Two Redundant Receptor-Like Cytoplasmic Kinases Function Downstream of Pattern Recognition Receptors to Regulate Activation of SA Biosynthesis.

Salicylic acid (SA) serves as a critical signaling molecule in plant defense. Two transcription factors, SARD1 and CBP60g, control SA biosynthesis through regulating pathogen-induced expression of Isochorismate Synthase1, which encodes a key enzyme for SA biosynthesis. Here, we report that Pattern-Triggered Immunity Compromised Receptor-like Cytoplasmic Kinase1 (PCRK1) and PCRK2 function as key regulators of SA biosynthesis. In the pcrk1 pcrk2 double mutant, pathogen-induced expression of SARD1, CBP60g, and ICS1 is greatly reduced. The pcrk1 pcrk2 double mutant, but neither of the single mutants, exhibits reduced accumulation of SA and enhanced disease susceptibility to bacterial pathogens. Both PCRK1 and PCRK2 interact with the pattern recognition receptor FLS2, and treatment with pathogen-associated molecular patterns leads to rapid phosphorylation of PCRK2. Our data suggest that PCRK1 and PCRK2 function downstream of pattern recognition receptor in a signal relay leading to the activation of SA biosynthesis.

NLR-associating transcription factor bHLH84 and its paralogs function redundantly in plant immunity.

In plants and animals, nucleotide-binding and leucine-rich repeat domain containing (NLR) immune receptors are utilized to detect the presence or activities of pathogen-derived molecules. However, the mechanisms by which NLR proteins induce defense responses remain unclear. Here, we report the characterization of one basic Helix-loop-Helix (bHLH) type transcription factor (TF), bHLH84, identified from a reverse genetic screen. It functions as a transcriptional activator that enhances the autoimmunity of NLR mutant snc1 (suppressor of npr1-1, constitutive 1) and confers enhanced immunity in wild-type backgrounds when overexpressed. Simultaneously knocking out three closely related bHLH paralogs attenuates RPS4-mediated immunity and partially suppresses the autoimmune phenotypes of snc1, while overexpression of the other two close paralogs also renders strong autoimmunity, suggesting functional redundancy in the gene family. Intriguingly, the autoimmunity conferred by bHLH84 overexpression can be largely suppressed by the loss-of-function snc1-r1 mutation, suggesting that SNC1 is required for its proper function. In planta co-immunoprecipitation revealed interactions between not only bHLH84 and SNC1, but also bHLH84 and RPS4, indicating that bHLH84 associates with these NLRs. Together with previous finding that SNC1 associates with repressor TPR1 to repress negative regulators, we hypothesize that nuclear NLR proteins may interact with both transcriptional repressors and activators during immune responses, enabling potentially faster and more robust transcriptional reprogramming upon pathogen recognition.

Regulation of transcription of nucleotide-binding leucine-rich repeat-encoding genes SNC1 and RPP4 via H3K4 trimethylation.

Plant nucleotide-binding leucine-rich repeat (NB-LRR) proteins serve as intracellular sensors to detect pathogen effectors and trigger immune responses. Transcription of the NB-LRR-encoding Resistance (R) genes needs to be tightly controlled to avoid inappropriate defense activation. How the expression of the NB-LRR R genes is regulated is poorly understood. The Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive 1 (snc1) mutant carries a gain-of-function mutation in a Toll/Interleukin1 receptor-like (TIR)-NB-LRR-encoding gene, resulting in the constitutive activation of plant defense responses. A snc1 suppressor screen identified modifier of snc1,9 (mos9), which partially suppresses the autoimmune phenotypes of snc1. Positional cloning revealed that MOS9 encodes a plant-specific protein of unknown function. Expression analysis showed that MOS9 is required for the full expression of TIR-NB-LRR protein-encoding RECOGNITION OF PERONOSPORA PARASITICA 4 (RPP4) and SNC1, both of which reside in the RPP4 cluster. Coimmunoprecipitation and mass spectrometry analyses revealed that MOS9 associates with the Set1 class lysine 4 of histone 3 (H3K4) methyltransferase Arabidopsis Trithorax-Related7 (ATXR7). Like MOS9, ATXR7 is also required for the full expression of SNC1 and the autoimmune phenotypes in the snc1 mutant. In atxr7 mutant plants, the expression of RPP4 is similarly reduced, and resistance against Hyaloperonospora arabidopsidis Emwa1 is compromised. Consistent with the attenuated expression of SNC1 and RPP4, trimethylated H3K4 marks are reduced around the promoters of SNC1 and RPP4 in mos9 plants. Our data suggest that MOS9 functions together with ATXR7 to regulate the expression of SNC1 and RPP4 through H3K4 methylation, which plays an important role in fine-tuning their transcription levels and functions in plant defense.

