This might enable the identification of mobile components acting in signal transmission to fine-tune the host defense reactions. LITERATURE CITED Akter S , Huang J, Bodra N, De Smet B, Wahni K, Rombaut D, Pauwels J, Gevaert K, Carroll K, Van Breusegem F, et al. ( 2015 ) DYn-2 based identification of Arabidopsis sulfenomes . Mol Cell Proteomics 14 : 1183 – 1200 Google Scholar Crossref Search ADS PubMed WorldCat Chaouch S , Queval G, Noctor G ( 2012 ) AtRbohF is a crucial modulator of defence-associated metabolism and a key actor in the interplay between intracellular oxidative stress and pathogenesis responses in Arabidopsis . Plant J 69 : 613 – 627 Google Scholar Crossref Search ADS PubMed WorldCat De Smet B , Willems P, Fernandez-Fernandez AD, Alseekh S, Fernie AR, Messens J, Van Breusegem F ( 2019 ) In vivo detection of protein cysteine sulfenylation in plastids . Plant J 97 : 765 – 778 Google Scholar Crossref Search ADS PubMed WorldCat Lamb C , Dixon RA ( 1997 ) The oxidative burst in plant disease resistance . Annu Rev Plant Physiol Plant Mol Biol 48 : 251 – 275 Google Scholar Crossref Search ADS PubMed WorldCat Liu P , Zhang H, Wang H, Xia Y ( 2014 ) Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method . Proteomics 14 : 750 – 762 Google Scholar Crossref Search ADS PubMed WorldCat McConnell EW , Berg P, Westlake TJ, Wilson KM, Popescu GV, Hicks LM, Popescu SC ( 2019 ) Proteome-wide analysis of cysteine reactivity during effector-triggered immunity . Plant Physiol 179 : 1248 – 1264 Google Scholar Crossref Search ADS PubMed WorldCat Mhamdi A , Noctor G ( 2016 ) High CO2 primes plant biotic stress defences through redox-linked pathways . Plant Physiol 172 : 929 – 942 Google Scholar PubMed OpenURL Placeholder Text WorldCat Moreau M , Westlake T, Zampogna G, Popescu G, Tian M, Noutsos C, Popescu S ( 2013 ) The Arabidopsis oligopeptidases TOP1 and TOP2 are salicylic acid targets that modulate SA-mediated signaling and the immune response . Plant J 76 : 603 – 614 Google Scholar Crossref Search ADS PubMed WorldCat Mou Z , Fan W, Dong X ( 2003 ) Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes . Cell 113 : 935 – 944 Google Scholar Crossref Search ADS PubMed WorldCat Slade WO , Werth EG, McConnell EW, Alvarez S, Hicks LM ( 2015 ) Quantifying reversible oxidation of protein thiols in photosynthetic organisms . J Am Soc Mass Spectrom 26 : 631 – 640 Google Scholar Crossref Search ADS PubMed WorldCat Waszczak C , Akter S, Eeckhout D, Persiau G, Wahni K, Bodra N, Van Molle I, De Smet B, Vertommen D, Gevaert K, et al. ( 2014 ) Sulfenome mining in Arabidopsis thaliana . Proc Natl Acad Sci USA 111 : 11545 – 11550 Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 Author for contact: [email protected]. 2 Senior author. www.plantphysiol.org/cgi/doi/10.1104/pp.19.00207 © 2019 American Society of Plant Biologists. All Rights Reserved. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
Exchanges at the Plant-Oomycete Interface That Influence DiseaseJudelson, Howard S.; Ah-Fong, Audrey M. V.
doi: 10.1104/pp.18.00979pmid: 30538168
The microbial eukaryotes known as oomycetes comprise more than 1,500 species, including many important phytopathogens. Most exhibit filamentous growth and feed osmotrophically. Oomycetes appear fungus-like but are classified as stramenopiles along with brown algae and diatoms (Beakes et al., 2012). Unlike most fungi, oomycetes are diploid, have cell walls made primarily of cellulose and β-glucans instead of chitin, make aseptate hyphae, undergo oogamous reproduction, and produce few secondary metabolites (Fawke et al., 2015). Open in new tabDownload slide Open in new tabDownload slide Oomycetes exhibit diverse lifestyles across terrestrial and aquatic niches. While best known as pathogens of leaves, stems, roots, and fruit, some oomycetes are endophytes, infect animals, or are saprophytes (Lamour and Kamoun, 2009; Ploch and Thines, 2011; Aram and Rizzo, 2018). Many are highly host adapted, unculturable on artificial media, and grow only on living plants as biotrophs. Examples include downy mildew pathogens such as Plasmopara viticola, which infects grapevine (Vitis vinifera), and Albugo candida, which causes white rust on crucifers (Kamoun et al., 2015). The obligate pathogens typically cause minimal damage to the plant but reduce yield and raise susceptibility to secondary infection or abiotic stress. Many oomycetes are hemibiotrophs, which start infections like biotrophs but cause necrosis late in the disease cycle. Most belong to the genus Phytophthora, including Phytophthora cinnamomi, which infects hundreds of agricultural, forest, and ornamental hosts; Phytophthora infestans, which blights potato (Solanum tuberosum) and tomato (Solanum lycopersicum); and Phytophthora sojae, which colonizes soybean (Glycine max) and lupines. Some species, such as Ph. cinnamomi, shift to necrotrophy early in infection, while others, such as Ph. infestans, make the transition much later, reflecting differences in how the species balance the two trophic behaviors. Unlike many other oomycetes, Phytophthora spp. are culturable and amenable to transformation; thus, they have been the subject of many molecular studies. The largest genus of necrotrophic oomycetes, which feed on nutrients from lysed cells, is Pythium. Most members of this group are opportunistic root pathogens with broad host ranges, such as Pythium ultimum, which infects vegetables, grains, and trees (Kamoun et al., 2015). Interestingly, some Pythium spp. also are mycoparasites (Benhamou et al., 2012). Also appearing to grow as a necrotroph is Aphanomyces euteiches, which causes root rot of legumes. This review focuses on events at the plant-oomycete interface, where exchanges of host and pathogen molecules play critical roles in determining the outcome of the association (Fig. 1). Oomycete pathogens sense, bind, and absorb nutrients from their hosts and also interact with other microbes in the phyllosphere and rhizosphere. Meanwhile, plants detect and deliver defenses against infection. Plant-oomycete interfaces can be dynamic, varying with infection stage and as immune responses are deployed. Here, we discuss insights into these topics yielded by advances in cell biology, genome analysis, transcriptomics, and protein structure analysis. Figure 1. Open in new tabDownload slide Interactions at plant-oomycete interfaces. Illustrated at center left is a biotrophic infection, starting from a sporangium and involving biflagellated zoospores, an appressorium formed from a germinated cyst, a primary infection vesicle (pv), an intercellular mycelium, and a haustorium. The effects of plant signals such as isoflavones, sucrose (suc), and amino acids (aa) on spore germination and/or homing are indicated. The bacterium at top right represents the effects of the microbiome on spores, as discussed in Box 2. The oval organelle marked “sequestered nutrients” represents a starch granule; this only releases significant carbohydrate to the pathogens during necrotrophy. The turquoise pentagon represents a nutrient such as sulfate that is located primarily in a plant vacuole. Yellow stars represent apoplastic effectors such as protease inhibitors (ae) and cytoplasmic effectors such as Crinklers (C) and RXLRs (R). The latter are shown inhibiting the delivery of defense materials, such as proteases and callose, to the apoplast and EHMx by secretory or autophagosomal vesicles of a mesophyll cell. These defense responses also occur in the epidermis. Shown at top right are the initial stages of infection initiated through a stomata (gray mycelium). Shown at right is an opportunistic necrotroph (spotted mycelium) entering through a wound, feeding from a lysed cell, and exiting into soil. Lysis of the host during infection by the necrotroph occurs due to the absence of defense-suppressing effectors, ROS generation, and early expression of NLPs, as discussed in Box 3. Figure 1. Open in new tabDownload slide Interactions at plant-oomycete interfaces. Illustrated at center left is a biotrophic infection, starting from a sporangium and involving biflagellated zoospores, an appressorium formed from a germinated cyst, a primary infection vesicle (pv), an intercellular mycelium, and a haustorium. The effects of plant signals such as isoflavones, sucrose (suc), and amino acids (aa) on spore germination and/or homing are indicated. The bacterium at top right represents the effects of the microbiome on spores, as discussed in Box 2. The oval organelle marked “sequestered nutrients” represents a starch granule; this only releases significant carbohydrate to the pathogens during necrotrophy. The turquoise pentagon represents a nutrient such as sulfate that is located primarily in a plant vacuole. Yellow stars represent apoplastic effectors such as protease inhibitors (ae) and cytoplasmic effectors such as Crinklers (C) and RXLRs (R). The latter are shown inhibiting the delivery of defense materials, such as proteases and callose, to the apoplast and EHMx by secretory or autophagosomal vesicles of a mesophyll cell. These defense responses also occur in the epidermis. Shown at top right are the initial stages of infection initiated through a stomata (gray mycelium). Shown at right is an opportunistic necrotroph (spotted mycelium) entering through a wound, feeding from a lysed cell, and exiting into soil. Lysis of the host during infection by the necrotroph occurs due to the absence of defense-suppressing effectors, ROS generation, and early expression of NLPs, as discussed in Box 3. PLANTS CAN ATTRACT UNWANTED GUESTS Oomycetes employ several types of spores for dissemination and host infection (Box 1). These include both asexual and sexual spores (McCarren et al., 2005; Granke et al., 2009). Colonization by the majority of oomycetes begins when an asexual sporangium releases zoospores, which encyst and form a germ tube (Fig. 1). As discussed below, many aspects of spore behavior are influenced by plant signals. The microbiome also affects spores and can attenuate or worsen disease, as described in Box 2 (Lioussanne et al., 2008; Windstam and Nelson, 2008; Raaijmakers et al., 2010; Schlatter et al., 2017; Jack and Nelson, 2018). Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Open in new tabDownload slide Host signals can be sensed by the asexual sporangia since they are fully hydrated and metabolically active prior to germination, unlike most fungal spores, which are desiccated. While sporangia require only free water to germinate, this can be hastened by plant signals. Studies have shown that Pl. viticola releases zoospores faster on leaves than in a host-free system (Kiefer et al., 2002) and that Pythium spp. germination is accelerated by volatiles, sugars, and amino acids from seeds (Nelson, 1987). Root exudates, or sprouted potato tubers in the case of Ph. infestans, also stimulate the germination of sexual spores (oospores), which typically stay dormant in soil until a host is present (El-Hamalawi and Erwin, 1986; Pittis and Shattock, 1994). Studies with Ap. euteiches indicated that its oospores respond more to host than nonhost exudates (Shang et al., 2000). It is intriguing to consider that in the future, it may be possible to use plant signal mimics to cause oospores to undergo suicide germination before a crop is planted. Zoospores exhibit several homing responses, including chemotaxis, electrotaxis, host-triggered encystment, and germ tube tropism (Deacon and Donaldson, 1993). These contribute to host specificity, especially with root pathogens. For instance, Ap. euteiches zoospores are attracted specifically to prunetin (Sekizaki et al., 1993), while Ph. sojae responds to daizein and genistein, which are produced by their respective hosts (Hosseini et al., 2014). These isoflavones also influence encystment and germ tube orientation (Morris et al., 1998). Recent data point to a role for G-proteins in these responses. Silencing of the Ph. sojae gene encoding its G-protein α-subunit interfered with zoospore motility and chemotaxis (Hua et al., 2008), and knockdowns of a G-protein α-subunit-interacting His triad protein inhibited chemotaxis (Zhang et al., 2016). In addition, encystment was stimulated and cyst germination was impaired by knocking down the expression of a protein that consists of a G-protein-coupled receptor domain coupled to a phosphatidylinositol phosphate kinase domain (Yang et al., 2013). Oomycetes express several novel G-protein-coupled receptor-like proteins with C-terminal accessory domains (van den Hoogen et al., 2018). Pharmacological studies have shown that calcium influences most aspects of zoospore behavior. This explains the biology behind the strategy of reducing root diseases by adding gypsum (calcium sulfate) to soil, which impairs zoosporogenesis or causes encystment before a plant is reached (Mostowfizadeh-Ghalamfarsa et al., 2018). Many spore-specific calcium channels and calcium-regulated protein phosphatases and kinases have been identified, although none have been tested for function (Ah-Fong et al., 2017b). Chemotaxis also occurs in foliar pathogens, where amino acids such as Gln attract zoospores, a process that also appears to involve G-proteins (Latijnhouwers et al., 2004). Amino acid signaling may explain why zoospores of Ph. infestans and many relatives concentrate near stomata (Dale and Irwin, 1991). Few Pl. viticola zoospores were drawn to stomata closed by exogenous abscisic acid, suggesting that the attractants are soluble or volatile substomatal chemicals. Such behavior is critical to Pl. viticola, which enters leaves only through stomata (Kiefer et al., 2002). OOMYCETES ENTER PLANTS THROUGH MULTIPLE ROUTES As water molds, most oomycetes prefer to grow in moist environments such as the apoplast. Entry into the plant may occur when zoospores or germ tubes pass through stomata or other natural openings, transit through wounds, or grow between root epidermal cells. Examples include Pl. viticola, which enters through stomata, Ph. cinnamomi, which can move through peridermal gaps, and Ph. infestans, which often enters tubers through lenticels. Ph. infestans, downy mildews, white rusts, and many Pythium spp. also penetrate tissue using appressoria. These swellings form when cyst germ tubes contact hydrophobic surfaces such as the cuticle, especially if epidermal cell boundaries or their topographic mimics are sensed (Bircher and Hohl, 1997). Insight into the biology of oomycete appressoria has lagged behind that of fungi. However, a study in Ph. infestans using GFP-labeled F-actin identified an aster-like structure where appressoria contact the leaf, which may focus cargo transport to the penetration peg (Kots et al., 2017). Also, a basic leucine zipper domain transcription factor and mitogen-activated protein kinase were shown to regulate appressorium formation (Blanco and Judelson, 2005; Li et al., 2010). Genes induced in the appressorium stage by Phytophthora spp. include cell wall-degrading enzymes (CWDEs), defense-suppressing effectors, and potential adhesion proteins (Kebdani et al., 2010). Mirroring the complexity of the plant cell wall, a typical oomycete expresses CWDEs belonging to as many as 28 glycosyl hydrolase groups (Blackman et al., 2015). A typical species of Phytophthora expresses about 200 genes encoding such proteins. Some of the (hemi)cellulases are predicted to bear glycophosphatidylinositol anchors and probably serve to expand the oomycete wall, which contains mostly cellulose plus β-1,3- and β-1,6-glucans (Mélida et al., 2013). Fewer types of CWDEs are expressed by biotrophs, as in the case of Albugo laibachii, which lacks pectate lyase and pectin