Molecular Plant Advance Access originally published online on April 15, 2008
Molecular Plant 2008 1(3):510-527; doi:10.1093/mp/ssn011
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A Lesion-Mimic Syntaxin Double Mutant in Arabidopsis Reveals Novel Complexity of Pathogen Defense Signaling
a Plant and Soil Science, Dept of Agricultural Sciences, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
b Dept of Plant Biology, Carnegie Institution, Stanford, CA 94305, USA
c Plant Pathology, Dept of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
d Research Laboratory, Carlsberg Research Center, Gl. Carlsbergvej 10, DK-2500 Valby, Copenhagen, Denmark
1 To whom correspondence should be addressed. E-mail htc{at}life.ku.dk, fax +45 35333460, tel +45 35333443.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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The lesion-mimic Arabidopsis mutant, syp121 syp122, constitutively expresses the salicylic acid (SA) signaling pathway and has low penetration resistance to powdery mildew fungi. Genetic analyses of the lesion-mimic phenotype have expanded our understanding of programmed cell death (PCD) in plants. Inactivation of SA signaling genes in syp121 syp122 only partially rescues the lesion-mimic phenotype, indicating that additional defenses contribute to the PCD. Whole genome transcriptome analysis confirmed that SA-induced transcripts, as well as numerous other known pathogen-response transcripts, are up-regulated after inactivation of the syntaxin genes. A suppressor mutant analysis of syp121 syp122 revealed that FMO1, ALD1, and PAD4 are important for lesion development. Mutant alleles of EDS1, NDR1, RAR1, and SGT1b also partially rescued the lesion-mimic phenotype, suggesting that mutating syntaxin genes stimulates TIR-NB-LRR and CC-NB-LRR-type resistances. The syntaxin double knockout potentiated a powdery mildew-induced HR-like response. This required functional PAD4 but not functional SA signaling. However, SA signaling potentiated the PAD4-dependent HR-like response. Analyses of quadruple mutants suggest that EDS5 and SID2 confer separate SA-independent signaling functions, and that FMO1 and ALD1 mediate SA-independent signals that are NPR1-dependent. These studies highlight the contribution of multiple pathways to defense and point to the complexity of their interactions.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plants have developed several different inducible defense mechanisms against microbial pathogens. These defenses are controlled by a number of signaling cascades. The best described are the salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signaling pathways (Glazebrook, 2005). Signaling components in each of these pathways have been discovered primarily through mutant screens in Arabidopsis thaliana (Durrant and Dong, 2004; Glazebrook, 2005; Beckers and Spoel, 2006; Broekaert et al., 2006). However, the different pathways do not function independently. The SA pathway, for instance, can act antagonistically to the JA pathway. This antagonism requires the SA-activated NPR1 (Spoel et al., 2003). Disease resistance is manifested downstream of these signaling pathways, through the expression of proteins that counteract the invading pathogen. In addition to SA-, JA-, and ET-regulated defenses, a major defense mechanism is the hypersensitive necrosis response (HR). This pathogen-induced programmed cell death (PCD) of one or more host cells confers effective protection against biotrophic pathogens, which require living host tissue for their survival. The HR is an important element of resistance (R) gene-mediated resistance (Glazebrook, 2005).
Mutants that constitutively express defense responses, many of which are lesion-mimic mutants with spontaneous PCD, have been essential for unraveling defense signaling pathways (Lorrain et al., 2003). Most of these mutations are recessive, suggesting that their wild-type alleles encode negative regulators of defense signaling. Many of the lesion-mimic mutants have more than one permanently activated signaling pathway. However, the SA pathway often predominates in part due to its suppression of other pathways. Therefore, when the SA pathway is silenced in lesion-mimic mutants, up-regulation of the JA and ET pathways is often observed (Clarke et al., 2000). The spontaneous PCD in lesion-mimic mutants is often used as a model for HR-cell death reactions. However, while R-gene-mediated HR appears to be stimulated by SA (Rairdan and Delaney, 2002; Raffaele et al., 2006), the role of SA in spontaneous PCD is ambiguous. There are examples of both SA-dependent (Brodersen et al., 2005; Torres et al., 2005) and SA-independent spontaneous PCD (Hunt et al., 1997).
Plants have developed a specific mechanism to block entry of fungi that attack by penetrating through the plant epidermal cell wall. In Arabidopsis, we identified a series of pen mutants that are compromised in penetration resistance to the non-adapted barley powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh) (Collins et al., 2003; Lipka et al., 2005; Stein et al., 2006). Cloning of the Arabidopsis PEN1 gene and the homologous barley ROR2 gene showed that they encoded SYP121 syntaxins (Collins et al., 2003). SYP121 is localized in the plasma membrane and was subsequently found to be required for timely formation of the cell wall appositions, which are important for this defense (Assaad et al., 2004). Syntaxins are essential proteins of the SNARE machinery, controlling vesicle traffic and bulk transport of cargo in cells (Lipka et al., 2007). SYP121 is thought to influence the secretion of components required for formation of pathogen-induced cell wall appositions.
We have recently demonstrated that syntaxin double mutants, of any of four alleles of syp121 and a T-DNA insertion allele of syp122, become dwarfed and develop severe necrosis after a period of 2–3 weeks of normal growth. These lesion-mimic plants are affected in several different pathogen defense pathways. The syntaxin double mutants lack penetration resistance to powdery mildew fungi. At the same time, the SA, JA, and ET signaling pathways, as well as a pathway that potentiates an HR-like defense to powdery mildew fungi, are elevated in syp121 syp122 plants (Zhang et al., 2007). The enhanced SA signaling is responsible for a significant part, but not all, of the lesion-mimic phenotype. The powdery mildew-triggered HR-like response appears to be independent of the SA signaling pathway based on studies in which mutations in SA signaling genes are introduced one by one. This response, which may also contribute to the lesion-mimic phenotype, occurs after attack by either Bgh or the virulent powdery mildew fungus (Golovinomyces cichoracearum, Gc). The response provides complete resistance to Gc (Zhang et al., 2007). Other data implicating syntaxins in pathogen defense include the demonstration that SYP121 and SYP122 are phosphorylated in response to flg22, an elicitor of basal defenses (Nühse et al., 2003; Benschop et al., 2007), and that a closely related tobacco syntaxin is phosphorylated during R-gene-mediated resistance (Heese et al., 2005). In addition, tobacco SYP132 is involved in resistance to bacteria and secretion of antimicrobial proteins (Kalde et al., 2007).
Our previous work has shown that silencing the SA pathway, using mutations in genes for SA signal components, does not fully rescue the necrosis and dwarfism in the syntaxin double mutant. Here, we report a detailed survey of the defense signaling in syp121 syp122 indicating that multiple defense pathways are active in this mutant. In addition, we show that different pathways lead to the spontaneous cell death and to the pathogen-triggered cell death reactions. Using global transcript profiling, we show that many SA responses and other defense-related transcripts are induced after mutation of SYP121 and SYP122. We observe rescuing effects of mutations in a number of well-described defense pathways, indicating that these are active in the syntaxin double mutant. We also demonstrate that SID2 and EDS5, which are key elements in SA biosynthesis, each have additional roles in defense signaling. We provide evidence that the recently described FMO1 and ALD1-defined pathways contribute to the phenotype independent of SA. However, we propose that these pathways share the NPR1 signaling node. We present data suggesting R-gene-mediated signaling contributes to the syntaxin double mutant phenotypes and that the ET pathway may serve to protect against necrosis development. These results emphasize the importance of syntaxins in defense and establish the syntaxin double mutant as a useful tool for studying defense signaling networks.
