A Pelota-like gene regulates root development and defence responses in rice

A Pelota-like gene regulates root development and defence responses in rice Abstract Background and Aims Pelota (Pelo) are evolutionarily conserved genes reported to be involved in ribosome rescue, cell cycle control and meiotic cell division. However, there is little known about their function in plants. The aim of this study was to elucidate the function of an ethylmethane sulphonate (EMS)-derived mutation of a Pelo-like gene in rice (named Ospelo). Methods A dysfunctional mutant was used to characterize the function of OsPelo. Analyses of its expression and sub-cellular localization were performed. The whole-genome transcriptomic change in leaves of Ospelo was also investigated by RNA sequencing. Key Results The Ospelo mutant showed defects in root system development and spotted leaves at early seedling stages. Map-based cloning revealed that the mutation occurred in the putative Pelo gene. OsPelo was found to be expressed in various tissues throughout the plant, and the protein was located in mitochondria. Defence responses were induced in the Ospelo mutant, as shown by enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae, coupled with upregulation of three pathogenesis-related marker genes. In addition, whole-genome transcriptome analysis showed that OsPelo was significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. Detailed analysis showed that activation of a number of innate immunity-related genes might be responsible for the enhanced disease resistance in the Ospelo mutant. Conclusions These results demonstrate that OsPelo positively regulates root development while its loss of function enhances pathogen resistance by pre-activation of defence responses in rice. Short root, spotted-leaf mutant, defence responses, RNA-seq, Oryza sativa INTRODUCTION Pelota (PELO) is an evolutionarily conserved protein and has been identified in a number of species, including Drosophila (Eberhart and Wasserman, 1995), archaebacteria (Ragan et al., 1996), yeast (Davis and Engebrecht, 1998), Arabidopsis thaliana (Caryl et al., 2000), human (Shamsadin et al., 2000), mouse (Shamsadin et al., 2002) and tomato (Lapidot et al., 2015). The PELO proteins contain 347–395 amino acid residues and RNA-binding domains similar to that found in the family members of the eukaryotic release factor 1 (eRF1) family members, which play roles in termination of protein synthesis (Davis and Engebrecht, 1998). The study in Drosophila first identified that the cell cycle is arrested during the meiotic G2/M transition phase in germline cells of Pelo male homozygotes, while only mitotic division is affected during oogenesis (Eberhart and Wasserman, 1995). In addition, the eyes of Pelo homozygotes are smaller than those of their heterozygous siblings. Further study revealed a critical role for PELO proteins in regulating self-renewal of germline stem cells (GSCs) by repressing the differentiation pathway (Xi et al., 2005). Disruption of the balance between self-renewal and differentiation of GSCs impaired the fertility of loss-of-function pelo mutant females. A similar role for PELO protein in meiotic and mitotic division was also found in Saccharomyces cerevisiae, where the disruption Dom34, the orthologue gene of Pelo, resulted in growth retardation and defective sporulation (Davis and Engebrecht, 1998). In mice, disruption of the Pelo gene caused early embryonic lethality and cell cycle defects (Adham et al., 2003). Further analysis found that PELO mediated gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes (Raju et al., 2015). PELO is also found to regulate extraembryonic endoderm development and epidermal differentiation, and to inhibit tumour progression and invasion (Nyamsuren et al., 2014; Pedersen et al., 2014; Elkenani et al., 2016). In addition, PELO is involved in high efficiency viral replication (Wu et al., 2014), and regulates a resistance reaction to begomovirus in tomato (Lapidot et al., 2015). Extensive studies have been conducted in yeast to characterize the function of PELO at the molecular level. The PELO-coding orthologue Dom34 together with its interacting partner Hbs1 participate in an RNA quality control mechanism called no-go decay (NGD) for the recycling of stalled ribosomes (Doma and Parker, 2006; Shoemaker and Green, 2011; Guydosh and Green, 2014). The structure of the DOM34–HBS1 complex is similar to that of eRF1 and eRF3, but Dom34 does not have motifs for codon recognition and peptide release (Chen et al., 2010; Becker et al., 2011). It was proposed that the DOM34–HBS1 complex binds to the ribosomal A site to promote dissociation of ribosome subunits (Shoemaker et al., 2010). In addition to the RNA quality control in the NGD pathway, DOM34–HBS1 is also important for non-stop decay (NSD), i.e. decay of non-functional 18S rRNAs and mRNAs with premature stop codons (Cole et al., 2009; Tsuboi et al., 2012; Saito et al., 2013). The DOM34–HBS1 complex also mediates dissociation of inactive 80S ribosomes to promote the restart of translation after stress (van den Elzen et al., 2014). The roles of DOM34/PELO in mRNA quality control are conserved in Drosophila and human (Ikeuchi et al., 2016; Hashimoto et al., 2017). The PELO–HBS1 complex is also involved in transposon silencing in the Drosophila germline (Yang et al., 2015). Recently, studies showed that defects in GTPBP2, a PELO-binding partner in mammals, resulted in ribosome stalling in a tRNAArg (UCU) mutant background and the death of mouse neurons (Ishimura et al., 2014; Kirmizitas et al., 2014). Little is yet known about the overall function of Pelo genes in plants. In this study, a Pelo-deficient rice mutant was isolated from an ethylmethane sulphonate (EMS)-mutagenized library. We describe a root development defect, a spotted-leaf phenotype and enhanced pathogen resistance for Pelo null rice. MATERIALS AND METHODS Plant materials and growth conditions The rice mutant Ospelo was isolated from an EMS-mutagenized rice (Oryza sativa L. indica, ‘Kasalath’) mutant library. Hydroponic experiments were conducted in normal rice culture solution with the pH adjusted to 5.5 (Zhu et al., 2012). In all hydroponic experiments, plants were grown in a greenhouse with a 12 h light (30 °C)/12 h dark (22 °C) cycle (16 000 lux) and a humidity of 70 %. Acetocarmine and 5-ethynyl-2’-deoxyuridine (EdU) staining Primary root tips of 4-day-old wild type (WT) and Ospelo plants were stained with 1 % acetocarmine solution for 10 min. After washing with 45 % acetic acid solution, they were mounted on glass slides and examined with a stereo microscope (Leica MZ95). EdU staining was conducted using an EdU kit (C10350, Click-iT EdU Alexa Fluor 488 HCS assay; Invitrogen) according to the manufacturer’s instruction. Roots of 4-day-old WT and Ospelo plants were immersed in 20 mm EdU solution for 2 h and then fixed for 30 min in 3.7 % formaldehyde solution in phosphate buffer (pH 7.2) with 0.1 % Triton X-100. After that they were incubated with EdU detection cocktail for 30 min and examined with the green fluorescent protein (GFP) channel on a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany). More than ten root samples of each genotype were examined, and the experiment was repeated twice. Trypan blue staining Trypan blue staining was performed on fresh leaves as previously described (Yin et al., 2000). In brief, leaf samples were submerged in lactic acid–phenol–trypan blue (LPTB) solution [2.5 mg mL–1 trypan blue, 25 % (w/v) lactic acid, 23 % water-saturated phenol and 25 % glycerol in H2O] at 30 °C for 12 h. The LPTB solution was then replaced with a chloral hydrate solution (50 g in 20 mL of H2O) for destaining. After multiple changes of chloral hydrate solution for 4 d, leaf samples were washed with H2O and mounted on glass slides before being examined with a stereo microscope (Leica MZ95). More than ten leaf samples were examined for each genotype, and the experiment was repeated three times. Pollen activity staining assay Spikelets about to flower from the WT and Ospelo were chosen for examination. After carefully opening the hull, anthers were placed in several drops of 1 % I2–KI solution on glass slides and crumbled to release pollens. Then they were examined with a light microscope (Nikon eclipse 80i). Samples from more than five plants of each genotype were examined, and the experiment was repeated twice. Histological observation Root tips from 4-day-old plants were fixed overnight at 4 °C in 2.5 % glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.2, and washed three times for 30 min in the same buffer. The samples were then refixed in OsO4 for 4 h at room temperature and washed for 30 min in the same buffer. Samples were dehydrated in a gradient ethanol, embedded in pure Spurr resin and polymerized overnight at 70 °C. Semi-thin sections (2 μm thick) were made using diamond knives on a power Tome XL microtome (RMC-Boeckeler Instruments, Tucson, AZ, USA) and stained with 0.1 % methylene blue for 3–5 min at 70 °C. The samples were rinsed with distilled water and visualized with a microscope (Nikon 90i, Japan). Mapping and cloning of OsPelo A mapping population was generated from crosses between the homozygous Ospelo mutant and japonica variety Nipponbare. A total of 30 and 538 short root plants from the F2 population were used for primary and fine mapping of OsPelo, respectively. The OsPelo gene was localized to a region of 411 kb between the sequence-tagged site (STS) markers STS1 and STS2 on chromosome 4. Primers used are listed in Supplementary Data Table S1. The OsPelo gene was selected from 52 putative protein-coding genes as one of the candidate genes. To identify the mutation site, genes were amplified by PCR from the genomic DNA of both WT and Ospelo plants and used for Sanger sequencing analysis. Construction of vectors and plant transformation The coding region of OsPelo was PCR amplified and put into the pUCM-T vector (Takara). After sequencing confirmation, the fragment was excised from the pUCM-T vector by BamHI and PstI digestion and ligated into the corresponding site of pCAMBIA1300. A 2264 bp promoter of OsPelo was obtained by PCR and inserted into the HindIII/BamHI site in front of the OsPelo coding region to drive its expression. The promoter was also put into the HindIII/BamHI site of vector pCAMBIA1300NH-GUS to create a transcriptional fusion of the OsPelo promoter and the β-glucuronidase (GUS) coding sequence, OsPelop::GUS. The above constructs were used for Agrobacterium tumefaciens-mediated rice transformation of the WT or Ospelo as described (Chen et al., 2003). Histochemical analysis and GUS assay Histochemical GUS staining was performed as previously described (Ding et al., 2015). Transgenic plant samples and freehand cross-section samples were incubated with GUS staining solution (100 mmol L–1 NaH2PO4 buffer pH 7.0, 0.5% Triton X-100, 0.5 mg mL–1 X-Gluc and 20 % methanol) overnight at 37 °C. Tissues were subsequently rinsed and mounted on slides, and photographed using a stereo microscope (Leica MZ95, Nussloch, Germany). Sub-cellular localization of OsPELO The full-length coding sequence of OsPelo with the eliminated stop codon was inserted in-frame before the coding sequence of a soluble modified GFP (smGFP4). The OsPELO–GFP fusion-coding sequence was subcloned into the binary vector 35S-pCAMBIA1301. The resulting construct was sequenced to verify in-frame fusion and used for transient transformation of onion epidermis using a biolistic PDS-1000/He particle delivery system (Bio-Rad, Hercules, CA, USA). Alternatice oxidase (AOX)–red fluorescent protein (RFP) located to the mitochondria was co-transformed as a mitochondrial marker. The GFP and RFP were visualized using a confocal laser-scanning microscope (Zeiss LSM 510). The experiment was repeated twice. Determination of resistance to bacterial blight in Ospelo Three races of Xanthomonas oryzae pv. oryzae (Xoo) were used for evaluation of bacterial blight resistance. The Philippines races PXO71, PXO99 and PXO145 were kindly provided by Dr Jie Zhou of the Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, China. New fully expanded leaves of ten independent WT and Ospelo plants at the maximum tillering stage were inoculated for each race of Xoo using the clipping leaf