TY - JOUR
AU - Glawischnig, Erich
AB - Abstract Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism. INTRODUCTION Cytochrome P450 enzymes are found in all domains of life but are particularly diversified in plants. In Arabidopsis (Arabidopsis thaliana), 244 P450-encoding genes are annotated, and individual enzymes have been shown to play roles in processes such as the biosynthesis of phytohormones or compounds involved in defense (Bak et al., 2011). However, the biological functions of the vast majority of Arabidopsis P450 enzymes remain unclear. Typically, eukaryotic P450 enzymes are anchored to the membrane of the endoplasmic reticulum (ER) with their catalytic centers facing the cytosolic side. These enzymes are able to form homomers and heteromers (Reed and Backes, 2012), and there is growing evidence that these interactions have an effect on the catalytic activities of the respective enzymes. This has been shown in detail for the human cytochrome P450 enzymes CYP2E1, CYP3A4, and CYP3A5 (Davydov et al., 2015). In contrast to human/animal systems, there is little information on potential functional interactions of plant P450 enzymes. For CYP73A5 and CYP98A3, physical interactions with each other and with additional enzymes of the phenylpropanoid biosynthetic pathway have been demonstrated by copurification and Förster resonance energy transfer (FRET; Bassard et al., 2012). Also, for CYP703 and CYP704 isoforms involved in sporopollenin biosynthesis, interactions with other pathway enzymes have been demonstrated by pulldown, yeast two-hybrid, and FRET experiments (Lallemand et al., 2013). Furthermore, there is evidence for complex formation of flavonoid biosynthetic enzymes (Crosby et al., 2011; Dastmalchi et al., 2016). It was recently shown in detail that the cyanogenic glucoside Dhurrin is synthesized by a protein complex of two cytochrome P450 enzymes, a P450 reductase and a glucosyl transferase (Laursen et al., 2016). These examples point to the formation of transient enzyme complexes, also referred to as metabolons, which allow for efficient channeling of intermediates, particularly for the biosynthesis of secondary metabolites (Fujino et al., 2018; Hawes and Kriechbaumer, 2018; Knudsen et al., 2018). In Arabidopsis, cytochrome P450 enzymes play a crucial role in the biosynthesis of indolic defense compounds, such as indole glucosinolates, camalexin, 4-hydroxyindole-3-carbonyl nitrile, and derivatives of indole-3-carboxylic acid (Figure 1; Rauhut and Glawischnig, 2009; Sønderby et al., 2010; Böttcher et al., 2014; Rajniak et al., 2015). For the biosynthesis of these specialized metabolites, Trp is converted to indole-3-acetaldoxime (IAOx) by CYP79B2 and CYP79B3. cyp79b2 cyp79b3 double mutants, in which Trp-derived defense compounds are essentially absent, are significantly more susceptible to a variety of pathogens than the wild type (Zhao et al., 2002; Glawischnig et al., 2004; Böttcher et al., 2009; Schlaeppi et al., 2010; Frerigmann et al., 2016). In healthy plants, IAOx is predominantly oxidized by CYP83B1 (also known as SUPERROOT2 or RUNT1) to the corresponding nitrile oxides or aci-nitro compound (Bak et al., 2001; Hansen et al., 2001), the precursors of indole glucosinolates. Pathogen infection or treatment with high dosages of UV light or heavy metals, such as silver nitrate, induce the production of CYP71A12 and CYP71A13, which in contrast dehydrate IAOx to form indole-3-acetonitrile (IAN) during the biosynthesis of camalexin (Nafisi et al., 2007; Müller et al., 2015), the major metabolite synthesized in response to these stresses. During camalexin biosynthesis, IAN is then activated, presumably to indole cyanohydrin, which also involves CYP71A12 and CY71A13, and conjugated with glutathione (Parisy et al., 2007), yielding GS-IAN. Glutathionylations are catalyzed by glutathione transferases (GSTs), which are found in all eukaryotes. Figure 1. Open in new tabDownload slide Biosynthetic Pathway of Camalexin and Related Metabolites. Enzymes are marked in red, detected compounds are labeled in blue, and biosynthetic intermediates in black. ICOOH, indole-3-carboxylic acid; IMG, indole-3-methylglucosinolate. Figure 1. Open in new tabDownload slide Biosynthetic Pathway of Camalexin and Related Metabolites. Enzymes are marked in red, detected compounds are labeled in blue, and biosynthetic intermediates in black. ICOOH, indole-3-carboxylic acid; IMG, indole-3-methylglucosinolate. Arabidopsis contains 54 GST genes belonging to 7 different classes (Krajewski et al., 2013). A number of GSTs have been shown to be capable of metabolizing xenobiotics (Dixon et al., 2002; Wagner et al., 2002), but with a few exceptions (Kitamura et al., 2004), information on their endogenous functions is limited, and it is unclear to which degree they are functionally redundant (Czerniawski and Bednarek, 2018). For GS-IAN formation, Su et al. (2011) suggested the involvement of GSTF6, a member of a small subfamily together with GSTF2, GSTF3, and GSTF7. Interestingly, camalexin concentrations detected in response to silver nitrate were not significantly different with respect to the wild type even in gstf2 gstf3 gstf6 triple knockout mutants or a gstf2 gstf3 gstf6 gstf7 knockdown line (Rauhut, 2009). This observation suggests that alternative GSTs might also participate in this step. Subsequently, GS-IAN is shortened to Cys(IAN), a process involving γ-glutamyl peptidase1 (GGP1), and Cys(IAN) is then converted to camalexin by the unique bifunctional P450 enzyme, CYP71B15 (Figure 1). cyp71b15 mutants (phytoalexin deficient3 [pad3]) are camalexin-deficient and accumulate camalexin precursors, such as Cys(IAN), dihydrocamalexic acid, and derivatives thereof (Glazebrook and Ausubel, 1994; Zhou et al., 1999; Bednarek et al., 2005; Schuhegger et al., 2006; Böttcher et al., 2009). Even though camalexin is a major sink for Trp in response to various stresses, intermediates such as IAOx do not accumulate, suggesting possible metabolite channeling between interacting proteins. Therefore, we hypothesized that camalexin is produced by a metabolon. In this study, we provide evidence that the cytochrome P450 enzymes of the camalexin biosynthetic pathway physically interact, and we systematically analyze the potential functions of GSTs in camalexin formation. RESULTS Cellular and Subcellular Localization of CYP71B15 We generated a construct expressing CYP71B15 as a C-terminal GFP fusion protein under the control of its own promoter, used it to transform an Arabidopsis pad3 knockout mutant, and selected lines complementing the camalexin-deficient phenotype. We monitored the expression of the CYP71B15-GFP protein by protein gel blot analysis and selected a line for further analysis in which a strong GFP signal was observed in response to Botrytis cinerea infection, whereas the signal was absent in untreated leaves (Supplemental Figure 1A). As a next step, we analyzed the cellular distribution and subcellular localization of CYP71B15-GFP in response to the fungal pathogens B. cinerea, Alternaria brassicicola, and Erysiphe cruciferarum (Figure 2). In accordance with its biological function in phytoalexin biosynthesis, CYP71B15-GFP was only observed in cells in close proximity to successful pathogen infection. We observed strong accumulation of CYP71B15-GFP around the site of B. cinerea infection (24 h after infection [hai]; Figures 2A to 2F), surrounding an area where the necrotrophic fungus had apparently already started to macerate the leaf tissue and where no CYP71B15-GFP was detected, possibly because these cells were no longer metabolically active. For necrotrophic A. brassicicola (18 hai), CYP71B15-GFP expression was only observed in cells in direct contact with the fungus (Figures 2G to 2L). In E. cruciferarum-infected leaves (24 hai), the highest CYP71B15-GFP abundance was observed in cells next to cells that had been attacked or penetrated by the biotrophic fungus (Figures 2M to 2R). Note that E. cruciferarum spores that had not germinated did not induce CYP71B15-GFP expression (Figures 2M to 2O). In all cases, CYP71B15-GFP was located in the ER, which also surrounds the nucleus. This observation is in accordance with the detected ER localization in the heterologous Nicotiana benthamiana system (see below). No focal protein accumulations at sites of plant-microbe interactions were detected. Figure 