esterase (Kemen et al., 2011). Studies in Ph. infestans and relatives show that CWDEs are expressed in stages during sporulation, germination, and in planta growth (Kebdani et al., 2010; Blackman et al., 2015). A less ordered pattern of expression was reported for Py. ultimum, which also expressed fewer CWDEs (Ah-Fong et al., 2017b). Other differences between Phytophthora and Pythium spp. are highlighted in Box 3. The pattern of CWDE expression in Py. ultimum suggests that the enzymes of this necrotroph may be used primarily to burst host cells rather than to digest plant walls for carbon. Indeed, cellulose is a poor carbon source for most oomycetes (Zerillo et al., 2013). Perhaps advanced imaging techniques such as superresolution confocal microscopy with specific organic fluorophores could be employed to obtain information about carbohydrate structure at penetration sites, the effects of CWDEs at different stages of infection, and polymer rearrangements resulting from plant defenses. Open in new tabDownload slide Open in new tabDownload slide Most stages of infection require adherence of the pathogen to the host. Zoospores turn their ventral grooves toward the host prior to encystment, allowing vesicles to discharge a glue-like thrombospondin repeat protein toward the plant interface (Robold and Hardham, 2005). A protein containing a Sushi domain, which in animals mediates cell-cell adhesion, reaches the plant surface from other zoospore vesicles by kiss-and-run exocytosis (Zhang et al., 2013). Sticky substances also are released from germ tubes. The downy mildew Hyaloperonospora arabidopsidis was shown to secrete proteins and fibrillar β-1,3-glucans that bind its germ tubes to the substratum (Carzaniga et al., 2001). This may help resist detachment by wind or rain or protect against desiccation. Other potential adhesion proteins include mucin-like proteins (Larousse et al., 2014), jacalin-like and cellulose-binding elicitor (CBEL) lectins (Gaulin et al., 2002), and the ACWP family of acidic wall proteins (Resjö et al., 2017). Abnormal appressoria resulted from the knockdown of ACWP genes, suggesting that they contribute to adhesion or wall integrity. Knockdowns of CBEL showed that it helped hyphae bind to cellulosic substances but was not essential for pathogenicity (Gaulin et al., 2002). One of the few oomycete proteins known to concentrate in haustoria, Hmp1, is membrane anchored and weakly resembles lectins. Silencing Hmp1 in Ph. infestans impaired the formation of infection vesicles in epidermal cells and haustoria, suggesting that the protein helps the pathogen bind to host interfaces (Avrova et al., 2008). PLANTS CAN DETECT OOMYCETES AND BRING DEFENSES TO THE INTERFACE Plants have evolved sophisticated systems for detecting microbes. One involves the binding of pathogen-associated molecular patterns (PAMPs) to plasmalemma-spanning pattern recognition receptors (PRRs), which activates PAMP-triggered immunity (PTI; Saijo et al., 2018). The salicylic acid (SA) and jasmonic acid pathways both participate in PTI against necrotrophic and (hemi)biotrophic oomycetes (Halim et al., 2009). Disrupting jasmonate production in Arabidopsis (Arabidopsis thaliana) allowed Pythium irregulare, which typically infects wounded or otherwise compromised hosts, to become a more severe pathogen (Staswick et al., 1998). Constituents of the cell wall or plasma membrane were among the first oomycete PAMPs to be identified. These include β-1,3- and β-1,6-glucans and arachidonic acid (Fawke et al., 2015; Robinson and Bostock, 2015). Medicago truncatula also responds to a chitosaccharide from Ap. euteiches; most other oomycete phytopathogens lack this PAMP, since they do not make chitin (Mélida et al., 2013; Nars et al., 2013). Proteinaceous PAMPs include a cell wall transglutaminase (Brunner et al., 2002), the glycosyl hydrolase domain of the oligopeptide elicitor (OPEL) protein (Chang et al., 2015), the cellulose-binding protein CBEL, the elicitin family of sterol-binding proteins (Takenaka et al., 2011; Derevnina et al., 2016), and the XEG1 endoglucanase (Wang et al., 2018). The latter are proposed to be used for sterol acquisition by Phytophthora and Pythium spp., which are sterol autotrophs. It is notable that Nep1-like proteins (NLPs) were classified recently as PAMPs by some researchers. Most NLPs in Phytophthora spp. are expressed late in infection and have been linked to necrotrophic growth (Feng et al., 2014). Analysis of crystal structures identified similarity with pore-forming cytotoxins of sea anemones, which suggests that NLPs destabilize the host plasmalemma (Lenarčič et al., 2017). Oomycetes are immune to NLPs, since the latter are specific for dicotyledonous sphingolipids. An NLP from Phytophthora parasitica was shown to elicit defenses in crucifers, which suggests that some NLPs affect plant cells both as pore-forming toxins and inducers of PTI (Böhm et al., 2014). Receptors for three oomycete PAMPs are known. The infestin elicitin of Ph. infestans and related proteins are recognized in potato by elicitin response protein (ELR), a plasmalemma-associated factor that associates with SUPPRESSOR OF BIR1-1 (SOBIR1), which is a leucine-rich repeat (LRR) receptor kinase (Domazakis et al., 2018). This pairing is needed since ELR lacks an intracellular kinase domain. When infestin is detected, the ELR-SOBIR1 complex recruits the LRR receptor-like kinase BRI1-ASSOCIATED KINASE-1, which is a known hub in defense responses. SOBIR1 also participates in the reaction of Arabidopsis to NLPs, which are recognized by the LRR receptor RLP23 (Albert et al., 2015). Recently identified was Response to XEG1 (RXEG1), an LLR protein that recognizes XEG1, a glycoside hydrolase 12 endoglucanase that is made by Phytophthora spp. RXEG1also forms a complex with BRI1-ASSOCIATED KINASE-1 and SOBIR1 to transduce the defense signal (Wang et al., 2018). Interestingly, fungal glycoside hydrolase 12 proteins also have been shown to serve as PAMPS and act through the same signaling hub (Gui et al., 2017). Once PTI is activated, defense molecules are delivered to plant-oomycete interfaces, including pathogenesis-related (PR) proteins, callose for thickening cell walls, and microbial toxins. Effector-triggered immunity reinforces and expands these responses and often leads to hypersensitive cell death. Since PTI and effector-triggered immunity are not oomycete specific, readers seeking more information are directed to other reviews (Kourelis and van der Hoorn, 2018; Saijo et al., 2018). However, oomycetes were used in many early studies of the cytoskeletal dynamics that occur during infection, which showed that plant actin microfilaments focused rapidly near penetration sites (Takemoto et al., 2003). This causes peroxisomes, nuclei, and the endomembrane transport network to move toward the infection, which may help deliver defenses (Li and Staiger, 2018). Some (hemi)biotrophic oomycetes have evolved counter defenses against these trafficking pathways and may have hijacked some to support haustoria. While the delivery of proteases, glucanases, and callose to oomycete-plant interfaces through canonical secretory and exocytosis pathways is long established, autophagic vesicles were shown recently to surround Ph. infestans haustoria and also may convey defenses (Dagdas et al., 2018). It is unknown whether plants use exosomes against oomycetes, for example by transporting inhibitory small RNAs, as shown recently with fungi (Cai et al., 2018). Nevertheless, there are reports of lettuce (Lactuca sativa) and potato being engineered to resist Bremia lactucae and Ph. infestans by host-induced gene silencing using small RNAs targeting oomycete genes (Govindarajulu et al., 2015; Jahan et al., 2015). Reactive oxygen species (ROS) are delivered to plant-oomycete interfaces through several pathways. ROS are derived from wall-bound peroxidases, respiratory burst oxidase homologs in plasmalemma, and glycolate oxidase in peroxisomes, which move to infection sites during cytoskeletal remodeling (Marino et al., 2012). ROS from tobacco (Nicotiana tabacum) roots have been implicated in blocking infection by Ph. parasitica zoospores, which interestingly die through programmed cell death (Galiana et al., 2005). Besides being antimicrobial, ROS strengthens cell walls by initiating lignin polymerization (Barros et al., 2015). Several other enzymes that fortify plant walls also are induced during PTI against oomycetes, including cinnamyl alcohol dehydrogenase and callose synthase (Wang et al., 2013; Hosseini et al., 2015). Toxic isoflavonoids, sesquiterpenes, polyacetylenes, and other molecules that are collectively named phytoalexins are believed to be delivered to the pathogen by ATP-binding cassette (ABC) transporters. The export of capsidiol during the elicitin-triggered defense response of Nicotiana benthamiana against Ph. infestans involves ABCG1 and ABCG2, which are up-regulated during PTI (Rin et al., 2017). Some phytoalexins, such as α-tomatine of tomato, are preformed in plants, while others are induced by infection, such as capsidiol of Nicotiana spp. and pepper (Capsicum annuum), glyceollin of soybean, and camalexin of crucifers (Hahn et al., 1985; Bednarek et al., 2005). These defenses may combine to produce apoplastic (or intracellular) environments that are unfavorable to oomycetes. This may explain why necrotrophy begins earlier in some Phytophthora-plant pathosystems than others, although the water soaking that is often associated with plant cell death may keep the pathogen hydrated. The low level of free water resulting from silicon polymerization in the apoplast also was invoked to explain why soybean grown at high silicon concentrations was less susceptible to Ph. sojae (Rasoolizadeh et al., 2018). MANY OOMYCETES HAVE EVOLVED ELABORATE COUNTER DEFENSES Oomycetes exhibit stealthy behaviors during biotrophic growth that minimize the immune response and maintain host integrity, which helps these pathogens feed from living cells. This is not an issue for necrotrophs such as Pythium spp. (Box 3). The (hemi)biotrophs resist host defenses using cytoplasmic and apoplastic effector proteins that are secreted toward their interface with plants. Oomycetes also produce enzymes that may degrade phytoalexins or immune-response hormones. The existence of these enzymes and effectors highlights the power of selection in the pathogen and the importance of their plant targets to the host defense response. One example involves the plant apoplastic Cys proteases Rcr3 and C14, which were shown by mutation and knockdowns in tomato and N. benthamiana to defend against Ph. infestans (Song et al., 2009; Kaschani et al., 2010). Ph. infestans and relatives antagonize these using effectors such as extracellular cystatin-like protease inhibitor 1 (EPIC1). Studies of EPIC1 from Ph. infestans and Phytophthora mirabilis (which infects Mirabilis jalapa) and the host proteases were performed, guided by the crystal structure of a related protease-inhibitor complex. Amino acid changes in EPIC1 were implicated in helping the pathogens jump to new host species (Dong et al., 2014). Orthologs of EPIC1 genes occur in downy mildew, white rust, and Pythium spp. genomes. Most oomycetes also can inhibit plant Ser proteases, and one from Phytophthora palmivora was shown to contribute to virulence against the rubber tree (Hevea brasiliensis; Ekchaweng et al., 2017). Another example of host-pathogen coevolution in the apoplast comes from studies of the endoglucanase XEG1 from Ph. sojae. Soybean produces an inhibitor of this CWDE, which binds XEG1 and blocks its contribution to virulence. To counteract the plant, Ph. sojae secretes an inactive enzyme as a decoy (Ma et al., 2017). With the defense protein unproductively bound to this trap, Ph. sojae can invade soybean more easily. Orthologs of XEG1 and its decoy are conserved throughout the Phytophthora genus. Another apoplastic effector that counteracts host defenses is the in planta-induced protein (IPI-O) of Ph. infestans. IPI-O contains an Arg-Gly-Asp motif that is believed to disrupt adhesion between the plant’s cell wall and plasmalemma by binding the lectin receptor kinase LecRK-I.9, thus promoting disease by reducing wall integrity (Bouwmeester et al., 2011). Intriguingly, IPI-O contains an RxLR motif (Arg-x-Leu-Arg) that is shared by many oomycete effectors that enter plant cells to interfere with plant defenses. In planta expression of IPI-O minus its signal peptide caused expanded lesions, suggesting that IPI-O acts both at the host plasmalemma and intracellularly (Chen and Halterman, 2017). The intracellular target, apparently, is resistance protein Rpi-blb1 (Champouret et al., 2009). RxLRs along with CRN (Crinkler) proteins represent the known cytoplasmic effectors of oomycetes. These are absent from Pythium spp. (Box 3) but are encoded by large gene families in Phytophthora spp., downy mildews, and white rusts, albeit with divergent signature motifs in some species (Kemen et al., 2011). How these proteins move into plants is not fully clarified, but RxLR uptake may involve binding a receptor on lipid rafts, as shown for a host-targeted protein from the animal pathogenic oomycete Saprolegnia parasitica (Trusch et al., 2018). RxLRs and CRNs are known to defeat plant immune responses through many routes, which include reprogramming host gene expression, altering RNA metabolism, and binding to host proteins involved in signaling (Wang and Wang, 2018). In this review, mention will be made only of RxLRs that act at the oomycete-plant interface. Many RxLRs affect the trafficking of defense molecules. AVRblb2 accumulates in plants near haustoria and blocks the secretion of C14 protease (Bozkurt et al., 2011). RxLR24 of Phytophthora brassicae interferes with the delivery of antimicrobial proteins such as PR-1 by attaching to a GTPase involved in exocytosis (Tomczynska et al., 2018). Trafficking also is blocked by Avr1 of Ph. infestans, which binds exocyst protein SEC5 (Du et al., 2015), REX3 of Ph. palmivora, which interferes with brefeldin-sensitive secretion (Evangelisti et al., 2017), and PexRD54 of Ph. infestans, which depletes the Joka2 cargo protein from the autophagosomal membrane-forming ATG8 complex (Dagdas et al., 2016). The latter is interesting since the pathogen may be hijacking autophagosomes to destroy defense molecules through selective autophagy. Although their functions are unidentified, the RxLRs Avh241 of Ph. sojae and HaRxL77 of H. arabidopsidis localize to the plant plasmalemma and are hypothesized to bind PRRs at the plant-oomycete interface (Caillaud et al., 2012; Yu et al., 2012). Causing auxin levels to rise at the interface is Penetration Specific Effector1 of Ph. parasitica, which is made in appressoria and interferes with auxin carriers (Evangelisti et al., 2013). This may elevate plant susceptibility since auxin inhibits SA signaling. Interestingly, some species of Pythium are known to produce the auxin indole-3-acetic acid (Gravel et al., 2007). While there is no proof that oomycetes make other plant hormones, the sunflower (Helianthus annuus) downy mildew Plasmopara halstedii apparently encodes all enzymes for synthesizing cytokinin, which some bacteria make to direct host nutrients to infection sites (Sharma et al., 2015). Pl. halstedii also seems capable of producing brassinolide from phytosterols, which would negatively regulate the immune response. Many oomycetes also encode a predicted isochorismatase, which may disrupt SA signaling by breaking down its precursor. Interestingly, the enzyme in Ph. sojae localizes to haustoria (Liu et al., 2014). Unlike fungi, most