method (Kauffman et al., 1973). The lesion length on inoculated plants was measured 3 weeks after inoculation. Quantitative real-time PCR (qRT-PCR) analysis RNA was extracted from three biological replicates of leaf samples of 20-day-old WT and Ospelo plants using RNAiso plus (Takara). Three technical replicates were performed for each sample. The first-strand cDNA was synthesized from total RNA using Superscript II (Invitrogen, Carlsbad, CA, USA) and used as the qRT-PCR template. Real-time PCR analysis was performed using a Roche Lightcycler480 real-time PCR system with SYBR Premix Ex Taq (Takara). Primers used are listed in Supplementary Data Table S1. The Ubiquitin gene (LOC_Os03g13170) in rice was used as a reference gene. Transcriptome sequencing Total RNA was extracted from leaves of 20-day-old WT and Ospelo plants under normal conditions using the RNeasy Plant Mini Kit (Qiagen, USA). Three biological replicates for each genotype were collected. RNA was quantified using the Nanodrop-2000 (ThermoFisher, USA) and RNA quality was then examined using a 2100 Bioanalyzer (Agilent Technologies, USA). High-quality RNA samples for library construction were selected based on the 260/280 nm ratio and RNA integrity number (RIN) above 2.0 and 7.0, respectively. Sequencing libraries were prepared using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were subjected to 125 cycles of paired-end sequencing with the Illumina HiSeq2500 system according to the manufacturer’s instructions. The raw sequencing data have been uploaded to the SRA (Sequence Read Archive; https://www.ncbi.nlm.nih.gov/sra) database (accession no. SRP117240). Differentially expressed gene analysis Raw reads were first processed using in-house Perl scripts. In this step, clean reads were obtained by removing reads containing adaptor, reads containing ploy-N and low-quality reads (the number of bases with quality value ≤20 is more than 35). The retained high-quality reads, i.e. clean reads, were then analysed by the TopHat–Cufflinks pipeline (Trapnell et al., 2012). Briefly, clean reads were mapped to the rice genome (MSU version 7, http://rice.plantbiology.msu.edu/) using TopHat. Cufflinks was then used for transcriptome assembly and assessment of the FPKM (fragments per kilobase of transcript per million mapped reads) value. Counts of mapped reads to genes were obtained with HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) and differentially expressed genes (DEGs) were determined using DESeq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html) by the Negative Binomial Distribution test (Love et al., 2014; Anders et al., 2015). Genes with a false discovery rate (FDR)-adjusted P-value <0.05 were assigned as DEGs. Gene Ontology (GO) annotation was conducted by querying Swiss-Prot (http://www.uniprot.org) with transcripts of DEGs and enrichment analysis was performed by the hypergeometric distribution test using R with a threshold FDR-adjusted P-value <0.01. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted by PlantGSEA with a threshold FDR-adjusted P-value <0.01 (Yi et al., 2013). The functional categorization of DEGs was conducted by MapMan (Thimm et al., 2004). RESULTS The rice Ospelo mutant showed root and leaf growth defects A mutant showing short roots and spotted leaves was isolated from an EMS-mutagenized rice mutant library (Oryza sativa L. indica ‘Kasalath’). The mutant was designated as Ospelo, which will be described later on (Fig. 1A). After growth in culture solution for 7 d, the primary root length of Ospelo (8.91 ± 1.06 cm) was only 57.9 % of that of the WT (15.38 ± 0.88 cm) (Fig. 1B). Moreover, the length of adventitious roots and lateral roots of Ospelo was also significantly shorter than that of those of the WT (Fig. 1B), while shoot height was slightly shorter than that of the WT (Fig. 1B). Fig. 1. View largeDownload slide Phenotypic characterization of Ospelo. (A) Growth phenotype of 7-day-old WT and Ospelo plants. Scale bar = 2 cm. (B) The primary, lateral and adventitious root length of the 7-day-old WT and Ospelo plants. The three longest adventitious roots and the 20 longest lateral roots on each primary root were counted. Error bars represent the s.d. (n = 20). (C) Longitudinal sections of the maturation zone (top) and root tip (bottom) of 4-day-old WT and Ospelo plants. Scale bars = 100 µm. (D) S-phase entry of 4-day-old WT and Ospelo root tips visualized by EdU staining. Scale bar = 100 µm. (E) Size of EdU-labelled root apical meristems (RAMs) of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10). (F) Acetocarmine staining of root tips of 4-day-old WT and Ospelo plants. Scale bar = 0.5 mm. (G) Size of acetocarmine-stained root apical meristems of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10) (H) Base, middle and tip regions of leaves from 30- and 60-day-old WT and Ospelo plants. Scale bars = 1 cm. (I) Trypan blue staining of leaves from 60-day-old WT and Ospelo plants Leaves decolorized directly by ethanol were included as control. Scale bars = 1 mm. The asterisks in (B), (E) and (G) indicate a significant difference between the WT and Ospelo (P < 0.01, by Student’s t-test). Fig. 1. View largeDownload slide Phenotypic characterization of Ospelo. (A) Growth phenotype of 7-day-old WT and Ospelo plants. Scale bar = 2 cm. (B) The primary, lateral and adventitious root length of the 7-day-old WT and Ospelo plants. The three longest adventitious roots and the 20 longest lateral roots on each primary root were counted. Error bars represent the s.d. (n = 20). (C) Longitudinal sections of the maturation zone (top) and root tip (bottom) of 4-day-old WT and Ospelo plants. Scale bars = 100 µm. (D) S-phase entry of 4-day-old WT and Ospelo root tips visualized by EdU staining. Scale bar = 100 µm. (E) Size of EdU-labelled root apical meristems (RAMs) of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10). (F) Acetocarmine staining of root tips of 4-day-old WT and Ospelo plants. Scale bar = 0.5 mm. (G) Size of acetocarmine-stained root apical meristems of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10) (H) Base, middle and tip regions of leaves from 30- and 60-day-old WT and Ospelo plants. Scale bars = 1 cm. (I) Trypan blue staining of leaves from 60-day-old WT and Ospelo plants Leaves decolorized directly by ethanol were included as control. Scale bars = 1 mm. The asterisks in (B), (E) and (G) indicate a significant difference between the WT and Ospelo (P < 0.01, by Student’s t-test). To investigate the cause of the short root phenotype of Ospelo at a cellular level, root longitudinal section analysis was conducted. It showed that the radial organization patterns and the quiescent centre (QC) of the stem cell niche in Ospelo were comparable with those of the WT (Fig. 1C). The cell length of maturation zones of Ospelo was also similar to that in the WT (Fig. 1C). To determine the root meristem activity of Ospelo, 4-day-old seedlings were cultured for 2 h in the presence of the thymidine analogue EdU, and in situ incorporation of EdU into DNA during active DNA synthesis in the root tip was visualized (Kotogány et al., 2010). Compared with the WT, Ospelo had reduced levels of EdU labelling in the root meristem (Fig. 1D, E). Consistent with this, the traditional acetocarmine staining also showed that the meristematic region in Ospelo was reduced compared with the WT (Fig. 1F, G). Taken together, these results suggested that the meristematic activity in the Ospelo root is compromised. Moreover, lesion-mimic spots appeared on Ospelo leaves after being grown in culture solution for about 20 d. The spotted-leaf phenotype became more severe as plants grew and expanded into whole leaves of 60-day-old Ospelo (Fig. 1H). In order to examine the occurrence of cell death or irreversible membrane damage, leaves of the WT and Ospelo were stained with trypan blue. As expected, there were clearly dyed blue spots on Ospelo leaves, while no detectable staining was observed on WT leaves (Fig. 1I). Critical roles of OsPelo in development and fertility The mature stage development and fertility of Ospelo were severely impaired (Fig. 2A, B). The plant height, tiller number and seed setting rate of Ospelo were all significantly decreased compared with the WT (Fig. 2C). However, the 1000-grain weight of Ospelo was similar to that of the WT (data not shown). To find the cause of the fertility defect, reproductive organs of Ospelo were further examined. There was no significant difference in pistils of Ospelo and the WT (Fig. 2D), and a successful cross using Ospelo as the female for the mapping population also confirmed its normal female fertility (data not shown). However, anthers of Ospelo were found to be pale compared with the healthy yellow anthers of the WT, indicating a severe defect in pollen development (Fig. 2E). Further staining analysis showed that pollen development of Ospelo was dysfunctional and their fertility was dramatically lower than in the WT (Fig. 2F). Fig. 2. View largeDownload slide Comparison of agronomic traits and reproductive organs in the WT and Ospelo. (A) Mature WT and Ospelo plants. Scale bar = 20 cm. (B) Spikes of the WT and Ospelo. Scale bar = 4 cm. (C) Plant height, tiller number and seed setting rate of the WT and Ospelo at the maturation stage. Error bars represent the s.d. (n = 20). (D) Pistils of the WT and Ospelo. Scale bars = 0.5 mm. (E) Stamens of the WT and Ospelo. Scale bars = 0.5 mm. (F) I2–IK solution staining of pollen from the WT and Ospelo. Scale bars = 50 µm Fig. 2. View largeDownload slide Comparison of agronomic traits and reproductive organs in the WT and Ospelo. (A) Mature WT and Ospelo plants. Scale bar = 20 cm. (B) Spikes of the WT and Ospelo. Scale bar = 4 cm. (C) Plant height, tiller number and seed setting rate of the WT and Ospelo at the maturation stage. Error bars represent the s.d. (n = 20). (D) Pistils of the WT and Ospelo. Scale bars = 0.5 mm. (E) Stamens of the WT and Ospelo. Scale bars = 0.5 mm. (F) I2–IK solution staining of pollen from the WT and Ospelo. Scale bars = 50 µm Map-based cloning of OsPelo To identify the mutated gene, an F2 population was developed by crossing the Ospelo mutant with Nipponbare (japonica). The F1 seedlings displayed the WT phenotype and their F2 progeny showed segregation of WT and Ospelo phenotypes at a ratio close to 3:1 (257:81, χ2 = 0.32, P < 0.05), indicating that the Ospelo phenotype was controlled by a single recessive nuclear gene. The OsPelo locus was first mapped to chromosome 4 between simple sequence repeat (SSR) markers RM3217 and RM567 using 30 F2 mutant plants (Fig. 3A). The OsPelo gene was further mapped to a 411 kb region between two new STS markers STS1 and STS2 using 538 F2 mutant plants (Fig. 3A). Fifty-two open reading frames (ORFs) were predicted in this region (http://rice.plantbiology.msu.edu/). Sanger sequencing analysis for both the WT and Ospelo mutant identified one single-base insertion after 1630 bp from the start codon on the fourth exon of LOC_Os04g56480 (Fig. 3A). The insertion introduced a premature stop codon and putatively yielded a peptide with 89 amino acid residues (Fig. 3B). LOC_Os04g56480 encodes rice Pelota, which is the homologue of Pelota in Drosophila (Eberhart and Wasserman, 1995). Therefore, we named it OsPelo. The OsPelo gene is 7945 bp in length, and contains 15 exons and 14 introns. The protein-coding region of OsPelo is 1137 bp and encodes a 378 amino acid protein. The protein structure is consistent with the annotation generated by pfam (pfam.xfam.org), with three conserved eRF1 domains (Fig. 3B). A search in the rice genome with the full-length OsPELO protein sequence using BLASTp showed that it is a single-copy gene. Fig. 3. View largeDownload slide OsPelo encodes the PELO protein in rice. (A) Map-based cloning of OsPelo. Black boxes represent exons, and lines represent introns. White boxes indicate untranslated regions. The arrowhead shows the site of the single-base insertion after the nucleotide 1630 bp downstream of ATG. (B) Predicted domains of OsPELO by the pfam database (pfam.xfam.org). The arrowhead indicates the insertion within the eRF1_1 domain, which results in the production of a truncated peptide with 89 amino acid residues. (C) The ribbon diagram of the 3-D structure of OsPELO predicted by SWISS-MODEL (https://swissmodel.expasy.org). The other two ribbon diagrams are published crystal structures of PELO from human (SWISS-MODEL Template Library ID: 5LZW.78) and Dom34 from yeast (PDB ID: 2VGM). (D) Complementation analysis of Ospelo. The root and leaf phenotype of Ospelo was completely recovered by transformation of OsPelo driven by its native promoter. Two independent lines of transgenic plants (Comp1 and Comp2) in the Ospelo mutant background are shown. Scale bar left = 4 cm; right = 1 cm. Fig. 3. View largeDownload slide OsPelo encodes the PELO protein in rice. (A) Map-based cloning of OsPelo. Black boxes represent exons, and lines represent introns. White boxes indicate untranslated regions. The arrowhead shows the site of the single-base insertion after the nucleotide 1630 bp downstream of ATG. (B) Predicted domains of OsPELO by the pfam database (pfam.xfam.org). The arrowhead indicates the insertion within the eRF1_1 domain, which results in the production of a truncated peptide with 89 amino acid residues. (C) The ribbon diagram of the 3-D structure of OsPELO predicted by SWISS-MODEL (https://swissmodel.expasy.org). The other two ribbon diagrams are published crystal structures of PELO from human (SWISS-MODEL Template Library ID: 5LZW.78) and Dom34 from yeast (PDB ID: 2VGM). (D) Complementation analysis of Ospelo. The root and leaf phenotype of Ospelo was completely recovered by transformation of OsPelo driven by its native promoter. Two independent lines of transgenic plants (Comp1 and Comp2) in the Ospelo mutant background are shown. Scale bar left = 4 cm; right = 1 cm. PELO is a conserved protein involved in the mRNA NGD pathway based on its structural similarity to tRNA (Kobayashi et al., 2010). The putative tertiary structure of OsPELO was predicted using the online SWISS-MODEL server (https://swissmodel.expasy.org) (Biasini et al., 2014). As expected, its structure showed high similarity to those of human and yeast PELOs, with three conserved domains arranged in an L-shape (Fig. 3C). This further confirms that OsPELO is the rice homologue of PELO. Functional complementation test of Ospelo To confirm that the single nucleotide insertion in OsPelo is responsible for the mutant phenotype, complementation analysis was conducted using A. tumefaciens-mediated transformation. The protein-coding region of OsPelo was cloned into a binary vector driven by its 2264 bp native promoter and used for transformation of Ospelo. More than 20 independent transgenic lines were obtained. The short root and spotted-leaf phenotype was restored in all the positive transformants (Fig. 3D). These results demonstrate that the single base insertion in OsPelo causes the short root and spotted-leaf phenotype in Ospelo. Expression pattern and sub-cellular localization analysis of OsPelo To determine the tissue-specific expression pattern of OsPelo, a 2264 bp native promoter was fused to the GUS reporter gene. This chimeric gene cassette was used to transform WT plants via the A. tumefaciens-mediated transformation method. Histochemical staining for GUS activity in T2 plants showed that OsPelo was ubiquitously expressed in plant organs, including the primary root tip, tip and base of lateral roots, leaf vein and guard cells, stem and auricle, ligule, lemma, anther, stigma, glume, peduncle, pollen and paddle (Fig. 4A–H). Fig. 4. View largeDownload slide Expression pattern of OsPelo and sub-cellular localization of OsPELO. (A–H) Histochemical staining analysis of expression of the OsPelo promoter–GUS fusion in various tissues. GUS signals were detected in the primary root tip (A), the tip and base of lateral roots (B), leaf vein and guard cells (C), stem and auricle (D), ligule (E), young spikelet (F), pollen (G) and paddle (H). Scale bars = 0.2 mm in (A–F, H), 10 µm in (G). (I) OsPELO targets GFP to mitochondria in transiently transformed onion epidermal cells. The AOX–RFP is used as the mitochondrial marker. Scale bars = 10 µm. Fig. 4. View largeDownload slide Expression pattern of OsPelo and sub-cellular localization of OsPELO. (A–H) Histochemical staining analysis of expression of the OsPelo promoter–GUS fusion in various tissues. GUS signals were detected in the primary root tip (A), the tip and base of lateral roots (B), leaf vein and guard cells (C), stem and auricle (D), ligule (E), young spikelet (F), pollen (G) and paddle (H). Scale bars = 0.2 mm in (A–F, H), 10 µm in (G). (I) OsPELO targets GFP to mitochondria in transiently transformed onion epidermal cells. The AOX–RFP is used as the mitochondrial marker. Scale bars = 10 µm. Furthermore, online prediction tools were employed to predict the sub-cellular localization of OsPELO. A mitochondrial localization was suggested with a high probability by MitoProt II (https://ihg.gsf.de/ihg/mitoprot.html;Claros et al., 1996) and WoLF PSORT (https://wolfpsort.hgc.jp;Horton et al., 2007) and a lower probability by TargetP (http://www.cbs.dtu.dk/services/TargetP;Emanuelsson et al., 2000). To examine the sub-cellular localization of OsPELO experimentally, a chimeric fusion gene of the coding region of OsPelo and GFP under the control of the 35S promoter was constructed and delivered into onion epidermal cells for transient expression. Fluorescence analysis showed that the fusion protein co-localized with a co-transformed mitochondrial marker (Fig. 4I), indicating that OsPELO is located in mitochondria. Enhanced disease resistance in Ospelo The occurrence of necrotic spots in Ospelo resembles the hypersensitive response (HR) after infection by pathogens. A number of spotted-leaf mutants showed enhanced resistance to bacterial and/or fungal pathogens (Fekih et al., 2015; Wang et al., 2017). To examine whether Ospelo also gains disease resistance, WT and Ospelo plants were inoculated with three races of Xoo, the causal agent of rice bacterial blight. The Ospelo plants exhibited significantly enhanced resistance to all tested Xoo strains (PXO71, PXO99 and PXO145) compared with the WT (Fig. 5A, B). Fig. 5. View largeDownload slide Detection of bacterial blight pathogen resistance and expression of resistance-related genes. (A) Reactions of the WT and Ospelo to three Xanthomonas oryzae pv. oryzae (Xoo) isolates. Scale bar = 2 cm. (B) Lesion lengths of the WT and Ospelo produced by three Xoo isolates measured 3 weeks after infection. Data are means ± s.d. of ten plants. (C) Relative expression of pathogenesis-related genes. WT and Ospelo leaves were collected from seedlings at the tillering stage. Data are means ± s.d. of three biological replicates (Student’s t-test: *P < 0.01). Fig. 5. View largeDownload slide Detection of bacterial blight pathogen resistance and expression of resistance-related genes. (A) Reactions of the WT and Ospelo to three Xanthomonas oryzae pv. oryzae (Xoo) isolates. Scale bar = 2 cm. (B) Lesion lengths of the WT and Ospelo produced by three Xoo isolates measured 3 weeks after infection. Data are means ± s.d. of ten plants. (C) Relative expression of pathogenesis-related genes. WT and Ospelo leaves were collected from seedlings at the tillering stage. Data are means ± s.d. of three biological replicates (Student’s t-test: *P < 0.01). Defence response genes were commonly induced during lesion development in a number of rice spotted-leaf mutants (Fekih et al., 2015; Wang et al., 2015). Therefore, we detected expression of three pathogenesis-related (PR) marker genes (PR1b, PR10 and PO-C1) associated with defence response. The results showed that all these PR marker genes were highly upregulated in Ospelo (Fig. 5C). Whole-genome expression analysis of Ospelo PELO is a conserved key member of the RNA surveillance pathway and known to be involved in ribosome rescue, spermatogenesis, cell cycle control and meiotic cell division. However, almost all this knowledgs was from studies in yeast and mammals, with little information in plants. To gain further insight into the in planta function of OsPelo, transcriptome sequencing analysis of WT and Ospelo plants was conducted using RNA sequencing (RNA-seq). As lesion-mimic spots started to emerge on 20-day-old Ospelo leaves, this stage was selected for the profiling analysis. Three replicates of each genotype were used, yielding six libraries in total. Each of these libraries generated >20 million 125 bp paired-end reads after quality control, and about 86 % of them were uniquely mapped onto the rice reference genome (Supplementary Data Table S2). A total of 4990 DEGs were identified with a cut-off of the FDR-adjusted P-value <0.05. Among them, 2914 DEGs showed higher expression in Ospelo than in the WT, and were termed upregulated genes, while 2076 DEGs showed lower expression in Ospelo than in the WT and were termed downregulated genes (Fig. 6A; Supplementary Data Table S3). Among these DEGs, more than half of upregulated and downregulated genes showed a fold change <2 (Fig. 6B). This is in line with what was expected when considering that the leaf samples selected were at the very early stage of phenotypic change. Fig. 6. View largeDownload slide Analysis and Gene Ontology (GO) enrichment of differentially expressed genes (DEGs) between the WT and Ospelo by RNA-seq. (A) The number of up- and downregulated DEGs between the WT and Ospelo. (B) The fold change distribution of DEGs between the WT and Ospelo. (C–E) GO term enrichment analysis of up- and downregulated DEGs in Biological process (C), Molecular function (D) and Cellular component (E). Fig. 6. View largeDownload slide Analysis and Gene Ontology (GO) enrichment of differentially expressed genes (DEGs) between the WT and Ospelo by RNA-seq. (A) The number of up- and downregulated DEGs between the WT and Ospelo. (B) The fold change distribution of DEGs between the WT and Ospelo. (C–E) GO term enrichment analysis of up- and downregulated DEGs in Biological process (C), Molecular function (D) and Cellular component (E). To analyse further the effects of OsPelo mutation on the transcriptomes, GO classification analysis of the up- and downregulated DEGs was conducted (Fig. 6C–E; Supplementary Data Table S4). Within the category of biological process, upregulated genes were largely associated with response to stress, secondary metabolic process and cell death, indicating that the stress response in Ospelo was activated (Fig. 6C). Genes involved in post-embryonic development, reproduction and embryo development were significantly enriched among downregulated genes, which was consistent with the defects in root development and fertility in Ospelo. Moreover, genes involved in translation, metabolic process, transport, photosynthesis and biosynthesis were also enriched in downregulated genes. Within the category of molecular function, genes involved in catalytic activity, kinase activity and oxygen binding activity were enriched among upregulated genes, while genes involved in transcription factor activity and RNA binding were specifically enriched among downregulated genes (Fig. 6D). In terms of cellular component, only the plasma membrane was significantly enriched among upregulated genes, while downregulated genes showed association with the plastid, mitochondrion, cytosol, nucleolus and cytoskeleton, suggesting a broad range of functional repression of organelles in Ospelo (Fig. 6E). To explore further the biological pathways in which OsPelo may be involved, we performed KEGG pathway enrichment analysis for the DEGs between Ospelo and the WT. Twenty-three pathways were significantly enriched for upregulated genes and 53 for downregulated genes (Supplementary Data Table S5). Among the top 15 enriched pathways, the highly enriched upregulated pathways were mainly related to plant–pathogen interaction, protein processing, carbohydrate metabolism (amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis), amino acid metabolism (phenylalanine and glutathione metabolism), lipid metabolism (fatty acid metabolism, fatty acid elongation, biosynthesis of unsaturated fatty acids, α-linolenic acid metabolism, peroxisome) and secondary metabolism (biosynthesis of secondary metabolites, phenylpropanoid and flavonoid) (Fig. 7A). This was consistent with the enhanced pathogen resistance in Ospelo (Fig. 5A, B). The highly enriched pathways associated with down-regulated genes were mainly related to translation (aminoacyl-tRNA biosynthesis, RNA transport, ribosome