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RESULTS |
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Defense-Related Transcripts Are Up-Regulated as a Result of Mutations in SYP121 and SYP122
A global transcript profiling experiment was performed to further characterize the activated defense signaling in the syntaxin double mutant, syp121 syp122. Gene expression was assayed in three biological replicates of wild-type Col-0, syp121–1, syp122–1, and syp121–1 syp122–1 plants at 2.5 weeks of age, prior to the appearance of visible spontaneous lesions. Affymetrix ATH1 GeneChips, carrying 22 500 probe sets, were used (Redman et al., 2004). Two-way ANOVA analysis identified 596 genes up-regulated (P
0.001) in the syntaxin double mutant relative to wild-type, with 365 of these being up-regulated more than two-fold. These 365 genes and their fold change in the syp121 syp122 double mutant relative to wild-type are listed in Supplemental Table 1. One gene (At3g30720) was also up-regulated in both of the syntaxin single mutants, syp121–1 and syp122–1, relative to wild-type. This gene, which encodes a unique 59 amino acid-protein of unknown function, was up-regulated five-fold in syp121–1 and 13-fold in syp122–1. The At3g30720 transcript was the only one to be significantly up-regulated in syp122–1. Remarkably, this is also the only gene which was significantly constitutively up-regulated in the pen3 mutant (Stein et al., 2006). The syp121–1 mutation was associated with the up-regulation of another six genes, one of which was SYP122. Interestingly, only one gene (At4g30140) was found to be significantly (P 0.001) down-regulated (about three-fold) in syp121–1 syp122–1 compared to wild-type. This gene encodes a lipase/hydrolase with a GDSL-motif. We used the Meta-Analyzer tool from Genevestigator (www.genevestigator.ethz.ch; Zimmermann et al., 2004) for a more detailed analysis of the 365 genes up-regulated two-fold or more. We calculated the mean of log2 of the change in expression of these 365 genes for each of the 83 categories in the Meta-Analyzer. For ten of the 83 categories, mean log2 values exceed 1, suggesting that the genes identified in our experiment are also frequently up-regulated in other stress-related experiments (Table 1). The gene expression profile of ozone-treated plants is most similar to that of the syp121 syp122 mutant, with 328 of the 365 genes up-regulated two-fold or more in response to ozone. This reactive oxygen molecule has been reported to induce an HR-like PCD (Overmyer et al., 2000). The overlapping response suggests a high similarity of the spontaneous PCD of syp121 syp122 and ozone-induced PCD. Moreover, the ten matching categories also include inoculation with Botrytis cinerea, Phytophthora infestans, and Erysiphe cichoracearum (= Gc). The two necrotrophic pathogens, B. cinerea and P. infestans, elicit up-regulation of 241 and 273 of the 365 genes, respectively. Even though the powdery mildew fungus, Gc, does not induce cell death, it surprisingly elicits up-regulation of 285 of the 365 genes. As expected, SA treatment was found among the ten categories and this hormone up-regulates 232 of the 365 genes. Subsequently, we assessed the expression patterns of the 365 genes in published Arabidopsis transcriptomics data (Table 2). R-gene-mediated responses to Pseudomonas syringae pv. tomato DC3000 (Pst) (Bartsch et al., 2006) of the 365 genes were highly similar to those observed in the syntaxin double mutant. When resistance is mediated by RPM1 or RPS4, 223 and 200, respectively, of the 365 genes are up-regulated. Of the 365 genes, 188 are up-regulated in response to attack by virulent Pst (www.genevestigator.ethz.ch). These observations suggest that at the 2.5-weeks time-point, the syntaxin double mutant is preparing for the initiation of PCD, which becomes visible a few days later. Furthermore, the similarity to the response of plants inoculated with avirulent Pst strains may suggest that R-gene defenses are constitutively activated in the syntaxin double mutant. This is supported below by the observation that the R-gene signaling genes, PAD4, EDS1, NDR1, SGT1b, and RAR1, are partially responsible for the spontaneous lesion-mimic phenotype of syp121 syp122.
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We also evaluated whether pathogen-associated molecular pattern (PAMP)-induced defenses were activated in the syntaxin double mutant by assessing the expression of the 365 genes in plants following treatments with various PAMPs. Of the 365 genes, 162 responded to chitin (www.genevestigator.ethz.ch), 50 to Escherichia coli and 59 to Pst hrpA (Thilmony et al., 2006) (see Table 2). The hrpA mutation of Pst eliminates the secretion of effectors, which otherwise interfere with PAMP-induced signaling. Even though these numbers are lower than those for PCD-inducing stresses (see above), they suggest that PAMP-like responses are also activated in the syp121 syp122 mutant.
In our previous analyses of the syntaxin double mutant, we found that alleviating the SA signaling in syp121 syp122 by introducing SA signaling mutations led to a significant increase in PDF1.2 expression. This indicates that the JA signaling pathway is activated in syp121 syp122, but suppressed by the antagonistic effect of high SA levels. Since optimal PDF1.2 expression also requires ET signaling (Penninckx et al., 1998), this pathway is likely to be activated in the syntaxin double mutants as well. The lack of expression of the JA signaling markers PDF1.2, THI2.1 (Bohlmann et al., 1998), and VSP2 (Feys et al., 1994) in the present transcriptomics analysis is consistent with the antagonism between SA and JA signaling in the syp121 syp122 mutant, as noted previously (Zhang et al., 2007). Two marker genes for ET signaling–HEL (Potter et al., 1993) and CHI-B (Norman-Setterblad et al., 2000)–are up-regulated 6.1 and 2.2-fold, respectively. Expression of CHI-B is thought to depend on both JA and ET signaling, which points to the complexity of the interplay among the SA, JA, and ET signaling pathways in syp121 syp122. No enhanced expression of genes involved in ET biosynthesis is observed in syp121 syp122, even though these genes were previously reported to be induced by ET (van der Straeten et al., 1992; Zhong and Burns, 2003). However, 101 and 87 of the genes up-regulated after treatment with ET and MeJA, respectively (www.genevestigator.ethz.ch), were found among the 365 genes (Table 2; Supplemental Table 1). This observation may suggest that JA and ET signaling are active, perhaps at a low level, in syp121 syp122, despite high SA levels.
Screen for Mutations Rescuing syp121 syp122
In order to search for novel factors that contribute to the lesion formation of syp121 syp122, a forward genetics approach was taken. Approximately 100 000 mutagenized syp121–1 syp122–1 M2 plants were screened for individuals rescued from the lesion-mimic phenotype. Approximately 240 candidate mutants were retained. Improved growth and reduced necrosis of all the selected mutant lines were confirmed in the M3 generation. We suggest that these 240 lines carry mutant alleles of genes required for lesion formation. These alleles we denote suppressors of syntaxin-related death (ssd).
