Suppressor Of Npr1-1 Research Articles

Multiple chromatin-associated modules regulate expression of an intracellular immune receptor gene in Arabidopsis.

The expression of an intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1, CONSTITUTIVE 1) is regulated by multiple chromatin-associated proteins for tuning immunity and growth in Arabidopsis. Whether and how these regulators coordinate to regulate SNC1 expression under varying environmental conditions is not clear. Here, we identified two activation and one repression regulatory modules based on genetic and molecular characterizations of five chromatin-associated regulators of SNC1. Modifier of snc1 (MOS1) constitutes the first module and is required for the interdependent functions of ARABIDOPSIS TRITHORAX-RELATED 7 (ATXR7) and HISTONE MONOUBIQUITINATION 1 (HUB1) to deposit H3K4me3 and H2Bub1 at the SNC1 locus. CHROMATIN REMODELING 5 (CHR5) constitutes a second module and works independently of ATXR7 and HUB1 in the MOS1 module. HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 15 (HOS15) constitutes a third module responsible for removing H3K9ac to repress SNC1 expression under nonpathogenic conditions. The upregulation of SNC1 resulting from removing the HOS15 repression module is partially dependent on the function of the CHR5 module and the MOS1 module. Together, this study reveals both the distinct and interdependent regulatory mechanisms at the chromatin level for SNC1 expression regulation and highlights the intricacy of regulatory mechanisms of NLR expression under different environment.

The intracellular immune receptor like gene SNC1 is an enhancer of effector-triggered immunity in Arabidopsis.

Plants contain many nucleotide-binding leucine-rich repeat (NLR) proteins that are postulated to function as intracellular immune receptors but do not yet have an identified function during plant-pathogen interactions. SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1) is one such NLR protein of the Toll-interleukin 1 receptor (TIR) type, despite its well-characterized gain-of-function activity and its involvement in autoimmunity in Arabidopsis (Arabidopsis thaliana). Here, we investigated the role of SNC1 in natural plant-pathogen interactions and genetically tested the importance of the enzymatic activities of its TIR domain for its function. The SNC1 loss-of-function mutants were more susceptible to avirulent bacterial pathogen strains of Pseudomonas syringae containing specific effectors, especially under constant light growth condition. The mutants also had reduced defense gene expression induction and hypersensitive responses upon infection by avirulent pathogens under constant light growth condition. In addition, genetic and biochemical studies supported that the TIR enzymatic activity of SNC1 is required for its gain-of-function activity. In sum, our study uncovers the role of SNC1 as an amplifier of plant defense responses during natural plant-pathogen interactions and indicates its use of enzymatic activity and intermolecular interactions for triggering autoimmune responses.

Differential requirement of TIR enzymatic activities in TIR-type immune receptor SNC1-mediated immunity.

Arabidopsis thaliana TIR-type immune receptor SNC1 (Suppressor of npr1-1, constitutive 1) requires NADase, but not the 2′,3′-cAMP/cGMP synthetase activity to trigger in planta immune responses.

Integrated SRNA-Seq and RNA-Seq Analysis Reveals the Regulatory Roles of miRNAs in the Low-Temperature Responses of Canarium album

Chinese olive (Canarium album), a characteristic fruit tree in tropical and subtropical areas, suffers greatly from low-temperature stress (LTS). The regulatory roles of microRNA (miRNA) in plant LTS responses have been confirmed in many plant species but not in C. album. In this study, a cold-tolerant cultivar ‘Rui’an 3′ (RA) and a susceptible cultivar ‘Qinglan 1’ (QL) treated at 25 °C (control, CK) and −3 °C (cold temperature treatment, CT) were subjected to small RNA (sRNA) and transcriptome sequencing for the exploration of the cold responses of C. album. Comparative sRNA sequencing analysis identified much fewer LTS-responsive, differentially expressed miRNAs (DEMs) in RA (4 DEMs) than in QL (23 DEMs). Cal-miR482-22 was found to be specifically induced by LTS in RA. Cal-miR397-3 was upregulated, while cal-miR398_2-3 and cal-undef-190 were downregulated after LTS only in QL. However, when compared with QL, a higher basic expression of cal-miR397-3, and lower expression of cal-miR398_2-3 and cal-undef-190 were found in RA, suggesting that they may contribute to the cold tolerance of RA. Comparative transcriptome analysis showed that the number of LTS-responsive differentially expressed genes (DEGs) identified in QL was larger than that in RA, and some DEGs were also predicted as the target genes of the identified DEMs, forming multiple differentially expressed miRNA–target gene pairs, such as cal-miR397-3_laccase 2, 4, 17, cal-miR482-22_suppressor of npr1-1, etc. Quantitative real time PCR results showed that the expression changes of DEGs and DEMs in different samples were generally consistent with the sequencing results. Our study indicated that the basic expression levels of some miRNAs (especially the cal-miR397-3, cal-miR398_2-3, and cal-miR482-22), and their target genes contribute greatly to the cold-tolerance characteristics of C. album. Our study is helpful for understanding the roles of miRNAs in the cold resistance and responses of C. album.

