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O-acetylserine (thiol) lyases (OASTLs) are evolutionarily conserved proteins among many prokaryotes and eukaryotes that perform sulfur acquisition and synthesis of cysteine. A mutation in the cytosolic OASTL-A1 protein ONSET OF LEAF DEATH3 (OLD3) was previously shown to reduce the OASTL activity of the old3-1 protein in vitro and cause auto-necrosis in specific Arabidopsis accessions. Here we investigated why a mutation in this protein causes auto-necrosis in some but not other accessions. The auto-necrosis was found to depend on Recognition of Peronospora Parasitica 1 (RPP1)-like disease resistance R gene(s) from an evolutionarily divergent R gene cluster that is present in Ler-0 but not the reference accession Col-0. RPP1-like gene(s) show a negative epistatic interaction with the old3-1 mutation that is not linked to reduced cysteine biosynthesis. Metabolic profiling and transcriptional analysis further indicate that an effector triggered-like immune response and metabolic disorder are associated with auto-necrosis in old3-1 mutants, probably activated by an RPP1-like gene. However, the old3-1 protein in itself results in largely neutral changes in primary plant metabolism, stress defence and immune responses. Finally, we showed that lack of a functional OASTL-A1 results in enhanced disease susceptibility against infection with virulent and non-virulent Pseudomonas syringae pv. tomato DC3000 strains. These results reveal an interaction between the cytosolic OASTL and components of plant immunity.
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O-acetylserine (thiol) lyase (OASTL; EC 184.108.40.206) or cysteine synthase is involved in two related functions across prokaryotes and eukaryotes: assimilation of inorganic sulfur and synthesis of the amino acid cysteine. In plants, the first step in cysteine synthesis is production of O-acetylserine (OAS) from serine and acetyl CoA via a hetero-oligomeric complex consisting of inactive OASTL and active serine acetyltransferase (SERAT) enzymes (Wirtz and Hell, 2006). OAS and reduced sulfide are then processed into cysteine by an active OASTL dimer. OASTL activity depends on the pyridoxal-phosphate binding site (PLP), which performs β-substitution of OAS by sulfide to form cysteine (Bonner et al., 2005).
In the model plant Arabidopsis thaliana, nine OASTL-like genes were identified, which represent the β-substituting Ala synthase (BSAS) gene family (Arabidopsis Genome Initiative, 2000; Hatzfeld et al., 2000). However, only three BSAS isoforms contribute to the majority of total cellular cysteine synthesis (Heeg et al., 2008; Watanabe et al., 2008). These are the cytosolic (OASTL-A1), plastidic (OASTL-B) and mitochondrial (OASTL-C) OASTLs. The other OASTLs either contribute very low OASTL activity or have acquired different functions during evolution. Examples of these functional divergences are OASTL-like β-cyanoalanine synthases (CAS), which are involved in cyanide detoxification, l-Cys desulfhydrases (DES), which degrades cysteine, and S-sulfocysteine synthases (CS26), which are involved in synthesis of sulfocysteine (Hatzfeld et al., 2000; Álvarez et al., 2010; Bermúdez et al., 2010). Lack of either of the major isoforms, OASTL-A1 or OASTL-B, has been shown to be largely neutral for growth and development under non-stressed growth conditions (Heeg et al., 2008; Watanabe et al., 2008). However, plants with mutations in two other BSAS genes, DES and CS26, displayed early senescence and cell death (Álvarez et al., 2010; Bermúdez et al., 2010). This suggests functional redundancy in cysteine biosynthesis and an intricate link between cysteine homeostasis and stress responses.
We have previously identified an EMS-induced single nucleotide substitution in the cytosolic OASTL-A1 gene in the Ler-0 accession of Arabidopsis (Shirzadian-Khorramabad et al., 2010). The mutation causes early senescence and death in plants, and is referred to as onset of leaf death3 (old3-1). The old3-1 mutation results in a glycine to glutamic acid substitution at residue 162 of OASTL-A1/OLD3 that abolishes the OASTL activity of the enzyme in vitro (Shirzadian-Khorramabad et al., 2010). The old3-1 mutation is co-dominant, and plants heterozygous for the old3-1 mutation (old3-1 OLD3) grow and produce viable seeds but show premature leaf yellowing. The old3-1-associated auto-necrosis phenotype is observed in the genetic background of its parent accession Ler-0, but not in the reference accession Col-0. Thus, the old3-1 phenotype in the Ler-0 accession depends on a natural variant region that was designated odd-ler (old3 determinant locus of the Ler accession) (Shirzadian-Khorramabad et al., 2010). Here we investigated the effects of mutations in the OASTL-A1 gene in two Arabidopsis accessions. Our results show that mutations in O-acetylserine (thiol) lyase regulate innate immune responses and disease susceptibility.