oomycetes have a limited capacity to degrade phytoalexins. Perhaps to compensate, oomycetes encode many more ABC transporters, which may expel the toxins (Ah-Fong et al., 2017b). While many fungi can degrade α-tomatine, Phytophthora and Pythium spp. that are pathogenic on tomato fail to degrade this glycoalkaloid (Sandrock and Vanetten, 1998). Ph. sojae can break down some soybean phytoalexins but not the most bioactive, glyceollin (Stossel, 1983). Whether Pythium spp. have special mechanisms to counteract plant defenses is unknown. However, during tuber infection, mRNA levels of Py. ultimum ABC transporters were about twice those of their counterparts in Ph. infestans, suggesting that the transporters might help eliminate phytoalexins liberated from lysing cells (Ah-Fong et al., 2017b). Cytochrome P450 enzymes also were more highly expressed in Py. ultimum. HAUSTORIA REPRESENT A SPECIALIZED INTERFACE Biotrophic and hemibiotrophic oomycetes form intimate associations with their hosts using haustoria (Fig. 1). These specialized hyphae breach host cell walls and become enveloped by a host membrane called the extrahaustorial membrane (EHM). Between the haustorium and EHM is a carbohydrate-rich amorphous layer called the extrahaustorial matrix (EHMx), which likely is of plant and pathogen origin (Caillaud et al., 2014). Little is known about how haustoria form and function in oomycetes, including how the host machinery is coopted during their genesis and what limits their expansion; most haustoria are less than 25 µm long. Recent studies with Ph. infestans and H. arabidopsidis indicated that the EHM is assembled de novo, as suggested for fungi (Lu et al., 2012; Bozkurt et al., 2015). Secretory vesicles are abundant near developing haustoria, along with trans-Golgi and late endosomal markers such as Rab5 and Rab7 GTPases (Caillaud et al., 2014; Inada et al., 2016). Within the EHM are plasmalemma proteins such as the Pen1 syntaxin, synaptotagmin, and remorin, which would be needed to deliver membrane material to growing haustoria. Some plant proteins are excluded from the EHM, including a calcium ATPase and at least some PRRs (Lu et al., 2012). Reduced ATPase activity could favor nutrient flow to the pathogen by reducing the plant’s capacity to retrieve nutrients from the EHMx, while PRR exclusion may minimize defense responses. Haustoria accommodation also causes host cells to reorganize their contents, with changes including endoplasmic reticulum aggregation, Golgi accumulation, and nuclear migration toward the haustoria (Lu et al., 2012). The nuclear shift might be part of a defense response or may be induced by the pathogen to facilitate the transport of CRN effectors, many of which act by reprogramming transcription (Song et al., 2015). Whether the reorganized endomembrane system delivers more transporters and/or nutrients to the EHM is an interesting question. There are dissimilarities between haustoria of different species. While haustoria made by Phytophthora spp. are typically short and finger-like, those of downy mildews and white rusts are bulbous. Moreover, while Ph. infestans haustoria are anucleate and contain few mitochondria and endoplasmic reticulum, those of H. arabidopsidis have nuclei and many mitochondria and Golgi bodies (Mims et al., 2004). While the FLS2 PRR was excluded from the EHM with Ph. infestans, this was not the case with H. arabidopsidis (Lu et al., 2012). Downy mildew haustoria also are more likely to be surrounded by a callose collar than those of Phytophthora spp. Several aspects of haustoria formation resemble plant defense responses. Deposition of the β-glucans that form callose collars involves secretory vesicles, multivesicular bodies, and Plasmodesmata-located-protein1 (PDLP1), which also is used to seal plasmodesmata during infection by other pathogens (Caillaud et al., 2014). Also possibly related to defense are haustorial encasements, which are double-layered membranes that often surround older haustoria (Lu et al., 2012). These are common with H. arabidopsidis but are seen less with Ph. infestans. Encasements might restrict the pathogen’s uptake of nutrients, impair effector translocation, or concentrate plant-derived antimicrobials. The EHM appears to have small invaginations, which also may promote its stability (Mims et al., 2004). The formation of these convolutions appears to involve PDLP1, since they increased when PDLP1 was overexpressed (Caillaud et al., 2014). Although the contribution of oomycete haustoria to nutrient uptake is unclear, as discussed in the next section, the role of this structure in transporting proteins to the EHMx is demonstrated. Effectors are discharged from haustoria through at least two mechanisms. Secretion of the EPIC1 protease inhibitor was blocked by brefeldin A, indicating that it reaches the apoplast by the classic Golgi-mediated pathway (Wang et al., 2017). However, the delivery of RxLR Pi04314 was brefeldin A insensitive, suggesting that this cytoplasmic effector follows an alternative route even though it contains a classic signal peptide. Isochorismatases also are secreted but lack signal peptides, suggesting that they use the unconventional secretion pathway that has been documented in nonoomycetes (Liu et al., 2014). NUTRIENT ACQUISITION AT THE PLANT-OOMYCETE INTERFACE Although not proven, oomycete haustoria often are assumed to play a major role in nutrition. Nevertheless, they lack the neckband that encircles fungal haustoria, which is thought to help establish electrochemical gradients for nutrient transport by sealing the EHMx (Mims et al., 2004). Al. candida and H. arabidopsidis contain an electron-dense layer near their callose collars, which might function like a neckband, however (Soylu, 2004). In Ph. infestans, EHMx continuity with the apoplast was confirmed by studying the distribution of fluorescently tagged Avr3a (Whisson et al., 2007). Also unlike fungi, no haustoria-specific transporters are identified in oomycetes. While Ph. infestans and Py. ultimum both express ∼410 nutrient transporters, very few are specific to the haustoria-forming species (Ah-Fong et al., 2017b). Most nutrients may be drawn from the apoplast, considering that analyses of images of potato leaves infected by Ph. infestans indicate that its haustoria represent only about 2% of the total pathogen surface area (H. Judelson, unpublished data). Regardless of where nutrients are acquired, plants contain myriad compounds to support pathogen growth. Metabolic models based on genome data indicate that most oomycetes can utilize the major plant hexoses, disaccharides, organic acids, starch, and sugar alcohols, although pentose utilization is restricted by the absence of arabinose isomerase (Rodenburg et al., 2018). Most oomycetes also can use the major nitrogen sources found in planta, including amino acids, ammonium, and nitrate. However, the biotrophs have reduced metabolic capabilities and, thus, a greater reliance on the host. While species of Phytophthora and Pythium each encode approximately 850 enzyme activities based on Enzyme Commission numbers, H. arabidopsidis and Al. candida encode only about 740 and 650, respectively (Judelson, 2017). These obligate biotrophs lack genes for nitrate assimilation and have impaired abilities to incorporate inorganic sulfur due to a lack of sulfite oxidase or reductase. The metabolic deficiencies in the (hemi)biotrophs may help suppress immune responses, besides providing potential energy savings to the pathogen. The biotrophs are unable to make unsaturated fatty acids such as arachidonate, which are PAMPs in Phytophthora and Pythium spp. (Robinson and Bostock, 2015). The haustoria-forming oomycetes lack molybdopterin-utilizing pathways and consequently must acquire thiamine from the host. This may be beneficial, since this vitamin can up-regulate plant defenses, as demonstrated in the Pl. viticola-grape system (Boubakri et al., 2013). It is important to differentiate the theoretical metabolism of oomycetes from what occurs in planta, since not all nutrients are at each plant-oomycete interface. While biotrophs are restricted to apoplastic nutrients, necrotrophs can access all compounds. Examples include starch and sulfate, which are sequestered within starch granules and vacuoles, respectively. Data from a study of Ph. infestans and Py. ultimum on potato tubers (Ah-Fong et al., 2017b) showed that while both encode α-amylase for starch utilization and adenylyl-sulfate kinase for incorporating sulfate, the Py. ultimum genes were expressed at greater than 10-fold higher levels (Box 3). This is the logical outcome of substrate-level induction. This situation changed during late infection when tissue colonized by Ph. infestans became necrotic, and mRNA levels for these enzymes equalized between the two pathogens. Similar patterns were observed for enzymes that act on other nutrients sequestered during biotrophic growth, such as phytate and lipids. This indicates that the terminal lifestyle of Ph. infestans is necrotrophic and not just necrogenic, thus addressing a debate in Phytophthora-host interactions. One position has been that plant necrosis does not benefit the pathogen and occurs simply because the pathogen no longer needs to suppress host defenses. The other viewpoint, which is supported by these results, is that necrosis is induced to liberate additional nutrients. OOMYCETES HAVE AN EXIT STRATEGY The final chapter in disease involves the pathogen’s egress from its host. Necrotrophs such as Pythium spp. can simply extend hyphae from macerated plant tissue into soil and transition to survival through saprophytic growth; sporulation is optional. In contrast, most (hemi)biotrophs must produce asexual spores. These are typically formed by root-rotting species at the crown, surface-exposed roots, or subterranean spaces adjoining roots. The task is more complicated for foliage-infecting (hemi)biotrophs, which usually sporulate from sporangiophores that pass through stomata (Farrell et al., 1969; Allègre et al., 2007). Most foliage-infecting oomycetes sporulate at night. This maximizes survival of the spores, which are prone to desiccation and lack UV-blocking pigments. Nocturnal sporulation is proposed to be regulated by cryptochromes in response to blue light (Xiang and Judelson, 2014) and requires modulating guard cell behavior, since stomata would normally be closed at night. Stomatal deregulation in the Pl. viticola-grape leaf interaction was proposed to be determined by a secreted glycoprotein, which caused stomata in colonized areas to remain open during darkness, water stress, and abscisic acid treatment (Allègre et al., 2007; Guillier et al., 2015). This effect resembles that caused by the bacterial toxin coronatine (Melotto et al., 2006). Substantial genomic resources are devoted to sporulation. In Ph. infestans, this involves the up-regulation of more than 3,000 genes (∼20% of the total), including those encoding storage, effector, and adhesion proteins, and several hundred components of flagella (Judelson et al., 2012; Ah-Fong et al., 2017a). Genes proven to regulate sporulation include MADS box and Myb transcription factors, a mitogen-activated protein kinase, and a cell cycle phosphatase (Ah-Fong and Judelson, 2011; Li et al., 2014; Xiang and Judelson, 2014). However, the primary trigger for sporulation is unknown. Nutrient limitation is thought to play a role, which is concordant with the finding that the nitrogen metabolite repression regulator NMRA is down-regulated near the onset of sporulation in Ph. infestans (Ah-Fong et al., 2017a). NMRA also was proposed to control the transcription of late-induced effectors in Phytophthora capsici (Pham et al., 2018). Spiking at the same time are mRNAs for genes used to assimilate nitrate, which is a nonpreferred nitrogen source compared with amino acids (Abrahamian et al., 2016). Since a study in Phytophthora cactorum found that adding amino acids or ammonium to media did not retard sporulation, the process also may be prompted by an accumulated metabolite (Elliott, 1989). The plant also may affect sporulation, since its metabolic pathways are linked to those of the pathogen during colonization. A dual-species systems approach to metabolism may help understand what influences sporulation, effector expression, phytohormone levels, and other aspects of plant-oomycete interactions. Open in new tabDownload slide Open in new tabDownload slide CONCLUSION Oomycetes have developed diverse lifestyles over their 400+ million years of evolution (Taylor et al., 2006). The (hemi)biotrophs have learned to coopt their hosts by suppressing defenses and coercing plants to form interfaces for effector and nutrient delivery. Such lifestyles may have evolved by exploiting pathways used by plants to harbor mutualists, since mutants of M. truncatula deficient in mycorrhizae formation were shown to have reduced susceptibility to Ph. palmivora (Rey et al., 2015). Many oomycetes have become host-adapted to the extent that they depend on plant metabolites for growth or reproduction. In contrast, necrotrophs have less-specialized lifestyles, as they can grow as saprophytes or pathogens, overpowering their hosts and perhaps even profiting from the plant immune response. Most oomycetes also have retained a flagellated life stage, which expands their access to new hosts, although this comes with a large genomic burden. Meanwhile, plants have evolved complex multilayered defenses that balance survival against the growth penalty that comes from activating the immune response (Ning et al., 2017). Many of the defenses, counter defenses, spore behaviors, and interactions with other microbes that we have described have small individual effects on disease outcomes but are significant from an epidemiological perspective. The infection potential of an oomycete spore on plant tissue is usually much less than 100%, similar to the situation in fungi (Mellersh and Heath, 2002; Kong and Hong, 2016). The progress of an epidemic will be influenced by factors that raise or lower this infection potential or the time between penetration and sporulation (Willocquet et al., 2017). While many plant scientists aim to develop strong resistance against pathogens, natural defenses (as well as changes in pathogens that enhance fitness) need not have blockbuster effects to be retained during evolution. Our knowledge of interactions involving Phytophthora spp. has grown dramatically due to the availability of genome sequences and tools for functional genomics, but research into other oomycetes has lagged. It is unfortunate that Pythium spp. have remained little studied despite their large agricultural impact, for example. Most Pythium spp. are easily cultured, so it should be possible for a new generation of researchers to improve our understanding of the genus. Studying the breadth of oomycetes is important since crop protection solutions developed for one group may not translate to others. LITERATURE CITED Abrahamian M , Ah-Fong AM, Davis C, Andreeva K, Judelson HS ( 2016 ) Gene expression and silencing studies in Phytophthora infestans reveal infection-specific nutrient transporters and a role for the nitrate reductase pathway in plant pathogenesis . PLoS Pathog 12 : e1006097 Google Scholar Crossref Search ADS PubMed WorldCat Ah-Fong AM , Judelson HS ( 2011 ) New role for Cdc14 phosphatase: Localization to basal bodies in the oomycete Phytophthora and its evolutionary coinheritance with eukaryotic flagella . 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Phytoparasitic Nematode Control of Plant Hormone PathwaysGheysen, Godelieve; Mitchum, Melissa G.