2. Open in new tabDownload slide CYP71B15-GFP Accumulates in Cells Surrounding Fungal Infection Sites. CYP71B15pro:CYP71B15-GFP-expressing plants were inoculated with spores of B. cinerea, A. brassicicola, or E. cruciferarum, and the expression of CYP71B15-GFP at fungal infections sites was detected. (A) to (C) Sites of infection with nectrotrophic B. cinerea (central transparent leaf area) 24 hai. (D) to (F) Higher magnification of the areas indicated by the squares in (A) to (C). Asterisks indicate spores from which hyphae emerged. (G) to (L) Sites of infection with A. brassicicola 18 hai. Asterisks indicate spores from which hyphae emerged. (M) to (R) Sites of successful infection by the biotrophic powdery mildew fungus E. cruciferarum 24 hai. Arrowheads indicate sites of fungal attack/penetration; asterisks indicate nongerminated spores. The first column shows transmission channel images ([A], [D], [G], and [J]) or red staining of fungal structures after staining with FM4-64 ([M] and [P]); images in the second column show CYP71B15-GFP accumulation, and the third column shows the overlay images of the two. Bars = 50 μm. Figure 2. Open in new tabDownload slide CYP71B15-GFP Accumulates in Cells Surrounding Fungal Infection Sites. CYP71B15pro:CYP71B15-GFP-expressing plants were inoculated with spores of B. cinerea, A. brassicicola, or E. cruciferarum, and the expression of CYP71B15-GFP at fungal infections sites was detected. (A) to (C) Sites of infection with nectrotrophic B. cinerea (central transparent leaf area) 24 hai. (D) to (F) Higher magnification of the areas indicated by the squares in (A) to (C). Asterisks indicate spores from which hyphae emerged. (G) to (L) Sites of infection with A. brassicicola 18 hai. Asterisks indicate spores from which hyphae emerged. (M) to (R) Sites of successful infection by the biotrophic powdery mildew fungus E. cruciferarum 24 hai. Arrowheads indicate sites of fungal attack/penetration; asterisks indicate nongerminated spores. The first column shows transmission channel images ([A], [D], [G], and [J]) or red staining of fungal structures after staining with FM4-64 ([M] and [P]); images in the second column show CYP71B15-GFP accumulation, and the third column shows the overlay images of the two. Bars = 50 μm. Untargeted Screen for Interaction Partners of CYP71B15 Applying this CYP71B15 pro:CYP71B15-GFP (pad3) line, we set up an untargeted proteomics screen to identify proteins that interact with CYP71B15 in B. cinerea-infected plants, as a model system for pathogen interactions, or in UV-irradiated plants. Rosette leaves of 6-week-old CYP71B15 pro:CYP71B15-GFP (pad3) and pad3 plants were infected with B. cinerea. After 24 h, we prepared and solubilized microsomes, performed coimmunoprecipitations (co-IPs), and subjected the eluates to trypsin digestion and mass spectrometry (MS) analysis. We also analyzed an aliquot of starting material to determine the composition of microsomal proteins in response to B. cinerea infection. Along with the bait protein CYP71B15, which was the protein corresponding to the highest signal intensity, a total of 71 proteins significantly accumulated with respect to the control IPs. Strikingly, among these, 22 cytochrome P450 enzymes (e.g., CYP71B23, CYP84A1, and CYP706A1) were highly overrepresented (Figure 3; Supplemental Figure 2A). CYP71A13, the enzyme channeling IAOx into the camalexin biosynthetic pathway, was among the interacting proteins that accumulated with the highest intensity (average log2 intensity = 25.4) and highest specificity (109-fold enrichment, P = 0.00014). Interestingly, the P450 enzyme CYP83B1, which competes with camalexin-specific enzymes for the intermediate IAOx (Figure 1; Bak et al., 2001; Hansen et al., 2001), and CYP71B6, which is involved in IAN metabolism (Bak et al., 2001; Hansen et al., 2001; Böttcher et al., 2014; Müller et al., 2019), were also enriched. CYP71A12 was also significantly enriched, with a label-free quantification (LFQ) intensity approximately 12-fold lower than that of CYP71A13. CYP79B2 and Arabidopsis P450 Reductase1 (ATR1) were detected in the co-IP, but the respective enrichments (4.5-fold, P = 0.17 and 5.0-fold, P = 0.022, respectively) were below the threshold of significance, pointing to a weak or transient interaction with the observed CYP71B15-containing protein complex. Interestingly, PDR12/ABCG40, which was recently identified as a camalexin transporter (He et al., 2019), was significantly enriched (7.8-fold, P = 0.00029), suggesting that the biosynthesis and transport of camalexin might be physically linked to some extent. For a comprehensive overview of the proteomics data, see Supplemental Data Set 1. Figure 3. Open in new tabDownload slide Proteins Copurified with CYP71B15-GFP from Leaves Infected with B. cinerea. CYP71B15-GFP was expressed under the control of its endogenous promoter in the pad3 background. The enrichment of interacting proteins in co-IP experiments (log2 fold change) is plotted against the significance of the change (–log10 P value). Cytochrome P450 enzymes are represented by blue circles, all other proteins are represented by open squares, and their respective size represents log2. LFQ indicates label-free quantification intensities. P450 enzymes were strongly enriched, including CYP71A13 and CYP71B6. P450 proteins above log2 LFQ intensity of 25 and those mentioned in the text are indicated. n = 3. Figure 3. Open in new tabDownload slide Proteins Copurified with CYP71B15-GFP from Leaves Infected with B. cinerea. CYP71B15-GFP was expressed under the control of its endogenous promoter in the pad3 background. The enrichment of interacting proteins in co-IP experiments (log2 fold change) is plotted against the significance of the change (–log10 P value). Cytochrome P450 enzymes are represented by blue circles, all other proteins are represented by open squares, and their respective size represents log2. LFQ indicates label-free quantification intensities. P450 enzymes were strongly enriched, including CYP71A13 and CYP71B6. P450 proteins above log2 LFQ intensity of 25 and those mentioned in the text are indicated. n = 3. To evaluate the extent to which this result depends on the trigger of camalexin biosynthesis, we also performed IPs with UV-irradiated leaves. The general outcome was similar (Supplemental Figure 2B): besides the bait, which showed the highest abundance, P450 enzymes such as CYP71A13, CYP83B1, and CYP71B6 were highly enriched, but CYP79B2 and CYP71A12 also copurified with CYP71B15 (Supplemental Data Set 1). Screen for Inducible Physical Interactors of CYP71A13 CYP71A13 was consistently identified as interactor in an untargeted screen with CYP71B15 as bait. As a complementary approach, we expressed CYP71A13-yellow fluorescent protein (YFP) fusion protein in Arabidopsis under the control of the 35S promoter. We isolated and solubilized microsomes of UV-irradiated or untreated rosette leaves and performed co-IP to address (1) whether the CYP71B15-CYP71A13 interaction is independent of the choice of baits and (2) which interaction partners specifically bind CYP71A13 in response to induction. A total of 875 proteins were reproducibly detected in the co-IPs of UV light-treated samples (Supplemental Data Set 2), including 26 cytochrome P450 enzymes and the cytochrome P450 reductases ATR1 and ATR2. Constitutive expression of the bait allows binding partners to also be detected under control conditions, where concentrations of CYP71A13 expressed under the control of its native promoter are too low for quantitative work. As this approach can also yield unspecific binding partners, the analysis was focused on the differences under UV light treatment versus control conditions. Strikingly, only one protein, CYP71B15, was significantly enriched in the UV light-treated versus nontreated sample (Figure 4). Five proteins were significantly depleted in the UV light-treated versus nontreated sample, including Nitrilase3 (approximately 7-fold), which is thought to convert IAN to the auxin indole-3-acetic acid upon sulfur starvation (Kutz et al., 2002). Figure 4. Open in new tabDownload slide Proteins Copurified from Leaves Overexpressing CYP71A13-YFP with or without UV Irradiation. The log2 fold change (UV-irradiated versus untreated leaves) is plotted against the significance of the change (–log10 P value). Cytochrome P450 enzymes