Auto-necrosis in the old3-1 mutant is modulated by day length
The old3-1 mutation causes temperature-dependent auto-necrosis when grown at 21°C (non-permissive temperature), and this phenotype is rescued when grown at the permissive temperature of 28°C (Shirzadian-Khorramabad et al., 2010). We found that auto-necrosis was reduced when the old3-1 mutant was grown at low temperature under short-day conditions (Figure 1). Thus both temperature and day length modulate the onset of leaf death and necrosis in the old3-1 mutant.
RPP1 gene(s) from an evolutionarily divergent disease resistance R gene cluster show negative epistasis to the old3-1 mutation
The old3-1 mutation causes auto-necrosis in combination with the odd-ler allele, which is present in the Ler-0 background (Shirzadian-Khorramabad et al., 2010). To identify the nature of the odd-ler allele, the odd-ler region was mapped using approximately 5000 F2 seedlings with an auto-necrosis phenotype from a cross between the old3-1 mutant and Col-0. The odd-ler allele was fine-mapped using SNP-based markers to an approximately 52 kb genomic region on chromosome 3, flanking the At3g44600 and At3g44630 genes in the Col-0 genome (Figure S1). In Col-0, this region is referred to here as odd-col and contains five annotated open reading frames, of which two are annotated as RECOGNITION OF PERONOSPORA PARASITICA1 (RPP1)-like disease resistance (R) genes. The odd-ler region falls within quantitative trait locus 3 (QTL3), which contains multiple polymorphisms within the genes present compared to the Col-0 sequence, including a cluster containing eight RPP1-like disease resistance R genes (Alcázar et al., 2009). The candidate genes At3g44610 (serine/threonine kinase), At3g44620 (tyrosine phosphatase) and the RPP1-like R gene family were therefore silenced in the old3-1 mutant using an RNAi approach. Transformation of the RNAi construct targeting At3g44610 or At3g44620 into the old3-1 mutant did not rescue the phenotype (Figure S2). However, targeting of the RPP1-like R genes rescued the mutant phenotype (Figures 2a and S2). Measurements of the relative gene expression of RPP1-like R alleles using quantitative RT-PCR showed that the rescued lines had decreased expression of the full-length RPP1-like genes R1, R2, R3, R4, R5, R7 and R8, whereas their expression was up-regulated in the old3-1 mutant, probably as part of the auto-necrosis response (Figure 2c). The rescued phenotype was furthermore found to be associated with reduced expression of defence associated PATHOGENESIS RELATED GENE 1 (PR-1) (Figures 2b and S2). These results suggest that negative epistasis between one or more than one RPP1-like R genes and the old3-1 mutation results in auto-necrosis.
The old3-1 mutation results in auto-necrosis in Arabidopsis accessions that are positive for an odd-ler like marker but not in accessions that are positive for an odd-col like marker (Table S1). This suggests that naturally variant disease resistance R gene(s) are involved in a similar interaction in these accessions. RPP1 genes contain Toll interleukin-1 resistance/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR; TNL) domains and have been identified as involved in disease resistance against oomycete pathogens (Botella et al., 1998). The genic antagonism between old3-1 and the R genes is the result of independent evolution of the odd locus in the Col-0 and Ler-0 accessions. Therefore, we determined the extent of recombination occurring within the RPP1-like R gene family (R1-R8) at the odd-ler region and the two RPP1-like R genes present in the odd-col region (At3g44630 and At3g44670) using progressiveMauve alignment software (Darling et al., 2010) (Figures 3 and S3). We found that the genetic sequence blocks encoding TNL domains in the eight RPP1-like R alleles in the odd-ler region show extensive recombination in LRR domains but not in NBS- and TIR-encoding genetic sequence blocks (Figures 3 and S3), in comparison with the two RPP1-like R genes in the odd-col region. Complex evolution of TNL domains in RPP1-like R genes may have occurred due to unequal crossing-over and gene conversions (McDowell and Simon, 2006). As more than one R gene may be involved in the negative epistasis, we attempted to identify the candidate R gene(s) at the odd-ler locus using an RNAi approach. Due to high homology between RPP1 genes, we selected the polymorphic putative 5 and 3′ untranslated regions (UTRs) flanking the predicted open reading frames of the R genes for the RNAi constructs. RNAi knockdown using a unique sequence present only in the putative 3′ end of R4, R7 and R8 consistently rescued the old3-1 phenotype. Suppression of R7 significantly correlated with the rescued phenotype compared to the two other targeted genes R4 and R8 (Figures 4 and S4). However, the transcript abundance of non-targeted full-length R genes was not significantly reduced in the rescued lines in comparison with the old3-1 mutant (Figure S5). The findings highlight an epistatic interaction between a mutation in a BSAS isoform and an R gene. RPP1-like R7 is therefore suggested as a candidate gene involved in auto-necrosis, but at present we cannot rule out the possibility that other R genes may also contribute to this genetic interaction.