doi: 10.1104/pp.18.01067pmid: 30397024
Plant-parasitic nematodes are microscopically small animals that cause global annual crop losses of at least 80 billion dollars (Nicol et al., 2011). The evolution of nematodes into plant parasites occurred several times, resulting in diverse interaction modes with the plant (Smant et al., 2018). We will focus this review on the sedentary cyst nematodes (CN) and root-knot nematodes (RKN), as they are the foremost studied due to their economic importance (Jones et al., 2013) and fascinating liaison with plants in the form of nematode feeding sites (Box 1). Open in new tabDownload slide Open in new tabDownload slide Figure 1. Open in new tabDownload slide Plant-parasitic nematodes and their impact on plant gene regulation. A, Two females of the beet CN H. schachtii feeding on Arabidopsis roots. Picture courtesy of Anju Verma. B, Female of the rice RKN M. graminicola on rice roots, with egg mass, stained pink with acid fuchsin staining. Picture courtesy of Zobaida Lahari. C and D, GUS assays on Arabidopsis roots infected with H. schachtii showing up-regulation of the DR5-promoter (blue color; C) at 24 h after inoculation and the IAA14-promoter (D) at 2 d post inoculation. Black arrows point to nematode heads. Reprinted with permission from Grunewald et al., 2008. E, Up-regulation of the LAX3-promoter after H. schachtii infection in Arabidopsis roots with LAX3-YFP construct: (left) fluorescence image; (right) brightfield image. Pictures courtesy of Chris Lee. Figure 1. Open in new tabDownload slide Plant-parasitic nematodes and their impact on plant gene regulation. A, Two females of the beet CN H. schachtii feeding on Arabidopsis roots. Picture courtesy of Anju Verma. B, Female of the rice RKN M. graminicola on rice roots, with egg mass, stained pink with acid fuchsin staining. Picture courtesy of Zobaida Lahari. C and D, GUS assays on Arabidopsis roots infected with H. schachtii showing up-regulation of the DR5-promoter (blue color; C) at 24 h after inoculation and the IAA14-promoter (D) at 2 d post inoculation. Black arrows point to nematode heads. Reprinted with permission from Grunewald et al., 2008. E, Up-regulation of the LAX3-promoter after H. schachtii infection in Arabidopsis roots with LAX3-YFP construct: (left) fluorescence image; (right) brightfield image. Pictures courtesy of Chris Lee. Nematodes establish feeding sites by recruiting specific plant developmental pathways, involving hormonal cross talk. At the same time, nematodes need to suppress plant defense and its interacting hormone pathways. This interface between development and defense results in a complex pattern in which it is difficult to unravel the specific roles of different plant hormones. Therefore, we present a simplified model for describing the roles of hormones in plant-nematode interactions in this review. Auxin, as the key regulator, and cytokinin, as its modulator, are the primary plant hormones involved in cell division and differentiation (Benková et al., 2003; Pernisová et al., 2009). Jasmonate (JA) and salicylate (SA) are the principal plant defense hormones (Mur et al., 2006). Other hormones modulate and cross talk with these principal hormones and with each other, and can have different effects depending on the specific host-nematode interaction. Besides the classical phytohormones, small, secreted peptide hormones shape plant development; remarkably, nematodes have evolved plant peptide hormone (PPH) effector mimics to facilitate parasitism. Recent studies have revealed a diversity of these peptides, which we will discuss in the latter half of this review. Open in new tabDownload slide Open in new tabDownload slide NEMATODES MANIPULATE PHYTOHORMONE PATHWAYS FOR FEEDING SITE FORMATION Auxin is Key to NFS Formation Auxin, or indole-3 acetic acid (IAA), is a key regulator of plant organogenesis. Hence, it is not surprising that the initiation and maturation of NFS is associated with local accumulation of auxin (Karczmarek et al., 2004). Congruently, auxin mutants are significantly less susceptible to both CN and RKN (for review, see Grunewald et al., 2009b; Gleason et al., 2016). Auxin could be underpinning many of the changes occurring during feeding site development, such as hypertrophy, cell wall ingrowths, and cell cycle activation (de Almeida Engler et al., 1999). Auxin is known for its role in cell expansion via the up-regulation of cell wall-modifying proteins and plasma membrane proton pumps that regulate acid growth (Majda and Robert, 2018). During transfer cell formation, auxin and ethylene (ET) cooperatively bring about the development of cell wall ingrowths (Yuan et al., 2016). In addition, auxin is not only an important trigger for cell-cycle entry but also acts in various other cell cycle phases (Perrot-Rechenmann, 2010). Transcriptome and promoter-reporter analyses of NFS reveal a complex temporal and spatial pattern of up- and down-regulation of auxin biosynthesis and signaling-related genes and miRNAs influencing mRNA stability (e.g., Ithal et al., 2007; Barcala et al., 2010; Ji et al., 2013; Hewezi et al., 2014; Cabrera et al., 2015, 2016). Soon after nematode infection, auxin biosynthesis and auxin-response genes are mainly up-regulated, whereas genes encoding repressors are turned off, supporting a role for auxin at this early stage of infection. The accumulation of auxin at the initiating NFS could be due to secretion by the nematodes, locally induced plant biosynthesis, and changes in auxin transport. Auxin, mainly in its conjugated form, has been detected in RKN (Meloidogyne incognita) and beet CN (Heterodera schachtii) secretions (De Meutter et al., 2005), but its impact on NFS formation is unknown. The role of auxin transport during nematode infection of Arabidopsis (Arabidopsis thaliana) roots has been established by analyses of the involved genes at the levels of expression, protein localization, and mutant phenotypes (Grunewald et al., 2009a; Lee et al., 2011; Kyndt et al., 2016). A substantial amount of auxin is produced in plant shoots, with polar auxin transport generating morphogenic auxin gradients. An interacting network of influx (AUXIN/LIKE AUXIN [AUX/LAX]) and efflux (PIN-formed [PIN]) transmembrane proteins with temporally and spatially adjusted subcellular locations mediate this auxin flow (Vieten et al., 2007). The AUX1 and LAX3 influx proteins appear to be essential to the formation of galls and syncytia, as the expression of corresponding genes is strongly up-regulated in the early infection stages, and mutants are less susceptible to infection. PIN4 is needed for proper expansion of both syncytia and galls, with pin4 mutants resulting in the development of smaller cysts (Grunewald et al., 2009a; Kyndt et al., 2016). However, the contributions of some other PIN proteins appear to be quite different between the two types of feeding sites. PIN1 is necessary for delivering auxin from the shoots to the initiating syncytium, where its expression is strongly down-regulated to prevent the pumping out of auxin. Inside syncytia, PIN3 is relocated from the basal to the lateral plasma membranes to redirect auxin to the neighboring cells, stimulating the radial expansion of the syncytium. While a pin1 mutant has fewer and smaller cysts than wild-type plants, the pin3 mutation only affects syncytium and female cyst size (Grunewald et al., 2009a). In contrast to syncytia, giant cells express PIN1. The pin1 mutants only show a slightly reduced number of galls and no difference in nematode development. In contrast to CN, PIN2 and PIN3 appear to be more important for the delivery of auxin into the initiating giant cells induced by RKN, as illustrated by a nearly halved number of galls in the pin2 and pin3 mutants but no difference in female development (Kyndt et al., 2016). How do nematodes manipulate auxin transport and signaling to provoke the necessary changes for NFS development? For the CN H. schachtii, two effector proteins have been pinpointed as facilitators of auxin effects in syncytia. The effector 19C07 targets the Arabidopsis LAX3 auxin import protein, possibly increasing its activity and thus enhancing auxin influx into the syncytium and adjacent cells (Lee et al., 2011). The effector 10A07 interacts with the auxin regulator protein INDOLEACETIC ACID-INDUCED16 (IAA16) from Arabidopsis (Hewezi et al., 2015). AUX/IAA constitute a gene family of 29 members in Arabidopsis that negatively regulate the auxin response factors (ARFs). Upon removal of specific AUX/IAA by the proteasome, the corresponding ARFs can activate auxin-responsive genes (Chapman and Estelle, 2009). Therefore, binding of 10A07 to IAA16 could prevent it from repressing auxin response genes. Congruently, plants overexpressing 10A07 show enhanced expression of ARF6-8 and 19 and are more susceptible to H. schachtii than control plants. Unexpectedly, however, overexpression of IAA16 has similar effects, indicating a more complex regulation than anticipated from an IAA repressor. Cytokinins Modulate NFS Formation Cytokinins are N6-substituted adenine derivatives that, in concert with auxin, control cell division and differentiation in plants (Schaller et al., 2014; Di Mambro et al., 2017). Cytokinins are critical for cell cycle control, and the timing and amplitude of their oscillating levels may be important for the decision of cells to go into mitosis or endoreduplication. Cytokinins delay senescence and convert tissues into sinks by modulating nutrient translocation. Due to their involvement in cell cycle control and nutrient mobilization, cytokinins have long been assumed to play a role in NFS development. De Meutter et al. (2003) detected cytokinins in secretions from the CN H. schachtii and the RKN M. incognita. For H. schachtii, this finding was corroborated with the identification of a cytokinin-synthesizing nematode gene being expressed in the early infection stages (Siddique et al., 2015); silencing of this gene results in reduced infectivity by the nematode. On the other hand, cytokinin biosynthesis Arabidopsis mutants show significantly smaller syncytia compared to wild-type plants (Siddique et al., 2015). This observation implies that both plant- and nematode-produced cytokinins are needed for the optimal formation of syncytia. Such detailed analyses have not been performed for RKN, but a similar scenario is very likely. Cytokinin signaling mutants and plants with reduced cytokinin levels are less susceptible to both types of nematodes (Lohar et al., 2004; Siddique et al., 2015; Shanks et al., 2016; Dowd et al., 2017). Nevertheless, expression of cytokinin biosynthesis, signaling, and catabolism genes is different in syncytia and galls (Dowd et al., 2017), which could be underlying divergent types of cell cycle progression. This hypothesis was confirmed by the analyses of cytokinin perception mutants, demonstrating that Ahk4 is the main Ahk gene (coding for Arabidopsis His kinases) involved in syncytium development, while Ahk2 and Ahk3 are important for gall formation (Siddique et al., 2015; Dowd et al., 2017). Comparing gene expression of young syncytia and galls with callus, Cabrera et al. (2015) found that, due to a higher cytokinin/auxin ratio, syncytia resemble shoot-forming calli and galls are similar to solid callus. However, it is still unknown how cytokinin signaling relates to the different abnormal cell cycles in syncytia and giant cells. ET has Diverse Roles in Plant Susceptibility to CN and RKN ET (H2C = CH2) is a gaseous hormone involved in many plant processes, but is famous for its role in senescence and fruit ripening (including the activation of cell wall degradation). In other plant processes, ET can result in different outcomes through its positive cross talk with either the auxin pathway (Strader et al., 2010) or the JA pathway (Nahar et al., 2011). The available information on the role of ET in nematode infection seems contradictory, but some major features can be distinguished. ET consistently inhibits RKN infection but has a positive effect on CN infection. Early reports by Glazer et al. (1983, 1985) showed that ET has a positive effect on gall weight and giant cell hypertrophy, but this effect does not necessarily equate to increased nematode infection. Indeed, all later studies across multiple plant species convincingly show that ET inhibits RKN infection, possibly through a decrease in nematode attraction to the roots (Nahar et al., 2011; Fudali et al., 2013; Mantelin et al., 2013). Consistent with ET playing a role in plant defense to RKN infection, resistant plants show more up-regulation of ET biosynthesis and response genes than susceptible plants (Kumari et al., 2016; Shukla et al., 2018). In contrast, ET enhances the attraction of H. schachtii to Arabidopsis roots as shown by higher levels of infection in plants with more ET (response), while mutants in ET response (or treated with ET inhibitors) have fewer nematodes (Goverse et al., 2000; Wubben et al., 2001; Kammerhofer et al., 2015). Higher ET levels also have been linked to increased syncytium expansion (Goverse et al., 2000) and Bent et al. (2006) found fewer soybean CN (SCN) H. glycines females developing on ET-insensitive soybean roots. On the other hand, detailed attraction studies using SCNs yielded dissimilar results (Hu et al., 2017b). H. glycines juveniles are attracted more to soybean root tips pretreated with an ET biosynthesis inhibitor than to control roots. The attraction of H. glycines to roots of Arabidopsis (nonhost for SCN) is enhanced in ET-insensitive mutants and diminished in ET-overproducing mutants (Hu et al., 2017b). Recent work by Piya et al. (2018) elucidates how ET perception in Arabidopsis can result in higher or lower susceptibility to H. schachtii (measured as the number of developing females), depending on the receptor and its downstream pathway. The canonical ET signaling pathway causes suppression of SA-based defense, resulting in higher susceptibility to the CN, fitting the idea that ET enhances CN infection. The second pathway acts via the ETHYLENE RECEPTOR1, with ET inhibiting cytokinin signaling and thus reducing susceptibility to H. schachtii infection (Piya et al., 2018). Depending on the specific host-nematode interaction and timing or location of ET effects, cross talk with other hormone pathways could, therefore, have different effects on the host response to CN infection. Habash et al. (2017) identified a tyrosinase-like protein secreted by H. schachtii (Hs-Tyr) that, upon ectopic expression in Arabidopsis, increases susceptibility to the CN but not to the RKN M. incognita. Hs-Tyr expression in the plant is correlated with higher auxin (IAA-conjugates) and 1-aminocyclopropane-1-carboxylic acid (ET-precursor) levels, two hormones involved in susceptibility to CN. DEFENSE HORMONE PATHWAYS ACTIVATED BY THE PLANT AND DAMPENED BY THE PARASITE An investigation of the plant response to organisms invading aerial plant parts identified SA and JA as important defense hormones interacting either antagonistically or synergistically (Mur et al., 2006). Findings from Arabidopsis led to the paradigm that SA generally protects against biotrophic pathogens, whereas JA inhibits necrotrophic micro-organisms and munching insects (Glazebrook, 2005; Beckers and Spoel, 2006). Gutjahr and Paszkowski (2009) concluded that SA also appears to activate defense against biotrophic pathogens in roots, but JA presented a complex picture, and more research was needed to dissect the role of both hormones in root defense signaling. Ten years and many publications later, this conclusion still stands true for plant-nematode interactions. SA Activates Basal Defenses Against Nematodes The application of SA, or chemicals with similar action, reduces nematode infection (e.g., Wubben et al., 2008; Nahar et al., 2011; Molinari et al., 2014; Kammerhofer et al., 2015; Molinari, 2016 ). In many cases, the effect is modest and has been explained by the capability of the nematodes to suppress the SA pathway (Sanz-Alférez et al., 2008; Barcala et al., 2010; Uehara et al., 2010; Ji et al., 2013; Shukla et al., 2018). Although the effect is not always significant, mutants and transgenics with lower SA levels or signaling generally are more susceptible to nematodes (Wubben et al., 2008; Nahar et al., 2011), whereas enhanced SA levels or signaling results in lower nematode infections (Priya et al., 2011; Lin et al., 2013; Youssef et al., 2013). Nguyen et al. (2016), however, did not find enhanced susceptibility to H. schachtii in Arabidopsis SA signaling mutants. Many nematode effectors suppress plant defenses (Haegeman et al., 2012), but in only a few cases has this effect been specifically linked to suppression of the SA pathway. Effectors of fungal and oomycete pathogens have been implicated in the manipulation of SA biosynthesis. Some of these microbes secrete chorismate mutase and isochorismatase that convert chorismate and isochorismate, respectively, away from the main SA biosynthesis pathway, in this way lowering SA levels and defenses (Djamei et al., 2011; Liu et al., 2014). Similar genes have been identified in plant-parasitic nematodes (see Table 1). Wang et al. (2018) demonstrated that transient expression of an M. incognita chorismate mutase effector in Nicotiana benthamiana causes a decline in SA levels and larger lesions upon infection with Phytophthora capsici. Transgenic N. benthamiana plants