are represented by blue circles, all other proteins are represented by open squares, and their respective size represents log2. Proteins enriched significantly in UV or in control samples are labeled. UV-dependent copurification of CYP71B15 was observed. n = 3. Figure 4. Open in new tabDownload slide Proteins Copurified from Leaves Overexpressing CYP71A13-YFP with or without UV Irradiation. The log2 fold change (UV-irradiated versus untreated leaves) is plotted against the significance of the change (–log10 P value). Cytochrome P450 enzymes are represented by blue circles, all other proteins are represented by open squares, and their respective size represents log2. Proteins enriched significantly in UV or in control samples are labeled. UV-dependent copurification of CYP71B15 was observed. n = 3. In summary, we conclude from the untargeted co-IP experiments that the core camalexin biosynthetic enzymes CYP71B15 and CYP71A13 physically interact with each other in challenged Arabidopsis rosette leaves. Also, CYP71B6, which specifically converts IAN to indole-3-aldehyde and ICOOH (Böttcher et al., 2014), was consistently identified as a member of the protein complex. In the untargeted screens, CYP79B2 was identified as a binding partner of CYP71B15, although the specificity of this interaction was not significant. No interaction in an untargeted screen with CYP71A13 was observed. This finding indicates that the binding of CYP79B2 to the proposed camalexin biosynthetic protein complex is weaker and more transient than the interaction between the camalexin-specific enzymes CYP71A13 and CYP71B15. Physical Interaction of Camalexin Biosynthetic Enzymes Is Confirmed by Targeted Co-IP To confirm the physical interaction of the camalexin biosynthetic enzymes CYP71A12, CYP71A13, CYP71B15, and ATR1, we transiently expressed different combinations of these proteins in N. benthamiana as C-terminally YFP- and FLAG-tagged proteins. Solubilized microsomes were applied to α-GFP beads, and IP and co-IP were monitored by protein gel blot analysis with GFP- and FLAG-specific antibodies, respectively (Figure 5; Supplemental Figure 3). As negative controls, all proteins were also coexpressed with membrane-bound GFP in order to exclude the possibility that protein interactions were detected due to the YFP tag or unspecific binding of the FLAG-tagged proteins to the polysaccharide chains of the GFP trap beads used for targeted co-IP. CYP71A13 interacted with CYP71B15 and ATR1, and CYP71B15 interacted with CYP71A12. In addition, we observed interactions of CYP71A13 and CYP71B15 with GSTU4 (see below). Figure 5. Open in new tabDownload slide Co-IP Analysis of the Physical Association of Camalexin-Specific Enzymes. YFP- and FLAG-tagged fusion proteins were transiently expressed in N. benthamiana, and microsomal proteins were extracted 4 d after infiltration. CYP71B15 (B15) in combination with CYP71A13 (A13; lane 1), CYP71A12 (A12; lane 2), or GSTU4 (U4; lane 4) and CYP71A13 in combination with GSTU4 (lane 3) or ATR1 (lane 5) are shown. (A) Protein gel blot analysis of input samples. (B) Protein gel blot analysis of IP samples. IP was performed with anti-GFP antibody, and interacting proteins were analyzed with an anti-FLAG antibody. Interaction is shown for CYP71A13-FLAG with CYP71B15-YFP (lane 1), GSTU4-YFP (lane 3), and ATR1-YFP (lane 5) and for CYP71B15-YFP with CYP71A12-FLAG (lane 2) and GSTU4-FLAG (lane 4). The experiment was repeated at least three times, with similar results. Combinations of fusion proteins where no co-IP was observed are shown in Supplemental Figure 3. Figure 5. Open in new tabDownload slide Co-IP Analysis of the Physical Association of Camalexin-Specific Enzymes. YFP- and FLAG-tagged fusion proteins were transiently expressed in N. benthamiana, and microsomal proteins were extracted 4 d after infiltration. CYP71B15 (B15) in combination with CYP71A13 (A13; lane 1), CYP71A12 (A12; lane 2), or GSTU4 (U4; lane 4) and CYP71A13 in combination with GSTU4 (lane 3) or ATR1 (lane 5) are shown. (A) Protein gel blot analysis of input samples. (B) Protein gel blot analysis of IP samples. IP was performed with anti-GFP antibody, and interacting proteins were analyzed with an anti-FLAG antibody. Interaction is shown for CYP71A13-FLAG with CYP71B15-YFP (lane 1), GSTU4-YFP (lane 3), and ATR1-YFP (lane 5) and for CYP71B15-YFP with CYP71A12-FLAG (lane 2) and GSTU4-FLAG (lane 4). The experiment was repeated at least three times, with similar results. Combinations of fusion proteins where no co-IP was observed are shown in Supplemental Figure 3. Confocal Laser Scanning Microscopy and FRET-Fluorescence-Lifetime Imaging Microscopy Analysis Demonstrate the Physical Interaction of Biosynthetic Enzymes in Vivo We analyzed the subcellular localization of CYP71A12, CYP71A13, CYP71B15, and CYP79B2, as well as GGP1 and the GSTU2 and GSTU4 (Figure 6), by confocal microscopy 3 d after transient expression of the corresponding C-terminal GFP and red fluorescent protein (RFP) fusion proteins in N. benthamiana (Figure 6; Supplemental Figure 4). CYP71A12 (Supplemental Figure 4A), CYP71A13 (Supplemental Figure 4B), and CYP71B15 (Supplemental Figure 4C) were localized to the ER and showed colocalization with the ER lumenal marker RFP-HDEL and with each other (Figures 6A to 6F). Interestingly, although all experimental conditions were identical for all P450 enzymes analyzed, the expression of CYP79B2-RFP was always weaker (Figure 6G). Nevertheless, colocalization with CYP71A13 was observed (Figure 6I). It appears that GGP1 was localized to the cytosol, and to some extent the mislocalization of CYP71A13 to the cytoplasm was induced by coexpression with GGP1 (Figures 6J to 6L). Figure 6. Open in new tabDownload slide Colocalization of Camalexin-Specific Enzymes in N. benthamiana Leaves. In each case, two GFP- or RFP-labeled P450 enzymes ([A], [B], [D], [E], [G], [H], [K], [N], and [Q]) in different combinations or together with either GGP1-RFP (J), GSTU4-RFP (M), or GSTU2-RFP (P) were expressed transiently in N. benthamiana and analyzed for localization and colocalization 3 d after infiltration. Fluorescence signals for CYP71B15, CYP71A13, CYP71A12, and CYP79B2 fusion proteins were detected at the ER ([A], [B], [D], [E], [G], and [H]), with CYP79B2 expression levels substantially lower than those of the other proteins (G). GSTU4 (J) and GSTU2 (M) are localized to the cytosol (see Supplemental Figures 4D and 4E), here shown by both constructs labeling the nucleoplasm, which is typical for cytosolic localization, rather than the nuclear envelope, which is typical for ER-localized proteins such as CYP71A13 ([N] and [Q]). Colocalization for CYP71A13 with CYP71B15 (C) and CYP79B2 (I) is shown in the merged images. Furthermore, CYP71B15 colocalizes with CYP71A12 (F), whereas no signal overlap is detectable when CYP71A13 is coexpressed with the cytosolic proteins GSTU4 (O) or GSTU2 (R); see Supplemental Figure 4. Bars = 10 μm. Figure 6. Open in new tabDownload slide Colocalization of Camalexin-Specific Enzymes in N. benthamiana Leaves. In each case, two GFP- or RFP-labeled P450 enzymes ([A], [B], [D], [E], [G], [H], [K], [N], and [Q]) in different combinations or together with either GGP1-RFP (J), GSTU4-RFP (M), or GSTU2-RFP (P) were expressed transiently in N. benthamiana and analyzed for localization and colocalization 3 d after infiltration. Fluorescence signals for CYP71B15, CYP71A13, CYP71A12, and CYP79B2 fusion proteins were detected at the ER ([A], [B], [D], [E], [G], and [H]), with CYP79B2 expression levels substantially lower than those of the other proteins (G). GSTU4 (J) and GSTU2 (M) are localized to the cytosol (see Supplemental Figures 4D and 4E), here shown by both constructs labeling the nucleoplasm, which is typical for cytosolic localization, rather than the nuclear envelope, which is typical for ER-localized proteins such as CYP71A13 ([N] and [Q]). Colocalization for CYP71A13 with CYP71B15 (C) and CYP79B2 (I) is shown in the merged images. Furthermore, CYP71B15 colocalizes with CYP71A12 (F), whereas no signal overlap is detectable when CYP71A13 is coexpressed with the cytosolic proteins GSTU4 (O) or GSTU2 (R); see Supplemental Figure 4. Bars = 10 μm. We analyzed physical interactions by FRET (Förster, 1948), as measured by fluorescence-lifetime imaging microscopy (FLIM) of the excited state of the donor (Becker, 2012; Schoberer and Botchway, 2014). The reduction in the lifetime of GFP (donor) fluorescence occurs only when an acceptor fluorophore (monmeric RFP) is within a distance of 10 nm, indicating a very high proximity and most likely direct physical contact between the two proteins of interest. We therefore quantified the fluorescence lifetimes of CYP71A12-GFP (Figure 7A), CYP71A13-GFP (Figure 7B), and CYP71B15-GFP (Figure 7C) in combination with various potential binding partners. Interactions were shown for CYP71A12 with CYP71B15, CYP79B2, GSTU4, and the soluble camalexin-biosynthetic enzyme GGP1. CYP71A13 binds to CYP71A12, CYP71B15, CYP79B2, GSTU4, and GGP1. Furthermore, the fluorescence lifetime of CYP71B15-GFP in the presence of CYP71A12, CYP79B2, GSTU4, or GGP1 was significantly reduced, which indicates an interaction of these enzymes. Figure 7. Open in new tabDownload slide The Tight Physical Interaction of Camalexin Biosynthesis Enzymes Is Supported by FRET-FLIM. GFP-tagged CYP71A12 (A), CYP71A13 (B), or CYP71B15 (C) was transiently expressed in N. benthamiana alone (black bars) or in combination with different RFP-tagged proteins (white bars). Three days after inoculation, protein-protein interaction was determined by measuring the GFP fluorescence lifetime via FLIM. In the case of FRET, a significant reduction of GFP fluorescence lifetime was detectable compared with the donor-only sample. Physical interaction could be observed for CYP71A12, CYP71A13, and CYP71B15 with each other and with CYP79B2, GGP1, and GSTU4. No interaction with GSTU2 and no homodimerization of CYP71A12 or CYP71B15 were observed. Error bars indicate sd of at least three independent replicates. One-way ANOVA for independent samples, standard weighted-means analysis, with Tukey’s honestly significant difference posthoc test: *, P < 0.05; **, P < 0.01. Figure 7. Open in new tabDownload slide The Tight Physical Interaction of Camalexin Biosynthesis Enzymes Is Supported by FRET-FLIM. GFP-tagged CYP71A12 (A), CYP71A13 (B), or CYP71B15 (C) was transiently expressed in N. benthamiana alone (black bars) or in combination with different RFP-tagged proteins (white bars). Three days after inoculation, protein-protein interaction was determined by measuring the GFP fluorescence lifetime via FLIM. In the case of FRET, a significant reduction of GFP fluorescence lifetime was detectable compared with the donor-only sample. Physical interaction could be observed for CYP71A12, CYP71A13, and CYP71B15 with each other and with CYP79B2, GGP1, and GSTU4. No interaction with GSTU2 and no homodimerization of CYP71A12 or CYP71B15 were observed. Error bars indicate sd of at least three independent replicates. One-way ANOVA for independent samples, standard weighted-means analysis, with Tukey’s honestly significant difference posthoc test: *, P < 0.05; **, P < 0.01. Taking the co-IP and FRET-FLIM data together, we demonstrated that the known camalexin biosynthetic enzymes form a protein complex in vivo. Interestingly, no CYP71A12 or CYP71B15 homodimer formation was observed. This also demonstrates that the observed interactions between, for example, CYP71A12/A13 and CYP71B15 are not due to unspecific dimerization of the cytochrome P450s. Enzymatic Parameters of CYP79B2 Indicate Allosteric Interaction with CYP71A13 To examine potential metabolic channeling, we coexpressed the first two pathway enzymes, CYP79B2 and CYP71A13, together with ATR1 in yeast (Saccharomyces cerevisiae). As a control, the CYP71A13 expression construct was replaced by an empty vector. We monitored Trp conversion by the corresponding microsomes. A striking shift of the product spectrum toward the formation of IAN was observed for CYP79B2/CYP71A13 with respect to microsomes harboring CYP79B2/empty vector (Figure 8A). In addition, coexpression of CYP79B2 and CYP71A13 reduced the apparent K m value of CYP79B2 for Trp more than twofold (6.9 ± 0.9 µM versus 17.5 ± 1.9 µM; Figure 8B). Figure 8. Open in new tabDownload slide Higher Apparent Substrate Affinity of CYP79B2 in the Presence of CYP71A13. CYP79B2 was expressed in S. cerevisiae together with CYP71A13 or the vector control. (A) Turnover of Trp with NADPH as cosubstrate by corresponding microsomes. Detection of substrate and products was by HPLC; chromatogram at 278 nm. (B) Apparent K m values for Trp. CYP79B2: K m = 17.5 ± 1.9 μM, R 2 = 0.95; CYP79B2/CYP71B13: K m = 6.9 ± 0.9 μM, R 2 = 0.90 (n = 16). Figure 8. Open in new tabDownload slide Higher Apparent Substrate Affinity of CYP79B2 in the Presence of CYP71A13. CYP79B2 was expressed in S. cerevisiae together with CYP71A13 or the vector control. (A) Turnover of Trp with NADPH as cosubstrate by corresponding microsomes. Detection of substrate and products was by HPLC; chromatogram at 278 nm. (B) Apparent K m values for Trp. CYP79B2: K m = 17.5 ± 1.9 μM, R 2 = 0.95; CYP79B2/CYP71B13: K m = 6.9 ± 0.9 μM, R 2 = 0.90 (n = 16). GSTU4 Physically Interacts with CYP71A13 and Is Relevant for the Camalexin Response During camalexin biosynthesis, activated IAN, presumably indole cyanohydrin, is glutathionylated, a process likely involving a GST (Klein et al., 2013). The untargeted co-IP screens (Supplemental Data Sets 1 and 2) revealed only a few GSTs as copurified proteins, with very low signal intensities. GSTF6, which was previously proposed to be involved in camalexin biosynthesis (Su et al., 2011), was not detected. Perhaps the interaction of the cytosolic GSTs with the P450 enzymes is not sufficiently strong to persist in the presence of the Triton X-100 concentration utilized. To evaluate which Arabidopsis GSTs are capable of this conversion, we performed a qualitative screening in a yeast strain in which four endogenous GSTs and three genes of glutathione conjugate catabolism were deleted (GTO1, GTO2, GTO3, TEF4, CPC, CPY, and CIS2; Krajewski et al., 2013; Kowalski, 2016) and in which expression plasmids for ATR1 and CYP71A13 were introduced. These yeast cells were transformed with each of the 54 Arabidopsis GSTs and, after selection, screened for the biotransformation of IAN and glutathione, yielding GS-IAN. When an empty vector was used instead of the CYP71A13 expression plasmid, no activity was detected. Also, when no Arabidopsis GST was expressed, no GS-IAN was synthesized. Strikingly, for 41 enzymes, including most ϕ- and τ-class GSTs, product formation was observed (Supplemental Figure 5). As an approach to identify which of the active GSTs is relevant in planta, we surveyed transcriptomics data for coexpression with camalexin biosynthetic genes. In particular, GSTU4 is strongly induced by pathogens, and its expression is correlated with the genes of camalexin biosynthesis (CYP71B15, r = 0.85; CYP71A13, r = 0.77; expression angler, B. cinerea set [Toufighi et al., 2005]; see also Supplemental Table 1). We coexpressed GSTU4 with CYP71A13 in N. benthamiana as RFP/GFP fusion proteins and monitored their subcellular localization (Figure 6; Supplemental Figures 4 and 6). As a control, GSTU2 was included, which is closely related to GSTU4 and a member of the same gene cluster and is only weakly transcriptionally coregulated with genes of camalexin biosynthesis (CYP71B15, r = 0.53; CYP71A13, r = 0.62; expression angler, B. cinerea set [Toufighi et al., 2005]). We tested the physical interactions of both pairs of proteins by FRET-FLIM. For GSTU4-RFP, a strong reduction in the lifetimes of CYP71A12-GFP, CYP71A13-GFP, and CYP71B15-GFP was detected (Figure 7). All three CYP71s and GSTU2 did not physically interact (Figure 7). The interaction of GSTU4 with CYP71A13 and CYP71B15 was also demonstrated via co-IP analysis (Figure 5). To evaluate the potential functions of GSTU2 and GSTU4 in camalexin biosynthesis, we analyzed gstu2 and gstu4 knockout as well as GSTU4 overexpression lines for camalexin formation in response to UV-C light (Supplemental Figure 7A), silver nitrate (heavy metal) treatment (Supplemental Figure 7B), and B. cinerea infection (Supplemental Figure 7C). While no difference in camalexin levels relative to the wild-type control was observed for gstu2, the gstu4 knockout mutants typically showed elevated camalexin concentrations. Strikingly, in response to B. cinerea infection, the 35S pro:GSTU4 overexpression lines accumulated less camalexin than wild-type plants. To statistically evaluate these effects, we combined data from four