The presence of the old3-1 mutation and not the lack of functional OASTL-A1 is required for auto-necrosis
The mutant old3-1 protein does not have detectable OASTL enzyme activity in vitro (Shirzadian-Khorramabad et al., 2010). Therefore, the auto-necrosis phenotype may be the result of reduced enzyme activity in combination with the RPP1-like R gene(s). To test for this possibility, we first silenced OASTL-A1/OLD3 in Ler-0 using an RNAi approach. We also generated recombinant OASTL-A1 functional knockout lines by crossing Ler-0 with the OASTL-A1 T-DNA knockout mutant old3-2, which is identical to the previously described lines oas.a1.1 and bsas1;1 (López-Martín et al., 2008; Watanabe et al., 2008). F2 seedlings homozygous for both old3-2 and odd-ler were selected, and are referred to here as old3-2 odd-ler. Neither silenced Ler-0 lines with up to 97% reduced OASTL-A1 transcript levels, nor old3-2 odd-ler plants displayed auto-necrosis (Figure 5a,c). This suggests that lack or reduced cytosolic OASTL activity does not result in development of auto-necrosis in combination with the R gene(s), and that the specific old3-1 mutation causes the auto-necrosis phenotype in combination with R gene(s). To test this, the old3-1 protein was suppressed in old3-1 mutants using an OASTL-A1-specific RNAi construct. The auto-necrosis phenotype was completely rescued in all transformants, consistent with the reduction in expression levels of the old3-1 gene (Figure 5b). Thus, the G/E substitution at residue 162 of OASTL-A1 results in activation of auto-necrosis in response to R gene(s) present in the odd-ler region.
Mutations in the OASTL-A1 gene cause a major reduction in total OASTL activity in planta, but not a reduction in total SERAT activity and OAS, cysteine and glutathione contents
We next aimed to understand the mechanism of the auto-necrosis and uncouple the effect of the old3-1 mutation on OASTL activity from that of its interaction with R protein(s). Five genotypes as described in Figure 6 were used, including the old3-1 mutant together with its wild-type parent Ler-0, the OASTL-A1 T-DNA knockout mutant old3-2, and its Col-0 wild-type parent. In addition, to analyse the effect of the old3-1 mutation in the absence of the haplotype RPP1-like R genes from the odd-ler region, which is present on chromosome 3, we used the previously described line old3-1 odd-col. This line has a mixed Col-0/Ler-0 genetic background but is homozygous for old3-1 and odd-col and therefore does not show an auto-necrosis phenotype (Shirzadian-Khorramabad et al., 2010). This line is referred to here as old3-1rec.
As high temperature suppresses the old3-1 auto-necrosis phenotype, all five genotypes were grown under long-day conditions on soil at 28°C for 16 days (Figures 6b and S6). One experimental set of plant lines was subsequently grown for an additional 5 days at 28°C, and another set was transferred to 20°C (non-permissive temperature for old3-1), after which only the old3-1 mutant exhibited chlorophyll loss and onset of auto-necrosis within 5 days of the temperature change (Figure S7). In contrast, the old3-2 and old3-1rec mutants were indistinguishable from wild-type plants at both temperatures (Figures 6b and S6). Measurements were obtained 0, 2 and 5 days into the temperature-shift experiment (Figure S6).