We previously demonstrated that knockout mutations in EDS5, SID2, and NPR1 only partially suppressed the lesion-mimic phenotype of the syp121 syp122 double mutant (Zhang et al., 2007). The triple mutant, syp121–1 syp122–1 sid2-1, had a characteristic yellow-green color, and complementation tests with syp121–1 syp122–1 ssd mutants with a similar phenotype led to the identification of 15 new sid2 mutants. Also, syp121–1 syp122–1 npr1-1 had a distinct bleaching phenotype (see below), but none of the novel suppressor mutants resembled this triple mutant in appearance. Furthermore, crossing syp121–1 syp122–1 npr1-1 to 70 randomly selected suppressor mutants did not reveal any npr1 mutants among the ssd mutants. Unlike the triple mutants with sid2 and npr1, syp121–1 syp122–1 eds5-3 mutants did not have an immediately recognizable phenotype. Crosses of syp121–1 syp122–1 eds5-3 to 16 random suppressor mutants revealed two novel eds5 mutations. We subsequently performed inter-mutant crosses among a subset of approximately 100 of the suppressor mutants to place them in complementation groups. Three groups were established consisting of ten, seven and seven alleles, respectively. These were named ssd1-1 to ssd1-10, ssd2-1 to ssd2-7 and ssd3-1 to ssd3-7. Representative triple mutants of these three complementation groups are shown in Figure 1A and in Supplemental Figure 1. The mutant alleles of these three SSD genes are stronger suppressors of the lesion-mimic phenotype of syp121–1 syp122–1 than sid2-1, eds5-3, and npr1-1 (Figure 1A; Zhang et al., 2007).
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FMO1, ALD1, and PAD4 Contribute to the Lesion-Mimic Phenotype of syp121 syp122
Initial mapping placed SSD1 on the top of chromosome 1 between markers F21M12 and ciw12. Subsequent fine-mapping, using an additional 520 F2 plants, localized SSD1 to an interval of seven genes (At1g19230 to At1g19310). One gene in this interval, At1g19250, was highly up-regulated in the syp121 syp122 double mutant (22-fold; Supplemental Table 1). At1g19250 encodes the FLAVIN-DEPENDENT MONO-OXYGENASE1 (FMO1) that recently has been implicated in defense (Mishina and Zeier, 2006; Koch et al., 2006; Bartsch et al., 2006; Olszak et al., 2006). We PCR amplified and sequenced the coding region of FMO1 from the genomic DNA of the ten ssd1 suppressor mutants. In each of them, either a premature stop codon, a base-change resulting in an amino acid substitution or a splice site mutation was identified (Figure 1B; Supplemental Figure 2). It is discussed whether the first 45 nucleotides of exon 4 belong to the adjacent intron. Supporting Bartsch et al. (2006), the mutation in fmo1-10 is five base pairs away from the potential splice site at base pair 1548 in exon 4 (Supplemental Figure 2). This is too far away to affect splicing. This documents that the mutation in fmo1-10 must be a premature stop-codon in exon 4 and therefore the longer splice-version contributes to the lesion formation and dwarfing of syp121 syp122. Subsequently, the T-DNA insertion mutant allele fmo1-1 (Bartsch et al., 2006) in line SALK_026163 was introduced into syp121–1 syp122–1. The phenotype of the resulting triple mutant was indistinguishable from that of syp121 syp122 ssd1 mutants (data not shown).
SSD2 was initially mapped to the top of chromosome 2 between markers nga1145 and ciw3 and then fine-mapped in a population of 230 F2 plants to a 25-gene interval from At2g13590 to At2g13850. One of these 25 genes (At2g13810) was up-regulated in the syp121 syp122 mutant (11-fold; Supplemental Table 1). At2g13810 encodes the AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1), which has been shown to contribute to defense (Song et al., 2004a, 2004b). The coding region of ALD1 was PCR amplified and sequenced using genomic DNA from the seven syp121 syp122 ssd2 triple mutants. In all of them, alterations resulting in modification of the ALD1 protein were encountered (Figure 1C; Supplemental Figure 3). Subsequently, the T-DNA insertion mutant allele ald1-T2 (Song et al., 2004a) in line SALK_007673 was introduced into syp121–1 syp122–1. The phenotype of the resulting triple mutant was indistinguishable from that of syp121 syp122 ssd2 mutants (data not shown). An alignment of the Arabidopsis ALD1 amino acid sequence to the most similar protein for which a 3-D structure is available (i.e. aromatic aminotransferase from Pyrococcus horikoshii Ot3 gi|14278622) revealed that the five amino acid substitutions found in the ssd2 mutant proteins occur in structurally important and well-conserved amino acids. Meanwhile, ssd2-3 has a premature stop codon, and ssd2-4 a splice site mutation.
A map-position was not found for SSD3 on chromosomes 1, 2, 4, or 5, suggesting that it is located on chromosome 3, and thus could not be mapped using the initial Ler syp121 syp122 line we created. To determine whether SSD3 was located on chromosome 3 near either SYP121 or SYP122, crosses were made between syp121–1 syp122–1 ssd3-7 and syp121–1 and between syp121–1 syp122–1 ssd3-7 and syp122–1. While 21.8% of the F2's from the cross to syp122–1 had the syp121 syp122 lesion-mimic phenotype (not significantly different from a 3/16 segregation ratio), no plants with this phenotype were recorded in the cross to syp121–1, demonstrating ssd3 is closely linked to syp122. Subsequently, approximately 10 000 F2's of a cross between syp121–1 syp122–1 ssd3-7 and Col-0 wild-type were analyzed. Only two F2 plants had the syp121 syp122 phenotype. This indicated exceptionally close linkage between SYP122 and SSD3. Near SYP122, only one gene (At3g52430) is up-regulated in syp121 syp122 (10-fold; Supplemental Table 1). At3g52430, which is three genes distal from SYP122 (At3g52400), encodes PHYTOALEXIN DEFICIENT 4 (PAD4). Based on PAD4's close functional association with EDS1 (Wiermer et al., 2005) and the ability of an eds1 mutation to partially suppress the lesion-mimic phenotype of syp121 syp122 (Zhang et al., 2007; Figure 1A), this result was not unexpected. We sequenced the coding region of PAD4 from genomic DNA of the seven syp121 syp122 ssd3 triple mutants. In six of them, base changes resulting in premature stop codons and, in one of them, a base change resulting in an amino acid substitution were encountered (Figure 1D; Supplemental Figure 4).
We conclude that FMO1, ALD1, and PAD4 are constitutively active in syp121 syp122 syntaxin double mutants and that these signaling components, and their downstream elements, contribute to lesion formation and dwarfing. In Figure 1, the ssd allele names are converted to fmo1, ald1, and pad4 allele names.
SID2 and EDS5 Each Confer Activities in Addition to SA Signaling
Numerous signaling genes are essential as positive regulators for pathogen defense in Arabidopsis. Some of these are listed in Supplemental Table 2. To assay which of these genes are involved in development of lesions and dwarfism, mutant alleles were crossed into syp121 syp122 (Table 3; Supplemental Figure 5). Most of these defense regulators clearly influence the development of the lesion-mimic phenotype of syp121 syp122. Thus, by combining the different mutations in the syntaxin double mutant background, it is possible to study whether the defense pathways, defined by these, genes interact.