biogenesis in eukaryotes), mismatch repair and primary metabolism including nucleotides, carbohydrates and amino acids (Fig. 7B). Fig. 7. View largeDownload slide Pathway enrichment and MapMan analysis of DEGs between the WT and Ospelo. (A, B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of up- (A) and downregulated (B) DEGs. (C) Mapping of DEGs associated with the KEGG aminoacyl-tRNA biosynthesis pathway. Boxes labelled with blue colour indicate downregulated DEGs between the WT and Ospelo. (D–F) MapMan analysis of DEGs associated with RNA–protein synthesis (D), lignin synthesis (E) and JA synthesis (F). Fig. 7. View largeDownload slide Pathway enrichment and MapMan analysis of DEGs between the WT and Ospelo. (A, B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of up- (A) and downregulated (B) DEGs. (C) Mapping of DEGs associated with the KEGG aminoacyl-tRNA biosynthesis pathway. Boxes labelled with blue colour indicate downregulated DEGs between the WT and Ospelo. (D–F) MapMan analysis of DEGs associated with RNA–protein synthesis (D), lignin synthesis (E) and JA synthesis (F). In total, 30 DEGs were found to be involved in the aminoacyl-tRNA biosynthesis pathway, among which 28 DEGs responsible for synthesis of most aminoacyl-tRNAs were significantly downregulated in Ospelo (Fig. 7C; Supplementary Data Table S6). Further MapMan classification of DEGs also showed that a number of gene bins in the RNA–protein synthesis pathway were greatly downregulated, including RNA transcription and processing, protein amino acid activation, and protein synthesis initiation, elongation and release (Fig. 7D). These results were consistent with the putative role of OsPelo in stalled ribosome release in the mRNA decay pathway, whose dysfunction would result in the repression of translation. Among the enriched KEGG pathways for upregulated genes, there were several biotic stress-related pathways, including plant–pathogen interaction, α-linolenic acid metabolism and phenylpropanoid biosynthesis (Fig. 7A). The α-linolenic acid metabolism pathway is responsible for jasmonic acid (JA) synthesis, and the phenylpropanoid biosynthesis pathway produces lignin. JA is one of major signalling pathways in plant disease resistance (Nahar et al., 2011; Xie et al., 2011). Lignin is a non-degradable mechanical barrier for most micro-organisms, and an increase in lignification is a common response to pathogen attack to block parasite invasion and reduce the susceptibility of hosts (Moura et al., 2010). Consistent with these findings, MapMan analysis clearly showed that the synthesis pathways for JA and lignin were both significantly activated (Fig. 7E, F). DISCUSSION In the present study, a rice mutant, Ospelo, was isolated from an EMS-mutagenized population of rice (indica, ‘Kasalath’). The mutation caused loss of function of Pelota, a rice homologue of a key component in the NGD pathway. The mutant showed defects in root system development and spotted leaves from the early seedling stage, semi-dwarfness and defective pollen development (Figs 1 and 2). Functional complementation with WT OsPelo rescued the mutant phenotype observed in Ospelo (Fig. 3). We further conducted transcriptome sequencing of Ospelo and the WT, and found that DEGs were significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. OsPELO belongs to a family of evolutionarily conserved proteins called PELO, with their primary function in the regulation of translation and cell cycle progression. In Drosophila, Pelo has been shown to be required to control meiotic cell cycle progression and self-renewal and division of GSCs in the ovary (Eberhart and Wasserman, 1995; Xi et al., 2005). The yeast homologue of PELO, DOM34, functions in protein translation to promote G1 progression and differentiation, and the dom34 mutants grow slowly and have defects in meiosis and sporulation (Davis and Engebrecht, 1998). In mice, disruption of the Pelo gene results in early embryonic lethality and defects in cell cycle progression (Adham et al., 2003). In rice Ospelo mutants, the root meristem activity was repressed, and pollen fertility and seed setting rate were dramatically decreased, suggesting a conserved role for OsPelo in cell cycle control through translation. Genes involved in translation, including aminoacyl-tRNA biosynthesis, protein amino acid initiation, elongation and release, were significantly enriched among the downregulated genes, suggesting the repression of translation in Ospelo (Fig. 7C, D; Supplementary Data Table S5). The mitochondrial localization of OsPELO suggests its possible involvement in the translation process taking place in mitochondria, one of the only two organelles containing their own genomes in cells (Fig. 4I). It has been reported that the PELO proteins are located in the cytoplasm of Drosophila (Xi et al., 2005) and in the cytoskeleton of mammalian cells (Burnicka-Turek et al., 2010). The difference in sub-cellular localization of PELO proteins among different species might suggest their functional divergence during evolution. The cell cycle is controlled by a complex machinery composed of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors (CKIs), E2F transcription factors and a number of other proteins (Inzé and Veylder, 2006; Guo et al., 2007). Among them, the A-type CDKs (CDKAs) are essential for G1 to S and G2 to M transition, and the B-type CDKs (CDKBs) show maximum activity at the G2 to M transition and the M-phase (De Veylder et al., 2007; Endo et al., 2012). D-type cyclins (CYCDs) mainly regulate the G1 to S transition through association with CDKs. In addition, the binding of CKI proteins could also adjust CDK activity (Polyn et al., 2015). The overexpression of one CKI gene, KRP1, could result in reduced cell production during leaf development and seed filling, and disturbed production of endosperm cells (Barrôco et al., 2006). Consistent with this, several key regulatory components of the cell cycle were found to be downregulated, including two cyclin genes (CycD3;1 and CycF2;3), seven CDK genes (CDKA;1, CDKB1;1, CDKB2;1, CDKD;1, CKL1, CKL6 and CKL7) and one E2F transcription factor gene (E2F2) (Supplementary Data Table S7). Moreover, KRP1 was found to be upregulated. These data suggested that the cell cycle progression in Ospelo was repressed, which might explain the observed short root phenotype. Defence response might be activated without pathogen attack in spotted-leaf mutants, and contribute to enhanced resistance to pathogen infection (Wang et al., 2017). Recently PELO has been reported to be involved in general antiviral activity in Drosophila and resistance to begomovirus in tomato (Wu et al., 2014; Lapidot et al., 2015). Mutation or silencing of Pelo similarly resulted in virus resistance in both Drosophila and tomato, and the critical role of PELO in highly efficiently translating viral proteins of infective viruses was suggested. The loss-of-function OsPelo mutation results in HR-like lesion spots on leaves and enhanced resistance to bacterial blight (Fig. 5A, B). The expression of three PR marker genes, PR1b, PR10 and PO-C1, was significantly upregulated in Ospelo during the development of lesion spots (Fig. 5C), indicating activation of PR genes and their possible roles in the enhanced pathogen resistance. Furthermore, whole-genome transcriptome analysis showed that there were 40 PR genes in the DEGs and all but one of them were significantly upregulated in Ospelo (Supplementary Data Table S8). Salicylic acid (SA) and JA are two conserved positive regulators of defence response in plants and are proposed to activate a common pathogen defence system in rice (Tamaoki et al., 2013; Berens et al., 2017). SA-mediated redox status changes control the nucleocytoplasmic localization of NPR1, and it interacts with TGA transcription factors upon localization to the nucleus and activates SA-responsive genes encoding PR proteins (Dong, 2004; Koornneef and Pieterse, 2008). Analysis of DEGs in Ospelo identified seven SA biosynthesis-related PAL genes, two NPR genes (NPR1 and NPR4), four TGA transcription factor genes, 20 JA biosynthesis-associated genes and three JAZ genes (Supplementary Data Table S9). All but three of them were upregulated in Ospelo, suggesting that OsPelo plays a negative role in both SA and JA biosynthesis and/or signalling and its loss of function might cause higher accumulation of SA and JA, thus enhancing plant defence against pathogens. Moreover, WRKY transcription factors are also proposed to be critical components in SA-dependent defence responses and control PR gene expression (Koornneef and Pieterse, 2008; Wei et al., 2013). In our study, there were 37 WRKY genes showing differential expression in Ospelo compared with the WT, and 34 of them were upregulated (Supplementary Data Table S10). Among them there were a number of WRKYs which have been reported to regulate pathogen resistance in rice (Liu et al., 2005; Chujo et al., 2007; Qiu et al., 2007; Shimono et al., 2007; Peng et al., 2008; Chujo et al., 2013; Yokotani et al., 2013). The KEGG enrichment analysis indicated the constitutive activation of the plant–pathogen interaction pathway (Fig. 7A). A total of 41 genes in the pathway were found to be differentially expressed in Ospelo compared with the WT, and all of them except two CNGC genes were upregulated (Supplementary Data Table S11). These genes participated in pathogen-associated molecular patterning (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) pathways in innate immunity. Within the PTI pathway, there were two CNGC genes (CNGC12 and CNGC10), one CAM gene (Cam1-1), 15 CML genes and four CPK genes (CPK10, CPK20, CPK21 and CPK23) involved in calcium signalling; two Rboh genes (Rboh5 and Rboh7) involved in generation of reactive oxygen species (ROS) and NOS1 for nitric oxide production; and two PR1 genes as antimicrobial components. Within the ETI pathway, RIN4, RPS2, SGT1 and three RPM1 genes were involved in recognition of avirulent effectors; and there were two HSP90 genes for HR. The loss-of-function mutation of CNGC in arabidopsis and barley resulted in high levels of SA, constitutive expression of PR genes and enhanced resistance to pathogens (Clough et al., 2000; Balagué et al., 2003; Rostoks et al., 2006; Kaplan et al., 2007). Overexpression of OsCPK10 and OsCPK20 in rice activated both SA- and JA-dependent defence responses and enhanced the resistance of transgenic plants to pathogens (Fu et al., 2013, 2014). These results showed that both the PTI- and ETI-related signalling components were significantly upregulated in Ospelo, suggesting that the loss of function of OsPelo resulted in activation of both PTI and ETI, reinforcement of cell walls and induction of PR proteins, thus enhancing resistance of Ospelo to pathogens. In conclusion, we report herein that OsPelo functions in development and defence in rice. We characterized the roles of this rice homologue of PELO protein, and confirmed that loss of function of OsPelo resulted in defects in root system development and enhanced pathogen resistance in rice. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: primers used in the study. Table S2: RNA-seq output and mapping results. Table S3: differentially expressed genes (DEGs) between the WT and Ospelo. Table S4: GO enrichment analysis of DEGs. Table S5: KEGG pathway enrichment analysis of DEGs. Table S6: list of DEGs associated with aminoacyl-tRNA biosynthesis. Table S7: list of DEGs associated with cell cycle regulation. Table S8: list of 40 PR genes found in DEGs. Table S9: list of DEGs associated with SA and JA biosynthesis and signalling. Table S10: list of 37 WRKY genes found in DEGs. Table S11: list of 41 DEGs involved in plant–pathogen interaction. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China [grant nos 31371595 and 31300246], the Zhejiang Provincial Natural Science Foundation of China [grant nos LY17C020002 and LQ16C020001], the Natural Science Foundation of Ningbo [grant no. 2017A610291] and the K. C. Wong Magna Fund in Ningbo University. LITERATURE CITED Adham IM, Sallam MA, Steding G, et al.   2003. 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A Pelota-like gene regulates root development and defence responses in rice

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Abstract