Functional requirement of the Arabidopsis importin-α nuclear transport receptor family in autoimmunity mediated by the NLR protein SNC1.

IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.

SIZ1-Mediated SUMOylation of TPR1 Suppresses Plant Immunity in Arabidopsis

Plant immune responses are tightly regulated to ensure their appropriate deployment. Overexpression of TOPLESS-RELATED 1 (TPR1), a SUPPRESSOR OF npr1-1, CONSTITUTIVE 1 (SNC1)-interacting protein, results in autoimmunity that reduces plant growth and development. However, how TPR1 activity is regulated remains unknown. Loss of function of SIZ1, a (SUMO) E3 ligase, induces an autoimmune response, partially due to elevated SNC1 levels. Here we show that SNC1 expression is upregulated in Arabidopsis thaliana siz1-2 due to positive-feedback regulation by salicylic acid. SIZ1 physically interacts with TPR1 and facilitates its SUMO modification. The K282 and K721 residues in TPR1 serve as critical SUMO attachment sites. Simultaneous introduction of K282R and K721R substitutions in TPR1 blocked its SUMOylation, enhanced its transcriptional co-repressor activity, and increased its association with HISTONE DEACETYLASE 19 (HDA19), suggesting that SUMOylation of TPR1 represses its transcriptional co-repressor activity and inhibits its interaction with HDA19. In agreement with this finding, the simultaneous introduction of K282R and K721R substitutions enhanced TPR1-mediated immunity, and the tpr1 mutation partially suppressed autoimmunity in siz1-2. These results demonstrate that SIZ1-mediated SUMOylation of TPR1 represses plant immunity, which at least partly contributes to the suppression of autoimmunity under non-pathogenic conditions to ensure proper plant development.

Modulation of ACD6 dependent hyperimmunity by natural alleles of an Arabidopsis thaliana NLR resistance gene.

Plants defend themselves against pathogens by activating an array of immune responses. Unfortunately, immunity programs may also cause unintended collateral damage to the plant itself. The quantitative disease resistance gene ACCELERATED CELL DEATH 6 (ACD6) serves to balance growth and pathogen resistance in natural populations of Arabidopsis thaliana. An autoimmune allele, ACD6-Est, which strongly reduces growth under specific laboratory conditions, is found in over 10% of wild strains. There is, however, extensive variation in the strength of the autoimmune phenotype expressed by strains with an ACD6-Est allele, indicative of genetic modifiers. Quantitative genetic analysis suggests that ACD6 activity can be modulated in diverse ways, with different strains often carrying different large-effect modifiers. One modifier is SUPPRESSOR OF NPR1-1, CONSTITUTIVE 1 (SNC1), located in a highly polymorphic cluster of nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes, which are prototypes for qualitative disease resistance genes. Allelic variation at SNC1 correlates with ACD6-Est activity in multiple accessions, and a common structural variant affecting the NL linker sequence can explain differences in SNC1 activity. Taken together, we find that an NLR gene can mask the activity of an ACD6 autoimmune allele in natural A. thaliana populations, thereby linking different arms of the plant immune system.

Salicylic acid-mediated alleviation in NO2 phytotoxicity correlated to increased expression levels of the genes related to photosynthesis and carbon metabolism in Arabidopsis