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Both SID2 and EDS5 affect SA accumulation and null mutants contain very low SA levels after pathogen attack (Heck et al., 2003). EDS5 encodes a MATE-type transporter required for SA signaling (Nawrath et al., 2002), and SID2 encodes an isochorismate synthase (Wildermuth et al., 2001). Inactivation of either SID2 or EDS5 rescued the syp121 syp122 lesion-mimic phenotype to approximately the same level and eliminated the constitutive SA level (Figure 2A) and PR-1 gene expression (Zhang et al., 2007). However, the two different triple mutant lines were easily differentiated visually. syp121–1 syp122–1 eds5-3 was relatively more necrotic than syp121–1 syp122–1 sid2-1, which was light green with distinct necrotic lesions surrounded by chloroses. Interestingly, the quadruple mutant, syp121–1 syp122–1 eds5-3 sid2-1, performed markedly better than either of the two triple mutants (Figures 1A and 2B; Table 3; Supplemental Figure 5). These observations suggest that EDS5 and SID2 have additional, unique roles, aside from SA biosynthesis, and that all these distinct roles contribute additively to lesion development and dwarfism in syp121 syp122.
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SA-Dependent, NPR1-Independent Signaling
The triple mutants syp121–1 syp122–1 eds5-3 and syp121–1 syp122–1 sid2-1 contained very little or no SA. In contrast, syp121–1 syp122–1 npr1-1 plants contained SA amounts exceeding those of syp121 syp122 (Figure 2A). The high SA levels are apparently caused by the loss of the two syntaxins, which leads to continuous SA production. In the absence of functional NPR1, SA perception may be compromised and therefore the effects of high SA levels are diminished. However, the triple mutant syp121–1 syp122–1 npr1-1 did develop characteristic bleaching of leaf and stem tissues, which remained alive for some additional time. To test the possibility that the bleaching is a phenomenon restricted to the npr1-1 allele, the NPR1 mutant alleles npr1-2, npr1-3 (Cao et al., 1997), npr1-5 (Shah et al., 1997), and npr1-P4-7 (Xiao et al., 2005) were crossed into syp121–1 syp122–1. The resulting triple mutant lines all show the same bleaching phenotype (data not shown). Interestingly, we also observed bleaching when npr1 single mutants were sprayed with SA (data not shown). The bleaching phenotype thus appears to be associated with high SA levels. Introducing mutant alleles of SID2 and EDS5 into syp121 syp122 npr1, thereby abolishing SA biosynthesis, generated quadruple mutants with improved performance (Figure 2B; Table 3; Supplemental Figure 5). Importantly, these syp121 syp122 npr1 eds5 and syp121 syp122 npr1 sid2 quadruple mutants do not have the bleaching phenotype of syp121 syp122 npr1. These observations suggest SA-dependent, NPR1-independent processes in Arabidopsis.
ALD1 and FMO1-Mediated Signals, SA and NPR1
In order to determine how defense pathways controlled by ALD1 and FMO1 interact with SA signaling, a number of combinations were made between alleles of these genes and of EDS5, SID2, NPR1, and PAD4, all in the syp121 syp122 mutant background. The partially rescued syp121 syp122 fmo1 and syp121–1 syp122–1 ald1 performed even better when SA biosynthesis or signaling also was inactivated (i.e. in the quadruple mutants including mutations in EDS5, SID2, or PAD4) (Table 3). These observations were made in F2 populations segregating for mutant alleles of the following six gene-pairs: FMO1/ESD5, FMO1/SID2, FMO1/PAD4, ALD1/SID2, ALD1/ESD5, and ALD1/PAD4. Approximately 1/16 of the plants became remarkably larger, as exemplified in Figure 3, suggesting that ALD1 and FMO1 mediate signals that are distantly associated to SA signaling. However, it is interesting that no notably larger plants were found in F2 populations of crosses between syp121 syp122 npr1 and syp121 syp122 fmo1 or between syp121 syp122 npr1 and syp121 syp122 ald1, as exemplified in Figure 3. All these phenotypes have been recorded using several alleles, as indicated in Table 3. This suggests that FMO1 and ALD1 contribute to the lesion-mimic phenotype of syp121 syp122 by mechanisms that are partially parallel to SA signaling and to PAD4-dependent signaling. Interestingly, the ALD1 and FMO1 signals also appear to act separately from the SA-independent signals suggested above to be conferred by EDS5 and SID2. However, ALD1 and FMO1 seem to mediate signals that depend on NPR1. All in all, these observations suggest the existence of SA-independent, NPR1-dependent signaling.
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R-Gene-Mediated Signaling Pathways in syp121 syp122
In general, R-gene-dependent resistance mediated by toll-interleukin-1 receptor (TIR)-type nucleotide binding-leucine-rich repeat (NB-LRR) proteins depends on EDS1 and PAD4, whereas resistance mediated by coiled-coil (CC)-type NB-LRR proteins is NDR1-dependent (Hammond-Kosack and Parker, 2003; Wiermer et al., 2005). The proteins RAR1 and SGT1b are important for the stability of the NB-LRR proteins and are thus required for R-gene-mediated resistance in general (Holt et al., 2005).
Given the contribution of PAD4 and EDS1 to the syp121 syp122 phenotype (Figure 1; Table 3; Supplemental Figure 5; Zhang et al., 2007), we sought to analyze whether R-gene-mediated signals were functioning in the syp121 syp122 mutant and contributing to its lesion-mimic phenotype. Since EDS1 and PAD4 act upstream of the SA pathway (Hammond-Kosack and Parker, 2003; Wiermer et al., 2005), which clearly contributes to the syp121 syp122 lesion-mimic phenotype, the question remains whether the SA pathway activation in syp121 syp122 is the only signal dependent on EDS1/PAD4 function. Improved performance of the syntaxin double mutant in an eds1 or pad4 background compared to a sid2, eds5, or npr1 background indicates that these latter three SA signaling components control only a subset of the pathways triggered by EDS1 and PAD4 (Figure 1). Remarkably, inactivation of SID2, EDS5, or NPR1 in the syp121–1 syp122–1 pad4-19 genetic background led to further improved performance of the resulting quadruple mutant relative to the triple mutant (Figure 4A; Table 3; Supplemental Figure 5). This result suggests either that the SA signaling is independent of PAD4 or, more likely, that SID2, EDS5, and NPR1 also control SA- and PAD4-independent signals.
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Further support for a role of R-gene-mediated signaling in the lesion-mimic phenotype of syp121 syp122 was provided by the partial suppression of this mutant phenotype by mutations in NDR1, RAR1, or SGT1b (Figure 4B; Table 3). These mutations rescued to approximately the same degree as mutations in SA signaling genes until the plants reached the 3-week stage. In older plants, mutations of NDR1, RAR1, or SGT1b resulted in only slightly enhanced performance compared to syp121 syp122 (Figure 4C; Table 3; Supplemental Figure 5). To analyze whether NDR1-mediated signaling interacts with the SA pathway, we introduced the ndr1-1 allele into syp121–1 syp122–1 npr1-1 and syp121–1 syp122–1 sid2-1. The resulting quadruple mutants performed significantly better than either of the triple mutants, suggesting that NDR1 activates SA-independent defense processes (Figure 4C; Table 3). Interestingly, the SA-related bleaching in syp121–1 syp122–1 npr1-1 was maintained in syp121–1 syp122–1 npr1-1 ndr1-1 in contrast to syp121–1 syp122–1 npr1-1 sid2-1, where no bleaching is observed. This indicates that NDR1-mediated signaling does not influence SA accumulation. Finally, the quintuple mutant syp121–1 syp122–1 npr1-1 sid2-1 ndr1-1 was obtained. Even though older plants of this genotype became necrotic, the quintuple mutant grew almost as well as wild-type Col-0.