Abstract Background and Aims Pelota (Pelo) are evolutionarily conserved genes reported to be involved in ribosome rescue, cell cycle control and meiotic cell division. However, there is little known about their function in plants. The aim of this study was to elucidate the function of an ethylmethane sulphonate (EMS)-derived mutation of a Pelo-like gene in rice (named Ospelo). Methods A dysfunctional mutant was used to characterize the function of OsPelo. Analyses of its expression and sub-cellular localization were performed. The whole-genome transcriptomic change in leaves of Ospelo was also investigated by RNA sequencing. Key Results The Ospelo mutant showed defects in root system development and spotted leaves at early seedling stages. Map-based cloning revealed that the mutation occurred in the putative Pelo gene. OsPelo was found to be expressed in various tissues throughout the plant, and the protein was located in mitochondria. Defence responses were induced in the Ospelo mutant, as shown by enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae, coupled with upregulation of three pathogenesis-related marker genes. In addition, whole-genome transcriptome analysis showed that OsPelo was significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. Detailed analysis showed that activation of a number of innate immunity-related genes might be responsible for the enhanced disease resistance in the Ospelo mutant. Conclusions These results demonstrate that OsPelo positively regulates root development while its loss of function enhances pathogen resistance by pre-activation of defence responses in rice. Short root, spotted-leaf mutant, defence responses, RNA-seq, Oryza sativa INTRODUCTION Pelota (PELO) is an evolutionarily conserved protein and has been identified in a number of species, including Drosophila (Eberhart and Wasserman, 1995), archaebacteria (Ragan et al., 1996), yeast (Davis and Engebrecht, 1998), Arabidopsis thaliana (Caryl et al., 2000), human (Shamsadin et al., 2000), mouse (Shamsadin et al., 2002) and tomato (Lapidot et al., 2015). The PELO proteins contain 347–395 amino acid residues and RNA-binding domains similar to that found in the family members of the eukaryotic release factor 1 (eRF1) family members, which play roles in termination of protein synthesis (Davis and Engebrecht, 1998). The study in Drosophila first identified that the cell cycle is arrested during the meiotic G2/M transition phase in germline cells of Pelo male homozygotes, while only mitotic division is affected during oogenesis (Eberhart and Wasserman, 1995). In addition, the eyes of Pelo homozygotes are smaller than those of their heterozygous siblings. Further study revealed a critical role for PELO proteins in regulating self-renewal of germline stem cells (GSCs) by repressing the differentiation pathway (Xi et al., 2005). Disruption of the balance between self-renewal and differentiation of GSCs impaired the fertility of loss-of-function pelo mutant females. A similar role for PELO protein in meiotic and mitotic division was also found in Saccharomyces cerevisiae, where the disruption Dom34, the orthologue gene of Pelo, resulted in growth retardation and defective sporulation (Davis and Engebrecht, 1998). In mice, disruption of the Pelo gene caused early embryonic lethality and cell cycle defects (Adham et al., 2003). Further analysis found that PELO mediated gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes (Raju et al., 2015). PELO is also found to regulate extraembryonic endoderm development and epidermal differentiation, and to inhibit tumour progression and invasion (Nyamsuren et al., 2014; Pedersen et al., 2014; Elkenani et al., 2016). In addition, PELO is involved in high efficiency viral replication (Wu et al., 2014), and regulates a resistance reaction to begomovirus in tomato (Lapidot et al., 2015). Extensive studies have been conducted in yeast to characterize the function of PELO at the molecular level. The PELO-coding orthologue Dom34 together with its interacting partner Hbs1 participate in an RNA quality control mechanism called no-go decay (NGD) for the recycling of stalled ribosomes (Doma and Parker, 2006; Shoemaker and Green, 2011; Guydosh and Green, 2014). The structure of the DOM34–HBS1 complex is similar to that of eRF1 and eRF3, but Dom34 does not have motifs for codon recognition and peptide release (Chen et al., 2010; Becker et al., 2011). It was proposed that the DOM34–HBS1 complex binds to the ribosomal A site to promote dissociation of ribosome subunits (Shoemaker et al., 2010). In addition to the RNA quality control in the NGD pathway, DOM34–HBS1 is also important for non-stop decay (NSD), i.e. decay of non-functional 18S rRNAs and mRNAs with premature stop codons (Cole et al., 2009; Tsuboi et al., 2012; Saito et al., 2013). The DOM34–HBS1 complex also mediates dissociation of inactive 80S ribosomes to promote the restart of translation after stress (van den Elzen et al., 2014). The roles of DOM34/PELO in mRNA quality control are conserved in Drosophila and human (Ikeuchi et al., 2016; Hashimoto et al., 2017). The PELO–HBS1 complex is also involved in transposon silencing in the Drosophila germline (Yang et al., 2015). Recently, studies showed that defects in GTPBP2, a PELO-binding partner in mammals, resulted in ribosome stalling in a tRNAArg (UCU) mutant background and the death of mouse neurons (Ishimura et al., 2014; Kirmizitas et al., 2014). Little is yet known about the overall function of Pelo genes in plants. In this study, a Pelo-deficient rice mutant was isolated from an ethylmethane sulphonate (EMS)-mutagenized library. We describe a root development defect, a spotted-leaf phenotype and enhanced pathogen resistance for Pelo null rice. MATERIALS AND METHODS Plant materials and growth conditions The rice mutant Ospelo was isolated from an EMS-mutagenized rice (Oryza sativa L. indica, ‘Kasalath’) mutant library. Hydroponic experiments were conducted in normal rice culture solution with the pH adjusted to 5.5 (Zhu et al., 2012). In all hydroponic experiments, plants were grown in a greenhouse with a 12 h light (30 °C)/12 h dark (22 °C) cycle (16 000 lux) and a humidity of 70 %. Acetocarmine and 5-ethynyl-2’-deoxyuridine (EdU) staining Primary root tips of 4-day-old wild type (WT) and Ospelo plants were stained with 1 % acetocarmine solution for 10 min. After washing with 45 % acetic acid solution, they were mounted on glass slides and examined with a stereo microscope (Leica MZ95). EdU staining was conducted using an EdU kit (C10350, Click-iT EdU Alexa Fluor 488 HCS assay; Invitrogen) according to the manufacturer’s instruction. Roots of 4-day-old WT and Ospelo plants were immersed in 20 mm EdU solution for 2 h and then fixed for 30 min in 3.7 % formaldehyde solution in phosphate buffer (pH 7.2) with 0.1 % Triton X-100. After that they were incubated with EdU detection cocktail for 30 min and examined with the green fluorescent protein (GFP) channel on a confocal laser-scanning microscope (Zeiss LSM 510, Jena, Germany). More than ten root samples of each genotype were examined, and the experiment was repeated twice. Trypan blue staining Trypan blue staining was performed on fresh leaves as previously described (Yin et al., 2000). In brief, leaf samples were submerged in lactic acid–phenol–trypan blue (LPTB) solution [2.5 mg mL–1 trypan blue, 25 % (w/v) lactic acid, 23 % water-saturated phenol and 25 % glycerol in H2O] at 30 °C for 12 h. The LPTB solution was then replaced with a chloral hydrate solution (50 g in 20 mL of H2O) for destaining. After multiple changes of chloral hydrate solution for 4 d, leaf samples were washed with H2O and mounted on glass slides before being examined with a stereo microscope (Leica MZ95). More than ten leaf samples were examined for each genotype, and the experiment was repeated three times. Pollen activity staining assay Spikelets about to flower from the WT and Ospelo were chosen for examination. After carefully opening the hull, anthers were placed in several drops of 1 % I2–KI solution on glass slides and crumbled to release pollens. Then they were examined with a light microscope (Nikon eclipse 80i). Samples from more than five plants of each genotype were examined, and the experiment was repeated twice. Histological observation Root tips from 4-day-old plants were fixed overnight at 4 °C in 2.5 % glutaraldehyde in 0.1 m sodium phosphate buffer, pH 7.2, and washed three times for 30 min in the same buffer. The samples were then refixed in OsO4 for 4 h at room temperature and washed for 30 min in the same buffer. Samples were dehydrated in a gradient ethanol, embedded in pure Spurr resin and polymerized overnight at 70 °C. Semi-thin sections (2 μm thick) were made using diamond knives on a power Tome XL microtome (RMC-Boeckeler Instruments, Tucson, AZ, USA) and stained with 0.1 % methylene blue for 3–5 min at 70 °C. The samples were rinsed with distilled water and visualized with a microscope (Nikon 90i, Japan). Mapping and cloning of OsPelo A mapping population was generated from crosses between the homozygous Ospelo mutant and japonica variety Nipponbare. A total of 30 and 538 short root plants from the F2 population were used for primary and fine mapping of OsPelo, respectively. The OsPelo gene was localized to a region of 411 kb between the sequence-tagged site (STS) markers STS1 and STS2 on chromosome 4. Primers used are listed in Supplementary Data Table S1. The OsPelo gene was selected from 52 putative protein-coding genes as one of the candidate genes. To identify the mutation site, genes were amplified by PCR from the genomic DNA of both WT and Ospelo plants and used for Sanger sequencing analysis. Construction of vectors and plant transformation The coding region of OsPelo was PCR amplified and put into the pUCM-T vector (Takara). After sequencing confirmation, the fragment was excised from the pUCM-T vector by BamHI and PstI digestion and ligated into the corresponding site of pCAMBIA1300. A 2264 bp promoter of OsPelo was obtained by PCR and inserted into the HindIII/BamHI site in front of the OsPelo coding region to drive its expression. The promoter was also put into the HindIII/BamHI site of vector pCAMBIA1300NH-GUS to create a transcriptional fusion of the OsPelo promoter and the β-glucuronidase (GUS) coding sequence, OsPelop::GUS. The above constructs were used for Agrobacterium tumefaciens-mediated rice transformation of the WT or Ospelo as described (Chen et al., 2003). Histochemical analysis and GUS assay Histochemical GUS staining was performed as previously described (Ding et al., 2015). Transgenic plant samples and freehand cross-section samples were incubated with GUS staining solution (100 mmol L–1 NaH2PO4 buffer pH 7.0, 0.5% Triton X-100, 0.5 mg mL–1 X-Gluc and 20 % methanol) overnight at 37 °C. Tissues were subsequently rinsed and mounted on slides, and photographed using a stereo microscope (Leica MZ95, Nussloch, Germany). Sub-cellular localization of OsPELO The full-length coding sequence of OsPelo with the eliminated stop codon was inserted in-frame before the coding sequence of a soluble modified GFP (smGFP4). The OsPELO–GFP fusion-coding sequence was subcloned into the binary vector 35S-pCAMBIA1301. The resulting construct was sequenced to verify in-frame fusion and used for transient transformation of onion epidermis using a biolistic PDS-1000/He particle delivery system (Bio-Rad, Hercules, CA, USA). Alternatice oxidase (AOX)–red fluorescent protein (RFP) located to the mitochondria was co-transformed as a mitochondrial marker. The GFP and RFP were visualized using a confocal laser-scanning microscope (Zeiss LSM 510). The experiment was repeated twice. Determination of resistance to bacterial blight in Ospelo Three races of Xanthomonas oryzae pv. oryzae (Xoo) were used for evaluation of bacterial blight resistance. The Philippines races PXO71, PXO99 and PXO145 were kindly provided by Dr Jie Zhou of the Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, China. New fully expanded leaves of ten independent WT and Ospelo plants at the maximum tillering stage were inoculated for each race of Xoo using the clipping leaf method (Kauffman et al., 1973). The lesion length on inoculated plants was measured 3 weeks after inoculation. Quantitative real-time PCR (qRT-PCR) analysis RNA