We first measured OASTL and SERAT enzyme activities in the plant lines. Depending on growth conditions, the old3-2 mutant showed a significant reduction of 40–80% in total OASTL activity in comparison with its wild-type Col-0 (Figure 6c). Similar to old3-2, the old3-1 and old3-1rec mutants also showed significantly reduced total OASTL activity of 40–60% in comparison with the wild-type Ler-0 and Col-0 plants (Figure 6c). The total SERAT activity was unaltered in old3-1 compared to Ler-0 at both growth temperatures. The total SERAT activity was unaltered in old3-2 compared to Col-0. However, a significant reduction was observed in the total SERAT activity in old3-2 plants only at day 2 of the temperature shift (Figure 6c). Thus the shift in growth temperature affects total SERAT activity in the Col-0 genetic background only.
OASTL-A1 interacts with the cytosolic protein SERAT1;1/SAT5 in vivo, as shown by the yeast two-hybrid system and co-immunoprecipitation studies (Bogdanova and Hell, 1997; Jost et al., 2000; Heeg et al., 2008; Wirtz et al., 2010). We tested the interaction between the old3-1 mutant protein and cytosolic SERAT1;1/SAT5 using the yeast two-hybrid system. The results show that, in contrast to wild-type OASTL-A1, there is a loss of interaction between the old3-1 mutant protein and SERAT1;1 (Figure S8). This suggest that the old3-1 mutant protein does not interact with the cytosolic SERAT1;1 protein in planta either. However, it remains possible that the mutant protein behaves differently in the plants compared to yeast.
Because OASTL-A1 possesses β-cyanoalanine synthase (CAS), which is involved in cyanide detoxification (Watanabe et al., 2008), as a side activity, we also tested CAS activity. A significant reduction in total CAS activity was observed in old3-2 and old3-1rec mutants, but not in the old3-1 mutant and the wild-types (Figure S9), suggesting that CAS activity is not affected by the old3-1 mutation per se. The reduced CAS activity in old3-1rec therefore appears to be an effect of the mixed Ler/Col-0 background. Thus, a comparable reduction in OASTL activity was seen in all old3 mutant lines, regardless of growth temperature and the presence of the auto-necrosis phenotype.
Next, the effects of reduced OASTL activity on sulfur assimilation were analysed in all five lines. The results show that total cellular sulfate, OAS, cysteine and glutathione (GSH) levels were not significantly reduced in the old3-2 and old3-1rec mutants in comparison with the wild-type Col-0 at either high or low temperatures (Figure 6d). However, a significant reduction in total GSH level was found only in the old3-2 mutant at 5 days after the temperature change, but not at 28°C. None of these metabolites were reduced in the old3-1 mutant at either temperature compared to Ler-0 (Figure 6d). However, the total cysteine, OAS and sulfate levels were increased in the old3-1 mutant within 2 days after a shift to low temperature. (Figure 6d). Thus cysteine and GSH levels are not reduced due to the old3-1 mutation, and the temperature-dependent increase in sulfate, OAS and cysteine levels is strictly associated with auto-necrosis. In conclusion, the results show that the old3-1 mutation results in reduced OASTL activity in vivo, and are consistent with the notion that reduced OASTL activity per se does not cause the auto-necrosis phenotype.
Auto-necrosis in the old3-1 mutant coincides with changes in metabolic pathways and activation of abiotic and biotic stress responses
Auto-necrosis in the old3-1 mutant coincided with accumulation of sulfate, OAS and cysteine. We next determined which other metabolic processes are also affected, to further understand the mechanism of auto-necrosis. For this purpose, all five genotypes were characterized by profiling over 70 metabolites at 0, 2 and 5 days in the temperature-shift experiment as described above. Hierarchical clustering analysis (HCA) was performed to monitor variation in biological replicates within each genotype under two temperature regimes (Figure S10). The data show that, at high temperature, changes in primary metabolic pathways such as the tricarboxylic acid cycle and glycolysis, and levels of anions and amino acids were largely similar in old3-2 and old3-1 mutants in comparison with their wild-types (Figure S11). As the old3-1rec mutant has a mixed genetic background, the metabolic view represents a mixed pattern of metabolic changes. However, after growth at low temperature, the old3-1 mutant specifically showed perturbations in primary metabolism, including higher levels of free sugars and amino acids (Figure 7). Starch levels remained unchanged, but a slight but significant reduction in total soluble protein was observed in the old3-1 mutant only (Figures 7 and S12). Various defence metabolites associated with abiotic stress, such as proline, succinic acid, maltose, pyroglutamic acid, putrescine, β-alanine, ascorbic acid and γ-aminobutyric acid (GABA), also accumulated in old3-1 mutant tissues only (Figure S11). The old3-1rec and old3-2 mutant and wild-type plants did not exhibit similar accumulation of amino acids and sugars and stress-associated metabolites, although temperature-dependent changes in the metabolic profile were observed (Figures 7 and S11). We also found that camalexin and 4-methoxy-indol-3-ylmethyl-glucosinolate, which are synthesized de novo upon pathogen infection (Bednarek et al., 2009; Clay et al., 2009), accumulated in the absence of pathogen infection specifically in the old3-1 mutant, but not in the old3-2 and old3-1rec mutants, (Figure 7). These changes coincided with an increase in the levels of salicylic acid, which is involved in disease resistance responses, in the old3-1 mutant only. These results support the view that lack of functional OASTL-A1 is largely neutral for stress metabolism under permissive growth conditions. The results also indicate that R-mediated autoimmunity induces accumulation of amino acids and sugars, and is associated with activation of pathways of specific secondary metabolites that are involved in basal immune responses and abiotic stress resistance.