Stability of plant immune-receptor resistance proteins is controlled by SKP1-CULLIN1-F-box (SCF)-mediated protein degradation.

The nucleotide-binding domain and leucine-rich repeats containing proteins (NLRs) serve as immune receptors in both plants and animals. Overaccumulation of NLRs often leads to autoimmune responses, suggesting that the levels of these immune receptors must be tightly controlled. However, the mechanism by which NLR protein levels are regulated is unknown. Here we report that the F-box protein CPR1 controls the stability of plant NLR resistance proteins. Loss-of-function mutations in CPR1 lead to higher accumulation of the NLR proteins SNC1 and RPS2, as well as autoactivation of immune responses. The autoimmune responses in cpr1 mutant plants can be largely suppressed by knocking out SNC1. Furthermore, CPR1 interacts with SNC1 and RPS2 in vivo, and overexpressing CPR1 results in reduced accumulation of SNC1 and RPS2, as well as suppression of immunity mediated by these two NLR proteins. Our data suggest that SKP1-CULLIN1-F-box (SCF) complex-mediated stability control of plant NLR proteins plays an important role in regulating their protein levels and preventing autoimmunity.

Roles of microbiota in autoimmunity in Arabidopsis leaves.

Over the past three decades, researchers have isolated plant mutants that show constitutively activated defence responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. Here, from a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes an S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high-humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, showing lesions, and having a high basal level of defence gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several 'lesion mimic'-type autoimmune mutants in Arabidopsis. It is worth noting that autoimmune phenotypes caused by mutations in two Nucleotide-Binding, Leucine-Rich Repeat (NLR) genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of at least two classes of autoimmunity (microbiota-dependent versus microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom. These parallels highlight the intricate relationship between host immunity and microbial communities across various biological systems.

Putative members of the Arabidopsis Nup107-160 nuclear pore sub-complex contribute to pathogen defense.

In eukaryotic cells, transduction of external stimuli into the nucleus to induce transcription and export of mRNAs for translation in the cytoplasm is mediated by nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups). We previously reported that Arabidopsis MOS3, encoding the homolog of vertebrate Nup96, is required for plant immunity and constitutive resistance mediated by the de-regulated Toll interleukin 1 receptor/nucleotide-binding/leucine-rich repeat (TNL)-type R gene snc1. In vertebrates, Nup96 is a component of the conserved Nup107-160 nuclear pore sub-complex, and implicated in immunity-related mRNA export. Here, we used a reverse genetics approach to examine the requirement for additional subunits of the predicted Arabidopsis Nup107-160 complex in plant immunity. We show that, among eight putative complex members, beside MOS3, only plants with defects in Nup160 or Seh1 are impaired in basal resistance. Constitutive resistance in the snc1 mutant and immunity mediated by TNL-type R genes also depend on functional Nup160 and have a partial requirement for Seh1. Conversely, resistance conferred by coiled coil-type immune receptors operates largely independently of both genes, demonstrating specific contributions to plant defense signaling. Our functional analysis further revealed that defects in nup160 and seh1 result in nuclear accumulation of poly(A) mRNA, and, in the case of nup160, considerable depletion of EDS1, a key positive regulator of basal and TNL-triggered resistance. These findings suggest that Nup160 is required for nuclear mRNA export and full expression of EDS1-conditioned resistance pathways in Arabidopsis.

Arabidopsis resistance protein SNC1 activates immune responses through association with a transcriptional corepressor.

In both plants and animals, nucleotide-binding (NB) domain and leucine-rich repeat (LRR)-containing proteins (NLR) function as sensors of pathogen-derived molecules and trigger immune responses. Although NLR resistance (R) proteins were first reported as plant immune receptors more than 15 years ago, how these proteins activate downstream defense responses is still unclear. Here we report that the Toll-like/interleukin-1 receptor (TIR)-NB-LRR R protein, suppressor of npr1-1, constitutive 1 (SNC1) functions through its associated protein, Topless-related 1 (TPR1). Knocking out TPR1 and its close homologs compromises immunity mediated by SNC1 and several other TIR-NB-LRR-type R proteins, whereas overexpression of TPR1 constitutively activates SNC1-mediated immune responses. TPR1 functions as a transcriptional corepressor and associates with histone deacetylase 19 in vivo. Among the target genes of TPR1 are Defense no Death 1 (DND1) and Defense no Death 2 (DND2), two known negative regulators of immunity that are repressed during pathogen infection, suggesting that TPR1 activates R protein-mediated immune responses through repression of negative regulators.