Nitrogen dioxide (NO2) is one of the major air pollutants affecting plant growth, development and yields. Salicylic acid (SA)-mediated alleviation in NO2-induced plant damage has been reported in Arabidopsis. However, the involved mechanisms are far from clear. In this study, Arabidopsis wild type (WT) and its SA-altering mutants, snc1 (suppressor of npr1-1, constitutive 1) with high SA, npr1-1 (nonexpressor of PR gene) with SA signaling blockage, and transgenic line nahG with low SA, were used to decipher the SA action mechanisms. NO2 fumigation at a concentration of 10 ppm for 8 h led to more severe injuries on nahG and npr1-1 plants, whereas a lesser on snc1 plants compared with WT plants. The expressions of SA biosynthesis-related genes were up-regulated leading to more SA accumulations in all the tested plants after NO2 treatment. The expression patterns of ethylene, oxylipin and nitric oxide biosynthesis-related genes were regularly altered among NO2-treated genotypes, among which the expression of dioxygenase-encoding gene was dramatically increased in snc1 plants. Total chlorophyll contents, net photosynthetic rate, maximum quantum and actual quantum efficiency of PSII were decreased, to a greater degree in nahG and npr1-1 plants, whereas a lesser in snc1 plants compared with WT plants after NO2 treatment. We analyzed the expression patterns of the selected genes related to chlorophyll biosynthesis, photosystem I and II structure and functions, electron transport, ATP synthesis, Calvin cycle, as well as photorespiration, pentose phosphate pathway and starch degradation. The expression levels of the most genes were up-regulated in snc1 and WT plants, whereas down-regulated in nahG and npr1-1 plants after NO2 treatment, among which the expression pattern of a photorespiration-related gene was particularly specific. The plant tolerant phenotypes were positively correlated with the gene expressions, suggesting that SA-mediated alleviation in NO2 phytotoxicity was associated with photosynthesis and carbon metabolisms, also with other signal molecules.

BABA-Primed Histone Modifications in Potato for Intergenerational Resistance to Phytophthora infestans.

In this paper we analyzed β-aminobutyric acid (BABA)-primed epigenetic adjustment of potato cv. “Sarpo Mira” to Phytophthora infestans. The first stress-free generation of the potato genotype obtained from BABA-primed parent plants via tubers and seeds showed pronounced resistance to the pathogen, which was tuned with the transcriptional memory of SA-responsive genes. During the early priming phase before the triggering stress, we found robust bistable deposition of histone marks (H3K4me2 and H3K27me3) on the NPR1 (Non-expressor of PR genes) and the SNI1 gene (Suppressor of NPR1, Inducible), in which transcription antagonized silencing. Switchable chromatin states of these adverse systemic acquired resistance (SAR) regulators probably reprogrammed responsiveness of the PR1 and PR2 genes and contributed to stress imprinting. The elevated levels of heritable H3K4me2 tag in the absence of transcription on SA-dependent genes in BABA-primed (F0) and its vegetative and generative progeny (F1) before pathogen challenge provided evidence for the epigenetic mark for intergenerational memory in potato. Moreover, our study revealed that histone acetylation was not critical for maintaining BABA-primed defense information until the plants were triggered with the virulent pathogen when rapid and boosted PRs gene expression probably required histone acetyltransferase (HAT) activity both in F0 and F1 progeny.

MOS6 and TN13 in plant immunity

ABSTRACTThe Arabidopsis nuclear transport receptor IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for constitutive defense responses of the auto-immune mutant snc1 (suppressor of npr1-1, constitutive 1) and contributes to basal disease resistance, suggesting a role in nuclear import of defense-regulatory cargo proteins. We recently showed that MOS6 selectively interacts with TN13, a TIR-NBS protein involved in basal resistance to Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the effectors AvrPto and AvrPtoB. Consistent with a predicted N-terminal transmembrane domain, TN13 localizes to the endoplasmic reticulum (ER) and the nuclear envelope (NE) where it interacts with MOS6 in a transient expression assay. Here, we propose a model that summarizes the subcellular localization, association and function of TN13 and MOS6 in plant defense signaling.

DNA damage as a consequence of NLR activation

DNA damage observed during plant immune responses is reported to be an intrinsic component of plant immunity. However, other immune responses may suppress DNA damage to maintain host genome integrity. Here, we show that immunity-related DNA damage can be abrogated by preventing cell death triggered by Nucleotide-binding, Leucine-rich-repeat immune Receptors (NLRs). SNI1 (suppressor of npr1-1, inducible 1), a subunit of the structural maintenance of chromosome (SMC) 5/6 complex, was reported to be a negative regulator of systemic acquired resistance (SAR) and to be necessary for controlling DNA damage. We find that cell death and DNA damage in sni1 loss-of-function mutants are prevented by mutations in the NLR signaling component EDS1. Similar to sni1, elevated DNA damage is seen in other autoimmune mutants with cell death lesions, including camta3, pub13 and vad1, but not in dnd1, an autoimmune mutant with no visible cell death. We find that as in sni1, DNA damage in camta3 is EDS1-dependent, but that it is also NLR-dependent. Using the NLR RPM1 as a model, we also show that extensive DNA damage is observed when an NLR is directly triggered by effectors. We also find that the expression of DNA damage repair (DDR) genes in mutants with cell death lesions is down regulated, suggesting that degraded DNA that accumulates during cell death is a result of cellular dismantling and is not sensed as damaged DNA that calls for repair. Our observations also indicate that SNI1 is not directly involved in SAR or DNA damage accumulation.