A temperature-dependent immune response was shown to be associated with auto-necrosis activated by R genes (Bomblies et al., 2007). Therefore, we measured expression levels of hallmarks of the innate immune response in old3-1 mutants and the wild-type within 24 h of temperature shift. The transcript levels of ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), WRKY18, ISOCHORISMATE SYNTHASE 1 (ICS1) and PR-1 increased greatly in the old3-1 mutant only, within 8–16 h of the change in temperature (Figures 8a and S13). Microscopic observation of leaf tissues stained with trypan blue and diaminobenzidine furthermore showed an oxidative burst and cell death in old3-1 mutants (Figure 8b). In contrast, no increase in PR-1 expression was found in old3-1rec tissues, even 24 h after the temperature change, consistent with no indication of cell death or an oxidative burst in this mutant line (Figure 8c,d). In conclusion, the results indicate that growth of the old3-1 mutant at the non-permissive temperature leads to an innate immune response and metabolic disorder, ultimately leading to hypersensitive response-like cell death. This response depends on the R gene(s) present in the odd-ler region as no autoimmune metabolic changes were found in old3-1rec.
Mutation in OASTL-A1 interacts with disease resistance against the pathogen Pseudomonas syringae pv. tomato DC3000
The results suggest an intricate relationship between OASTL-A1 and immunity. We therefore assessed the disease resistance ability in plants lacking OASTL-A1 against the pathogen Pseudomonas syringae pv. tomato DC3000. Pst DC3000 causes effector-triggered susceptibility by releasing effector molecules that dampen PAMP-triggered immunity (PTI) (Katagiri et al., 2002). Arabidopsis furthermore lacks effector-triggered immunity (ETI) against Pst DC3000, rendering this pathogen virulent. Arabidopsis Ler-0 plants in which OASTL-A1 expression was suppressed using RNAi, and the old3-2 mutant, together with their respective wild-types, were infiltrated with Pst DC3000. Upon infection, mutants lacking functional OASTL-A1 in both genetic backgrounds showed an enhanced infection rate, concomitant with higher bacterial counts compared to their wild-types (Figures 9 and S14). Thus, OASTL-A1 contributes to resistance against a virulent pathogen in Arabidopsis. The results are consistent with recent findings showing that the oas.a1.1 mutant, which is identical to the old3-2 mutant, shows disease susceptibility as well as an impaired ETI response highlighting the role of OASTL-A1 in ETI (Álvarez et al., 2012). We also determined whether OASTL-A1 is involved in the PTI-driven resistance phenotype. For this purpose, we used the Pst DC3000 hrpA− strain for infections, which lacks the ability to deliver effector molecules inside plant cells to cause effector-triggered susceptibility. Therefore, Arabidopsis successfully triggers PTI against this strain without showing disease-induced chlorosis (Roine et al., 1997; Wei et al., 2000). Upon infiltration or spray inoculation, old3-2 mutants showed development of enhanced disease-induced chlorosis in response to Pst hrpA− compared to wild-type Col-0 plants (Figure S14). Here, we also included an old3-1 RNAi-RPP1 line, in which expression of the RPP1-like genes was suppressed. Both old3-2 and old3-1 RNAi-RPP1 plants showed enhanced chlorosis compared to Ler-0 (Figure S14). These results highlight the role of OASTL-A1 in the PTI-driven disease resistance response, and indicate that the function of cytosolic OASTL-A1 is indispensable in plant defence.