Control of salicylic acid synthesis and systemic acquired resistance by two members of a plant-specific family of transcription factors.

Salicylic acid (SA) is a defense hormone required for both local and systemic acquired resistance (SAR) in plants. Pathogen infections induce SA synthesis through up-regulating the expression of Isochorismate Synthase 1 (ICS1), which encodes a key enzyme in SA production. Here we report that both SAR Deficient 1 (SARD1) and CBP60g are key regulators for ICS1 induction and SA synthesis. Whereas knocking out SARD1 compromises basal resistance and SAR, overexpression of SARD1 constitutively activates defense responses. In the sard1-1 cbp60g-1 double mutant, pathogen-induced ICS1 up-regulation and SA synthesis are blocked in both local and systemic leaves, resulting in compromised basal resistance and loss of SAR. Electrophoretic mobility shift assays showed that SARD1 and CBP60g represent a plant-specific family of DNA-binding proteins. Both proteins are recruited to the promoter of ICS1 in response to pathogen infections, suggesting that they control SA synthesis by regulating ICS1 at the transcriptional level.

MOS11: a new component in the mRNA export pathway.

Nucleocytoplasmic trafficking is emerging as an important aspect of plant immunity. The three related pathways affecting plant immunity include Nuclear Localization Signal (NLS)-mediated nuclear protein import, Nuclear Export Signal (NES)-dependent nuclear protein export, and mRNA export relying on MOS3, a nucleoporin belonging to the Nup107-160 complex. Here we report the characterization, identification, and detailed analysis of Arabidopsis modifier of snc1, 11 (mos11). Mutations in MOS11 can partially suppress the dwarfism and enhanced disease resistance phenotypes of snc1, which carries a gain-of-function mutation in a TIR-NB-LRR type Resistance gene. MOS11 encodes a conserved eukaryotic protein with homology to the human RNA binding protein CIP29. Further functional analysis shows that MOS11 localizes to the nucleus and that the mos11 mutants accumulate more poly(A) mRNAs in the nucleus, likely resulting from reduced mRNA export activity. Epistasis analysis between mos3-1 and mos11-1 revealed that MOS11 probably functions in the same mRNA export pathway as MOS3, in a partially overlapping fashion, before the mRNA molecules pass through the nuclear pores. Taken together, MOS11 is identified as a new protein contributing to the transfer of mature mRNA from the nucleus to the cytosol.

Roles of microbiota in autoimmunity in Arabidopsis.

Over the past three decades, researchers have isolated plant mutants that display constitutively activated defense responses in the absence of pathogen infection. These mutants are called autoimmune mutants and are typically dwarf and/or bearing chlorotic/necrotic lesions. From a genetic screen for Arabidopsis genes involved in maintaining a normal leaf microbiota, we identified TIP GROWTH DEFECTIVE 1 (TIP1), which encodes a S-acyltransferase, as a key player in guarding leaves against abnormal microbiota level and composition under high humidity conditions. The tip1 mutant has several characteristic phenotypes of classical autoimmune mutants, including a dwarf stature, displaying lesions, and having a high basal level of defense gene expression. Gnotobiotic experiments revealed that the autoimmune phenotypes of the tip1 mutant are largely dependent on the presence of microbiota as axenic tip1 plants have markedly reduced autoimmune phenotypes. We found that the microbiota dependency of autoimmune phenotypes is shared by several "lesion mimic"-type autoimmune mutants in Arabidopsis. Interestingly, autoimmune phenotypes caused by mutations in NLR genes do not require the presence of microbiota and can even be partially alleviated by microbiota. Our results therefore suggest the existence of two classes of autoimmunity (microbiota-dependent vs. microbiota-independent) in plants. The observed interplay between autoimmunity and microbiota in the lesion mimic class of autoimmunity is reminiscent of the interactions between autoimmunity and dysbiosis in the animal kingdom.

A critical role of a eubiotic microbiota in gating proper immunocompetence in Arabidopsis.

Although many studies have shown that microbes can ectopically stimulate or suppress plant immune responses, the fundamental question of whether the entire preexisting microbiota is indeed required for proper development of plant immune response remains unanswered. Using a recently developed peat-based gnotobiotic plant growth system, we found that Arabidopsis grown in the absence of a natural microbiota lacked age-dependent maturation of plant immune response and were defective in several aspects of pattern-triggered immunity. Axenic plants exhibited hypersusceptibility to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and the fungal pathogen Botrytis cinerea. Microbiota-mediated immunocompetence was suppressed by rich nutrient conditions, indicating a tripartite interaction between the host, microbiota and abiotic environment. A synthetic microbiota composed of 48 culturable bacterial strains from the leaf endosphere of healthy Arabidopsis plants was able to substantially restore immunocompetence similar to plants inoculated with a soil-derived community. In contrast, a 52-member dysbiotic synthetic leaf microbiota overstimulated the immune transcriptome. Together, these results provide evidence for a causal role of a eubiotic microbiota in gating proper immunocompetence and age-dependent immunity in plants.