independent experiments (Figure 9; Supplemental File 1), finding that there is a significant negative effect of GSTU4 on the relative camalexin concentration accumulating in response to B. cinerea infection. Figure 9. Open in new tabDownload slide Camalexin Formation in Response to B. cinerea Infection in gstu4 Knockout and Overexpression Plants. Leaves of 6-week-old plants were treated with B. cinerea spores. After 48 h, camalexin was extracted and levels were analyzed via HPLC. In gstu4 lines, the camalexin level significantly increased, whereas a significant decrease was observed in GSTU4-overexpressing lines. Camalexin levels are shown as arithmetic means with se of 26 to 32 independent plants. Different letters indicate significant differences according to ANOVA (Scheffé’s test, P < 0.05; Supplemental File 1); asterisks indicate significant differences from Col-0 according to Student’s t test (P < 0.05). Figure 9. Open in new tabDownload slide Camalexin Formation in Response to B. cinerea Infection in gstu4 Knockout and Overexpression Plants. Leaves of 6-week-old plants were treated with B. cinerea spores. After 48 h, camalexin was extracted and levels were analyzed via HPLC. In gstu4 lines, the camalexin level significantly increased, whereas a significant decrease was observed in GSTU4-overexpressing lines. Camalexin levels are shown as arithmetic means with se of 26 to 32 independent plants. Different letters indicate significant differences according to ANOVA (Scheffé’s test, P < 0.05; Supplemental File 1); asterisks indicate significant differences from Col-0 according to Student’s t test (P < 0.05). DISCUSSION The physical interaction of enzymes is a powerful strategy to effectively channel biosynthetic pathways and avoid the release of reactive intermediates. Upon induction, camalexin is a major sink for Trp. Nevertheless, intermediates such as IAOx do not accumulate, indicating metabolite channeling. Camalexin biosynthesis involves several P450 enzymes that are bound to ER membranes. Membrane anchoring restricts diffusion, allowing P450 enzymes to serve as nuclei for the formation of metabolic complexes. In addition, the ER membrane can reorganize, bringing cytochrome P450 enzymes into contact with pathway enzymes in other organelles. For example, this process was observed for CYP81F2 in the interaction of Arabidopsis with nonadapted powdery mildew Blumeria graminis f sp hordei (Fuchs et al., 2016). For the ultimate enzyme of the camalexin biosynthetic pathway, CYP71B15 (PAD3), in the interaction with B. cinerea, A. brassicicola, and E. cruciferarum (Figure 2), we observed a strong induction of protein expression but no focal accumulation. Highly localized expression at sites of interaction together with metabolic channeling in multienzyme complexes may ensure highly controlled and safe production of camalexin on demand. We identified proteins that physically interact with CYP71B15 (PAD3) following an untargeted co-IP approach (Figure 3). The relative abundance of copurified proteins does not reflect the relative protein abundance of the corresponding solubilized microsomes, which served as the starting material. Based on LFQ intensities, P450 enzymes represent only a minor fraction of total microsomal proteins, while they were highly overrepresented in the co-IP samples and highly enriched with respect to control IPs. This finding indicates that the interaction between CYP71B15 and other P450 enzymes is not random. CYP71A12 and CYP71A13 were copurified with high significance, demonstrating the specific interaction of camalexin biosynthetic enzymes. In addition, enzymes involved in other pathways, such as phenylpropanoid and glucosinolate metabolism, were also significantly enriched, including CYP71B6, which degrades IAN to ICOOH and cyanide. Remarkably, the detected CYP71B15-CYP73A5 and CYP71B15-CYP98A3 interactions were also observed via a reverse approach with the two phenylpropanoid biosynthetic enzymes as baits in a tandem affinity purification-based screen (Bassard et al., 2012). Perhaps direct or indirect interactions of CYP71B15 with P450 enzymes of other biosynthetic pathways involve the mutual regulation of their catalytic activities. Alternatively, ER-bound P450s tend to interact, as they are dependent on the reductases ATR1 or ATR2 (Bassard et al., 2012). However, these P450 reductases were detected in solubilized microsomes but not significantly enriched by co-IP. In addition, a number of membrane-bound kinases were enriched. Whether this interaction has functional significance (e.g., phosphorylation of the biosynthetic enzymes) remains to be investigated. We performed a second co-IP screen in order to identify interacting proteins that are specifically inducible. Here, constitutively expressed CYP71A13 was used as a bait and UV light-challenged leaves were compared with the untreated controls (Figure 4). Only one of the copurified proteins was significantly enriched: CYP71B15. In conclusion, CYP71A13-CYP71B15 was robustly identified as a core protein complex, and this interaction was confirmed by targeted co-IP and FRET-FLIM (Figures 5 and 7). The formation of biosynthetic complexes is typically a transient and reversible process (Perkins et al., 2010). For targeted co-IP, the bait and interacting proteins were transiently overexpressed, also enabling interactions with proteins of low abundance in planta. Here, a CYP71A13-ATR1 interaction was also observed. Furthermore, CYP71A12-CYP79B2 and CYP71A13-CYP79B2 interactions were revealed by FRET-FLIM analysis, as this method is most suitable for detecting transient protein interactions. As co-IP experiments with microsomal proteins as baits involve solubilization with mild detergents, cytosolic components of the complex will not directly be solubilized and therefore depleted relative to membrane-bound partners. This is likely the case for GGP1, which was not enriched using the untargeted approaches. Similarly, a soluble GST was proposed to be a component of the camalexin biosynthetic machinery, but apparently, no GSTs were significantly enriched in an untargeted co-IP with CYP71B15 as bait. FRET-FLIM analysis is a powerful approach for detecting interactions between known membrane-bound and soluble proteins, as it is not affected by differences in protein solubility. Here, in addition to the interactions of the camalexin biosynthetic cytochrome P450 enzymes, we observed interactions of CYP71A13 with GSTU4 and GGP1 (Figure 7). Camalexin biosynthesis involves glutathionylation of IAN. As most Arabidopsis GSTs are capable of catalyzing this reaction in vitro in concert with CYP71A13 (Supplemental Figure 5), it can be postulated that the functioning of a specific GST in camalexin biosynthesis is due to its ability to interact with the biosynthetic machinery or the local substrate concentration rather than by its substrate specificity. GSTU4 is transcriptionally coregulated with camalexin and Trp biosynthetic genes, and the corresponding protein was identified as a physical interactor of CYP71A13 (Figures 5 and 7; Supplemental Table 1). Therefore, GSTU4 was a prime candidate for being a key GST gene in camalexin biosynthesis. In contrast to this assumption, after infection with B. cinerea, gstu4 knockout plants had elevated concentrations of camalexin, whereas in overexpression plants, camalexin levels were reduced with respect to wild-type leaves. This observation is opposite that expected for a camalexin biosynthetic gene. The mechanism by which GSTU4 negatively interferes with camalexin biosynthesis remains unclear. One possibility is that a subcellular transport process is involved, as some GSTs such as GSTF12 (TRANSPARENT TESTA19) act as transporters between cellular compartments rather than as glutathione transferases (Kitamura et al., 2004; Sun et al., 2012). In this case, an intermediate of camalexin biosynthesis could be exported from the metabolon and metabolized. Alternatively, GSTU4 could have a regulatory function. The human GST Pi acts as an inhibitor of Jun N-terminal kinase (JNK). In response to UV light irradiation or H2O2 treatment, GSTp oligomerizes and dissociates from the GSTp-JNK complex (Adler et al., 1999). Whether such a GST-dependent activation mechanism in response to stress is also relevant in Arabidopsis remains to be investigated. Also, it is unclear whether the P450-GSTU4 interaction