Cysteine metabolism in oastl-a1/old3 mutants
The major contribution of cytosolic OASTL-A1 to total OASTL activity, the high protein abundance in comparison with other isoforms (Wirtz et al., 2010), interaction with a sulfur transporter (Shibagaki and Grossman, 2010), and the evolutionary conservation among Arabidopsis accessions suggests a principal function for this protein in plant sulfur and cysteine metabolism. Indeed, OASTL-A1 in Arabidopsis was found to be essential in providing resistance to oxidative stress and metal and pollutant toxicity (Barroso et al., 1999; Domı́nguez-Solı́s et al., 2001; Noji and Saito, 2002; Heeg et al., 2008; López-Martín et al., 2008; Watanabe et al., 2008; Shirzadian-Khorramabad et al., 2010). This suggests a function for OASTL-A1 under stress conditions, possibly due to increased sequestration of cysteine in various pathways involved in abiotic stress responses.
The results show that mutations in cytosolic OASTL-A1 have largely neutral effects on the plant phenotype, cysteine levels and metabolites of stress defence pathways in Arabidopsis when grown under permissive growth conditions. Remarkably, although the mean OASTL activity decreased by ∼50% among old3 mutants, no significant decrease in cysteine levels was found. Similarly, anti-sense RNA-mediated suppression of OASTL genes in potato (Solanum tuberosum) caused reduced OASTL activity without a reduction in cysteine levels (Riemenschneider et al., 2005). Nevertheless, it was previously reported that Arabidopsis oastl-a1/old3-2 mutants had reductions in cysteine levels ranging from 15 to 28% (Heeg et al., 2008; Watanabe et al., 2008). The reduced cysteine levels were reported to coincide with the absence of a growth phenotype (Heeg et al., 2008; Watanabe et al., 2008), but others noted an approximately 34% reduction in cysteine levels coinciding with lesions resulting from increased oxidative stress (López-Martín et al., 2008). We attribute these differences to variations in growth media, day length and age. We have detected a significant reduction in GSH levels at day 5 after a temperature shift in the old3-2 mutant only, which indicates that altered growth conditions or age may affect thiol levels. Thus, although cytosolic cysteine levels may be affected in old3 mutants, the results are consistent with the suggestion that facilitated diffusion of cysteine between subcellular compartments contributes to maintenance of the total cysteine and GSH contents at the cellular level, at least under non-stressed growth conditions (Heeg et al., 2008; Watanabe et al., 2008; Krueger et al., 2009).
Upon activation of autoimmunity in old3-1 mutants, levels of amino acids, including cysteine, increased markedly, despite the reduced total OASTL activity. An increase in free sugars and amino acids during the immune response to pathogen infection has been reported across species (Scharte et al., 2005; Swarbrick et al., 2006; Dulermo et al., 2009), probably because amino acids are precursors of plant defence compounds (Halkier and Gershenzon, 2006; Bolton, 2009; Stuttmann et al., 2011). The increase in cysteine and other amino acids may also result from protein degradation as total protein content decreased during autoimmunity. However, the possibility cannot be excluded that cysteine is synthesized de novo as a result of remaining functional OASTL activity in plastids and mitochondria. Indeed, the oastl-ab mutant lacks functional OASTL-A1 and B but accumulates significantly higher cysteine levels, probably because of residual OASTL activity (Heeg et al., 2008). The oastl-ab mutant displays growth retardation, and the higher cysteine accumulation in this mutant may be part of a stress response. Furthermore, cysteine may accumulate during leaf senescence without an increase in GSH levels (Figure S15).