expressing M. incognita chorismate mutase effector are more susceptible to M. incognita. Overexpression of a Hirschmanniella oryzae chorismate mutase or an isochorismatase in rice also enhances susceptibility to this nematode (L. Bauters, unpublished data). Nematode effectors mimicking PPHs and influencing phytohormone physiology and signaling at feeding sites Table 1. Nematode effectors mimicking PPHs and influencing phytohormone physiology and signaling at feeding sites Effector Mimics of PPHs . CLE-like Peptides . HgCLE Heterodera glycines Wang et al., 2001, 2005, 2010a; Gao et al., 2003 HsCLE H. schachtii Wang et al., 2011 GrCLE Globodera rostochiensis Lu et al., 2009; Guo et al., 2011; Chen et al., 2015 RrCLE Rotylenchulus reniformis Wubben et al., 2015 MhCLE Meloidogyne hapla Bird et al., 2015 CEP-like Peptides MhCEP M. hapla Bobay et al., 2013; Bird et al., 2015 RrCEP R. reniformis Eves-Van Den Akker et al., 2016 IDA-like Peptides MiIDL M. incognita Tucker and Yang, 2013; Kim et al., 2018 MhIDL M. hapla Kim et al., 2018 MfIDL M. floridensis Kim et al., 2018 Effectors Influencing Phytohormone Physiology and Signaling Auxins Conjugated forms H. schachtii De Meutter et al., 2005 Conjugated forms M. incognita De Meutter et al., 2005 Cytokinins iP, Z, BA-types H. schachtii De Meutter et al., 2003; Siddique et al., 2015 iP, Z, BA-types M. incognita De Meutter et al., 2003 Chorismate Mutase HgCM H. glycines Bekal et al., 2003 HsCM H. schachtii Vanholme et al., 2009 GrCM G. rostochiensis Lu et al., 2008 GpCM G. pallida Jones et al., 2003; Yu et al., 2011 GtCM G. tabacum Yu et al., 2011 GeCM G. ellingtonae Chronis et al., 2014 MjCM M. javanica Lambert et al., 1999; Doyle and Lambert, 2003 MiCM M. incognita Huang et al., 2005; Wang et al., 2018 MaCM M. arenaria Long et al., 2006a, 2006b HoCM Hirschmanniella oryzae Bauters et al., 2014 PcCM Pratylenchus coffeae Haegeman et al., 2011 Tyrosinase HsTYR H. schachtii Habash et al., 2017 Isochorismatase GrICM G. rostochiensis Eves-Van Den Akker et al., 2016 MhICM M. hapla Opperman et al., 2008 RrICM R. reniformis Wubben et al., 2010 HoICM H. oryzae Bauters et al., 2014 Novel Proteins Hg19C07 H. glycines Gao et al., 2003 Hs19C07 H. schachtii Lee et al., 2011 Hg10A07 H. glycines Hewezi et al., 2015 Hs10A07 H. schachtii Hewezi et al., 2015 Effector Mimics of PPHs . CLE-like Peptides . HgCLE Heterodera glycines Wang et al., 2001, 2005, 2010a; Gao et al., 2003 HsCLE H. schachtii Wang et al., 2011 GrCLE Globodera rostochiensis Lu et al., 2009; Guo et al., 2011; Chen et al., 2015 RrCLE Rotylenchulus reniformis Wubben et al., 2015 MhCLE Meloidogyne hapla Bird et al., 2015 CEP-like Peptides MhCEP M. hapla Bobay et al., 2013; Bird et al., 2015 RrCEP R. reniformis Eves-Van Den Akker et al., 2016 IDA-like Peptides MiIDL M. incognita Tucker and Yang, 2013; Kim et al., 2018 MhIDL M. hapla Kim et al., 2018 MfIDL M. floridensis Kim et al., 2018 Effectors Influencing Phytohormone Physiology and Signaling Auxins Conjugated forms H. schachtii De Meutter et al., 2005 Conjugated forms M. incognita De Meutter et al., 2005 Cytokinins iP, Z, BA-types H. schachtii De Meutter et al., 2003; Siddique et al., 2015 iP, Z, BA-types M. incognita De Meutter et al., 2003 Chorismate Mutase HgCM H. glycines Bekal et al., 2003 HsCM H. schachtii Vanholme et al., 2009 GrCM G. rostochiensis Lu et al., 2008 GpCM G. pallida Jones et al., 2003; Yu et al., 2011 GtCM G. tabacum Yu et al., 2011 GeCM G. ellingtonae Chronis et al., 2014 MjCM M. javanica Lambert et al., 1999; Doyle and Lambert, 2003 MiCM M. incognita Huang et al., 2005; Wang et al., 2018 MaCM M. arenaria Long et al., 2006a, 2006b HoCM Hirschmanniella oryzae Bauters et al., 2014 PcCM Pratylenchus coffeae Haegeman et al., 2011 Tyrosinase HsTYR H. schachtii Habash et al., 2017 Isochorismatase GrICM G. rostochiensis Eves-Van Den Akker et al., 2016 MhICM M. hapla Opperman et al., 2008 RrICM R. reniformis Wubben et al., 2010 HoICM H. oryzae Bauters et al., 2014 Novel Proteins Hg19C07 H. glycines Gao et al., 2003 Hs19C07 H. schachtii Lee et al., 2011 Hg10A07 H. glycines Hewezi et al., 2015 Hs10A07 H. schachtii Hewezi et al., 2015 Open in new tab Table 1. Nematode effectors mimicking PPHs and influencing phytohormone physiology and signaling at feeding sites Effector Mimics of PPHs . CLE-like Peptides . HgCLE Heterodera glycines Wang et al., 2001, 2005, 2010a; Gao et al., 2003 HsCLE H. schachtii Wang et al., 2011 GrCLE Globodera rostochiensis Lu et al., 2009; Guo et al., 2011; Chen et al., 2015 RrCLE Rotylenchulus reniformis Wubben et al., 2015 MhCLE Meloidogyne hapla Bird et al., 2015 CEP-like Peptides MhCEP M. hapla Bobay et al., 2013; Bird et al., 2015 RrCEP R. reniformis Eves-Van Den Akker et al., 2016 IDA-like Peptides MiIDL M. incognita Tucker and Yang, 2013; Kim et al., 2018 MhIDL M. hapla Kim et al., 2018 MfIDL M. floridensis Kim et al., 2018 Effectors Influencing Phytohormone Physiology and Signaling Auxins Conjugated forms H. schachtii De Meutter et al., 2005 Conjugated forms M. incognita De Meutter et al., 2005 Cytokinins iP, Z, BA-types H. schachtii De Meutter et al., 2003; Siddique et al., 2015 iP, Z, BA-types M. incognita De Meutter et al., 2003 Chorismate Mutase HgCM H. glycines Bekal et al., 2003 HsCM H. schachtii Vanholme et al., 2009 GrCM G. rostochiensis Lu et al., 2008 GpCM G. pallida Jones et al., 2003; Yu et al., 2011 GtCM G. tabacum Yu et al., 2011 GeCM G. ellingtonae Chronis et al., 2014 MjCM M. javanica Lambert et al., 1999; Doyle and Lambert, 2003 MiCM M. incognita Huang et al., 2005; Wang et al., 2018 MaCM M. arenaria Long et al., 2006a, 2006b HoCM Hirschmanniella oryzae Bauters et al., 2014 PcCM Pratylenchus coffeae Haegeman et al., 2011 Tyrosinase HsTYR H. schachtii Habash et al., 2017 Isochorismatase GrICM G. rostochiensis Eves-Van Den Akker et al., 2016 MhICM M. hapla Opperman et al., 2008 RrICM R. reniformis Wubben et al., 2010 HoICM H. oryzae Bauters et al., 2014 Novel Proteins Hg19C07 H. glycines Gao et al., 2003 Hs19C07 H. schachtii Lee et al., 2011 Hg10A07 H. glycines Hewezi et al., 2015 Hs10A07 H. schachtii Hewezi et al., 2015 Effector Mimics of PPHs . CLE-like Peptides . HgCLE Heterodera glycines Wang et al., 2001, 2005, 2010a; Gao et al., 2003 HsCLE H. schachtii Wang et al., 2011 GrCLE Globodera rostochiensis Lu et al., 2009; Guo et al., 2011; Chen et al., 2015 RrCLE Rotylenchulus reniformis Wubben et al., 2015 MhCLE Meloidogyne hapla Bird et al., 2015 CEP-like Peptides MhCEP M. hapla Bobay et al., 2013; Bird et al., 2015 RrCEP R. reniformis Eves-Van Den Akker et al., 2016 IDA-like Peptides MiIDL M. incognita Tucker and Yang, 2013; Kim et al., 2018 MhIDL M. hapla Kim et al., 2018 MfIDL M. floridensis Kim et al., 2018 Effectors Influencing Phytohormone Physiology and Signaling Auxins Conjugated forms H. schachtii De Meutter et al., 2005 Conjugated forms M. incognita De Meutter et al., 2005 Cytokinins iP, Z, BA-types H. schachtii De Meutter et al., 2003; Siddique et al., 2015 iP, Z, BA-types M. incognita De Meutter et al., 2003 Chorismate Mutase HgCM H. glycines Bekal et al., 2003 HsCM H. schachtii Vanholme et al., 2009 GrCM G. rostochiensis Lu et al., 2008 GpCM G. pallida Jones et al., 2003; Yu et al., 2011 GtCM G. tabacum Yu et al., 2011 GeCM G. ellingtonae Chronis et al., 2014 MjCM M. javanica Lambert et al., 1999; Doyle and Lambert, 2003 MiCM M. incognita Huang et al., 2005; Wang et al., 2018 MaCM M. arenaria Long et al., 2006a, 2006b HoCM Hirschmanniella oryzae Bauters et al., 2014 PcCM Pratylenchus coffeae Haegeman et al., 2011 Tyrosinase HsTYR H. schachtii Habash et al., 2017 Isochorismatase GrICM G. rostochiensis Eves-Van Den Akker et al., 2016 MhICM M. hapla Opperman et al., 2008 RrICM R. reniformis Wubben et al., 2010 HoICM H. oryzae Bauters et al., 2014 Novel Proteins Hg19C07 H. glycines Gao et al., 2003 Hs19C07 H. schachtii Lee et al., 2011 Hg10A07 H. glycines Hewezi et al., 2015 Hs10A07 H. schachtii Hewezi et al., 2015 Open in new tab The JA Pathway has a Polemical Role in Nematode Infection The release of JA during plant defense was first discovered as a response to insect attack. JA enhances the expression of protease inhibitors and pathways producing secondary metabolites with antiherbivore activity. Protease inhibitors constrain the proteolytic activity of the insects’ digestive enzymes to debilitate their growth and reproduction. Nematodes, being animals, also rely on proteases for obtaining sufficient nutrients from their food source. Therefore, it is not surprising that JA would play a role in defense to plant-parasitic nematodes. However, data on the role of the JA pathway (see Fig. 2 for an overview) in nematode infection are not unequivocal, at least not for RKN. Figure 2. Open in new tabDownload slide Overview of JA biosynthesis pathway and related mutants. Only the main pathway of oxylipin synthesis to jasmonate is shown. Several branches occur that give rise to many other metabolites. In addition, several enzymes are encoded by multiple genes from a gene family, although only one is shown. Intermediates and derivates: 13-HPOT, 13-hydroperoxy-octadecatrienoic acid; 12,13-EOT, 12,13-epoxy octadecatrienoic acid; OPC-8:0, 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid; JA-Ile, jasmonoyl-Ile. Enzymes: LOX, 13-lipoxygenase; OPR, 12-oxophytodienoate reductase; JAR, jasmonate response locus encoding a jasmonic acid-amido synthetase that converts JA into the bio-active JA-Ile. Mutants: spr2, suppressor of prosystemin response 2 mutant; coi1, the mutant in COI F-box protein involved in jasmonate signaling; jai, jasmonate insensitive, also mutant in COI. Figure 2. Open in new tabDownload slide Overview of JA biosynthesis pathway and related mutants. Only the main pathway of oxylipin synthesis to jasmonate is shown. Several branches occur that give rise to many other metabolites. In addition, several enzymes are encoded by multiple genes from a gene family, although only one is shown. Intermediates and derivates: 13-HPOT, 13-hydroperoxy-octadecatrienoic acid; 12,13-EOT, 12,13-epoxy octadecatrienoic acid; OPC-8:0, 3-oxo-2-(2-pentenyl)-cyclopentane-1-octanoic acid; JA-Ile, jasmonoyl-Ile. Enzymes: LOX, 13-lipoxygenase; OPR, 12-oxophytodienoate reductase; JAR, jasmonate response locus encoding a jasmonic acid-amido synthetase that converts JA into the bio-active JA-Ile. Mutants: spr2, suppressor of prosystemin response 2 mutant; coi1, the mutant in COI F-box protein involved in jasmonate signaling; jai, jasmonate insensitive, also mutant in COI. For CN, the available data are consistent with JA enhancing defense. Application of Methyl-JA to Arabidopsis leaves reduces H. schachtii infection on the roots, and the JA biosynthesis mutants delayed-dehiscence2 (dde2) and lipoxygenase 6 (lox6) show enhanced female development compared to control plants (Kammerhofer et al., 2015). Arabidopsis mutants with higher JA levels/signaling are less susceptible to H. schachtii (Ali et al., 2013; Nguyen et al., 2016; Sidonskaya et al., 2016), and soybean roots overexpressing (E,E)-a-farnesene synthase (a gene up-regulated upon JA treatment) support lower levels of H. glycines infection, indicating an additional possible mechanism of JA action (Lin et al., 2017). At first sight, the results of Ozalvo et al. (2014) fit the “JA = defense” picture with the JA biosynthesis mutant lox4 being more susceptible to H. schachtii, but a closer look contradicts this conclusion (see below). Over the past 10 years, more than 20 papers have been published on the role of JA in RKN infections, and the data overwhelmingly support JA as a defense molecule. Application of MeJA on tomato (Solanum lycopersicum), rice (Oryza sativa), and soybean (Glycine max) invariably reduces RKN infection (Cooper et al., 2005; Shimizu and Mazzafera, 2007; Fujimoto et al., 2011; Nahar et al., 2011; Zhang et al., 2011; Zinovieva et al., 2013; Vieira Dos Santos et al., 2014; Zhou et al., 2015; Hu et al., 2017a; Kyndt et al., 2017), while inhibitors of JA biosynthesis enhance infection (Nahar et al., 2011; Zhou et al., 2015). In contrast, analyses of mutants and transgenics modified in JA signaling or biosynthesis yield brain-twisting results. The first indication of the complexity of the JA pathway was the report that a JA-insensitive mutant in tomato is less susceptible to M. incognita than the wild type (Bhattarai et al., 2008). This observation led to the conclusion that, whereas the hormone JA results in defense, JA signaling is needed for successful infection. However, other experiments with JA-signaling mutants do not support this conclusion: specifically, M. incognita infection of the Arabidopsis mutant coronatine insensitive (coi) does not differ from the wild type (Gleason et al., 2016), and the rice mutant jar1 is slightly more susceptible to the rice RKN Meloidogyne graminicola (T Kyndt and R singh, unpublished data). To augment the complexity, Gleason et al. (2016) demonstrated that coi is not needed for JA-induced defense against M. incognita infection in Arabidopsis. What about mutant/transgenic plants with changes in JA biosynthesis? Tomato suppressor of prosystemin-mediated responses2 mutants, affected in the production of linolenic acid needed for JA biosynthesis, are more susceptible to M. incognita (Sun et al., 2011; Fan et al., 2015). Tomato plants overexpressing miR319 show lower JA levels and are highly susceptible to M. incognita (Zhao et al., 2015). Rice plants overexpressing allene oxide synthase (AOS) are less susceptible to M. graminicola (Kyndt et al., 2017), and the Arabidopsis AOS mutant dde2 shows more galling by M. hapla than wild type (Gleason et al., 2016). However, not all mutants in JA biosynthesis corroborate the role of JA in defense. Depending on the Lox or allene oxide cyclase (Aoc) gene, mutants are more (lox4-1, Ozalvo et al., 2014; aoc-3, Naor et al., 2018) or less (lox3-1, Ozalvo et al., 2014) susceptible to RKN infection. As Naor et al. (2018) explain, the oxylipin biosynthesis pathway branches into many metabolites with differing levels of toxicity to RKN; therefore, mutants likely affect more than just the JA level. Gleason et al. (2016), for instance, showed that the intermediate 12-oxo-phytodienoic acid (OPDA) is much more important than JA for defense against RKN, which is consistent with JA and OPDA having different signaling roles (Dave and Graham, 2012). In contrast to Gleason et al. (2016), Naor et al. (2018) found the Arabidopsis dde2 mutant to be less susceptible to M. javanica.Ozalvo et al. (2014) add further to the confusion by demonstrating that the highly susceptible biosynthesis mutant lox4 has not lower but higher JA levels upon nematode infection and also higher JA, ET, and SA- regulated transcription, all thought to be involved in defense to RKN. It is difficult to compare the different results, as some papers report gall numbers (the initial infection stage) and others describe female numbers or measure percent female development. In addition, numbers per root system can give a different conclusion compared to numbers per gram of root, especially if using mutants that are affected in their root morphology. Unfortunately, most authors do not describe the root phenotype of the mutants. An example of these complications are the results of Gao et al. (2008) on the lox3-4 mutant in maize (Zea mays). This mutant has elevated JA, SA, and ET levels in its roots and is highly susceptible to M. incognita infection, based on increased nematode attraction to roots and a higher number of eggs per gram of root compared to wild type. The lox3-4 mutant has much shorter roots, but the number of root tips needed for nematode invasion is most likely unaltered or even higher (nematode attraction and invasion per plant are higher). As a consequence, calculation of the number of eggs per invaded nematode is much lower in the lox3-4 than in wild type, which could be interpreted as less susceptible if susceptibility is measured as the ability of the host to allow nematode multiplication. In conclusion, while spraying JA enhances plant defense to nematodes, it is not JA itself that is responsible, but its effects on the production of proteins (such as proteinase inhibitors) and metabolites (such as terpenes and oxylipins). Depending on how mutations in JA-related genes affect these antiherbivore compounds, the plant is rendered more or less susceptible to nematode infection. In view of the importance of JA in defense, we could expect nematode effectors that interfere with this pathway. Indeed, transcriptome analyses have found suppression of JA-related genes in syncytia and giant cells (Ithal et al., 2007; Ji et al., 2013). Nematode-secreted fatty acid and retinol (FAR)-binding proteins have been proposed to interfere with lipid signaling in host defense, for animal (e.g., Garofalo et al., 2003) as well as plant parasites. The FAR protein of the potato (Solanum tuberosum) CN Globodera pallida is located on the cuticle surface and interferes with plant LOX-mediated defense (Prior et al., 2001). Tomato roots expressing the M. javanica MjFAR are more susceptible to RKN infection, and this observation is correlated with lower expression of the JA-responsive proteinase inhibitor2 (Iberkleid et al., 2013); however, some genes in the JA pathway are expressed at higher levels in these roots (Iberkleid et al., 2015). OTHER PLANT HORMONES PLUG INTO THE DEFENSE CORE In contrast to the ample studies on the importance of auxin and jasmonate in