Multiple biological processes involved in the regulation of salicylic acid in Arabidopsis response to NO2 exposure

Salicylic acid (SA) has been extensively implicated in plant response to abiotic stresses. However, just a few studies addressed the regulatory role of SA in plants exposed to NO2. In this study, we used Arabidopsis wild type (WT) and its SA-related mutants, including snc1 (suppressor of npr1-1, constitutive 1) with high endogenous SA, transgenic line nahG with low SA level, and npr1-1 (nonexpressor of PR gene) with SA signaling blockage to explore the possible regulation mechanism of SA, focusing on NO2-induced gene expression patterns. Plants were fumigated with 10 ppm NO2 for 8 h. The results showed that NO2 fumigation led to oxidative stress damage to all the tested plants as indicated by productions of H2O2, superoxide anion (O2−) and malondialdehyde, and cell death shown by electrolyte leakage and Evans Blue staining. However, snc1 plants were injured to a much less degree, whereas nahG and npr1-1 plants to a larger degree as compared with WT plants. Correspondingly, the activities of superoxide dismutase, peroxidase, catalase and nitrate reductase were induced by NO2 fumigation to a higher extent in snc1 plants, and a less extent in nahG and npr1-1 plants relative to WT plants. The expressions of the genes related to antioxidative defense, disulfide bond reduction, SA-responsive and –biosynthesis pathway, flavonoid biosynthesis, nitrate metabolism and kinase were analyzed. The results showed that these genes were differentially expressed among the tested genotypes under NO2 fumigation, wherein the most were significantly up-regulated in snc1 plants relative to other tested genotypes. Based on the gene expression patterns, it was proposed that SA-mediated plant tolerance to NO2 stress was associated with multiple biological processes.

The Arabidopsis Chromatin-Remodeling Factor CHR5 Regulates Plant Immune Responses and Nucleosome Occupancy.

ATP-dependent chromatin-remodeling factors use the energy of ATP hydrolysis to alter the structure of chromatin and are important regulators of eukaryotic gene expression. One such factor encoded by CHR5 (Chromatin-Remodeling Factor 5) in Arabidopsis (Arabidopsis thaliana) was previously found to be involved in regulation of growth and development. Here we show that CHR5 is required for the up-regulation of the intracellular immune receptor gene SNC1 (SUPPRESSOR OF npr1-1, CONSTITUTIVE1) and consequently the autoimmunity induced by SNC1 up-regulation. CHR5 functions antagonistically with another chromatin-remodeling gene DDM1 (DECREASED DNA METHYLATION 1) and independently with a histone mono-ubiquitinase HUB1 (HISTONE MONOUBIQUITINATION 1) in SNC1 regulation. In addition, CHR5 is a positive regulator of SNC1-independent plant immunity against the bacterial pathogen Pseudomonas syringae. Furthermore, the chr5 mutant has increased nucleosome occupancy in the promoter region relative to the gene body region at the whole-genome level, suggesting a global role for CHR5 in remodeling nucleosome occupancy. Our study thus establishes CHR5 as a positive regulator of plant immune responses including the expression of SNC1 and reveals a role for CHR5 in nucleosome occupancy which probably impacts gene expression genome wide.

The truncated NLR protein TIR-NBS13 is a MOS6/IMPORTIN-α3 interaction partner required for plant immunity.

Importin-α proteins mediate the translocation of nuclear localization signal (NLS)-containing proteins from the cytoplasm into the nucleus through nuclear pore complexes (NPCs). Genetically, Arabidopsis IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is required for basal plant immunity and constitutive disease resistance activated in autoimmune mutant snc1 (suppressor of npr1-1, constitutive 1), suggesting that MOS6 plays a role in the nuclear import of proteins involved in plant defense signaling. Here, we sought to identify and characterize defense-regulatory cargo proteins and interaction partners of MOS6. We conducted both in silico database analyses and affinity purification of functional epitope-tagged MOS6 from pathogen-challenged stable transgenic plants coupled with mass spectrometry. We show that among the 13 candidate MOS6 interactors we selected for further functional characterization, the TIR-NBS-type protein TN13 is required for resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 lacking the type-III effector proteins AvrPto and AvrPtoB. When expressed transiently in N. benthamiana leaves, TN13 co-immunoprecipitates with MOS6, but not with its closest homolog IMPORTIN-α6, and localizes to the endoplasmic reticulum (ER), consistent with a predicted N-terminal transmembrane domain in TN13. Our work uncovered the truncated NLR protein TN13 as a component of plant innate immunity that selectively binds to MOS6/IMPORTIN-α3 in planta. We speculate that the release of TN13 from the ER membrane in response to pathogen stimulus, and its subsequent nuclear translocation, is important for plant defense signal transduction.