In conclusion, these results suggest that loss of SYP121 and SYP122 leads to the activation of similar signaling pathways as CC-NB-LRR and TIR-NB-LRR-type R-gene-mediated pathogen recognition. This observation is corroborated by the similarities between transcript profiles of syp121 syp122 and plants in which R-gene signaling was triggered (Table 2; Supplemental Table 1).
The autophagy component of PCD has been implicated in R-gene-mediated resistance (Lui et al., 2005). A number of autophagy genes (ATGs) have been identified in a variety of organisms. In Arabidopsis, ATG7 is a non-redundant gene (Doelling et al., 2002) and the T-DNA atg7 allele in SALK_057605 can be used to test the role of autophagy in the lesion-mimic phenotype of syp121 syp122. Blocking autophagy by this method did not cause any visible phenotypic effects (Table 3; Supplemental Figure 5). This result suggests that autophagy does not play a role in the lesion-mimic phenotype, the HR-like response to the powdery mildew fungi or the SA-hypersensitivity (see below) phenotypes of the syntaxin double mutant. We also examined the quadruple mutants syp121–1 syp122–1 npr1-1 atg7 and syp121–1 syp122–1 sid2-1 atg7, and were again unable to demonstrate any effect of the atg7 mutation. These observations are supported by introductions of mutant alleles of the non-redundant genes ATG3 (SALK_031693) and ATG6 (SALK_051168; Fujiki et al., 2007) into syp121–1 syp122–1. In both cases, all plants in syntaxin double mutant homozygous families that were segregating for the atg3 or atg6 mutations exhibited the typical syp121–1 syp122–1 phenotype (data not shown).
An oxidative burst, generated by NADPH oxidases, is an indispensable element of pathogen defense mechanisms in plants (Foyer and Noctor, 2005). In Arabidopsis, AtrbohD is the NADPH oxidase primarily responsible for this burst (Torres et al., 2002). We introduced the T-DNA atrbohD allele in SALK_070610 into the syp121 syp122 background. This mutation seemed to have no effect on the lesion-mimic phenotype, the HR-like response to the powdery mildew fungi or the SA-hypersensitivity (see below) of the syntaxin double mutant (Table 3; Supplemental Figure 5). However, both the atrbohD and syp121–1 syp122–1 atrhohD plants were hyper-sensitive to high light, which confirms the presence of the atrbohD mutant allele (data not shown).
Ethylene Signaling Antagonizes syp121 syp122 Lesion Formation
We previously suggested that the ET and JA signaling pathways are activated in syp121 syp122 (Zhang et al., 2007). This was documented by the increased expression of the PDF1.2 transcript, a marker for these two pathways (Penninckx et al., 1998), following interruption of the SA signaling pathway in the syp121 syp122 double mutant. To analyze the role of the ET signaling pathway in the double syntaxin mutant phenotypes, the mutant allele ein2-1 was crossed into syp121–1 syp122–1. The biochemical function of EIN2 is not known, but the protein is required for general ET signaling (Benavente and Alonso, 2006). EIN2 appeared to suppress necrosis in the syp121–1 syp122–1 double mutant, as suggested by the enhanced necrotic phenotype of syp121–1 syp122–1 ein2-1 triple mutant (Figure 5; Table 3; Supplemental Figure 5). This ein2-1 effect was even more obvious when NPR1 was also mutated, but not when SID2 was mutated (Table 3; Figure 5). The quadruple mutant syp121–1 syp122–1 npr1-1 ein2-1 died after approximately 6 weeks.
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JA Signaling Impacts Lesion Formation in syp121 syp122 Plants
The jar1-1 mutant allele was used to assess the importance of JA signaling for the syp121 syp122 phenotypes. JAR1 catalyses conjugation of JA to amino acids (Staswick and Tiryaki, 2004). When introduced into syp121–1 syp122–1, no phenotypic consequences were observed (Figure 5; Table 3; Supplemental Figure 5). This lack of effect was confirmed by using a coi1 mutant (Xie et al., 1998) and a mutant (SALK_017756) in the AOS gene (Park et al., 2002). None of the plants in an F2 population of syp121–1 syp122–1, segregating for the coi1-1 or the aos allele, deviated from the necrotic and dwarfed appearance of the syntaxin double mutant. During the flowering of these plants, expected pollen-sterile individuals were detected, confirming that these lines were homozygous for coi1-1 or aos (data not shown).
The absence of a detectable role for JA signaling could be due to the JA pathway being suppressed by the high SA level found in the syntaxin double mutant (Spoel et al., 2003; Zhang et al., 2007). To test this hypothesis, mutant combinations blocking both SA and JA signaling or biosynthesis were crossed into the syp121 syp122 background. However, this approach gave what appear to be conflicting results (Figure 5; Table 3; Supplemental Figure 5). syp121–1 syp122–1 npr1-1 jar1-1 showed more necrosis than syp121–1 syp122–1 npr1-1 plants, which is consistent with the suggestion by Rao et al. (2000) that JA signaling protects against cell death. However, syp121–1 syp122–1 sid2-1 jar1-1 plants were indistinguishable from syp121–1 syp122–1 sid2-1 plants. Surprisingly, the jar1-1 mutation resulted in improved performance of the quintuple mutants syp121–1 syp122–1 sid2-1 npr1-1 jar1-1 and syp121–1 syp122–1 sid2-1 eds5-3 jar1-1.
Gc-Induced HR-Like PCD
syp121 syp122 has a multi-cellular HR-like response to the two powdery mildew fungi Bgh and Gc. Introducing mutant alleles of gene in the SA pathway into the syntaxin double mutant demonstrated this HR-like response to be SA-independent (Zhang et al., 2007). The HR-like response confers complete resistance to Gc. Combining any two of the three SA pathway mutations, eds5-3, sid2-1, and npr1-1, in the syntaxin double mutant did not break this PCD-associated resistance (Figure 6).