susceptibility and defense, respectively, very little research has been done on the role of gibberellic acid (GA), abscisic acid (ABA), brassinosteroids, and strigolactones in nematode infection. The available knowledge is limited mainly to the rice-M. graminicola system. GA is well known for its role in stimulating plant growth by the degradation of DELLAs, a class of growth-repressing nuclear proteins. Studies in Arabidopsis revealed that GA antagonizes JA action and promotes SA signaling and/or perception (Navarro et al., 2008). In rice, GA interacts antagonistically with both JA and SA signaling pathways (De Vleesschauwer et al., 2016). Congruently, GA is important for susceptibility of rice to M. graminicola, as shown in a detailed study using the application of GA or a GA-biosynthesis inhibitor and a series of mutants (Yimer et al., 2018). In contrast, foliar application of GA to tomato increases resistance against M. javanica (Moosavi, 2017). However, these latter results were not confirmed by analysis of GA-mutants, and the GA concentration applied might have influenced the outcome (Bauters et al., 2018; Yimer et al., 2018). The application of ABA increases the susceptibility of rice and tomato to RKN infection (Kyndt et al., 2017; Moosavi, 2017), and brassinosteroids suppress rice defense to M. graminicola (Nahar et al., 2013). In rice, ABA (Kyndt et al., 2017), brassinosteroids (Nahar et al., 2013), and strigolactones (Lahari et al., 2018) all appear to enhance susceptibility to M. graminicola through antagonism with the JA pathway. NEMATODES SECRETE PPH EFFECTOR MIMICS FOR FEEDING SITE FORMATION Besides the classical phytohormones discussed so far, small, secreted peptide hormones are also potent modulators of plant growth and development. It has become increasingly evident that secreted peptides play critical roles in mediating a range of plant-microbe interactions, either by induction of PPH gene expression, for instance during legume-rhizobium symbioses, or by secreting PPH effector mimics (Yamaguchi et al., 2016; Ronald and Joe, 2018; Taleski et al., 2018). Here, we focus on PPH effector mimics secreted by nematodes, the first animal-pathogen model identified to secrete such molecules for parasitism. The different classes of PPH effector mimics identified from nematodes have expanded to include CLAVATA3/EMBRYO SURROUNDING REGION (CLE)-like, C-TERMINALLY ENCODED PEPTIDE (CEP)-like, and INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-like peptides. CLE-like Peptides Plant CLEs play important roles in shoot, root, and vascular meristem maintenance and are classified as either A-type or B-type peptides (for review, see Yamaguchi et al., 2016). The A-type peptides promote cell differentiation, whereas the B-type peptides suppress differentiation of tracheary elements and promote procambial cell division. Comprehensive clustering analysis has categorized plant CLEs into groups with potentially shared function (Goad et al., 2017). Aside from plants, CLE-like peptide effector mimics have been identified from multiple genera of CN, RKN, and more recently, from the reniform nematode (a semiendoparasite that induces syncytia; Fig. 3). In the case of CN and reniform nematodes, the domain architecture of CLE-like peptide effector mimics resembles that of plant CLE proteins (Lu et al., 2009; Wang et al., 2010a, 2011; Wubben et al., 2015). Plant CLEs are produced as prepropeptides harboring an N-terminal secretion signal peptide that directs them through the plant secretory pathway for delivery to the apoplast. A central “pro” domain, referred to as the “variable” domain because of its lack of sequence homology among family members, separates the secretion signal peptide and C-terminal CLE domain. Similarly, CN and reniform produce CLEs as prepropeptides, but this occurs in the dorsal esophageal gland cell of the nematode (Wang et al., 2010a; Wubben et al., 2015). The N-terminal secretion signal peptide directs these effector proteins through the gland cell secretory pathway for packaging into secretory granules. They are then delivered as propeptides (comprised of a central variable domain and a C-terminal CLE domain with homology to plant CLE peptides) to the cytoplasm of host root cells through the stylet (Lu et al., 2009; Wang et al., 2010a; Mitchum et al., 2012, 2013). Once in the cytoplasm of host root cells, they are redirected through the plant secretory pathway to the apoplast by an unknown posttranslational trafficking mechanism mediated by a conserved “cryptic signal peptide” sequence in the N-terminal portion of the variable domain (Wang et al., 2010b). The proteins subsequently undergo posttranslational modification by hydroxyproline (Hyp) arabinosylation and proteolytic cleavage down to the 12-amino acid CLE peptide to release one or more bioactive ligands (Chen et al., 2015). These ligands interact with plant Leu-rich repeat (LRR) receptor kinases, including CLV1, CLV2, and BARELY ANY MERISTEMs, to positively regulate NFS development (Guo et al., 2010, 2015; Chen et al., 2015). Silencing of nematode CLE genes or their cognate plant receptors delays nematode development by impairing NFS formation (Replogle et al., 2011, 2013; Chen et al., 2015; Guo et al., 2015, 2017). In contrast to CN, the absence of a “pro” domain from RKN CLEs suggests this nematode may deliver bioactive CLE peptide mimics directly into the apoplast (Mitchum et al., 2012; Bird et al., 2015). Figure 3. Open in new tabDownload slide Representative structures of nematode CLE-like, CEP-like, and IDA-like proteins. Cyst and reniform nematode CLE and CEP-like proteins contain an N-terminal signal peptide, a variable domain, and either single or multiple conserved C-terminal peptide motifs similar to plant CLE or CEPs, respectively. The green box in the variable domain of cyst and reniform nematode CLE and CEP-like proteins denotes a cryptic signal peptide sequence. Root-knot nematode CLE, CEP, and IDA-like proteins lack a variable domain sequence. Figure 3. Open in new tabDownload slide Representative structures of nematode CLE-like, CEP-like, and IDA-like proteins. Cyst and reniform nematode CLE and CEP-like proteins contain an N-terminal signal peptide, a variable domain, and either single or multiple conserved C-terminal peptide motifs similar to plant CLE or CEPs, respectively. The green box in the variable domain of cyst and reniform nematode CLE and CEP-like proteins denotes a cryptic signal peptide sequence. Root-knot nematode CLE, CEP, and IDA-like proteins lack a variable domain sequence. Based on the findings that nematode CLE peptide effector mimics belong to multigene families, are coordinately expressed, and can encode proteins with multiple CLE domains (Mitchum et al., 2012), it appears that nematodes may require the simultaneous secretion of a mixture of CLE peptide mimics for NFS formation. Multiple single-domain CLEs have been identified from Heterodera, RKN, and reniform, whereas Globodera species harbor multidomain CLEs. Another fascinating observation is that no two identical CLE sequences have been identified within a single genus or across genera. Whether these differences play a significant role in plant host adaptation is still unknown. Until recently, only A-type CLE-like peptide effector mimics had been identified from nematodes (Mitchum et al., 2012). However, mining of the H. glycines early parasitic stage transcriptome (Gardner et al., 2018) revealed B-type CLE peptide effector mimics nearly identical to tracheary element differentiation inhibitory factor (TDIF), encoded by CLE41 and CLE44, in Arabidopsis (Guo et al., 2017). In plants, the TDIF peptide regulates vascular stem cell maintenance through an interaction with the TDIF RECEPTOR (TDR)/PHLOEM INTERCALATED WITH XYLEM receptor kinase to activate two independent downstream pathways. The TDIF-TDR-WOX4 pathway promotes procambial cell proliferation, whereas the TDIF-TDR-Glycogen Synthase Kinase3-BRI1-EMS SUPPRESSOR1 pathway inhibits xylem differentiation from procambial cells. Procambial-associated genes are activated in both CN and RKN feeding sites (Guo et al., 2017; Yamaguchi et al., 2017). The TDIF-TDR-WOX4 procambial cell proliferation pathway is required for CN feeding site formation (Guo et al., 2017); however, further research is needed to assess whether the TDIF-TDR-Glycogen Synthase Kinase3-BRI1-EMS SUPPRESSOR1 signaling is equally important. As of yet, B-type CLE peptide effector mimics have not been identified from RKN, and a potential role of these vascular stem cell signaling pathways in RKN feeding site formation remains to be confirmed. Of note is the low abundance of nematode B-type CLEs relative to A-type CLEs in early CN parasitic stages (Guo et al., 2017). A detailed analysis assessing if nematodes tightly control the expression and release of specific peptide effectors during the phases of NFS formation will help gauge whether there is any potential biological significance of peptide synergism. Other than a role for WOX4, little is known about the downstream intracellular nematode peptide signaling cascades. Additional research is needed to dissect what appears to be a complex network of nematode CLE-receptor interactions to understand fully their specific contribution to NFS formation. CEP-like Peptides As the genomes and transcriptomes of more plant-parasitic nematodes have been released, computational scans have identified additional classes of PPH effector mimics, lending further support for peptide hormone mimicry as a signature adaptation to plant parasitism (Bird et al., 2015). CEP-like peptide effector mimics have been identified from Meloidogyne genomes and, more recently, from reniform nematode but not from CN genomes. In plants, CEPs are small, secreted peptide hormones implicated in nitrogen-demand signaling, nodulation, and lateral root development (reviewed by Taleski et al., 2018). CEP propeptides are cleaved by amino- and carboxypeptidases to release 15-amino acid bioactive peptides that signal through LRR-RK CEPR1. Like CLEs, CEP activity is regulated by posttranslational modifications in the form of Hyp arabinosylation. Despite the widespread identification of CEPs in plants, downstream signaling mediated by CEP-CEPR remains unknown. Twelve reniform CEP gene family members have been identified to date. They are unique in that they harbor one intron per domain sequence, whereas all other CEPs identified from animals and plants are encoded by a single exon, suggesting an independent evolutionary origin (Eves-Van Den Akker et al., 2016). Similar to plant CEPs, the reniform CEPs are produced as prepropeptides. Remarkably, the “pro” domain harbors a cryptic signal peptide with similarity to CN and reniform CLE-like effectors, not only suggesting that these effector proteins may be indirectly routed to the apoplast upon delivery as propeptides to host root cells but that the trafficking mechanism by which this occurs may be conserved across genera and span to different classes of effectors. CEPs are produced within the dorsal gland cell of sedentary reniform females, suggesting a prominent role in NFS formation. Interestingly, the RKN CEPs, like their CLE counterparts, lack the “pro” domain, lending further support for a mechanism of direct delivery to the apoplast to exert their function in giant cell formation (Bobay et al., 2013; Bird et al., 2015). Although the role of CEP-like effector mimics in nematode parasitism remains unknown, Arabidopsis primary root length and lateral root number are inhibited in a dose-dependent manner upon exogenous application of RrCEP1, similar to the application of plant CEP peptides. In addition, feeding sites induced by the CN H. schachtii are smaller in size in the RrCEP1-treated roots, suggesting that one potential function of nematode CEPs may be to regulate NFS size (Eves-Van Den Akker et al., 2016). Further studies are needed to clarify the unique role of CEP-like PPH effector mimics in plant-nematode interactions and any potential role in host nitrate uptake like their plant counterparts. IDA-like Peptides The broad spectrum of PPH effector mimics identified from Meloidogyne species may aid RKN to parasitize a broad range of host plant species. In addition to CLE-like and CEP-like PPH effector mimics, several IDA-like (IDL) family members have been identified from multiple Meloidogyne species (Tucker and Yang, 2013; Kim et al., 2018). An exhaustive search of CN and reniform sequence data for IDL peptides remains to be conducted; however, no IDL peptides were identified from existing sequence data for Heterodera and Globodera spp. (Kim et al., 2018). In plants, IDA signaling through the LRR-RKs HAESA (HAE) and HAESA-like2 activates a MAP kinase signaling cascade that leads to the expression of KNOX transcription factors, which regulate a suite of cell wall-modifying proteins important for cell separation during floral organ abscission and lateral root emergence. More recently, IDL peptides were shown to modulate plant stress and defense responses to pathogens (Vie et al., 2017; Wang et al., 2017). Like CLEs and CEPs, IDAs harbor an N-terminal secretion signal peptide and undergo proteolytic cleavage to 14-amino acid bioactive peptides in the apoplast. RKN IDL effector mimics have a similar domain architecture. A synthetic M. incognita IDL1 (MiIDL1) peptide applied exogenously to the Arabidopsis mutant ida is able to rescue floral abscission and lateral root phenotypes in an HAE/HAESA-like2-dependent manner (Kim et al., 2018). However, direct binding of MiIDL1 to these receptors has not been demonstrated. Similarly, transgenic Arabidopsis ida mutant plants expressing MiIDL1 exhibit wild-type floral abscission. Host-derived RNAi targeting of MiIDL1 leads to fewer and smaller galls compared to control plants, demonstrating a critical role in parasitism. Together, these data provide evidence of a specific role of IDL PPH mimics in giant cell formation and point to a potentially unique adaptation for RKN parasitism. INTEGRATION OF PEPTIDE AND HORMONE SIGNALING FOR NFS FORMATION Cross talk between peptide and hormone signaling regulates developmental processes and responses to external stimuli (for review, see Wang et al., 2016). Evidence for such cross talk governing NFS formation is amassing in the literature. Alterations to phytohormone physiology and signaling, induced in response to nematode feeding, may be coordinately regulated by PPH effector mimics and hormones to fine-tune root developmental programs in favor of NFS formation. Studies showing that a low Mr peptide (s) from G. rostochiensis secretions costimulates the proliferation of protoplasts together with auxin and cytokinin, provided some of the first evidence for potential cross talk between nematode-secreted peptides and hormonal signaling (Goverse et al., 1999). Recent studies suggest CN may be co-opting early signaling events in vascular cell patterning, a process controlled by CLE peptides and hormonal signaling, for the successful formation of NFS. For instance, the beet (Beta vulgaris) CN H. schachtii secretes HsCLE2, an A-type CLE peptide mimic that is identical to AtCLE5/6 while simultaneously secreting HsCLEB, a B-type CLE peptide mimic nearly identical to Arabidopsis CLE41/TDIF (Guo et al., 2017). These peptides act synergistically in an auxin-dependent manner to suppress differentiation and promote vascular stem cell proliferation. They also activate the expression of numerous auxin-responsive genes known to be up-regulated in NFS (Whitford et al., 2008). Though plant CLE peptides exhibit cell-type specific expression patterns, overlapping expression domains may be critical for developmental programs requiring the synergistic action of multiple CLE peptides. Nematodes appear to have adapted to exploit this by controlling both the timing and quantity of A- and B-type peptides secreted into a chosen cell to potentially bypass the plant’s own cell type-specific and negative feedback regulation mechanisms. Aside from auxin, there are also reports of intersections among CLE signaling and BR, CK, and GA signaling both locally and systemically. TDIF signaling suppresses xylem differentiation from procambial cells through integration with BR signaling (Kondo et al., 2014); GA positively regulates the expression of CLE6 and overexpression of this peptide partially rescues GA-deficiency (Bidadi et al., 2014); and CLEs are regulators of type-A ARRs to promote CK signaling (Kondo et al., 2011). A similarly complex cross talk is likely at play for other classes of plant peptides and hormones. For instance, the developmental programs underlying lateral root emergence requires the integration of auxin and IDA signaling to regulate cell-wall–modifying proteins involved in cell separation (Kumpf et al., 2013). These studies illuminate the incredibly complex network of peptide and hormone signaling pathways likely active in NFS formation. Open in new tabDownload slide Open in new tabDownload slide CONCLUSION Substantial progress has been made in our understanding of how plant hormones shape the interface between plants and nematodes and how nematode effector proteins contribute to this interaction. However, the few effectors that have been identified as participating in NFS formation cannot explain the myriad of complex changes that lead to a mature feeding cell (see "Outstanding Questions"). Undoubtedly, we still have much to learn about the interplay among peptide, phytohormone, and defense signaling pathways in NFS formation. Moreover, the field has expanded, as nematodes are no longer unique among plant pathogens in their ability to secrete mimics of PPHs (Ronald and Joe, 2018). It was recently discovered that the fungal pathogen Fusarium oxysporum and the bacterial pathogen Xanthomonas oryzae pv. oryzae secrete functional peptide mimics of plant rapid alkalinization factor and plant peptide containing sulfated tyrosine peptides, respectively (Masachis et al., 2016; Pruitt et al., 2017). Thus, as we continue to uncover the complex interplay between peptide and hormone signaling in plant-nematode interactions, the findings are likely to have much broader applicability in molecular plant-microbe interactions than previously thought. ACKNOWLEDGMENTS The authors wish to thank the various funding agencies that have supported their research programs on hormone and peptide signaling through the years, as well as the postdoctoral fellows, students, staff scientists, and many colleagues who have contributed to the research findings described in this review. The authors also thank Nagabhushana Ithal for the nematode picture of the vTOC icon. LITERATURE CITED Ali MA , Abbas A, Kreil DP, Bohlmann H ( 2013 ) Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots . BMC Plant Biol 13 : 47 Google Scholar Crossref Search ADS PubMed WorldCat Barcala M , García A, Cabrera J, Casson S, Lindsey K, Favery B, García-Casado G, Solano R, Fenoll C, Escobar C ( 2010 ) Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells . 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IOS-1456047), the U.S. Department of Agriculture National Institute of Food and Agriculture, and the United Soybean Board. 2 Author for contact: [email protected]. 3 Senior authors. 4 These authors contributed equally to this article. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Godelieve Gheysen ([email protected]). G.G. and M.G.M. conceived the structure and the contents of the paper. G.G. wrote the plant hormone part and M.G.M. focused on the effector part. G.G. and M.G.M. commented on and edited the final text. [OPEN] Articles can be viewed without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.18.01067 © 2019 American Society of Plant Biologists. All Rights Reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Plant NLRs with Integrated Domains: Unity Makes StrengthGrund, Elisabeth; Tremousaygue, Dominique; Deslandes, Laurent