A Role of Cytokinin Transporter in Arabidopsis Immunity.

The phytohormone cytokinin (CK) is not only essential for plant growth and development but also impacts plant immunity. A mutant screen in a constitutively active plant immune receptor mutant snc1 (suppressor of npr1, constitutive1) identified a suppressor mutation of SNC1-induced defense responses in an ABC transporter coding gene ABCG14. ABCG14 transports CK from roots to the shoots, and the suppression of the SNC1-mediated defense response by the loss of ABCG14 is due to a deficiency of trans-zeatin (tZ)-type CK in the shoot. In addition, exogenous application of the tZ-type CK enhances disease resistance associated with increased expression of the plant immune receptor gene SNC1. Taken together, this study further established the role of tZ-type CK in disease resistance and suggests a new intersection of CKs with plant immunity at the expression regulation of a plant immune receptor gene.

The putative kinase substrate MUSE7 negatively impacts the accumulation of NLR proteins.

Stringent modulation of immune signaling in plants is necessary to enable a rapid response to pathogen attack without spurious defense activation. To identify genes involved in plant immunity, a forward genetic screen for enhancers of the autoimmune snc1 (suppressor of npr1, constitutive 1) mutant was conducted. The snc1 mutant contains a gain-of-function mutation in a gene encoding a NOD-like receptor (NLR) protein. The isolated muse7 (mutant, snc1-enhancing, 7) mutant was shown to confer a reversion to autoimmune phenotypes in the wild-type-like mos4 (modifier of snc1, 4) snc1 background. Positional cloning revealed that MUSE7 encodes an evolutionarily conserved putative kinase substrate of unknown function. The muse7 single mutants display enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. While transcription of SNC1 is not enhanced, elevated SNC1 protein accumulation is associated with mutations in muse7. Accumulation of two additional NLR proteins, RPS2 (RESISTANCE TO PSEUDOMONAS SYRINGAE 2) and RPM1 (RESISTANCE TO PSEUDOMONAS SYRINGAE pv. MACULICOLA 1), was also observed in muse7 plants. Although proteasome-mediated degradation of NLR proteins is a well studied event in plant immunity, no interactions were detected between MUSE7 and selected components of this pathway. This study has demonstrated a role for MUSE7 in modulating plant immune responses through negatively affecting NLR accumulation, and will benefit future studies of MUSE7 homologs in other species.

Crystal structure of Arabidopsis thaliana SNC1 TIR domain

Plant immune response is initiated by Resistance proteins (R proteins). Toll/interleukin-1 receptor (TIR) domain in R proteins, which is responsible for the dimerization but has limited conservation in their primary structures. Suppressor of npr1-1, constitutive 1 (SNC1), a TIR-containing R protein, is involved in autoimmunity of plant, but the binding partner of SNC1 via the TIR domain and its specific cognate effector protein remain elusive. Here, we present the crystal structure of the TIR domain of Arabidopsis thaliana SNC1 (AtSNC1-TIR). The structure shows that AtSNC1-TIR domain is similar to those of other plant TIR domains including AtTIR, L6 and RPS4. Structural and sequence analysis on AtSNC1-TIR revealed that almost all conserved amino acids are located in the core of the structure, while the amino acids on the surface are highly variable, implicating that each TIR domain utilizes the variable surface for interacting its binding partner. In addition, the interaction between AtSNC1-TIR proteins in the crystal suggests two possible dimerization modes of AtSNC1-TIR domain. This study provides structural platform to investigate AtSNC1-TIR mediated signaling pathway of plant immune responses.

AtCDC48A is involved in the turnover of an NLR immune receptor.