Arabidopsis clade I TGA transcription factors regulate plant defenses in an NPR1-independent fashion.

Transcriptional reprogramming during induction of salicylic acid (SA)-mediated defenses is regulated primarily by NPR1 (NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1), likely through interactions with TGA bZIP transcription factors. To ascertain the contributions of clade I TGA factors (TGA1 and TGA4) to defense responses, a tga1-1 tga4-1 double mutant was constructed and challenged with Pseudomonas syringae and Hyaloperonospora arabidopsidis. Although the mutant displayed enhanced susceptibility to virulent P. syringae, it was not compromised in systemic acquired resistance against this pathogen or resistance against avirulent H. arabidopsidis. Microarray analysis of nonelicited and SA-treated plants indicated that clade I TGA factors regulate fewer genes than NPR1. Approximately half of TGA-dependent genes were regulated by NPR1 but, in all cases, the direction of change was opposite in the two mutants. In support of the microarray data, the NPR1-independent disease resistance observed in the autoimmune resistance (R) gene mutant snc1 is partly compromised by tga1-1 tga4-1 mutations, and a triple mutant of clade I TGA factors with npr1-1 is more susceptible than either parent. These results suggest that clade I TGA factors are required for resistance against virulent pathogens and avirulent pathogens mediated by at least some R gene specificities, acting substantially through NPR1-independent pathways.

SRFR1 negatively regulates plant NB-LRR resistance protein accumulation to prevent autoimmunity.

Plant defense responses need to be tightly regulated to prevent auto-immunity, which is detrimental to growth and development. To identify negative regulators of Resistance (R) protein-mediated resistance, we screened for mutants with constitutive defense responses in the npr1-1 background. Map-based cloning revealed that one of the mutant genes encodes a conserved TPR domain-containing protein previously known as SRFR1 (SUPPRESSOR OF rps4-RLD). The constitutive defense responses in the srfr1 mutants in Col-0 background are suppressed by mutations in SNC1, which encodes a TIR-NB-LRR (Toll Interleukin1 Receptor-Nucleotide Binding-Leu-Rich Repeat) R protein. Yeast two-hybrid screens identified SGT1a and SGT1b as interacting proteins of SRFR1. The interactions between SGT1 and SRFR1 were further confirmed by co-immunoprecipitation analysis. In srfr1 mutants, levels of multiple NB-LRR R proteins including SNC1, RPS2 and RPS4 are increased. Increased accumulation of SNC1 is also observed in the sgt1b mutant. Our data suggest that SRFR1 functions together with SGT1 to negatively regulate R protein accumulation, which is required for preventing auto-activation of plant immunity.

Activation of plant immune responses by a gain-of-function mutation in an atypical receptor-like kinase.

Arabidopsis (Arabidopsis thaliana) suppressor of npr1-1, constitutive1 (snc1) contains a gain-of-function mutation in a Toll/interleukin receptor-nucleotide binding site-leucine-rich repeat Resistance (R) protein and it has been a useful tool for dissecting R-protein-mediated immunity. Here we report the identification and characterization of snc4-1D, a semidominant mutant with snc1-like phenotypes. snc4-1D constitutively expresses defense marker genes PR1, PR2, and PDF1.2, and displays enhanced pathogen resistance. Map-based cloning of SNC4 revealed that it encodes an atypical receptor-like kinase with two predicted extracellular glycerophosphoryl diester phosphodiesterase domains. The snc4-1D mutation changes an alanine to threonine in the predicted cytoplasmic kinase domain. Wild-type plants transformed with the mutant snc4-1D gene displayed similar phenotypes as snc4-1D, suggesting that the mutation is a gain-of-function mutation. Epistasis analysis showed that NON-RACE-SPECIFIC DISEASE RESISTANCE1 is required for the snc4-1D mutant phenotypes. In addition, the snc4-1D mutant phenotypes are partially suppressed by knocking out MAP KINASE SUBSTRATE1, a positive defense regulator associated with MAP KINASE4. Furthermore, both the morphology and constitutive pathogen resistance of snc4-1D are partially suppressed by blocking jasmonic acid synthesis, suggesting that jasmonic acid plays an important role in snc4-1D-mediated resistance. Identification of snc4-1D provides us a unique genetic system for analyzing the signal transduction pathways downstream of receptor-like kinases.