doi: 10.1104/pp.18.01134pmid: 30530739
Unlike animals, plants lack an adaptive and circulating immune system. Thus, to detect pathogens and generate effective defense responses, plants rely on an elaborate innate immunity that involves different types of immune receptors (Cook et al., 2015). Conserved pathogen-associated molecular patterns are recognized in the extracellular compartment of the host by cell surface-localized receptors. This event triggers the activation of basal immune responses called pathogen-associated molecular pattern-triggered immunity (PTI). During evolution, pathogens have evolved sophisticated virulence strategies to overcome host defense responses. Host-adapted pathogens use an arsenal of virulence factors called effectors that are delivered into the plant cell in order to subvert diverse cellular functions (effector-triggered susceptibility) through interference with PTI signaling (Jones and Dangl, 2006). However, effector activities can turn against the pathogen, as they often betray its presence within the cell. To recognize pathogen effectors, plants use a repertoire of intracellular immune receptors that belong to a superfamily of nucleotide-binding domain and leucine-rich repeat (LRR)-containing proteins (NLRs). NLRs can mediate the specific recognition of pathogen effectors and initiate effector-triggered immunity (ETI). ETI involves transcriptional reprogramming overlapping with transcriptional regulations during PTI and often provokes localized host cell death at infection sites to limit pathogen spread (Jones and Dangl, 2006; Maekawa et al., 2011). Adapted pathogens can evade host recognition by reconfiguring their effector repertoires through various mechanisms including gain and loss of effector genes, modulation of expression, and rapid evolution of effectors by mutation (Arnold and Jackson, 2011; Lo Presti et al., 2015). Therefore, cycles of pathogen-induced effector-triggered susceptibility and plant-mediated ETI are considered as major forces driving plant host-pathogen coevolution (Jones and Dangl, 2006). Open in new tabDownload slide Open in new tabDownload slide NLR proteins belong to STAND (Signal Transduction ATPases with Numerous Domains) P-loop NTPases. Canonical plant NLRs possess a multidomain architecture composed of a central nucleotide-binding site (NBS) and a C-terminal LRR domain. The NBS is believed to function, through nucleotide-dependent conformational changes, as a molecular switch for NLR activation (Takken et al., 2006). Depending on the nature of their N-terminal domain, NLRs can be divided into two main classes: those having a Toll and IL-1 receptor (TIR) domain and those with a coiled-coil (CC) domain (Takken and Goverse, 2012). A third class, based on the presence of the N-terminal RPW8 domain, also can be defined. Over the last two decades, with the cloning of plant NLRs and their associated effectors, molecular characterization of the mechanisms employed by NLRs for specific effector recognition and signaling have been the subject of intensive research. NLR-mediated effector recognition often involves host components that bind to and/or are modified by effectors (Fig. 1). In the guard model, effector interference with a host target (cofactor or bait) is detected by the NLR, acting as a guard of modified self (Dodds and Rathjen, 2010; Maekawa et al., 2011). Many identified guarded host proteins (also referred to as guardees) represent hubs with key immune-related functions, including signaling or the regulation of gene expression, and therefore are commonly targeted by various effectors (Mukhtar et al., 2011; Weßling et al., 2014). An effector decoy model has been proposed for a number of effector-sensing NLRs. In this model, the guarded host protein has no defense role but mimics an operational effector target and, thus, acts as a decoy that lures the pathogen effector and diverts it from its real targets (van der Hoorn and Kamoun, 2008; Lewis et al., 2013; Ntoukakis et al., 2014). Studies have shown that several NLRs can both detect pathogens and initiate downstream signaling, whereas other NLR proteins form heterogenous protein complexes (Césari et al., 2014b; Williams et al., 2014). In the core of these complexes are NLR pairs in which the two members are encoded by genes arranged in a head-to-head orientation with a common promoter region, which strongly suggests their coregulation (Birker et al., 2009; Saucet et al., 2015). In several cases, the two proteins of NLR pairs form a heterocomplex receptor with each partner featuring specific attributes: one detects pathogen effectors (the sensor) while the other functions as an inducer of disease resistance (the transducer), and the signaling activity of the latter is repressed by the sensor (Césari et al., 2014b; Williams et al., 2014). To explain how effectors are recognized by NLR pairs, an extension of the decoy model has been proposed with the integrated decoy hypothesis (Cesari et al., 2014a). Indeed, sensor NLR partners were shown to contain, in addition to their conserved multidomain NLR architecture, unconventional domains that are able to interact physically with their corresponding effectors (Kanzaki et al., 2012; Cesari et al., 2013). Recent studies demonstrated that several of these integrated domains (IDs) act as decoys of effector targets, enabling the sensor NLR to specifically detect pathogens (Fig. 1; Le Roux et al., 2015; Maqbool et al., 2015; Ortiz et al., 2017). Perturbations of the sensor NLR are perceived by the signaling partner, which activates immune signaling (Le Roux et al., 2015; Sarris et al., 2015; Ortiz et al., 2017). Comparative analyses of plant immune receptor architectures suggest that the integration of unusual domains, which potentially serve as baits for pathogen effectors, is not restricted to paired NLRs and represent a widespread mechanism (Kroj et al., 2016; Sarris et al., 2016; Bailey et al., 2018). The identification of NLR-IDs signifies a breakthrough in plant NLR biology pathology, since it has profoundly changed our view of how plant NLRs can function and evolve. Figure 1. Open in new tabDownload slide A, NLRs can directly or indirectly detect the presence of pathogen effectors by monitoring the manipulation of their host targets (baits or decoys). B and C, According to the integrated decoy model, integrated domains (IDs) in NLRs behave as decoys of effector targets, enabling the recognition of effector activities. This recognition can be direct (B) or indirect (C). D, Different studies reported the existence of diverse IDs (in sequence and predicted molecular functions), which can be present at various positions within the modular structure of NLRs. E, NLR-IDs can be engineered using different strategies aimed at providing (1) extended specificity (i.e. specific point mutations in IDs enabling the recognition of various allelic forms of a pathogen effector), (2) multirecognition capabilities (by integrating IDs from different NLRs within a single NLR), or (3) new recognition specificities (by integrating previously characterized effector targets that then act as sensors). E, Effector; ET, previously characterized effector target; Nt, N-terminal domain; T, effector target. Figure 1. Open in new tabDownload slide A, NLRs can directly or indirectly detect the presence of pathogen effectors by monitoring the manipulation of their host targets (baits or decoys). B and C, According to the integrated decoy model, integrated domains (IDs) in NLRs behave as decoys of effector targets, enabling the recognition of effector activities. This recognition can be direct (B) or indirect (C). D, Different studies reported the existence of diverse IDs (in sequence and predicted molecular functions), which can be present at various positions within the modular structure of NLRs. E, NLR-IDs can be engineered using different strategies aimed at providing (1) extended specificity (i.e. specific point mutations in IDs enabling the recognition of various allelic forms of a pathogen effector), (2) multirecognition capabilities (by integrating IDs from different NLRs within a single NLR), or (3) new recognition specificities (by integrating previously characterized effector targets that then act as sensors). E, Effector; ET, previously characterized effector target; Nt, N-terminal domain; T, effector target. In this review, we summarize current knowledge of NLR-IDs with detailed examples, discuss their genetic and functional diversity, and illustrate how the study of NLR function and mode of action has led to advances in plant disease control. NLRS WITH INTEGRATED DECOYS: AN INGENIOUS PATHOGEN DETECTION MECHANISM Recent independent studies have provided convincing evidence that IDs enable the specific detection of pathogens by acting as molecular decoys that structurally mimic pathogen true virulence targets to monitor host immunosuppression attempts. How these IDs confer effector recognition and trigger the activation of immune signaling are very intriguing questions. Well-characterized examples of NLR-ID fusions in paired NLRs include the Arabidopsis (Arabidopsis thaliana) RRS1, which carries a WRKY domain (Le Roux et al., 2015), and the RGA5 and Pik-1 proteins from rice (Oryza sativa), both of which integrate a heavy metal-associated (HMA; RATX1) domain (Ortiz et al., 2017). These examples are described in detail below. The WRKY Domain of RRS1-R Experimental validation of the integrated decoy model was first provided for the Arabidopsis/Ralstonia solanacearum model. In 2001, Deslandes and colleagues cloned a resistance gene encoding RRS1-R (RESISTANCE TO RALSTONIA SOLANACEARUM1) conferring broad-spectrum resistance to the soil-borne bacterium R. solanacearum, the causal agent of bacterial wilt (Deslandes et al., 1998, 2002). RRS1-R contains at its C terminus a WRKY DNA-binding domain. This domain is conserved in defensive plant WRKY transcription factors that orchestrate biotic stress responses through the recognition of W-box cis-regulatory elements in gene promoters (Rushton et al., 2010). As the first cloned NLR with an extra domain, RRS1-R was initially considered an anomaly in the field. Later, RRS1-R was shown to cooperate genetically and molecularly with a second TIR-NB-LRR, namely RPS4 (RESISTANCE TO PSEUDOMONAS SYRINGAE4; Birker et al., 2009; Narusaka et al., 2009), to recognize effectors from different pathogens. These effectors included R. solanacearum PopP2, a member of the YopJ superfamily of acetyltransferase (Deslandes et al., 2003; Tasset et al., 2010), and AvrRps4, an unrelated effector from leaf-infecting Pseudomonas syringae pv pisi (Hinsch and Staskawicz, 1996; Sohn et al., 2012). Encoded by two coregulated genes present in a head-to-head orientation, RRS1-R and RPS4 TIR-NB-LRRs form a functional receptor recognition/signaling complex through homodimerization and heterodimerization involving their respective TIR domains (Williams et al., 2014). Two recent studies revealed that the RRS1-R/RPS4 NLR complex is activated through the targeting of the RRS1-R WRKY domain by PopP2 and AvrRps4 effectors (Le Roux et al., 2015; Sarris et al., 2015). Catalytically active PopP2 acetylates a key Lys residue (K1221) in the invariant heptad of the WRKY domain of RRS1-R, blocking its binding to W-box DNA sequences. Homology modeling predicts that K1221 acetylation disrupts WRKY domain electrostatic potential at the interface with DNA. In the absence of RRS1-R/RPS4 recognition, PopP2 uses this acetylation strategy to inhibit WRKY DNA-binding activities and transactivation functions needed for defense gene expression and basal resistance. Therefore, the RRS1-R WRKY domain represents a decoy that betrays the defense-suppressing abilities of PopP2 and AvrRps4 on their host virulence targets: the defensive WRKY transcription factors. The direct fusion of a WRKY decoy domain within the RRS1-R/RPS4 NLR complex creates an efficient monitoring system for the indispensable virulence activities of different pathogens. Recently, Ma et al. (2018) demonstrated that, prior to effector detection, the WRKY domain negatively regulates the RPS4-RRS1 complex through specific interactions with an adjacent domain in RRS1. Binding of AvrRps4 to the WRKY domain disrupts these intramolecular interactions, leading to the derepression of RRS1. Therefore, besides its effector-sensing function, the integrated WRKY domain of RRS1 also has an important regulatory role in preventing inappropriate receptor activation in the absence of pathogens. The HMA Domain of RGA5 and Pik-1 The study of the RGA4/RGA5 receptor NLR pair in rice has enabled significant progress in deciphering the mode of action of paired NLRs. This NLR pair cooperates genetically and physically in the recognition of AVR-PiA and AVR1-CO39, two unrelated effectors of the rice blast fungus Magnaporthe oryzae (Okuyama et al., 2011; Cesari et al., 2013). In the absence of the pathogen, constitutive disease resistance and cell death mediated by RGA4 is repressed by RGA5 through the formation of heteroprotein complexes. The C-terminal part of RGA5 contains an HMA domain, initially found in a cytoplasmic copper chaperone in Saccharomyces cerevisiae, which can interact directly with AVR-PiA and AVR1-CO39, enabling pathogen recognition. Physical association of the AVR-PiA effector with the HMA domain of RGA5 triggers cell death through RGA4 derepression (Césari et al., 2014b). Interestingly, recognition of the AVR-Pik effector of M. oryzae by the unrelated CC-NLR pair Pik-1/Pik-2 in rice also is triggered by direct binding to an HMA domain in Pik-1 that, contrary to RGA5, is integrated between its CC and nucleotide-binding domain regions (Ashikawa et al., 2008; Maqbool et al., 2015). The different locations of the HMA domain in RGA5 and Pik-1 suggests that these domains have been fused to those two unrelated NLRs through independent events (Cesari et al., 2013). Although HMA domain-containing proteins have not been described previously as effector targets, the presence of an HMA domain in the rice Pi21 factor, which is required for full susceptibility to the rice blast fungus (Fukuoka et al., 2009; Zhang et al., 2016), supports the idea that the HMA domains of RGA5 and Pik-1 are decoys for AVR-PiA, AVR1-CO39, AVR-Pik, and functionally related effectors. Determination of the crystal structure of AVR-PikD complexed with a dimer of the Pikp-1 HMA domain revealed that key residues at the interaction interface are required for effector binding and recognition (Maqbool et al., 2015). In addition, variations at binding interfaces between AVR-Pik effector variants and HMA domains of Pik alleles were found to determine recognition specificity. Such recent findings highlight how new receptor specificities arise from natural selection (De la Concepcion et al., 2018). How the binding of effectors to HMA domains can trigger the activation of immune signaling remains unknown. It is hypothesized that