is specific for the camalexin biosynthetic machinery or might play a more general role. In conclusion, CYP79B2, CYP71A12/A13, CYP71B15, and ATR1 form a metabolic complex (Figure 10). FRET-FLIM indicated that, in addition, GGP1 can be recruited to this complex. Based on the data from our untargeted co-IP screens, ATR1 and CYP79B2 are likely less tightly associated with the core camalexin biosynthetic complex. This notion is in accordance with their different biological functions. ATR1 is required for many different biosynthetic processes in Arabidopsis. CYP79B2 is also involved in the biosynthesis of indole glucosinolates (Hull et al., 2000; Mikkelsen et al., 2000), the biosynthesis of auxin under specific conditions (Brumos et al., 2014; Tivendale et al., 2014), and the remodeling of root architecture (Julkowska et al., 2017). A possible interaction of CYP79B2 with CYP83B1, which is involved in indole glucosinolate biosynthesis and competes with CYP71A12/A13 for IAOx, was not detected in previous co-IP or split-ubiquitin-based yeast two-hybrid screens (Nintemann et al., 2017), perhaps due to rather weak or temporary protein-protein binding. CYP79B2, a key branch-point enzyme that is recruited for various processes, might modify the activities of downstream enzymes. In yeast microsomes expressing CYP71A13 in addition to CYP79B2, the apparent binding constant for the substrate Trp was significantly reduced, indicating an allosteric interaction and possibly substrate channeling. A similar effect was observed for the entry enzymes of flavonoid biosynthesis (Crosby et al., 2011). For other P450 enzymes of the pathway, such an effect was not observed. However, these enzymes might require Arabidopsis components not present in the heterologous system. Substrate turnover numbers were not determined, as it is typically not possible to purify active membrane-bound P450s to homogeneity (Cobbett et al., 1998). Therefore, the amount of mutual activation of catalytic activities might be underestimated, and we hypothesize that the camalexin biosynthetic enzymes cooperatively interact to allow high flux to the end product. Figure 10. Open in new tabDownload slide Model of a Camalexin Biosynthetic Metabolon. CYP79B2, CYP71A12/A13, CYP71B15, and ATR1 form a metabolic complex at the ER surface. CYP71B15 interacts with CYP79B2, CYP71A13, and CYP71A12. CYP79B2 is rather loosely associated with the complex and might function as a branch-point enzyme that takes part in different protein complexes. Under stress conditions, the cytosolic component GSTU4 might be recruited to the complex. Its role in camalexin biosynthesis remains unsettled. Figure 10. Open in new tabDownload slide Model of a Camalexin Biosynthetic Metabolon. CYP79B2, CYP71A12/A13, CYP71B15, and ATR1 form a metabolic complex at the ER surface. CYP71B15 interacts with CYP79B2, CYP71A13, and CYP71A12. CYP79B2 is rather loosely associated with the complex and might function as a branch-point enzyme that takes part in different protein complexes. Under stress conditions, the cytosolic component GSTU4 might be recruited to the complex. Its role in camalexin biosynthesis remains unsettled. METHODS Plant Growth Conditions and Stress Treatment After stratification for 2 d, Arabidopsis (Arabidopsis thaliana) and Nicotiana benthamiana plants were grown in a growth chamber under long-day conditions (160 µmol m−2 s−1, white full-spectra LED modules, 16 h of light, 8 h of dark) at 21°C and 50% relative humidity. To induce phytoalexin biosynthesis Arabidopsis 6-week-old rosette leaves were sprayed with 5 mM AgNO3, treated with UV-C light for 2 h (Desaga UVVIS; λ= 254 nm, 8 W, distance of 20 cm), or infected with Botrytis cinerea spores (strain B05.10, 2 × 105 spores mL−1). Camalexin was extracted after 24 h (UV-C and AgNO3 treatment) or 48 h (B. cinerea infection). Constructs for the Expression of Fusion Proteins To generate CYP71B15-GFP under the control of the endogenous promoter, the CYP71B15 promoter (Schuhegger et al., 2006; Chapman et al., 2016) was cloned into pBSK, and the CYP71B15 coding sequence was introduced into this plasmid via NcoI/SmaI digestion. The total insert was cut out by EcoRI/SmaI and introduced into pEZS-NL (Carnegie Institution). The promoter-coding sequence-GFP sequence was cut out with EcoRI/XbaI and introduced into the EcoRI/XbaI pGPTV-BarB vector fragment (Becker et al., 1992). Constructs for YFP-, GFP-, RFP-, and FLAG-tagged proteins were created via the Gateway cloning system (Invitrogen; Karimi et al., 2005; Katzen, 2007). Genes were amplified from Arabidopsis cDNA with specific primers (Supplemental Table 2) and cloned into pDONR223. Plasmids were confirmed by sequencing. The LR reaction was performed, and the constructs were transferred into destination vectors containing the 35S promoter and a tag (YFP: pEarlyGate101, GFP: pB7FWG2, RFP: pB7RWG2, FLAG: pEarlyGate202). Generation of Transgenic Arabidopsis Lines Arabidopsis accession Columbia was transformed with Agrobacterium tumefaciens harboring the CYP71B15 pro:CYP71B15-GFP expression construct via the floral dip method (Clough and Bent, 1998). Phosphinothricin-resistant primary transformants were confirmed by PCR and qualitatively screened for GFP fluorescence in response to AgNO3 spraying. A high-expression line was crossed to the cyp71b15/pad3 T-DNA insertion line SALK_026585 (Xu et al., 2008; Lemarié et al., 2015), and homozygous pad3/CYP71B15 pro:CYP71B15-GFP plants that carried the construct and (at least partially) complemented the camalexin-deficient pad3 phenotype were selected from the F2 generation (Supplemental Figure 1B). The progeny of one individual was used for proteomics analysis. For constitutive expression of CYP71A13-YFP, a corresponding pEarleyGate101 construct was used. Replicates represent independent microsome preparations from independent plants. Analysis of CYP71B15-GFP Localization in Response to Pathogens B. cinerea strain B05.10 was cultivated on potato dextrose agar under UV light (12 h of dark, 12 h of light) at room temperature. The preparation of B. cinerea spore suspension and the inoculation procedure were as described by Gronover et al. (2001) using 10-μL droplets of a suspension of 8 × 105 conidia mL−1 on fully developed Arabidopsis leaves. Alternaria brassicicola was grown on synthetic nutrient-poor agar (Nirenberg, 1981) under UV light. Fully developed Arabidopsis leaves were inoculated with 10-μL droplets of a suspension of 5 × 104 spores mL−1 water/0.02% (v/v) Tween 20. Plants infected with B. cinerea or A. brassicicola were cultivated under normal growth conditions in a closed box to retain high humidity levels. For infection with Erysiphe cruciferarum, Arabidopsis plants were placed under an inoculation box covered with a polyamide net (0.2 mm2) and inoculated at a density of 3 to 5 conidia mm−2 by brushing conidia off of powdery mildew-infected plants. E. cruciferarum membranes were stained with 20 μM SynaptoRed C2 (also known as FM-464; Sigma-Aldrich) for 15 min in the dark. Images were taken with a confocal laser-scanning microscope (Leica SP5). GFP was excited with a 488-nm laser line and detected between 500 and 530 nm, and SynaptoRed was excited at 561 nm and detected between 600 and 645 nm. Transient Expression in N. benthamiana For transient protein expression in N. benthamiana, expression plasmids were transformed into A. tumefaciens GV3101(MP90). Correct transformants were confirmed by PCR specific for the transgene. Overnight cultures of 25 mL were centrifuged and resuspended in 10 mM MES, 10 mM MgCl2, and 150 µM acetosyringone, pH 5.6, at an OD600 of 0.5 to 0.6. The cells were then incubated in a shaker for 2 h (room temperature), and A. tumefaciens cultures expressing the possibly interacting proteins and the supporting strain p19 were mixed at a ratio of 1:1:1 (Sparkes et al., 2006). For each sample, four to six N. benthamiana leaves were infiltrated onto the abaxial side of the leaves with a 1-mL syringe. After infiltration and before harvesting the infiltrated leaves, the plants were incubated for 3 d in a growth chamber under long-day conditions (160 µmol m−2 s−1, 16 h of light, 8 h of dark) at 21°C. Plant Microsome Generation and Co-IP Infiltrated leaves were harvested and ground with a mix of sea sand and Polyklar AT (Serva; 1:1 ratio) and 5 mL of ice-cold buffer 1 (100 mM ascorbic acid, 50 mM