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In order to determine which defense components contribute to this powdery mildew-induced HR-like response, a series of triple mutants were examined macroscopically. Neither the HR-like response nor the resistance to Gc was significantly affected by inactivating the NDR1, RAR1, or SGT1b genes implicated in R-gene signaling (Table 3). However, mutations in EDS1, and, to a greater extent, in PAD4, partially restricted the HR-like response and disease resistance. This allowed weak colony growth occasionally to be observed on these triple mutants (Table 3). Interestingly, introducing each of the three SA pathway mutations, eds5-3, sid2-1, and npr1-1, into syp121–1 syp122–1 pad4-19 had an obvious negative effect on the HR-like response, and the quadruple mutants were susceptible to Gc during the first week after inoculation (Table 3; Figure 6). However, at later time-points, necrosis was observed, which arrested fungal growth (data not shown). We have no evidence to suggest that the following genes are required for the HR-like response and resistance to Gc resistance of the syp121 syp122 double mutant: JAR1, EIN2, RbohD, ATG7, FMO1, and ALD1. Furthermore, when mutations of some of these genes were introduced into triple mutants blocked in SA synthesis or signaling (syp121–1 syp122–1 npr1-1, syp121–1 syp122–1 eds5-3, and syp121–1 syp122–1 sid2-1), the HR-like response was not sufficiently suppressed to permit Gc growth (Table 3).
syp121 syp122 Mutants are Hypersensitive to SA
Wild-type plants respond to exogenous SA by induction of, for instance, the PR-1 gene (Durrant and Dong, 2004). However, even though SA is involved in responses leading to PCD, wild-type plants do not necessarily develop PCD, even when treated with high concentrations of SA. This suggests activation of parallel signaling is required for PCD, and, as such, SA is considered a potentiator rather than a trigger for PCD in R-gene-mediated HR (Shirasu et al., 1997; Tenhaken and Rubel 1997).
In contrast to wild-type plants, very young syp121 syp122 mutants underwent PCD in response to exogenous SA (Figure 7). This suggests that the syntaxin double mutant is hypersensitive to SA. To examine whether this SA-hypersensitivity was due in part to the high endogenous SA level in the syntaxin double mutant (Figure 2A; Zhang et al., 2007), mutations of the SID2 or EDS5 genes or the NahG gene were introduced into the syp121–1 syp122–1 background. The SA-hypersensitivity of the syntaxin double mutant was still observed in mutants unable to synthesize SA. However, the SA-hypersensitivity did require an intact NPR1 protein (Figure 7). The independence of endogenous SA and the uniqueness of this phenotype in syp121–1 syp122–1 were supported by an analysis of other dwarfed and/or necrotic mutants constitutively expressing the SA signaling pathway. None of the tested lines, acd2-2, acd11-1, cpr1-1, lsd1-1, or mpk4, all of which can be rescued by interruption of the SA signaling pathway, exhibits such an SA-hypersensitivity (Figure 7; Table 3).
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Having shown that a number of signaling pathways, other than the SA pathway, are active in syp121 syp122, we analyzed whether these other defense pathways contributed to the SA-hypersensitive phenotype. Among the pathways tested, none could be assigned to this phenotype. Furthermore, pathways that did not contribute to the lesion phenotype in syp121 syp122 were also not involved in the SA-hypersensitivity (Table 3).
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DISCUSSION |
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Activation of the different inducible defense mechanisms against microbial pathogens can be recognized by transcriptomics. Here, such analysis showed that inactivation of both SYP121 and SYP122 led to activation of 365 genes by more than two-fold prior to the visible appearance of cell death. Most of these genes are also induced by ozone, by a necrotrophic fungus and by a cell death-inducing oomycete. Therefore, the signals required for PCD are already present before the appearance of necroses. A strong overlap with genes induced by HR-inducing avirulent bacteria points to the operation of R-gene-mediated defense pathways in syp121 syp122. Previous evidence for a role of SA signaling in the phenotype development of this line is corroborated by the observation that more than half of the 365 genes in syp121 syp122 also respond to SA. However, it was surprising to discover that a large majority of these genes, otherwise related to induction of cell death, are also induced by a virulent isolate of the powdery mildew fungus Gc, which does not elicit host cell death. A possible explanation is that this fungal pathogen primes but does not fully activate PCD, or that Gc releases efficient anti-cell death effectors to the plant cell. A significant number of the 365 genes are PAMP-induced, suggesting that inactivation of SYP121 and SYP122 also activates PAMP-type signaling. Interestingly, only one gene is down-regulated after mutating the syntaxin genes. This is in contrast to the transcript analysis of the acd11 lesion-mimic mutant, where a large set of genes involved in primary metabolism is down-regulated (Brodersen et al., 2002). Inoculations with pathogens also cause a reduction in transcript levels of genes involved in primary metabolism, even before disease development (e.g. Zimmerli et al., 2004; Thilmony et al., 2006; Truman et al., 2006; Gjetting et al., 2007). This indicates that SYP121 and SYP122 function as regulators of a signaling node that controls defense pathways, without affecting primary metabolism. The log2 of the change of the 365 genes in all 83 Genevestigator categories was found to be 0.26, showing that this set of genes are not common response genes of the many different kinds of stresses in this collection. This demonstrates that the syp121 and syp122 mutations specifically elicit defense-like gene expression changes.
The role of the JA and ET signaling pathways in the syntaxin double mutant syp121 syp122 is less clear. A few marker genes indicative of constitutive ET signaling are up-regulated; however, ET biosynthesis genes are not up-regulated in syp121 syp122. Yet, mutating EIN2 in syp121 syp122 suggested that ET signaling plays a role in delaying lesion formation. ET seems to have conflicting regulatory functions in pathogen defense (van Loon et al., 2006). ET can apparently both promote and inhibit PCD. It is thought to potentiate senescence-related PCD (Oh et al., 1997), ozone-induced PCD (Overmyer et al., 2000), and vad1 mutant PCD (Bouchez et al., 2007). On the other hand, resistance to the necrotrophic fungus, B. cinerea, requires functional EIN2 and application of ET reduces susceptibility (Thomma et al., 1999), suggesting that ET acts antagonistically to PCD. Similar to the latter case, it is likely that introducing the ein2-1 allele into syp121–1 syp122–1 interrupts a constitutive ET signal that otherwise serves to protect against cell death. Interestingly, the lesion formation, delayed by ET, appears to depend on a SID2-mediated signal. This is seen from the fact that the ein2-1 allele does not promote lesion formation in syp121–1 syp122–1 sid2-1. However, it does not depend on NPR1, since ein2-1 does promote lesion formation in syp121–1 syp122–1 npr1-1 (see Figure 5). An NPR1-independent, SID2-mediated signal in syp121–1 syp122–1 that potentially can be antagonized by ET is described below.
JA can also have both anti-PCD (Overmyer et al., 2000) and pro-PCD (Mur et al., 2006) functions. However, while we did not observe an effect of interrupting JA signaling in syp121–1 syp122–1, using mutations in three different genes, there were potentially anti- and pro-PCD effects of JA signaling, depending on which SA signaling elements were knocked out (see Figure 5). Further analyses are required in order to explain these observations.
EDS1 is required for development of the syntaxin double mutant lesion-mimic phenotype (Figure 1; Zhang et al., 2007). Since EDS1 and PAD4 are closely associated functionally (Wiermer et al., 2005), PAD4 would also be expected to play a role. We were originally unable to introduce a mutation of PAD4 into syp121–1 syp122–1 due to extremely close genetic linkage. However, this barrier was overcome by isolation of induced mutations in PAD4 after mutagenesis of syp121–1 syp122–1 and selection of genotypes with improved performance. This confirmed the requirement of PAD4. In the same way, mutant alleles of FMO1 and ALD1 were encountered, demonstrating requirement of these genes for development of the syp121 syp122 lesion-mimic phenotype. All in all, we estimate that PAD4, FMO1, or ALD1 mutant alleles, as well as SID2 or EDS5 mutant alleles, each constitute 5–10% of the 240 ssd alleles recovered from the suppressor screen.