Plants rely on different immune receptors to recognize pathogens and defend against pathogen attacks. Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play a major role as intracellular immune receptors. Their homeostasis must be maintained at optimal levels in order to effectively recognize pathogens without causing autoimmunity. Previous studies have shown that the activity of the ubiquitin-proteasome system is essential to prevent excessive accumulation of NLR proteins such as Suppressor of NPR1, Constitutive 1 (SNC1). Attenuation of the ubiquitin E3 ligase SCFCPR1 (Constitutive expressor of Pathogenesis Related genes 1) or the E4 protein MUSE3 (Mutant, SNC1-Enhancing 3) leads to NLR accumulation and autoimmunity. In the current study, we report the identification of AtCDC48A as a negative regulator of NLR-mediated immunity. Plants carrying Atcdc48A-4, a partial loss-of-function allele of AtCDC48A, exhibit dwarf morphology and enhanced disease resistance to the oomycete pathogen Hyaloperonospora arabidopsidis (H.a.) Noco2. The SNC1 level is increased in Atcdc48A-4 plants and AtCDC48A interacts with MUSE3 in co-immunoprecipitation experiments, supporting a role for AtCDC48A in NLR turnover. While Arabidopsis contains four other paralogs of AtCDC48A, knockout mutants of these genes do not show obvious immunity-related phenotypes, suggesting functional divergence within this family. As an AAA-ATPase, AtCDC48A likely serves to process the poly-ubiquitinated NLR substrate for final protein degradation by the 26S proteasome.

IBR5 Modulates Temperature-Dependent, R Protein CHS3-Mediated Defense Responses in Arabidopsis.

Plant responses to low temperature are tightly associated with defense responses. We previously characterized the chilling-sensitive mutant chs3-1 resulting from the activation of the Toll and interleukin 1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR)-type resistance (R) protein harboring a C-terminal LIM (Lin-11, Isl-1 and Mec-3 domains) domain. Here we report the identification of a suppressor of chs3, ibr5-7 (indole-3-butyric acid response 5), which largely suppresses chilling-activated defense responses. IBR5 encodes a putative dual-specificity protein phosphatase. The accumulation of CHS3 protein at chilling temperatures is inhibited by the IBR5 mutation. Moreover, chs3-conferred defense phenotypes were synergistically suppressed by mutations in HSP90 and IBR5. Further analysis showed that IBR5, with holdase activity, physically associates with CHS3, HSP90 and SGT1b (Suppressor of the G2 allele of skp1) to form a complex that protects CHS3. In addition to the positive role of IBR5 in regulating CHS3, IBR5 is also involved in defense responses mediated by R genes, including SNC1 (Suppressor of npr1-1, Constitutive 1), RPS4 (Resistance to P. syringae 4) and RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1). Thus, the results of the present study reveal a role for IBR5 in the regulation of multiple R protein-mediated defense responses.

The Chromatin Remodeler SPLAYED Negatively Regulates SNC1-Mediated Immunity.

SNC1 (SUPPRESSOR OF NPR1, CONSTITUTIVE 1) is one of a suite of intracellular Arabidopsis NOD-like receptor (NLR) proteins which, upon activation, result in the induction of defense responses. However, the molecular mechanisms underlying NLR activation and the subsequent provocation of immune responses are only partially characterized. To identify negative regulators of NLR-mediated immunity, a forward genetic screen was undertaken to search for enhancers of the dwarf, autoimmune gain-of-function snc1 mutant. To avoid lethality resulting from severe dwarfism, the screen was conducted using mos4 (modifier of snc1, 4) snc1 plants, which display wild-type-like morphology and resistance. M2 progeny were screened for mutant, snc1-enhancing (muse) mutants displaying a reversion to snc1-like phenotypes. The muse9 mos4 snc1 triple mutant was found to exhibit dwarf morphology, elevated expression of the pPR2-GUS defense marker reporter gene and enhanced resistance to the oomycete pathogen Hyaloperonospora arabidopsidis Noco2. Via map-based cloning and Illumina sequencing, it was determined that the muse9 mutation is in the gene encoding the SWI/SNF chromatin remodeler SYD (SPLAYED), and was thus renamed syd-10. The syd-10 single mutant has no observable alteration from wild-type-like resistance, although the syd-4 T-DNA insertion allele displays enhanced resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Transcription of SNC1 is increased in both syd-4 and syd-10. These data suggest that SYD plays a subtle, specific role in the regulation of SNC1 expression and SNC1-mediated immunity. SYD may work with other proteins at the chromatin level to repress SNC1 transcription; such regulation is important for fine-tuning the expression of NLR-encoding genes to prevent unpropitious autoimmunity.