Our results also indicate that there is a loss of interaction between the old3-1 mutant protein and the cytosolic SERAT1;1 protein in yeast, and these enzymes may not interact in planta either. As SERAT activity depends on the interaction with OASTLs, cytosolic SERAT activity may be lower in old3 mutants. However, lack of functional OASTL-A1 did not affect total SERAT activity in the old3 mutant lines in different genetic backgrounds. This is consistent with a previous observation (Heeg et al., 2008), but an approximately 30% increase in SERAT activity was found by others (López-Martín et al., 2008). Unchanged total SERAT activity coincided with unaltered total OAS levels as reported here under permissive growth conditions and by Heeg et al. (2008). The observed higher OAS levels during the autoimmune response in the old3-1 mutant are probably the result of other functional SERAT-OASTL partners. Indeed, the oastl-ab mutant, but not the oastl-c mutant, has previously been shown to accumulate higher OAS levels (Heeg et al., 2008). This suggests that OAS levels may increase during stress given the presence of functional mitochondrial OASTL-C. This is consistent with the finding that mitochondria rather than the cytosol are the major site of OAS production (Heeg et al., 2008; Watanabe et al., 2008; Krueger et al., 2009). Higher OAS contents furthermore coincided with higher sulfate and cysteine levels in old3-1 mutants during autoimmunity.
Genetic interaction between OASTL-A1 and R genes and regulation of plant immunity
Here we showed that mutations in OASTL-A1 enhance disease susceptibility against a virulent pathogen. This is consistent with a recent finding which shows that the oas.a1.1 (old3-2) mutant exhibits an impaired ETI response (Álvarez et al., 2012). Further, our results suggest a role for OASTL-A1 in basal defence, as wild-type Arabidopsis successfully establishes PTI against Pst DC3000, hrpA− (Wei et al., 2000). Thus, recent evidence and our findings suggest that the cytosolic OASTL machinery is important in Arabidopsis immunity. The old3-2/oas.a1.1 mutant has an oxidized cellular redox state (López-Martín et al., 2008) and reduced total OASTL activity. Emerging evidence indicates that cytosolic redox status modulates Non-expresser of Pathogenesis Related 1 (NPR1)-mediated defence reprogramming in the nucleus, and cysteine-derived metabolites, including GSH, glucosinolates and camalexin, are involved in basal defence (Rausch and Wachter, 2005; Parisy et al., 2007; Tada et al., 2008; Bednarek et al., 2009; Clay et al., 2009). Thus the impaired disease resistance response in the old3-2/oas.a1.1 mutant may be linked to reduced compartment-specific levels of cysteine during biotic stress conditions (Álvarez et al., 2012). Hence OASTL-A1 may contribute to plant fitness during both abiotic and biotic stress. However, the old3-1 mutation in OASTL-A1 causes activation of plant defence and auto-necrosis in certain accessions. These findings are reminiscent of those for ACCELERATED CELL DEATH6 (ACD6), which is involved in plant fitness and disease resistance, but genetic variation at this locus activates immune responses and later onset of necrosis in specific Arabidopsis accessions (Todesco et al., 2010). Here we show that the old3-1 mutation in OASTL-A1 activates an innate immune response due to a genetic interaction with disease resistance RPP1-like R gene(s). Allelic variation in RPP1-like genes at the odd region in Arabidopsis is associated with auto-necrosis, and complex evolution of TNL domains has resulted in a pool of hybrid RPP1-like R genes that may be categorized as a type I evolving R gene cluster (Kuang et al., 2004). The rescue of auto-necrosis via RNAi-mediated suppression of OASTL-A1 or RPP1-like genes in the old3-1 mutant furthermore demonstrated that the R-mediated autoimmune response is caused by the mutation in OASTL-A1 in combination with the R protein(s) and is not the result of lower cysteine biosynthesis or ectopic free SERAT. The absence of noticeable differences between the old3-1rec and old3-2 metabolome (i.e. in the absence of the odd-ler region) shows that the old3-1 mutant protein itself does not cause major metabolic disturbances, and suggests that the old3-1 mutant protein is not involved in synthesis of compounds that activate immune responses. If the old3-1 mutant protein does have ectopic activity, an RPP1-like protein(s) is required for this activity to be detected.
Temperature has been shown a major environmental factor that affects R-mediated auto-necrosis (Yang and Hua, 2004). High temperature was found to be linked with suppression of expression of plant defence genes. Our results showed that, within hours after induction of auto-necrosis by changing the temperature, expression of defence-related markers and R genes increased. Thus high temperature-mediated rescue may be linked to suppression of R and other defence-related genes. A recent study revealed that temperature may modify conformational requirements in an NBS-LRR protein for downstream activation of the immune response (Zhu et al., 2010). Thus the effects of temperature on the immune response may be linked to post-translational changes in R proteins or their interaction with the old3-1 mutant protein. In addition to temperature, day length is also an important environmental factor that modulates immunity in plants (Roden and Ingle, 2009). Our results show that R-mediated auto-necrosis and dwarfism was far more suppressed under short-day conditions compared with long-day conditions. This may be due to dampening of the plant immune responses, as short-day conditions were shown to suppress constitutive accumulation of SA and camalexin in an Arabidopsis lesion-mimic mutant (Chaouch et al., 2010).