Plant-Microbe Interactions Facing Environmental Challenge.

In the past four decades, tremendous progress has been made in understanding how plants respond to microbial colonization and how microbial pathogens and symbionts reprogram plant cellular processes. In contrast, our knowledge of how environmental conditions impact plant-microbe interactions is less understood at the mechanistic level, as most molecular studies are performed under simple and static laboratory conditions. In this review, we highlight research that begins to shed light on the mechanisms by which environmental conditions influence diverse plant-pathogen, plant-symbiont, and plant-microbiota interactions. There is a great need to increase efforts in this important area of research in order to reach a systems-level understanding of plant-microbe interactions that are more reflective of what occurs in nature.

MOS2, a protein containing G-patch and KOW motifs, is essential for innate immunity in Arabidopsis thaliana.

Innate immunity is critical for sensing and defending against microbial infections in multicellular organisms. In plants, disease resistance genes (R genes) play central roles in recognizing pathogens and initiating downstream defense cascades. Arabidopsis SNC1 encodes a TIR-NBS-LRR-type R protein with a similar structure to nucleotide binding oligomerization domain (Nod) proteins in animals. A point mutation in the region between the NBS and LRR of SNC1 results in constitutive activation of defense responses in the snc1 mutant. Here, we report the identification and characterization of mos2-1, a mutant suppressing the constitutive defense responses in snc1. Analysis of mos2 single mutants indicated that it is not only required for resistance specified by multiple R genes, but also for basal resistance. Map-based cloning of MOS2 revealed that it encodes a novel nuclear protein that contains one G-patch and two KOW domains and has homologs across the animal kingdom. The presence of both G-patch and KOW domains in the MOS2 protein suggests that it probably functions as an RNA binding protein critical for plant innate immunity. Our discovery on the biological functions of MOS2 will shed light on functions of the MOS2 homologs in animals, where they may also play important roles in innate immunity.

Ubiquitination in NB-LRR-mediated immunity.

As a common protein modification, ubiquitination is used for regulating the fate of protein targets, notably in terms of stability. In recent years, it has emerged to play key roles in the regulation of plant defense responses. Given its flexibility and critical roles in signaling, primarily in the control of protein turnover, ubiquitination is probably targeting many major immune regulators for modification or degradation. In this review, we summarize the latest findings on how different components of the ubiquitination pathway are involved in NB-LRR R protein-mediated immunity.

Nucleoporin MOS7/Nup88 contributes to plant immunity and nuclear accumulation of defense regulators.

Controlled nucleocytoplasmic trafficking is an important feature for fine-tuning signaling pathways in eukaryotic organisms. Nuclear pore complexes (NPCs) composed of nucleoporin proteins (Nups) are essential for the exchange of macromolecules across the nuclear envelope. A recent genetic screen in our laboratory identified a partial loss-of-function mutation in Arabidopsis MOS7/Nup88 that causes defects in basal immunity, Resistance (R) protein-mediated defense and systemic acquired resistance. In Drosophila and mammalian cells, exportin-mediated nuclear export of activated Rel/NFκB transcription factors is enhanced in nup88 mutants resulting in immune response failure. Consistent with Nup88 promoting nuclear retention of NFκB, our functional analyses revealed that MOS7/Nup88 is required for appropriate nuclear accumulation of the autoactivated R protein snc1, as well as the key immune regulators EDS1 and NPR1. These results suggest that controlling the nuclear concentrations of specific immune regulators is fundamental for defining defense outputs.

Regulation of cell death and innate immunity by two receptor-like kinases in Arabidopsis.

Upon recognition of bacterial flagellin, the plant receptor FLS2 heterodimerizes with brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and activates plant defense responses. Because constitutive activation of defense responses is detrimental, plant resistance signaling pathways must be negatively controlled, although the mechanisms involved are unclear. We identified Arabidopsis BIR1 as a BAK1-interacting receptor-like kinase. Knocking out BIR1 leads to extensive cell death, activation of constitutive defense responses, and impairment in the activation of MPK4, a negative regulator of plant resistance (R) protein signaling, by flagellin. sobir1-1, a mutant obtained in a screen for suppressors of the bir1-1 phenotype, rescued cell death observed in bir1-1. SOBIR1 encodes another receptor-like kinase whose overexpression activates cell death and defense responses. Our data suggest that BIR1 negatively regulates multiple plant resistance signaling pathways, one of which is the SOBIR1-dependent pathway identified here.