The finding that EDS1, PAD4, NDR1, RAR1, and SGT1b were required for the lesion phenotype suggests that R-gene-mediated types of signals are activated when both SYP121 and SYP122 are mutated. The requirement for all five genes suggests that both CC-NB-LRR and TIR-NB-LRR types of signals are active, but the stronger effects of the EDS1 and PAD4 mutations suggest that the TIR-NB-LRR type may dominate. This was further supported by the HR-like response to Gc being dependent on EDS1 and PAD4, but not on NDR1. We have no direct evidence to support a direct role for R-proteins in signal activation in syp121 syp122, but the simplest explanation is that they are. Activation of NB-LRRs is not dependent on the presence of pathogens per se. In fact, NB-LRRs can be activated by amino acid-substitutions, truncations (Zhang et al., 2003; Howles et al., 2005; Takken et al., 2006; Weaver et al., 2006), or simply over-expression (Stokes et al., 2002). RAR1 and SGT1b are thought to be required for the stability of NB-LRRs (Holt et al., 2005), and the need for these genes in the syp121 syp122 lesion-mimic phenotype development may support that NB-LRRs themselves are involved. Therefore, the observation that the R-gene-mediated defenses may be activated by the syntaxin double mutant suggests that intact vesicle traffic is required for maintaining a stable balance between active and inactive NB-LRR proteins in the absence of recognizable effectors.
We found that the many active signaling pathways in syp121 syp122 make this genotype a highly useful tool for the study of interactions between the different pathogen defense pathways. For this purpose, we combined two or more knockout mutations in positive defense regulators in the syp121 syp122 genetic background. The most common observation was that this improved the rescue, which, in general, indicates that these regulators control independent signals that make separate contributions to the lesion-mimic phenotype. Yet, we also encountered improved rescue by combining presumed closely associated signaling components by what appears to be relief of signal compound accumulation. An example of this is probably what we observe when eds5-3 or sid2-1 improves the rescuing effect of npr1-1. In contrast, when no improved rescue is observed after combining two mutations, this strongly suggests that these signaling components are closely associated.
The result of combining knockouts in EDS5 and SID2 strongly indicates that each of these two genes are involved in mediating signals in addition to SA. Brodersen et al. (2005) suggested that SID2 is involved in the biosynthesis of an additional signaling molecule that might also be sensitive to the hydroxylase activity of NahG. This is consistent with our finding, in that expression of NahG in syp121–1 syp122–1 improved the performance significantly compared to inactivation of EDS5 or SID2 (Supplemental Figure 5). Heck et al. (2003) reach a similar conclusion regarding the effects of NahG expression.
Abolishing SA in syp121–1 syp122–1 npr1-1 by introducing mutant alleles of EDS5 or SID2 improved the performance of the plants. This suggests that SA contributes to the lesion-mimic phenotype also in the absence of functional NPR1, and that SA in itself has NPR1-independent effects. We correlate this phenomenon to tissue bleaching caused by SA in the absence of functional NPR1. It remains unclear whether this effect is caused by SA toxicity or whether a true signaling effect of SA is involved. A similar SA-induced bleaching in npr1-1 was observed by Bowling et al. (1997) and Cao et al. (1997). SA-dependent, NPR1-independent signaling has been suggested several times (Nandi et al., 2003; Yoshioka et al., 2001; Durrant and Dong, 2004; Desveaux et al., 2004; Uquillas et al., 2004; Tsutsui et al., 2006; Yaeno et al., 2006; Zhang et al., 2003), but further investigations are needed in order to establish the nature of the SA-related bleaching.
The recently discovered signaling element FMO1 is suggested (1) to be involved in TIR-NB-LRR resistance mediated through EDS1 (Bartsch et al., 2006), (2) to be essential for SA accumulation in distal but not local leaves during SAR (Mishina and Zeier, 2006), and (3) to be required for basal defense (Koch et al., 2006). The aminotransferase ALD1 has also been reported to be required for SAR and SA accumulation in distal leaves, as well as for resistance in local leaves. However, mutation in ALD1 only caused a minor reduction in the SA accumulation in local leaves. The loss of resistance in the ald1-T2 T-DNA mutant line could not be rescued by treatments with the SA analogue, BTH, suggesting that another mechanism is involved (Song et al., 2004b). Furthermore, ALD1 is suggested to function separately from PAD4 in terms of pathogen defense and PCD development in the lesion-mimic mutant acd6 (Song et al., 2004b).
We find it interesting that mutations in FMO1 and in ALD1 partially rescued the lesion-mimic phenotype of syp121 syp122. Firstly, this confirms once again that the syntaxin double mutant phenotype is caused by a concurrent activation of several defense signaling pathways. Secondly, the involvement of FMO1 in TIR-NB-LRR resistance supports that R-gene-mediated types of signals are activated in the syntaxin double mutant. Furthermore, it is noteworthy that the quadruple mutants, where we mutated FMO1 or ALD1 on one hand, and PAD4, EDS5, and SID2 on the other hand, all exhibited improved rescue relative to all five triple mutants. This suggests that both FMO1 and ALD1 may function in parallel to SA signaling. Regarding FMO1, this agrees with the additive effects of FMO1 and SID2 mutations on disease resistance (Bartsch et al., 2006) and failure of FMO1 mutations to affect local leaf SA level after pathogen attack (Bartsch et al., 2006; Mishina and Zeier, 2006). Regarding ALD1, it is comparable to the situation for the triple mutant pad4 ald1 acd6, described by Song et al. (2004b), that performs better than ald1 acd6 and pad4 acd6. In contrast, the fact that combinations of knockouts of FMO1 and NPR1, as well as of ALD1 and NPR1, do not give an improved rescue of the syntaxin double mutant may suggest that these two genes mediate signals that fully depend on NPR1.
A qPCR PR-1 transcript analysis has been conducted on most of the lines in Table 3 (data not shown). An inverse correlation between the level of rescue and the PR-1 transcript expression was found. While the jar1 and ein2 mutations did not reduce the high PR-1 transcript level in syp121 syp122, rar1, sgt1b, and ndr1 reduced it 10-fold. Meanwhile, eds5, sid2, npr1, fmo1, and NahG reduced the level approximately a 1000-fold, thereby approaching wild-type transcript level. The PR-1 transcript level in the quadruple mutants, having at least one of the eds5, sid2, or npr1 mutations, was obviously also reduced to wild-type levels. For that reason, this analysis was not able to provide support for the phenotype assay of the effect of combining mutations in two or more positive defense regulators.