effector binding promotes NLR domain rearrangements leading to immune complex activation (Césari et al., 2014b). The binding of AVR-PiA to the RGA5 HMA domain also is necessary for pathogen recognition, but protein-protein interaction analyses revealed that moderate affinity to mutated AVR-PiA proteins still confers recognition (Ortiz et al., 2017). Furthermore, additional sites in RGA5, outside the ID, are suspected to mediate interaction with the effector. Thus, the juxtaposition of integrated decoy domains with NLR sites having additional interacting properties creates a highly resilient surveillance system. The NOI/RIN4 Domain of Pii-2 Pathogen detection by paired NLR-IDs is not restricted to the direct recognition model. Indeed, IDs also might function in indirect recognition by perceiving modifications of a host protein targeted by an effector. This concept is supported by a study of the unconventional NOI/RIN4 domain of the rice NLR-ID Pii-2 that is hypothesized to monitor, through direct binding, the integrity of the OsExo70-F3 host protein, a target of the M. oryzae effector AVR-Pii (Fujisaki et al., 2017). NLR-IDS: A MECHANISM OF NLR DIVERSIFICATION The search for protein domains associated with typical NLR domains in public databases made it possible to identify entire NLR-ID directories and to analyze their structure in many plants. Already present in mosses, NLR-IDs occasionally represent a large proportion of NLRs in terrestrial plants (Kroj et al., 2016; Sarris et al., 2016; Bailey et al., 2018; Stein et al., 2018; Table 1). Kroj et al. (2016) detected 162 hypothetical IDs across 33 genomes by looking for interpro domains using the GreenPhyl database. Although their analysis was not exhaustive due to potential misannotations of the applied databases, they identified a high diversity of IDs (90 different domains). Sarris et al. (2016) reported 265 unique IDs fused to NLRs in 37 genomes of land plants. More recently, Bailey et al. (2018) identified NLR-IDs in nine grass species. Thirty-one types of different domains were represented mainly in these species. By focusing on 13 Oryza species, Stein et al. (2018) described a highly variable structure of genes coding for several hundreds of different NLR-ID proteins. They were able to detect a significant enrichment for these NLR-IDs within pairs of genes arranged in a head-to-head configuration in the genomes. The widespread distribution of NLR-IDs, despite their low abundance in some plant genomes (Table 1), suggests a successful evolutionary mechanism of NLR diversification commonly used by plants to expand their pathogen recognition capabilities, allowing them to cope with highly and rapidly adaptable pathogen-derived molecules. Accordingly, the IDs identified in these studies are derived from proteins that are extremely diverse in sequence and predicted molecular functions. The most frequent domains found in NLR-IDs include the WKRY and BED (BEAF and DREF proteins from Drosophila) zinc finger (Znf-BED) DNA-binding domains and the protein kinase domains. The decoy function of the WRKY domain in the RRS1-R NLR already has been validated (see above; Le Roux et al., 2015; Sarris et al., 2015). The Znf-BED domain was identified originally in transposases and transcription factors (Hayward et al., 2013), but, contrary to RRS1-R, the targeting by pathogen effectors remains to be demonstrated. Overview of NLR-ID repertoires in plant species Table 1. Overview of NLR-ID repertoires in plant species Reference . No. of Species Investigated . No. of Species with NLR-IDs . No. of NLR-IDs . No. of NLR-IDs per Species . Average Percentage of NLR-IDs (of All NLRs) . Kroj et al. (2016) 33 23 34 1 to 16 3.5 Sarris et al. (2016) 36 35 717 1 to 93 6.8 Bailey et al. (2018) 9 9 331 7 to 133 7.9 Funk et al. (2018) 1 1 24 24 14 Stein et al. (2018) 13 13 446 – 8.2 Reference . No. of Species Investigated . No. of Species with NLR-IDs . No. of NLR-IDs . No. of NLR-IDs per Species . Average Percentage of NLR-IDs (of All NLRs) . Kroj et al. (2016) 33 23 34 1 to 16 3.5 Sarris et al. (2016) 36 35 717 1 to 93 6.8 Bailey et al. (2018) 9 9 331 7 to 133 7.9 Funk et al. (2018) 1 1 24 24 14 Stein et al. (2018) 13 13 446 – 8.2 Open in new tab Table 1. Overview of NLR-ID repertoires in plant species Reference . No. of Species Investigated . No. of Species with NLR-IDs . No. of NLR-IDs . No. of NLR-IDs per Species . Average Percentage of NLR-IDs (of All NLRs) . Kroj et al. (2016) 33 23 34 1 to 16 3.5 Sarris et al. (2016) 36 35 717 1 to 93 6.8 Bailey et al. (2018) 9 9 331 7 to 133 7.9 Funk et al. (2018) 1 1 24 24 14 Stein et al. (2018) 13 13 446 – 8.2 Reference . No. of Species Investigated . No. of Species with NLR-IDs . No. of NLR-IDs . No. of NLR-IDs per Species . Average Percentage of NLR-IDs (of All NLRs) . Kroj et al. (2016) 33 23 34 1 to 16 3.5 Sarris et al. (2016) 36 35 717 1 to 93 6.8 Bailey et al. (2018) 9 9 331 7 to 133 7.9 Funk et al. (2018) 1 1 24 24 14 Stein et al. (2018) 13 13 446 – 8.2 Open in new tab However, Kroj et al. (2016) showed that the ZBED NLR protein from rice, containing three BED domains, is required for resistance to M. oryzae. In response to the pathogen, ZBED-overexpressing lines were more resistant, whereas a zbed null mutant showed increased susceptibility. These data strongly suggest that the BED domains in ZBED NLR proteins represents decoys that mimic host BED proteins targeted by M. oryzae effectors. The Xa1 NLR from rice, which confers resistance against isolates of the bacterial blight pathogen Xanthomonas oryzae by recognizing multiple transcription activator-like effectors (TALEs; Yoshimura et al., 1998; Ji et al., 2016), contains a Znf-BED domain in its N-terminal part. The mechanism that allows Xa1 to recognize TALEs remains to be elucidated. It is tempting to speculate that the Xa1 Znf-BED domain also might act as a decoy to lure TALEs that target host Znf-BED proteins for the subversion of host gene expression (Zuluaga et al., 2017). Also, the functionality of NLR IDs with predicted catalytically active protein kinase domains needs to be experimentally validated. However, their sensing abilities can be deduced from well-described examples of kinases acting as decoys that interact physically with classical NLRs (e.g. the kinases Pto and PBS5 interacting with the NLRs Prf and RPS5, respectively; Khan et al., 2016). It is noteworthy that, in primitive land plants, the fusion of kinase domains or DUF676 to NBS-LRRs that lack CC or TIR domains has been described (Gao et al., 2018), suggesting that these kind of IDs could ensure the signaling function of these missing domains. Therefore, such IDs in NLR proteins could fulfill either sensor or signaling functions, or both. Interestingly, there are significant overlaps between IDs and protein domains identified previously as interacting partners of effectors in interactome screens (Mukhtar et al., 2011; Weßling et al., 2014; Sarris et al., 2016), including well-characterized guardees or decoys. Examples are the exocyst complex factor Exo70, required for the recognition of AvrPii by NLR Pii in rice (Fujisaki et al., 2015), and RIN4, a target of multiple effectors that is guarded by RPS2 and RPM1 NLRs in Arabidopsis (Mackey et al., 2002, 2003; Kim et al., 2005). Such overlaps strongly suggest that IDs could act as sensor/decoy by mimicking effector targets. Since many IDs correspond to protein domains with unknown biological activity, they represent promising candidates to uncover host components targeted by effectors and whose participation in various layers of plant immunity has not been assigned yet. Whether all the putative IDs identified in the whole-genome analyses also serve as sensor/decoy in immune responses remains to be demonstrated. Moreover, detailed investigations on gene structure and function should help to reduce false-positive results among computationally predicted NLR-IDs (Giannakopoulou et al., 2016) and shed light on new resistance mechanisms. MECHANISMS INVOLVED IN NLR-ID FUSION EVENTS Within NLR-IDs, the majority of IDs appear as singular N- or C-terminal domains. However, in some cases, the fusion of several domains in the same protein is observed. For example, the AtWRKY19 NLR in Arabidopsis integrates both an N-terminal WRKY domain and a C-terminal kinase domain. In a minority of cases, including rice Pik-1, integration has occurred between the N-terminal signaling domain and the central nucleotide-binding domain of the NLR. These observations indicate that some NLRs can tolerate the integration of sensor domains at various positions in their modular architecture while maintaining their signaling functions. Bailey et al. (2018) recently investigated the evolutionary dynamics of NLR-IDs in the genomes of nine grass species. They concluded that NLR-IDs in grasses were not distributed evenly across their phylogeny, but a specific clade with up to 58% of NLRs containing IDs was observed. In this clade, they highlighted an amino acid sequence motif located immediately upstream of the fusion site, which could play an important role in the integration process. They proposed that DNA transposition and/or ectopic recombination is a major driving force behind domain integration in grasses; repeated independent integration events were observed, suggesting that integration occurred frequently and independently during evolution, giving rise to a high diversity of IDs. Similarly, Brabham et al. (2018) recently showed that orthologs of the RGH2 NLR from species across the grasses were subject to large variation in domain structure, including the presence/absence of an integrated Exo70 domain. These transspecies polymorphisms provided an opportunity to follow the molecular evolution of the Exo70 gene family and to investigate the role of Exo70 as an ID in the RGH2 NLR. This study showed that, upon pathogen pressure, nonintegrated Exo70 genes are under strong purifying selection, whereas they are under relaxed purifying selection when integrated into RGH2. Across the Oryza genomes, the presence of IDs in 17 different NLR subfamilies (from a total of 36) point to multiple and independent acquisition of IDs (Stein et al., 2018). NLR-IDS: TOWARD THE ELUCIDATION OF ADDITIONAL NLR FUNCTIONS? Besides the well-documented role of NLRs for innate immunity in both animals and plants, additional functions controlled by NLRs are currently discussed. In animals, NLRs play a role in developmental processes, such as spermatogenesis and fertility, suggesting a control of the reproductive system by NLR proteins (Meunier and Broz, 2017). In plants, inappropriate activation of NLRs caused by mutations or incompatible combinations also impacts development (Chae et al., 2014; Chen et al., 2016; Atanasov et al., 2018; Chakraborty et al., 2018). The integrated decoy model predicts an important role of IDs in pathogen detection. Beyond their function of effector sensor, some IDs might have retained the biological activities of the proteins from which they are derived. Thus, additional functions controlled by NLRs in plants could be revealed by looking at IDs. In this regard, the integration of a BED domain, one of the most frequently found IDs in plant species, gained attention. This domain appears to be shared by transposases and by proteins that perform critical cellular functions (Aravind, 2000; Hayward et al., 2013). The Znf-BED domain of the Tam3 (Transposase of Activator from maize) transposase has been shown to suppress the DNA TAM3 transposon activity in Anthirrhinum majus by relocalizing the Tam3 transposase out of the nucleus (Zhou et al., 2017). More globally, BED-related IDs could be sensors of cellular homeostasis perturbation by environmental stress that rely in part on DNA transposition control (Negi et al., 2016). In the case of RRS1, inhibition of its DNA-binding activity provoked by particular mutations in its WRKY domain leads to autoimmunity in an RPS4-dependent manner (Noutoshi et al., 2005; Sohn et al., 2014). Therefore, RRS1-R was initially considered a negative regulator of immune-related genes. The autoimmune phenotype of the RRS1-R slh1 variant is conditioned by low humidity, suggesting that RRS1-R, besides its function in pathogen recognition, also could sense particular environmental disturbances such as drought stress. In response to specific biotic and abiotic stresses, RRS1-R likely acts directly at genomic DNA and, together with RPS4, behaves as a reactive switch for transcriptional and signaling reprogramming. Whether NLR-IDs possess broader sensing functions remains to be determined, but almost certainly the functional characterization of their IDs will help to elucidate potential additional NLR functions. TOWARD THE ENGINEERING OF SYNTHETIC NLR-IDS WITH EXTENDED RECOGNITION CAPABILITIES The high diversity of NLRs provides plants with versatile options for effective plant immunity. However, on the one hand, the ability of effectors to evolve rapidly and, on the other hand, the necessary fine-tuning of NLR functions to avoid autoimmunity (Box 1) restrict the possibilities to transfer immune receptors into other plant genomes for improved crop protection. Hence, strategies for NLR engineering are of particular interest in plant immunity research. If successful, it could be possible to modify NLR recognition specificity or, alternatively, widen the range of effector recognition while making it less feasible for pathogens to bypass NLRs. One possibility is to alter the structure of NLR proteins itself. For example, a study conducted by Segretin et al. (2014) showed that a few amino acid changes in the LRR domain of R3a in potato (Solanum tuberosum) enabled this NLR to recognize another isoform of AVR3a from Phytophthora infestans. However, direct NLR effector recognition (modified self) is likely to be less tolerant to variations compared with the indirect and integrated decoy models. Therefore, decoy engineering represents a more suitable approach. A proof-of-concept study was performed recently by Kim et al. (2016), demonstrating successfully the modification of the Arabidopsis protein kinase PBS1 that represents a decoy for pathogen-derived proteases. PBS1 is involved in basal immune responses and acts as target/decoy for the effector AvrPphB from P. syringae, which can cleave PBS1 due to its protease activity (Shao et al., 2003; Ade et al., 2007). PBS1 cleavage products are recognized by the NLR protein RPS5 (DeYoung et al., 2012) that, in turn, initiates resistance signaling. For a modified PBS1 decoy variation, the proteolytic target site, which is normally cleaved by AvrPphB, is exchanged with another proteolytic site. This modification enables the cleavage of PBS1 by other effectors, such as AvrRpt2 of P. syringae, NIa protease of Tobacco etch virus, and Nla protease of Turnip mosaic virus. AvrRpt2 also triggers the activation of RPS2 NLR by cleaving a nitrate-induced (NOI) domain present in RIN4, a negative regulator of plant defense that is guarded by multiple NLR proteins (Mackey et al., 2002, 2003; Kim et al., 2005). Such protease cleavage sites are found in IDs present in a subset of NLRs, paving the way for the design of single NLR-IDs combining multiple protease recognition sequences. Open in new tabDownload slide Open in new tabDownload slide The modifications of NLR IDs, as well as the replacement of IDs with other identified effector targets from the host genome, display other promising tools to create novel effector traps. Considering that modifications of NLRs as well as of their IDs can compromise the homocomplex/heterocomplex formation required for their function and trigger either inactivation or autoactivation of the receptor, it is important for this approach to identify which IDs and which NLRs are best suited for fusion manipulation (e.g. ID swapping/shuffling). For this, we need to gain better knowledge of interaction sites within NLR and its IDs but also of the interaction of sensor and signaling NLRs. Furthermore, it is important to determine the layout of effector-ID complexes. Recently, the structures of several complexes have been resolved (Maqbool et al., 2015; Ortiz et al., 2017; Zhang et al., 2017). Determination of the molecular and structural bases of these interactions is crucial for the successful design of synthetic multiple-sensor NLR-IDs made from the juxtaposition of different IDs, giving them extended recognition capabilities. Open in new tabDownload slide Open in new tabDownload slide CONCLUSION The past 20 years have brought major advances in plant NLR biology. The discovery of NLR-IDs represents a significant step toward a better understanding of the mechanisms involved in the evolution and function of NLRs. During plant evolution, NLR-IDs appeared independently several times and in different configurations. Besides the sensing and regulating functions of IDs, important questions remain unanswered (see Outstanding Questions). There is still a critical need for further in-depth studies to establish the biological functions of IDs fused to NLRs and to elucidate the molecular events that link NLR-ID activation with immunity pathways. Overall, NLR-IDs provide promising tools for the design of new strategies to protect plants against pathogens. Nevertheless, it remains to be determined whether engineered synthetic NLR-IDs can provide sustainable disease resistance, especially in different crop species. 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