Na2SO4, 250 mM Tricine, 2 mM EDTA, 2 mM DTE, and 5 g/L BSA, pH 8.2). A total of 20 mL of buffer 1 was added, and the homogenate was centrifuged (20,000g, 4°C, 10 min). The supernatant was filtered through a gauze bandage and centrifuged again. Microsomes were pelleted by centrifugation (60,000g, 4°C, 2 h) and resuspended in 1.5 mL of buffer 2 (50 mM NaCl, 100 mM Tricine, 250 mM sucrose, 2 mM EDTA, and 2 mM DTE, pH 8.2). For solubilization, 500 μL of microsomes was mixed with Triton X-100 to a final concentration of 0.5% (v/v). The samples were incubated at 4°C for 1 h under constant shaking and centrifuged (20,000g, 1.5 h, 4°C). The supernatant was transferred to a new Eppendorf tube, and protein concentration was determined photometrically (Bio-Rad Protein Assay). For untargeted co-IP, GFP-Trap A beads (Chromotek; Rothbauer et al., 2008) were equilibrated with co-IP buffer (10 mM Tris, 150 mM NaCl, and 0.5 mM EDTA) and mixed with 1 volume of microsomes solubilized in 1% (v/v) Triton X-100 (100 μL of beads in a total volume of 4 mL for bait expressed under the control of the endogenous promoter and 50 μL of beads per 2 mL for bait expressed under the control of the 35S promoter). After incubation for 1 h at 4°C under constant shaking, the beads were centrifuged (2700g, 4°C, 2 min) and washed three times with co-IP buffer. The supernatant was replaced by 30 μL of NuPAGE LDS Sample Buffer (4×; Invitrogen) together with 30 μL of 100 mM DTT and incubated at 70°C for 15 min. Targeted co-IP was performed with 10 μL of GFP-Trap A beads each, in a total volume of 500 µL. The samples were analyzed via protein gel blot analysis using anti-FLAG (Sigma-Aldrich, F1804) and anti-GFP (Invitrogen, A-11122) antibodies followed by staining with goat anti-mouse HRP (Bio-Rad, 172-1011) or goat anti-rabbit HRP (Life Technologies, 65-6120), respectively (dilution of all antibodies, 1:3000). Replicates represent independent microsome preparations from independent plants. Protein Identification by Liquid Chromatography and Tandem Mass Spectrometry (LC-MS/MS) In-Gel Digestion Protein samples were reduced by 10 mM DTT and alkylated by 55 mM iodoacetamide (CYP71B15 data set) or 55 mM chloroacetamide (CYP71A13 data set). The proteins were run on a 4–12% NuPAGE gel to ∼1 cm to concentrate the sample prior to in-gel tryptic digestion. In-gel trypsin digestion was performed according to standard procedures (Shevchenko et al., 2006). LC-MS/MS Analysis of CYP71B15 Peptides generated by in-gel trypsin digestion were analyzed via LC-MS/MS on a nanoLC‐Ultra 1D+ (Eksigent) coupled to an LTQ‐Orbitrap Elite mass spectrometer (ThermoFisher Scientific). The peptides were delivered to a trap column (Reprosil-Pur C18 ODS3 5 µm resin, Dr. Maisch; 20 mm × 75 μm, self-packed) at a flow rate of 5 μL/min in 100% [v/v] solvent A0 (0.1% [v/v] formic acid in HPLC-grade water). The peptides were then transferred to an analytical column (Reprosil-Gold C18 120, 3 μm, Dr. Maisch; 400 mm × 75 μm, self-packed) and separated using a 110-min gradient from 4% to 32% (v/v) solvent B (0.1% [v/v] formic acid and 5% [v/v] DMSO in acetonitrile) in A (0.1% [v/v] formic acid and 5% [v/v] DMSO in HPLC-grade water) at a flow rate of 300 nL/min. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS2 spectra. Up to 15 peptide precursors were subjected to fragmentation by higher energy collision-induced dissociation and analyzed in the Orbitrap. Dynamic exclusion was set to 20 s. LC-MS/MS Analysis of CYP71A13 Peptides generated by in-gel trypsin digestion were analyzed via LC-MS/MS on a nanoLC-Ultra 1D+ (Eksigent) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). The peptides were delivered to a trap column (75 µm × 2 cm, packed in house with Reprosil-Pur C18 ODS3 5 µm resin, Dr. Maisch) for 10 min at a flow rate of 5 µL/min in 100% (v/v) solvent A0 (0.1% [v/v] formic acid in HPLC-grade water). The peptides were then separated on an analytical column (75 µm × 40 cm, packed in-house with Reprosil-Gold C18 120, 3 µm resin, Dr. Maisch) using a 120-min gradient ranging from 4 to 32% (v/v) solvent B (0.1% [v/v] formic acid and 5% [v/v] DMSO in acetonitrile) in A (0.1% [v/v] formic acid and 5% [v/v] DMSO in HPLC-grade water) at a flow rate of 300 nL/min. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS2 spectra. Up to 20 peptide precursors were subjected to fragmentation by higher energy collision-induced dissociation and analyzed in the Orbitrap. Dynamic exclusion was set to 20 s. Identification and Quantification of Peptides and Proteins Label-free quantification was performed using MaxQuant (version 1.6.1.0; Cox and Mann, 2008) by searching MS data against an Arabidopsis reference database derived from UniProt (version 09.07.2016, 31,424 entries) using the embedded search engine Andromeda (Cox et al., 2011). Carbamidomethylated Cys was used as the fixed modification; variable modifications included oxidation of Met and N-terminal protein acetylation. Trypsin/P was specified as proteolytic enzyme with up to two allowed missed cleavage sites. Precursor tolerance was set to 10 ppm, and fragment ion tolerance was set to 20 ppm. LFQ (Cox et al., 2014), match-between-runs, and intensity-based absolute quantification options were enabled, and the results were filtered for a minimal length of seven amino acids, 1% false discovery rate for peptides and proteins, as well as common contaminants and reverse identifications. Data Analysis and Visualization MaxQuant results were imported into the MaxQuant associated software suite Perseus (v.1.5.8.5; Tyanova and Cox, 2018). LFQs were filtered for at least three valid values for at least one experimental group and at least three peptides for identification per protein. Missing values were imputed from normal distribution (width 0.2, downshift 2.0). A two-sided unpaired Student’s t test was performed to assess statistical significance. Protein P values were corrected for multiple testing using a permutation-based 1% false discovery rate cutoff (1000 permutations). Standard functions in the SAM R package were used to adjust s0 for each data set (Tusher et al., 2001). For the CYP71B15 scatterplots, proteins were filtered for at least two valid values for at least one experimental group and at least three peptides for identification per protein. Means were calculated, and missing values were imputed by a constant (constant: 0). Yeast Transformation, Protein Expression, Yeast Microsomes, Enzyme Analysis, and Yeast Feeding Experiments Yeast (Saccharomyces cerevisiae) strain BY4741 (Brachmann et al., 1998), an auxotroph for His, Leu, Met, and Ura, was used for coexpression of ATR1 (on plasmid pGREG505), CYP79B2 (on plasmid pYeDP60), and CYP71A13 (on plasmid pSH62) or the corresponding vector control. Transformations were performed according to Gietz et al. (1992). Yeast cells were cultivated and microsomes were prepared essentially as described (Schuhegger et al., 2006), with the modification that instead of SGIW medium, the selection medium SD was used (Amberg et al., 2005). Microsomes were resuspended in TEG buffer (50 mM Tris, pH 7.5, 1 mM EDTA, 20% glycerol, and 2 mM DTE) and incubated with 0.5 mM NADPH and 5 to 200 µM Trp for 45 to 90 min. To stop the reaction, 2 volumes of 100% methanol was added, and the reaction mix was centrifuged twice to remove macroscopic contaminants. The conversion of Trp to IAOx was monitored by reverse-phase HPLC (Lichrosphere 100 RP-18, 250 × 3 mm, 5 µM, Merck; flow rate of 0.6 mL/min; solvents, 0.3% [v/v] formic acid in water [A] and acetonitrile [B]; gradient: 0 to 2 min, isocratic, 25% B; 2 to 10.5 min, linear from 25 to 45% B; 10.5 to 13 min, linear from 45 to 100% B; 13 to 15 min, isocratic, 100% B) and quantified based on a calibration curve of the authentic standard (Glawischnig et al., 2004). K m values were determined via GraphPad PrismGraph (Michaelis-Menten analysis). For feeding experiments, yeast cells carrying ATR1 (on plasmid pGREG505), CYP71A13 (on plasmid pYeDP60), and one of the 54 GSTs (on plasmid pSH62) were grown in SD medium with the appropriate supplements (-His, -Leu, -Met, -Ura) until OD600 = 0.6 was reached. Protein expression was induced by the addition of galactose for 16 h. Feeding was performed with 0.1 mM IAN and 0.2 mM GSH for 24 h. Subsequently, the yeast cells were harvested, washed in double distilled water, and combined with 350 μL of methanol:formic