Interaction between R and other host protein-encoding genes
R proteins are involved in activation of innate immunity and a hypersensitive response known as ETI, in response to pathogen effector-induced virulence. ETI is activated by R proteins upon sensing modifications in specific host proteins or by direct recognition of foreign effector molecules (Krüger et al., 2002; Jones and Dangl, 2006; Bomblies et al., 2007). Using metabolic profiling and transcriptional analysis, we show here that an RPP1-like gene(s) activates an innate immune response in a temperature-dependent manner, leading to an oxidative burst and cell death in the old3-1 mutant. The autoimmune response is associated with other changes in primary and secondary metabolism that may be part of the R-triggered hypersensitive response and programmed cell death. Thus RPP1-like mediated autoimmunity due to the old3-1 mutant protein mimics an ETI-like response during pathogen virulence, suggesting that an RPP1-like protein(s) senses modified OASTL-A1, directly or indirectly. It was suggested that RPP1 and other R proteins guard against modifications in various evolutionarily conserved plant proteins that are crucial in growth and immunity (Dangl and Jones, 2001; Krüger et al., 2002; Jones and Dangl, 2006; Alcázar et al., 2010). Our findings indicate that an RPP1-like R gene-encoded protein probably interacts with or guards the metabolic OASTL-A1/OLD3 enzyme directly or indirectly, possibly against an unknown virulence effector activity. However, this requires further investigation. Pathogen effector proteins were previously shown to target Arabidopsis enzymes to either modify the function of the host enzyme or use the host enzyme as a protein partner in enhancing virulence (Bernoux et al., 2008; Zhou et al., 2011). Consistent with this, it was recently shown that the bacterial CysK protein O-acetylserine sulfhydrylase is targeted by a foreign bacterial effector protein (Diner et al., 2012). The foreign tRNase/CdiA protein forms a complex with host CysK to activate the foreign nuclease, resulting in growth inhibition of the targeted host. Thus, the bacterial OASTL is a target of a foreign tRNase protein to suppress host growth. However, evidence for a protein–protein interaction between OASTL-A1/OLD3 and pathogen effectors is still lacking. It is furthermore important to note that, from a genetic point of view, OASTL-A1 is not a negative regulator of R-mediated immunity, as knockdown or knockout of OASTL-A1 in the odd-ler background does not cause auto-necrosis. However, it may still be possible that RPP1-like protein(s) guard or interact with multiple cytoplasmic OASTLs, and the R protein(s) do not detect absence of a single OASTL, but rather an altered OASTL conformation resulting from a point mutation and/or effector-induced modification. In this scenario, OASTLs together may act as negative regulators of R-mediated innate immunity.
Our finding that a mutation in a BSAS isoform activates RPP1-mediated autoimmunity provides evidence for a genetic epistatic interaction between R genes of the plant innate immune system and sulfur/cysteine metabolism. Recently, mutations in the BSAS-encoding genes DES and CS26 were shown to cause auto-activation of immune and biotic stress responses (Álvarez et al., 2010, 2012; Bermúdez et al., 2010). Therefore, it is tempting to speculate that these BSAS isoforms may interact with components of plant immunity as well. This is consistent with the emerging role of cysteine metabolism in plant immunity.
Details of the materials and methods are given in Data S1. This includes information on all plant lines used in the study, growth conditions in various experiments, the methodology for metabolic profiling, enzymatic activities, transcriptional analysis, gene mapping, pathogen infections, histochemical staining, in silico characterization of RPP1 gene sequences, preparation of RNAi constructs, vectors, transgenic Arabidopsis and Agrobacterium, and the yeast two-hybrid study. A metabolite reporting checklist and standards are given in Table S2. Primers used in this study are listed in Table S3.
We would like to acknowledge Higher Education Commission, Pakistan, for funding J.T. and the Massey University Research Fund for support of P.P.D. We would also like to thank the Max Planck Society for financial support. We are grateful to Xiao Song and Jay Jayaraman from Institute of Molecular BioSciences, Massey University, New Zealand) for help with the yeast two-hybrid experiment and pathogen assays.