was extracted from three biological replicates of leaf samples of 20-day-old WT and Ospelo plants using RNAiso plus (Takara). Three technical replicates were performed for each sample. The first-strand cDNA was synthesized from total RNA using Superscript II (Invitrogen, Carlsbad, CA, USA) and used as the qRT-PCR template. Real-time PCR analysis was performed using a Roche Lightcycler480 real-time PCR system with SYBR Premix Ex Taq (Takara). Primers used are listed in Supplementary Data Table S1. The Ubiquitin gene (LOC_Os03g13170) in rice was used as a reference gene. Transcriptome sequencing Total RNA was extracted from leaves of 20-day-old WT and Ospelo plants under normal conditions using the RNeasy Plant Mini Kit (Qiagen, USA). Three biological replicates for each genotype were collected. RNA was quantified using the Nanodrop-2000 (ThermoFisher, USA) and RNA quality was then examined using a 2100 Bioanalyzer (Agilent Technologies, USA). High-quality RNA samples for library construction were selected based on the 260/280 nm ratio and RNA integrity number (RIN) above 2.0 and 7.0, respectively. Sequencing libraries were prepared using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Libraries were subjected to 125 cycles of paired-end sequencing with the Illumina HiSeq2500 system according to the manufacturer’s instructions. The raw sequencing data have been uploaded to the SRA (Sequence Read Archive; https://www.ncbi.nlm.nih.gov/sra) database (accession no. SRP117240). Differentially expressed gene analysis Raw reads were first processed using in-house Perl scripts. In this step, clean reads were obtained by removing reads containing adaptor, reads containing ploy-N and low-quality reads (the number of bases with quality value ≤20 is more than 35). The retained high-quality reads, i.e. clean reads, were then analysed by the TopHat–Cufflinks pipeline (Trapnell et al., 2012). Briefly, clean reads were mapped to the rice genome (MSU version 7, http://rice.plantbiology.msu.edu/) using TopHat. Cufflinks was then used for transcriptome assembly and assessment of the FPKM (fragments per kilobase of transcript per million mapped reads) value. Counts of mapped reads to genes were obtained with HTSeq (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html) and differentially expressed genes (DEGs) were determined using DESeq2 (http://bioconductor.org/packages/release/bioc/html/DESeq2.html) by the Negative Binomial Distribution test (Love et al., 2014; Anders et al., 2015). Genes with a false discovery rate (FDR)-adjusted P-value <0.05 were assigned as DEGs. Gene Ontology (GO) annotation was conducted by querying Swiss-Prot (http://www.uniprot.org) with transcripts of DEGs and enrichment analysis was performed by the hypergeometric distribution test using R with a threshold FDR-adjusted P-value <0.01. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was conducted by PlantGSEA with a threshold FDR-adjusted P-value <0.01 (Yi et al., 2013). The functional categorization of DEGs was conducted by MapMan (Thimm et al., 2004). RESULTS The rice Ospelo mutant showed root and leaf growth defects A mutant showing short roots and spotted leaves was isolated from an EMS-mutagenized rice mutant library (Oryza sativa L. indica ‘Kasalath’). The mutant was designated as Ospelo, which will be described later on (Fig. 1A). After growth in culture solution for 7 d, the primary root length of Ospelo (8.91 ± 1.06 cm) was only 57.9 % of that of the WT (15.38 ± 0.88 cm) (Fig. 1B). Moreover, the length of adventitious roots and lateral roots of Ospelo was also significantly shorter than that of those of the WT (Fig. 1B), while shoot height was slightly shorter than that of the WT (Fig. 1B). Fig. 1. View largeDownload slide Phenotypic characterization of Ospelo. (A) Growth phenotype of 7-day-old WT and Ospelo plants. Scale bar = 2 cm. (B) The primary, lateral and adventitious root length of the 7-day-old WT and Ospelo plants. The three longest adventitious roots and the 20 longest lateral roots on each primary root were counted. Error bars represent the s.d. (n = 20). (C) Longitudinal sections of the maturation zone (top) and root tip (bottom) of 4-day-old WT and Ospelo plants. Scale bars = 100 µm. (D) S-phase entry of 4-day-old WT and Ospelo root tips visualized by EdU staining. Scale bar = 100 µm. (E) Size of EdU-labelled root apical meristems (RAMs) of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10). (F) Acetocarmine staining of root tips of 4-day-old WT and Ospelo plants. Scale bar = 0.5 mm. (G) Size of acetocarmine-stained root apical meristems of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10) (H) Base, middle and tip regions of leaves from 30- and 60-day-old WT and Ospelo plants. Scale bars = 1 cm. (I) Trypan blue staining of leaves from 60-day-old WT and Ospelo plants Leaves decolorized directly by ethanol were included as control. Scale bars = 1 mm. The asterisks in (B), (E) and (G) indicate a significant difference between the WT and Ospelo (P < 0.01, by Student’s t-test). Fig. 1. View largeDownload slide Phenotypic characterization of Ospelo. (A) Growth phenotype of 7-day-old WT and Ospelo plants. Scale bar = 2 cm. (B) The primary, lateral and adventitious root length of the 7-day-old WT and Ospelo plants. The three longest adventitious roots and the 20 longest lateral roots on each primary root were counted. Error bars represent the s.d. (n = 20). (C) Longitudinal sections of the maturation zone (top) and root tip (bottom) of 4-day-old WT and Ospelo plants. Scale bars = 100 µm. (D) S-phase entry of 4-day-old WT and Ospelo root tips visualized by EdU staining. Scale bar = 100 µm. (E) Size of EdU-labelled root apical meristems (RAMs) of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10). (F) Acetocarmine staining of root tips of 4-day-old WT and Ospelo plants. Scale bar = 0.5 mm. (G) Size of acetocarmine-stained root apical meristems of 4-day-old WT and Ospelo plants. Error bars represent the s.d. (n = 10) (H) Base, middle and tip regions of leaves from 30- and 60-day-old WT and Ospelo plants. Scale bars = 1 cm. (I) Trypan blue staining of leaves from 60-day-old WT and Ospelo plants Leaves decolorized directly by ethanol were included as control. Scale bars = 1 mm. The asterisks in (B), (E) and (G) indicate a significant difference between the WT and Ospelo (P < 0.01, by Student’s t-test). To investigate the cause of the short root phenotype of Ospelo at a cellular level, root longitudinal section analysis was conducted. It showed that the radial organization patterns and the quiescent centre (QC) of the stem cell niche in Ospelo were comparable with those of the WT (Fig. 1C). The cell length of maturation zones of Ospelo was also similar to that in the WT (Fig. 1C). To determine the root meristem activity of Ospelo, 4-day-old seedlings were cultured for 2 h in the presence of the thymidine analogue EdU, and in situ incorporation of EdU into DNA during active DNA synthesis in the root tip was visualized (Kotogány et al., 2010). Compared with the WT, Ospelo had reduced levels of EdU labelling in the root meristem (Fig. 1D, E). Consistent with this, the traditional acetocarmine staining also showed that the meristematic region in Ospelo was reduced compared with the WT (Fig. 1F, G). Taken together, these results suggested that the meristematic activity in the Ospelo root is compromised. Moreover, lesion-mimic spots appeared on Ospelo leaves after being grown in culture solution for about 20 d. The spotted-leaf phenotype became more severe as plants grew and expanded into whole leaves of 60-day-old Ospelo (Fig. 1H). In order to examine the occurrence of cell death or irreversible membrane damage, leaves of the WT and Ospelo were stained with trypan blue. As expected, there were clearly dyed blue spots on Ospelo leaves, while no detectable staining was observed on WT leaves (Fig. 1I). Critical roles of OsPelo in development and fertility The mature stage development and fertility of Ospelo were severely impaired (Fig. 2A, B). The plant height, tiller number and seed setting rate of Ospelo were all significantly decreased compared with the WT (Fig. 2C). However, the 1000-grain weight of Ospelo was similar to that of the WT (data not shown). To find the cause of the fertility defect, reproductive organs of Ospelo were further examined. There was no significant difference in pistils of Ospelo and the WT (Fig. 2D), and a successful cross using Ospelo as the female for the mapping population also confirmed its normal female fertility (data not shown). However, anthers of Ospelo were found to be pale compared with the healthy yellow anthers of the WT, indicating a severe defect in pollen development (Fig. 2E). Further staining analysis showed that pollen development of Ospelo was dysfunctional and their fertility was dramatically lower than in the WT (Fig. 2F). Fig. 2. View largeDownload slide Comparison of agronomic traits and reproductive organs in the WT and Ospelo. (A) Mature WT and Ospelo plants. Scale bar = 20 cm. (B) Spikes of the WT and Ospelo. Scale bar = 4 cm. (C) Plant height, tiller number and seed setting rate of the WT and Ospelo at the maturation stage. Error bars represent the s.d. (n = 20). (D) Pistils of the WT and Ospelo. Scale bars = 0.5 mm. (E) Stamens of the WT and Ospelo. Scale bars = 0.5 mm. (F) I2–IK solution staining of pollen from the WT and Ospelo. Scale bars = 50 µm Fig. 2. View largeDownload slide Comparison of agronomic traits and reproductive organs in the WT and Ospelo. (A) Mature WT and Ospelo plants. Scale bar = 20 cm. (B) Spikes of the WT and Ospelo. Scale bar = 4 cm. (C) Plant height, tiller number and seed setting rate of the WT and Ospelo at the maturation stage. Error bars represent the s.d. (n = 20). (D) Pistils of the WT and Ospelo. Scale bars = 0.5 mm. (E) Stamens of the WT and Ospelo. Scale bars = 0.5 mm. (F) I2–IK solution staining of pollen from the WT and Ospelo. Scale bars = 50 µm Map-based cloning of OsPelo To identify the mutated gene, an F2 population was developed by crossing the Ospelo mutant with Nipponbare (japonica). The F1 seedlings displayed the WT phenotype and their F2 progeny showed segregation of WT and Ospelo phenotypes at a ratio close to 3:1 (257:81, χ2 = 0.32, P < 0.05), indicating that the Ospelo phenotype was controlled by a single recessive nuclear gene. The OsPelo locus was first mapped to chromosome 4 between simple sequence repeat (SSR) markers RM3217 and RM567 using 30 F2 mutant plants (Fig. 3A). The OsPelo gene was further mapped to a 411 kb region between two new STS markers STS1 and STS2 using 538 F2 mutant plants (Fig. 3A). Fifty-two open reading frames (ORFs) were predicted in this region (http://rice.plantbiology.msu.edu/). Sanger sequencing analysis for both the WT and Ospelo mutant identified one single-base insertion after 1630 bp from the start codon on the fourth exon of LOC_Os04g56480 (Fig. 3A). The insertion introduced a premature stop codon and putatively yielded a peptide with 89 amino acid residues (Fig. 3B). LOC_Os04g56480 encodes rice Pelota, which is the homologue of Pelota in Drosophila (Eberhart and Wasserman, 1995). Therefore, we named it OsPelo. The OsPelo gene is 7945 bp in length, and contains 15 exons and 14 introns. The protein-coding region of OsPelo is 1137 bp and encodes a 378 amino acid protein. The protein structure is consistent with the annotation generated by pfam (pfam.xfam.org), with three conserved eRF1 domains (Fig. 3B). A search in the rice genome with the full-length OsPELO protein sequence using BLASTp showed that it is a single-copy gene. Fig. 3. View largeDownload slide OsPelo encodes the PELO protein in rice. (A) Map-based cloning of OsPelo. Black boxes represent exons, and lines represent introns. White boxes indicate untranslated regions. The arrowhead shows the site of the single-base insertion after the nucleotide 1630 bp downstream of ATG. (B) Predicted domains of OsPELO by the pfam database (pfam.xfam.org). The arrowhead indicates the insertion within the eRF1_1 domain, which results in the production of a truncated peptide with 89 amino acid residues. (C) The ribbon diagram of the 3-D structure of OsPELO predicted by SWISS-MODEL (https://swissmodel.expasy.org). The other two ribbon diagrams are published crystal structures of PELO from human (SWISS-MODEL Template Library ID: 5LZW.78) and Dom34 from yeast (PDB ID: 2VGM). (D) Complementation analysis of Ospelo. The root and leaf phenotype of Ospelo was completely recovered by transformation of OsPelo driven by its native promoter. Two independent lines of transgenic plants (Comp1 and Comp2) in the Ospelo mutant background are shown. Scale bar left = 4 cm; right = 1 cm. Fig. 3. View largeDownload slide OsPelo encodes the PELO protein in rice. (A) Map-based cloning of OsPelo. Black boxes represent exons, and lines represent introns. White boxes indicate untranslated regions. The arrowhead shows the site of the single-base insertion after the nucleotide 1630 bp downstream of ATG. (B) Predicted domains of OsPELO by the pfam database (pfam.xfam.org). The arrowhead indicates the insertion within the eRF1_1 domain, which results in the production of a truncated peptide with 89 amino acid residues. (C) The ribbon diagram of the 3-D structure of OsPELO predicted by SWISS-MODEL (https://swissmodel.expasy.org). The other two ribbon diagrams are published crystal structures of PELO from human (SWISS-MODEL Template Library ID: 5LZW.78) and Dom34 from yeast (PDB ID: 2VGM). (D) Complementation analysis of Ospelo. The root and leaf phenotype of Ospelo was completely recovered by transformation of OsPelo driven by its native promoter. Two independent lines of transgenic plants (Comp1 and Comp2) in the Ospelo mutant background are shown. Scale bar left = 4 cm; right = 1 cm. PELO is a conserved protein involved in the mRNA NGD pathway based on its structural similarity to tRNA (Kobayashi et al., 2010). The putative tertiary structure of OsPELO was predicted using the online SWISS-MODEL server (https://swissmodel.expasy.org) (Biasini et al., 2014). As expected, its structure showed high similarity to those of human and yeast PELOs, with three conserved domains arranged in an L-shape (Fig. 3C). This further confirms that OsPELO is the rice homologue of PELO. Functional complementation test of Ospelo To confirm that the single nucleotide insertion in OsPelo is responsible for the mutant phenotype, complementation analysis was conducted using A. tumefaciens-mediated transformation. The protein-coding region of OsPelo was cloned into a binary vector driven by its 2264 bp native promoter and used for transformation of Ospelo. More than 20 independent transgenic lines were obtained. The short root and spotted-leaf phenotype was restored in all the positive transformants (Fig. 3D). These results demonstrate that the single base insertion in OsPelo causes the short root and spotted-leaf phenotype in Ospelo. Expression pattern and sub-cellular localization analysis of OsPelo To determine the tissue-specific expression pattern of OsPelo, a 2264 bp native promoter was fused to the GUS reporter gene. This chimeric gene cassette was used to transform WT plants via the A. tumefaciens-mediated transformation method. Histochemical staining for GUS activity in T2 plants showed that OsPelo was ubiquitously expressed in plant organs, including the primary root tip, tip and base of lateral roots, leaf vein and guard cells, stem and auricle, ligule, lemma, anther, stigma, glume, peduncle, pollen and paddle (Fig. 4A–H). Fig. 4. View largeDownload slide Expression pattern of OsPelo and sub-cellular localization of OsPELO. (A–H) Histochemical staining analysis of expression of the OsPelo promoter–GUS fusion in various tissues. GUS signals were detected in the primary root tip (A), the tip and base of lateral roots (B), leaf vein and guard cells (C), stem and auricle (D), ligule (E), young spikelet (F), pollen (G) and paddle (H). Scale bars = 0.2 mm in (A–F, H), 10 µm in (G). (I) OsPELO targets GFP to mitochondria in transiently transformed onion epidermal cells. The AOX–RFP is used as the mitochondrial marker. Scale bars = 10 µm. Fig. 4. View largeDownload slide Expression pattern of OsPelo and sub-cellular localization of OsPELO. (A–H) Histochemical staining analysis of expression of the OsPelo promoter–GUS fusion in various tissues. GUS signals were detected in the primary root tip (A), the tip and base of lateral roots (B), leaf vein and guard cells (C), stem and auricle (D), ligule (E), young spikelet (F), pollen (G) and paddle (H). Scale bars = 0.2 mm in (A–F, H), 10 µm in (G). (I) OsPELO targets GFP to mitochondria in transiently transformed onion epidermal cells. The AOX–RFP is used as the mitochondrial marker. Scale bars = 10 µm. Furthermore, online prediction tools were employed to predict the sub-cellular localization of OsPELO. A mitochondrial localization was suggested with a high probability by MitoProt II (https://ihg.gsf.de/ihg/mitoprot.html;Claros et al., 1996) and WoLF PSORT (https://wolfpsort.hgc.jp;Horton et al., 2007) and a lower probability by TargetP (http://www.cbs.dtu.dk/services/TargetP;Emanuelsson et al., 2000). To examine the sub-cellular localization of OsPELO experimentally, a chimeric fusion gene of the coding region of OsPelo and GFP under the control of the 35S promoter was constructed and delivered into onion epidermal cells for transient expression. Fluorescence analysis showed that the fusion protein co-localized with a co-transformed mitochondrial marker (Fig. 4I), indicating that OsPELO is located in mitochondria. Enhanced disease resistance in Ospelo The occurrence of necrotic spots in Ospelo resembles the hypersensitive response (HR) after infection by pathogens. A number of spotted-leaf mutants showed enhanced resistance to bacterial and/or fungal pathogens (Fekih et al., 2015; Wang et al., 2017). To examine whether Ospelo also gains disease resistance, WT and Ospelo plants were inoculated with three races of Xoo, the causal agent of rice bacterial blight. The Ospelo plants exhibited significantly enhanced resistance to all tested Xoo strains (PXO71, PXO99 and PXO145) compared with the WT (Fig. 5A, B). Fig. 5. View largeDownload slide Detection of bacterial blight pathogen resistance and expression of resistance-related genes. (A) Reactions of the WT and Ospelo to three Xanthomonas oryzae pv. oryzae (Xoo) isolates. Scale bar = 2 cm. (B) Lesion lengths of the WT and Ospelo produced by three Xoo isolates measured 3 weeks after infection. Data are means ± s.d. of ten plants. (C) Relative expression of pathogenesis-related genes. WT and Ospelo leaves were collected from seedlings at the tillering stage. Data are means ± s.d. of three biological replicates (Student’s t-test: *P < 0.01). Fig. 5. View largeDownload slide Detection of bacterial blight pathogen resistance and expression of resistance-related genes. (A) Reactions of the WT and Ospelo to three Xanthomonas oryzae pv. oryzae (Xoo) isolates. Scale bar = 2 cm. (B) Lesion lengths of the WT and Ospelo produced by three Xoo isolates measured 3 weeks after infection. Data are means ± s.d. of ten plants. (C) Relative expression of pathogenesis-related genes. WT and Ospelo leaves were collected from seedlings at the tillering stage. Data are means ± s.d. of three biological replicates (Student’s t-test: *P < 0.01). Defence response genes were commonly induced during lesion development in a number of rice spotted-leaf mutants (Fekih et al., 2015; Wang et al., 2015). Therefore, we detected expression of three pathogenesis-related (PR) marker genes (PR1b, PR10 and PO-C1) associated with defence response. The results showed that all these PR marker genes were highly upregulated in Ospelo (Fig. 5C). Whole-genome expression analysis of Ospelo PELO is a conserved key member of the RNA surveillance pathway and known to be involved in ribosome rescue, spermatogenesis, cell cycle control and meiotic cell division. However, almost all this knowledgs was from studies in yeast and mammals, with little information in plants. To gain further insight into the in planta function of OsPelo, transcriptome sequencing analysis of WT and Ospelo plants was conducted using RNA sequencing (RNA-seq). As lesion-mimic spots started to emerge on 20-day-old Ospelo leaves, this stage was selected for the profiling analysis. Three replicates of each genotype were used, yielding six libraries in total. Each of these libraries generated >20 million 125 bp paired-end reads after quality control, and about 86 % of them were uniquely mapped onto the rice reference genome (Supplementary Data Table S2). A total of 4990 DEGs were identified with a cut-off of the FDR-adjusted P-value <0.05. Among them, 2914 DEGs showed higher expression in Ospelo than in the WT, and were termed upregulated genes, while 2076 DEGs showed lower expression in Ospelo than in the WT and were termed downregulated genes (Fig. 6A; Supplementary Data Table S3). Among these DEGs, more than half of upregulated and downregulated genes showed a fold change <2 (Fig. 6B). This is in line with what was expected when considering that the leaf samples selected were at the very early stage of phenotypic change. Fig. 6. View largeDownload slide Analysis and Gene Ontology (GO) enrichment of differentially expressed genes (DEGs) between the WT and Ospelo by RNA-seq. (A) The number of up- and downregulated DEGs between the WT and Ospelo. (B) The fold change distribution of DEGs between the WT and Ospelo. (C–E) GO term enrichment analysis of up- and downregulated DEGs in Biological process (C), Molecular function (D) and Cellular component (E). Fig. 6. View largeDownload slide Analysis and Gene Ontology (GO) enrichment of differentially expressed genes (DEGs) between the WT and Ospelo by RNA-seq. (A) The number of up- and downregulated DEGs between the WT and Ospelo. (B) The fold change distribution of DEGs between the WT and Ospelo. (C–E) GO term enrichment analysis of up- and downregulated DEGs in Biological process (C), Molecular function (D) and Cellular component (E). To analyse further the effects of OsPelo mutation on the transcriptomes, GO classification analysis of the up- and downregulated DEGs was conducted (Fig. 6C–E; Supplementary Data Table S4). Within the category of biological process, upregulated genes were largely associated with response to stress, secondary metabolic process and cell death, indicating that the stress response in Ospelo was activated (Fig. 6C). Genes involved in post-embryonic development, reproduction and embryo development were significantly enriched among downregulated genes, which was consistent with the defects in root development and fertility in Ospelo. Moreover, genes involved in translation, metabolic process, transport, photosynthesis and biosynthesis were also enriched in downregulated genes. Within the category of molecular function, genes involved in catalytic activity, kinase activity and oxygen binding activity were enriched among upregulated genes, while genes involved in transcription factor activity and RNA binding were specifically enriched among downregulated genes (Fig. 6D). In terms of cellular component, only the plasma membrane was significantly enriched among upregulated genes, while downregulated genes showed association with the plastid, mitochondrion, cytosol, nucleolus and cytoskeleton, suggesting a broad range of functional repression of organelles in Ospelo (Fig. 6E). To explore further the biological pathways in which OsPelo may be involved, we performed KEGG pathway enrichment analysis for the DEGs between Ospelo and the WT. Twenty-three pathways were significantly enriched for upregulated genes and 53 for downregulated genes (Supplementary Data Table S5). Among the top 15 enriched pathways, the highly enriched upregulated pathways were mainly related to plant–pathogen interaction, protein processing, carbohydrate metabolism (amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis), amino acid metabolism (phenylalanine and glutathione metabolism), lipid metabolism (fatty acid metabolism, fatty acid elongation, biosynthesis of unsaturated fatty acids, α-linolenic acid metabolism, peroxisome) and secondary metabolism (biosynthesis of secondary metabolites, phenylpropanoid and flavonoid) (Fig. 7A). This was consistent with the enhanced pathogen resistance in Ospelo (Fig. 5A, B). The highly enriched pathways associated with down-regulated genes were mainly related to translation (aminoacyl-tRNA biosynthesis, RNA transport, ribosome biogenesis in eukaryotes), mismatch repair and primary metabolism including nucleotides, carbohydrates and