acid (99.8:0.2%, v/v). After vortexing and incubation at room temperature for 15 min under constant shaking, the cell debris was removed and GS-IAN formation was analyzed via HPLC (Lichrosphere 100 RP-18, 250 × 3 mm, 5 µM, Merck; flow rate of 0.6 mL/min; solvents, 0.3% [v/v] formic acid in water [A] and acetonitrile [B]; gradient: 0 to 2 min, isocratic, 25% B; 2 to 19 min, linear from 25 to 50% B; 19 to 24 min, linear from 50 to 100% B; 24 to 26 min, isocratic, 100% B), calibrating with the authentic standard. Confocal Microscopy and FRET-FLIM Analysis For the colocalization experiments, leaf epidermal samples were imaged using a Zeiss PlanApo ×100/1.46 NA oil-immersion objective on a LSM880 confocal microscope (Zeiss) equipped with an Airyscan detector. A total of 512 × 512 images were collected in eight-bit with two-line averaging at an (x,y) pixel spacing of 20 to 80 nm with excitation at 488 nm (GFP) and 561 nm (RFP) and emission at 495 to 550 nm and 570 to 615 nm, respectively. Data were produced from at least three independent biological replicates, defined as separate plants independently infiltrated from glycerol stocks. At least 20 cells per combination were imaged in a randomized manner. FRET-FLIM analysis was performed according to Kriechbaumer et al. (2015). In brief, epidermal samples of infiltrated leaves were excised, and multiphoton FRET-FLIM data capture was performed by a two-photon microscope built around a Nikon TE2000-U inverted microscope with a modified Nikon EC2 confocal scanning system. Laser light at a wavelength of 920 nm was produced by a mode-locked titanium sapphire laser (Mira; Coherent Lasers), with 200-fs pulses at 76 MHz, pumped by a solid-state continuous wave 532-nm laser (Verdi V18; Coherent Laser). The laser beam was focused to a diffraction-limited spot using a water-immersion objective (Nikon VC; 360, numerical aperture of 1.2). Fluorescence emission was collected bypassing the scanning system and passed through a BG39 (Comar) filter to block the near-infrared laser light. Line, frame, and pixel clock signals were generated and synchronized with an external detector in the form of a fast microchannel plate photomultiplier tube (Hamamatsu, R3809U). Linking these signals via a time-correlated single-photon-counting PC module SPC830 (Becker and Hickl) generated the raw FLIM data. Prior to FLIM data collection, the GFP and monomeric RFP expression levels in the plant samples within the region of interest were confirmed using a Nikon EC2 confocal microscope with excitation at 488 and 543 nm, respectively. A 633-nm interference filter is used to minimize the contaminating effect of chlorophyll autofluorescence emission. Data were analyzed by obtaining excited-state lifetime values on a pixel-by-pixel basis, followed by values from a region of interest on the nuclear envelope, and calculations were made using SPCImage analysis software version 5.1. The distribution of lifetime values within the region of interest was generated and displayed as a curve. Only values with a χ2 between 0.9 and 1.4 were considered. The median lifetime and minimum and maximum values for one-quarter of the median lifetime values from the curve were taken to generate the range of lifetimes per sample. Data from a minimum of three independent biological replicates and at least five nuclei per replicate and per protein-protein combination were analyzed, and the average of the ranges was taken. Biological replicates were defined as separate plants independently infiltrated and analyzed. Camalexin Extraction Camalexin extraction was performed according to Müller et al. (2015). In brief, leaves were weighed and 400 μL of methanol:water (80:20, v/v) was added to the samples. After incubation for 1 h at 65°C under constant shaking, extracts were cleaned twice via centrifugation and analyzed by reverse-phase HPLC (MultoHigh 100 RP18, 5-mm particle size; Göhler Analytik). Accession Numbers Sequence data from this article can be found in the GenBank/EMBL libraries under the following accession numbers: CYP71B15, At3g26830; CYP71A13, At2g30770; CYP71A12, At2g30750; CYP79B2, At4g39950; ATR1, At4g24520; GGP1, At4g30530 ; GSTU2, At2g29480; GSTU4, At2g29460. Data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaíno et al., 2013) with the data set identifier PXD008812. Supplemental Data Supplemental Figure 1. CYP71B15 pro:CYP71B15-GFP is functional in the pad3 background. Supplemental Figure 2. Proteins co-purified with CYP71B15-GFP; log2 LFQ intensities plotted against log2 fold change. Supplemental Figure 3. Evaluation of unspecific complex formation of camalexin-specific enzymes in the co-IP experiments. Supplemental Figure 4. Localization of CYP71A12, CYP71A13, CYP71B15, GSTU2 and GSTU4 in N. benthamiana leaves. Supplemental Figure 5. Specificity of GST enzymes for conjugation of glutathione to IAN. Supplemental Figure 6. Cytosolic localization of GSTU2 and U4 compared with CYP71A13. Supplemental Figure 7. Camalexin formation in gstu2 and gstu4 knockout and GSTU4 overexpression lines under different stress conditions. Supplemental Table 1. Coexpression of GSTU4 with tryptophan and camalexin-related genes under B. cinerea stress. Supplemental Table 2. List of primers used for cloning. Supplemental Data Set 1. Mass-spectrometric measurement of co-IP experiments with CYP71B15 as bait protein. Supplemental Data Set 2. Mass-spectrometric measurement of co-IP experiments with CYP71A13 as bait protein. Supplemental File 1. ANOVA tables. DIVE Curated Terms The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper: ATR1 Gramene: AT4G24520 ATR1 Araport: AT4G24520 ATR2 Gramene: AT4G30210 ATR2 Araport: AT4G30210 GSTF7 Gramene: AT1G02920 GSTF7 Araport: AT1G02920 CYP71A12 Gramene: AT2G30750 CYP71A12 Araport: AT2G30750 CYP71A13 Gramene: AT2G30770 CYP71A13 Araport: AT2G30770 pad3 Gramene: AT3G26830 pad3 Araport: AT3G26830 CYP79B2 Gramene: AT4G39950 CYP79B2 Araport: AT4G39950 camalexin CHEBI: CHEBI:22990 ACKNOWLEDGMENTS We thank Alfons Gierl for hosting the Glawischnig lab during the early phase of the project, Thomas Rauhut for generating the CYP71B15-GFP construct, Vasko Veljanovski for generating the CYP79B2 and GGP1 expression constructs, Alexandra Chapman and Verena Stork for supporting co-IP establishment, Marion Lechner for the generation of yeast strains, Aaron Klepper for providing homozygous gstu4 T-DNA lines, Ramón Torres Ruiz (CALM) for supporting FRET-FLIM instrument handling, and Danièle Werck for providing the CYP73A5-YFP construct as a negative control. This work was supported by the Deutsche Forschungsgemeinschaft (DFG Heisenberg Fellowship grant GL346/5 to E.Gl. and GL346/8), the Hans-Fischer-Gesellschaft e.V., the TUM Junior Fellow Fund, and a Science and Technology Facilities Council Program (FRET-FLIM grant 14230008). AUTHOR CONTRIBUTIONS S.M. designed and conducted the majority of experiments; S.H. performed proteomics analysis under the guidance of B.K.; V.K. performed FRET-FLIM and colocalization studies; B.S. and E.Gl. performed P450 expression in yeast; C.K. and M.C. generated lines for untargeted co-IP; M.C. and E.Gl. performed untargeted co-IP; N.K., under the guidance of E.Gr., generated yeast strains expressing GSTs; R.E. performed confocal microscopic analysis of pathogen-infected material under the guidance of R.H.; E.Gl. designed and supervised the overall project; E.Gl. and S.M. wrote the article with contributions of all authors. REFERENCES 1. 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Plant Cell 11 : 2419 – 2428 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes 1 Current address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, United Kingdom. 2 Current address: School of Life Sciences, University of Warwick, Gibbet Hill Campus, Coventry CV4 7AL, United Kingdom. www.plantcell.org/cgi/doi/10.1105/tpc.19.00403 The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Erich Glawischnig (glawischnig@tum.de). © 2019 American Society of Plant Biologists. All rights reserved. © The Author(s) 2019. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
TI - The Formation of a Camalexin Biosynthetic Metabolon
JF - The Plant Cell
DO - 10.1105/tpc.19.00403
DA - 2019-11-14
UR - https://www.deepdyve.com/lp/oxford-university-press/the-formation-of-a-camalexin-biosynthetic-metabolon-mK2U6vptOw
SP - 2697
EP - 2710
VL - 31
IS - 11
DP - DeepDyve
ER -