Not surprisingly, the syntaxin double mutant is powdery mildew resistant. Unlike the single epidermal cell HR seen in Arabidopsis in response to Bgh, this resistance is manifested as a multi-cellular death reaction around the attacked epidermal cell, after the initial haustorium is established (Zhang et al., 2007). We have not yet encountered a signaling pathway that alone is required for this resistance. Rather, it appears to be dependent on the concerted action of a number of signaling pathways. Mutation of PAD4 reduces the resistance to the greatest extent. On the other hand, single or double mutations of EDS5, SID2, and NPR1 do not affect the resistance. However, when combined with a PAD4 mutation, they reduced resistance further (Figure 6). Deciphering the complete signaling events that lead to this resistance awaits future analyses. In the case of the SA-hypersensitivity of the syntaxin double mutant, we have found NPR1 to be strictly required. NPR1 possibly functions like a downstream ‘receptor’ for SA, and therefore may not be involved per se in making the plant hypersensitive. Interestingly, both the powdery mildew resistance and the SA-hypersensitivity have the potential to be mediated by R-gene-type of signaling, which would agree well with the notion above that such signaling appears active in syp121 syp122.
On the basis of well established signaling connections presented in the literature, we summarize some of our observations for the signaling network, activated by mutation of SYP121 and SYP122, in the model shown in Figure 8. We speculate that the broad activation of defense pathways reflects an equally broad activation of R-proteins, leaving many unknowns to be uncovered. Thereby, syp121 syp122 may express responses that otherwise would require several inoculation experiments to obtain. This underpins the usefulness of this line for studies of the signaling network. For instance, we find it intriguing that NPR1 is the only gene of those tested that appears to be needed for the signaling leading to the SA-hypersensitivity. This suggests that more active defense signaling pathways can still be found in the syntaxin double mutant.
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Plants
Arabidopsis thaliana plants were grown at 20°C, 125 µE sm–2 in short day (8 h light) conditions. Genotypes are described in Supplemental Table 2. The mutant lines jar1-1, coi1-1, ein2-1, ndr1-1, sgt1b-1, rar1-10, acd2-2, cpr1, as well as T-DNA insertion lines, were provided through NASC (http://arabidopsis.info). NahG and npr1-1 were provided by Xinnian Dong (Duke University, NC, USA), eds1-2 by Jane Parker (Max Planck Institute, Cologne, Germany), eds5-3 and sid2-1 by Jean-Pierre Métraux (University of Fribourg, Switzerland), mpk4 by John Mundy (University of Copenhagen), and npr1-N4-7 by Shunyuan Xiao (University of Maryland Biotechnology Institute, MD, USA). Triple mutants of syp121–1, syp122–1 and various positive defense signaling mutants were selected in F3 families derived from F2 plants showing the syp121 syp122-dwarf/necrosis-phenotype. Homozygous triple mutant F3 plants were identified either as partially rescued plants or using molecular markers for the mutation in the positive defense signaling gene. Subsequently, the mutations in positive defense signaling were confirmed using molecular markers. The alleles eds1-2, rar1-10, and sgt1b-1 are crossed in from Ler, and the rescuing effects of these are confirmed in four independent F3 descents for each (data not shown). Quadruple and quintuple mutants were obtained in the same manner by crossing triple and quadruple mutants.
Expression Profiling
Col-0, syp121–1, syp122–1, and syp121–1 pen122–1 were harvested in three replicas 2.5 weeks after sowing, prior to the appearance of lesions on syp121 syp122. Total RNA was extracted using an RNeasy kit (Qiagen GmbH, Hilden, Germany) and labeling and hybridization on the ATH1 microarrays (Thibaud-Nissen et al., 2006) (one sample per chip) were performed according to the manufacturers instructions (www.affymetrix.com/support/technical/manual/expression_manual.affx). Raw intensity data was normalized using the R (R Development Core Team, 2004) Affy package (Gautier et al., 2004) using the perfect match-only implementation of dCHIP (Li and Wong, 2001) and qspline (Workman et al., 2002). We applied one-way ANOVA in order to identify statistical significant differentially expressed genes. False-positive rates were estimated by recalculating P-values with permuted sample categories. This procedure was repeated four times, generating four sets of 22 810 permuted P-values. From this, the chosen P-value cut-off was estimated to contain a maximum of 15 false-positives (i.e. 
2.5%).
Mutagenesis of syp121 syp122
Approximately 30 000 Col-0 syp121–1 syp122–1 seeds were mutagenized using 0.2% ethyl methane-sulfonate (EMS) at room temperature for 12 h, followed by continuous rinsing with water for 3 h. The seeds were subsequently sown on soil for production of M2 seeds, which were harvested into ten pools. M2 plants, partially rescued for the lesion-mimic phenotype of syp121 syp122, were selected in the greenhouse.
Genetic Mapping
In order to generate mapping populations for the SSD genes, the syp121–1 and syp122–1 mutant alleles were first back-crossed into Ler. Thereby, a line with the typical syntaxin double mutant phenotype was obtained, which we confirmed to be homozygous Ler at several genetic marker loci on each of chromosomes 1, 2, 4, and 5. Both SYP121 and SYP122 are located on chromosome 3, and no effort was made to transfer Ler DNA to this chromosome. F2 populations were generated for each of the SSD genes (Col-0 syp121–1 syp122–1 ssdxLer syp121–1 syp122–1). Initial analyses of 22 rescued F2 plants in each population using published genetic markers (Bell and Ecker, 1994; Lukowitz et al., 2000) suggested map-positions. Subsequent fine-mappings were performed using the Cereon Col/Ler marker information (www.arabidopsis.org/cereon/index.jsp).
Salicylic Acid Measurements
Extraction and determination of total (free plus glycosylated) SA in shoots was performed by HPLC as described by Newman et al. (2001).
Plant Treatments
The Arabidopsis powdery mildew fungus (Golovinomyces cichoracearum, isolate UCSC1) was propagated on pumpkin plants. Inoculations were performed as described by Collins et al. (2003). SA treatment was performed by spraying plants with 250 µM SA once a day for five consecutive days. The treatment was initiated when the plants were 16 d old. Photos were taken 1 d after the last treatment.
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SUPPLEMENTARY DATA |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Financial support has been provided by the Danish Agricultural and Veterinary Research Council; the Danish Agency for Science, Technology and Innovation; Carl Tryggers Foundation, Sweden; the Carlsberg Foundation, Denmark; the Directorate for Food, Fisheries and Agri Business, Denmark; the US National Science Foundation; and the Carnegie Institution of Science, USA.
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Acknowledgements |
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We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. We thank Dr Peter Hagedorn for initial analyses of the global transcript profiles, and Dr Dale Godfrey for critically reading the manuscript. We thank the undergraduate students Louise Flagstad, Stine Petersen, Irene S. Rasmussen and Sabine B. Sørensen for mapping a mutant allele.
No conflict of interest declared.
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Assaad FF, et al. The PEN1 syntaxin defines a novel cellular compartment upon fungal attack and is required for the timely assembly of papillae. Mol. Biol. Cell (2004) 15:5118–5129.
Bartsch M, Gobbato E, Bednarek P, Debey S, Schultze JL, Bautor J, Parker JE. Salicylic acid-independent ENHANCED DISEASE SUSCEPTIBILITY1 signaling in Arabidopsis immunity and cell death is regulated by the monooxygenase FMO1 and the Nudix hydrolase NUDT7. Plant Cell (2006) 18:1038–1051.
Beckers GJ, Spoel SH. Fine-tuning plant defence signalling, salicylate versus jasmonate. Plant Biol. (Stuttg.) (2006) 8:1–10.
Bell CJ, Ecker JR. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics (1994) 19:137–144.[CrossRef][ISI][Medline]