amino acids (Fig. 7B). Fig. 7. View largeDownload slide Pathway enrichment and MapMan analysis of DEGs between the WT and Ospelo. (A, B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of up- (A) and downregulated (B) DEGs. (C) Mapping of DEGs associated with the KEGG aminoacyl-tRNA biosynthesis pathway. Boxes labelled with blue colour indicate downregulated DEGs between the WT and Ospelo. (D–F) MapMan analysis of DEGs associated with RNA–protein synthesis (D), lignin synthesis (E) and JA synthesis (F). Fig. 7. View largeDownload slide Pathway enrichment and MapMan analysis of DEGs between the WT and Ospelo. (A, B) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of up- (A) and downregulated (B) DEGs. (C) Mapping of DEGs associated with the KEGG aminoacyl-tRNA biosynthesis pathway. Boxes labelled with blue colour indicate downregulated DEGs between the WT and Ospelo. (D–F) MapMan analysis of DEGs associated with RNA–protein synthesis (D), lignin synthesis (E) and JA synthesis (F). In total, 30 DEGs were found to be involved in the aminoacyl-tRNA biosynthesis pathway, among which 28 DEGs responsible for synthesis of most aminoacyl-tRNAs were significantly downregulated in Ospelo (Fig. 7C; Supplementary Data Table S6). Further MapMan classification of DEGs also showed that a number of gene bins in the RNA–protein synthesis pathway were greatly downregulated, including RNA transcription and processing, protein amino acid activation, and protein synthesis initiation, elongation and release (Fig. 7D). These results were consistent with the putative role of OsPelo in stalled ribosome release in the mRNA decay pathway, whose dysfunction would result in the repression of translation. Among the enriched KEGG pathways for upregulated genes, there were several biotic stress-related pathways, including plant–pathogen interaction, α-linolenic acid metabolism and phenylpropanoid biosynthesis (Fig. 7A). The α-linolenic acid metabolism pathway is responsible for jasmonic acid (JA) synthesis, and the phenylpropanoid biosynthesis pathway produces lignin. JA is one of major signalling pathways in plant disease resistance (Nahar et al., 2011; Xie et al., 2011). Lignin is a non-degradable mechanical barrier for most micro-organisms, and an increase in lignification is a common response to pathogen attack to block parasite invasion and reduce the susceptibility of hosts (Moura et al., 2010). Consistent with these findings, MapMan analysis clearly showed that the synthesis pathways for JA and lignin were both significantly activated (Fig. 7E, F). DISCUSSION In the present study, a rice mutant, Ospelo, was isolated from an EMS-mutagenized population of rice (indica, ‘Kasalath’). The mutation caused loss of function of Pelota, a rice homologue of a key component in the NGD pathway. The mutant showed defects in root system development and spotted leaves from the early seedling stage, semi-dwarfness and defective pollen development (Figs 1 and 2). Functional complementation with WT OsPelo rescued the mutant phenotype observed in Ospelo (Fig. 3). We further conducted transcriptome sequencing of Ospelo and the WT, and found that DEGs were significantly associated with a number of biological processes, including translation, metabolism and biotic stress response. OsPELO belongs to a family of evolutionarily conserved proteins called PELO, with their primary function in the regulation of translation and cell cycle progression. In Drosophila, Pelo has been shown to be required to control meiotic cell cycle progression and self-renewal and division of GSCs in the ovary (Eberhart and Wasserman, 1995; Xi et al., 2005). The yeast homologue of PELO, DOM34, functions in protein translation to promote G1 progression and differentiation, and the dom34 mutants grow slowly and have defects in meiosis and sporulation (Davis and Engebrecht, 1998). In mice, disruption of the Pelo gene results in early embryonic lethality and defects in cell cycle progression (Adham et al., 2003). In rice Ospelo mutants, the root meristem activity was repressed, and pollen fertility and seed setting rate were dramatically decreased, suggesting a conserved role for OsPelo in cell cycle control through translation. Genes involved in translation, including aminoacyl-tRNA biosynthesis, protein amino acid initiation, elongation and release, were significantly enriched among the downregulated genes, suggesting the repression of translation in Ospelo (Fig. 7C, D; Supplementary Data Table S5). The mitochondrial localization of OsPELO suggests its possible involvement in the translation process taking place in mitochondria, one of the only two organelles containing their own genomes in cells (Fig. 4I). It has been reported that the PELO proteins are located in the cytoplasm of Drosophila (Xi et al., 2005) and in the cytoskeleton of mammalian cells (Burnicka-Turek et al., 2010). The difference in sub-cellular localization of PELO proteins among different species might suggest their functional divergence during evolution. The cell cycle is controlled by a complex machinery composed of cyclins, cyclin-dependent kinases (CDKs), CDK inhibitors (CKIs), E2F transcription factors and a number of other proteins (Inzé and Veylder, 2006; Guo et al., 2007). Among them, the A-type CDKs (CDKAs) are essential for G1 to S and G2 to M transition, and the B-type CDKs (CDKBs) show maximum activity at the G2 to M transition and the M-phase (De Veylder et al., 2007; Endo et al., 2012). D-type cyclins (CYCDs) mainly regulate the G1 to S transition through association with CDKs. In addition, the binding of CKI proteins could also adjust CDK activity (Polyn et al., 2015). The overexpression of one CKI gene, KRP1, could result in reduced cell production during leaf development and seed filling, and disturbed production of endosperm cells (Barrôco et al., 2006). Consistent with this, several key regulatory components of the cell cycle were found to be downregulated, including two cyclin genes (CycD3;1 and CycF2;3), seven CDK genes (CDKA;1, CDKB1;1, CDKB2;1, CDKD;1, CKL1, CKL6 and CKL7) and one E2F transcription factor gene (E2F2) (Supplementary Data Table S7). Moreover, KRP1 was found to be upregulated. These data suggested that the cell cycle progression in Ospelo was repressed, which might explain the observed short root phenotype. Defence response might be activated without pathogen attack in spotted-leaf mutants, and contribute to enhanced resistance to pathogen infection (Wang et al., 2017). Recently PELO has been reported to be involved in general antiviral activity in Drosophila and resistance to begomovirus in tomato (Wu et al., 2014; Lapidot et al., 2015). Mutation or silencing of Pelo similarly resulted in virus resistance in both Drosophila and tomato, and the critical role of PELO in highly efficiently translating viral proteins of infective viruses was suggested. The loss-of-function OsPelo mutation results in HR-like lesion spots on leaves and enhanced resistance to bacterial blight (Fig. 5A, B). The expression of three PR marker genes, PR1b, PR10 and PO-C1, was significantly upregulated in Ospelo during the development of lesion spots (Fig. 5C), indicating activation of PR genes and their possible roles in the enhanced pathogen resistance. Furthermore, whole-genome transcriptome analysis showed that there were 40 PR genes in the DEGs and all but one of them were significantly upregulated in Ospelo (Supplementary Data Table S8). Salicylic acid (SA) and JA are two conserved positive regulators of defence response in plants and are proposed to activate a common pathogen defence system in rice (Tamaoki et al., 2013; Berens et al., 2017). SA-mediated redox status changes control the nucleocytoplasmic localization of NPR1, and it interacts with TGA transcription factors upon localization to the nucleus and activates SA-responsive genes encoding PR proteins (Dong, 2004; Koornneef and Pieterse, 2008). Analysis of DEGs in Ospelo identified seven SA biosynthesis-related PAL genes, two NPR genes (NPR1 and NPR4), four TGA transcription factor genes, 20 JA biosynthesis-associated genes and three JAZ genes (Supplementary Data Table S9). All but three of them were upregulated in Ospelo, suggesting that OsPelo plays a negative role in both SA and JA biosynthesis and/or signalling and its loss of function might cause higher accumulation of SA and JA, thus enhancing plant defence against pathogens. Moreover, WRKY transcription factors are also proposed to be critical components in SA-dependent defence responses and control PR gene expression (Koornneef and Pieterse, 2008; Wei et al., 2013). In our study, there were 37 WRKY genes showing differential expression in Ospelo compared with the WT, and 34 of them were upregulated (Supplementary Data Table S10). Among them there were a number of WRKYs which have been reported to regulate pathogen resistance in rice (Liu et al., 2005; Chujo et al., 2007; Qiu et al., 2007; Shimono et al., 2007; Peng et al., 2008; Chujo et al., 2013; Yokotani et al., 2013). The KEGG enrichment analysis indicated the constitutive activation of the plant–pathogen interaction pathway (Fig. 7A). A total of 41 genes in the pathway were found to be differentially expressed in Ospelo compared with the WT, and all of them except two CNGC genes were upregulated (Supplementary Data Table S11). These genes participated in pathogen-associated molecular patterning (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) pathways in innate immunity. Within the PTI pathway, there were two CNGC genes (CNGC12 and CNGC10), one CAM gene (Cam1-1), 15 CML genes and four CPK genes (CPK10, CPK20, CPK21 and CPK23) involved in calcium signalling; two Rboh genes (Rboh5 and Rboh7) involved in generation of reactive oxygen species (ROS) and NOS1 for nitric oxide production; and two PR1 genes as antimicrobial components. Within the ETI pathway, RIN4, RPS2, SGT1 and three RPM1 genes were involved in recognition of avirulent effectors; and there were two HSP90 genes for HR. The loss-of-function mutation of CNGC in arabidopsis and barley resulted in high levels of SA, constitutive expression of PR genes and enhanced resistance to pathogens (Clough et al., 2000; Balagué et al., 2003; Rostoks et al., 2006; Kaplan et al., 2007). Overexpression of OsCPK10 and OsCPK20 in rice activated both SA- and JA-dependent defence responses and enhanced the resistance of transgenic plants to pathogens (Fu et al., 2013, 2014). These results showed that both the PTI- and ETI-related signalling components were significantly upregulated in Ospelo, suggesting that the loss of function of OsPelo resulted in activation of both PTI and ETI, reinforcement of cell walls and induction of PR proteins, thus enhancing resistance of Ospelo to pathogens. In conclusion, we report herein that OsPelo functions in development and defence in rice. We characterized the roles of this rice homologue of PELO protein, and confirmed that loss of function of OsPelo resulted in defects in root system development and enhanced pathogen resistance in rice. SUPPLEMENTARY DATA Supplementary data are available online at https://academic.oup.com/aob and consist of the following. Table S1: primers used in the study. Table S2: RNA-seq output and mapping results. Table S3: differentially expressed genes (DEGs) between the WT and Ospelo. Table S4: GO enrichment analysis of DEGs. Table S5: KEGG pathway enrichment analysis of DEGs. Table S6: list of DEGs associated with aminoacyl-tRNA biosynthesis. Table S7: list of DEGs associated with cell cycle regulation. Table S8: list of 40 PR genes found in DEGs. Table S9: list of DEGs associated with SA and JA biosynthesis and signalling. Table S10: list of 37 WRKY genes found in DEGs. Table S11: list of 41 DEGs involved in plant–pathogen interaction. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China [grant nos 31371595 and 31300246], the Zhejiang Provincial Natural Science Foundation of China [grant nos LY17C020002 and LQ16C020001], the Natural Science Foundation of Ningbo [grant no. 2017A610291] and the K. C. Wong Magna Fund in Ningbo University. LITERATURE CITED Adham IM, Sallam MA, Steding G, et al.   2003. 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Annals of BotanyOxford University Press

Published: May 16, 2018

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