RSI1/FLD and its epigenetic target RRTF1 are essential for the retention of infection memory in Arabidopsis thaliana.

Plants can retain a memory of previous pathogen infections to mount a more robust defense response during subsequent infections by developing systemic acquired resistance (SAR). However, the mechanism through which plants develop and retain infection memory is not known. Experiments have shown the association of epigenetic modifications of specific defense-related genes with SAR. RSI1/FLD codes for a histone demethylase and is required for the activation of SAR in Arabidopsis. Here we report the identification of RRTF1 as an epigenetic target of RSI1. RRTF1 expression is higher in pathogen-free distal tissues of the rsi1 mutant. Experiments with loss-of-function and overexpression lines suggest RRTF1 is a negative regulator of basal defense against virulent and avirulent pathogens as well as SAR. Enhanced expression of RRTF1 in a wild-type (WT) background specifically impairs SAR without impacting local resistance. RSI1 is recruited at the RRTF1 locus in a SAR-inducible manner and contributes to H3K4me2 and H3K4me3 demethylation. Introduction of the rrtf1 mutation rescues the loss-of-SAR phenotype of rsi1 plants. However, these plants fail to retain infection memory beyond 7 days post-primary inoculation, whereas WT plants retain memory for at least 11 days. Our results demonstrate that RSI1 and RRTF1 form a functional module for retaining infection memory in Arabidopsis.

POWERDRESS positively regulates systemic acquired resistance in Arabidopsis.

KEY MESSAGE: PWR, an epigenetic regulator, and PIF4, a transcription factor coordinately regulate both local resistance and systemic acquired resistance in Arabidopsis. A plant that gets infected once becomes resistant to subsequent infections through the development of systemic acquired resistance (SAR). Primary-infected tissues generate mobile signals that travel to systemic tissues and cause epigenetic changes associated with the SAR activation. Epigenetic regulators and the process of infection memory development are largely obscure for plants. POWERDRESS (PWR), a SANT domain-containing histone deacetylation (HDAC) promoting gene, is essential for thermomorphogenesis. Here we show that PWR is required for the SAR activation in Arabidopsis. The pwr mutants in Ler and Col-0 background possess normal local resistance but are defective in SAR. PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) genetically interacts with PWR for flowering and thermomorphogenesis and is a negative regulator of basal immunity. We found a cooperative function for suppressing basal immunity and SAR activation by PIF4 and PWR, respectively. PWR promotes the expression of SA biosynthesis genes and the accumulation of SA in the systemic tissues. RSI1/FLD, which influences histone methylation and acetylation, is essential to infection memory development in Arabidopsis. Our results show that PWR and RSI1 positively regulate each other's expression. Exogenous application of HDAC inhibitor sodium butyrate abolishes SAR-mediated SA accumulation, expression of PR1 gene, and protection against pathogens after challenge inoculation. The results indicate the possibility of the involvement of HDAC activity of PWR in the formation of infection memory development in Arabidopsis.

MTO1-RESPONDING DOWN 1 (MRD1) is a transcriptional target of OZF1 for promoting salicylic acid-mediated defense in Arabidopsis.

KEY MESSAGE: OZF1 promotes the transcription of MRD1, which is essential for SA-mediated defense against virulent and avirulent bacterial pathogens in Arabidopsis. Salicylic acid (SA) is critical for defense against biotrophic pathogens. A trans-activator protein NPR1 plays significant roles in SA-signaling. However, evidences suggest the existence of NPR1-independent pathways for SA signaling in plants. Previously, we reported Arabidopsis OXIDATION-RELATED ZN-FINGER PROTEIN1 (OZF1) as a positive regulator of NPR1-independent SA-signaling. However, the mechanism or components of OZF1-mediated SA signaling was not known. Through the analysis of differentially expressing genes, we report the identification of MTO1-RESPONDING DOWN 1 (MRD1) as a transcriptional target of OZF1. Expressions of MRD1 and its overlapping gene in Arabidopsis genome, HEI10 increase upon pathogen inoculation in an OZF1-dependent manner. Their mutants are susceptible to both virulent and avirulent bacterial pathogens and show compromised SA-mediated immunity. Overexpression of MRD1 but not the HEI10 rescues the loss-of-resistance phenotype of the ozf1 mutant. OZF1 physically associates at the MRD1 promoter area upon pathogen inoculation. Results altogether support that MRD1 is a transcriptional target of OZF1 for promoting SA-mediated defense in Arabidopsis.

TOPLESS in the regulation of plant immunity.

KEY MESSAGE: This review presents the multiple ways how topless and topless-related proteins regulate defense activation in plants and help in optimizing the defense-growth tradeoff. Eukaryotic gene expression is tightly regulated at various levels by hormones, transcription regulators, post-translational modifications, and transcriptional coregulators. TOPLESS (TPL)/TOPLESS-related (TPR) corepressors regulate gene expression by interacting with other transcription factors. TPRs regulate auxin, gibberellins, jasmonic acid, strigolactone, and brassinosteroid signaling in plants. In general, except for GA, TPLs suppress these signaling pathways to prevent unwanted activation of hormone signaling. The association of TPL/TPRs in these hormonal signaling reflects a wide role of this class of corepressors in plants' normal and stress physiology. The involvement of TPL in immune responses was first demonstrated a decade ago as a repressor of DND1 and DND2 that are negative regulators of plant immune response. Over the last decade, several research groups have established a larger role of TPL/TPRs in plant immunity during both pattern- and effector-triggered immunity. Very recent research unraveled the significant involvement of TPRs in balancing the growth and defense trade-off. TPRs, along with proteasomal degradation complex, miRNA, and phasiRNA, suppress the activation of autoimmunity in plants under normal conditions and promote defense under pathogen attack.

AtOZF1 positively regulates JA signaling and SA-JA cross-talk in Arabidopsis thaliana.

Plant hormones regulate growth, development, and defense against biotic and abiotic stresses. Salicylic acid (SA), ethylene (ET), and jasmonate (JA) are major phytohormones that control the defense against pathogens. SA and JA primarily regulate resistance against biotrophic and necrotrophic pathogens, respectively. NPR1 is the key regulator of SA signaling in plants. AtOZF1 function has recently been ascribed to promote both NPR1- dependent and -independent SA signaling. However, the role of AtOZF1 in JA signaling was not known. Here we report AtOZF1 as a positive regulator of JA signaling in Arabidopsis. The atozf1 mutants are more susceptible to the necrotrophic pathogen Botrytis cinerea than wildtype (WT) plants. AtOZF1 positively regulates the expression of JA inducible genes like PDF1.2, VSP2, THI2.1, and ORA59. AtOZF1 takes part in SA-JA cross-talk to an extent similar to that of NPR1. AtOZF1 is essential for the activation of PDF1.2 expression upon exogenous methyl-jasmonate (MeJA) application. Intriguingly, SA can significantly promote MeJA-induced PDF1.2 expression in the absence of AtOZF1. Altogether our results reveal a novel SA-JA interaction pathway in plants.

MYC2 influences salicylic acid biosynthesis and defense against bacterial pathogens in Arabidopsis thaliana.

Arabidopsis MYC2 is a basic helix-loop-helix transcription factor that works both as a negative and positive regulator of light and multiple hormonal signaling pathways, including jasmonic acid and abscisic acid. Recent studies have suggested the role of MYC2 as a negative regulator of salicylic acid (SA)-mediated defense against bacterial pathogens. By using myc2 mutant and constitutively MYC2-expressing plants, we further show that MYC2 also positively influences SA-mediated defense; whereas, myc2 mutant plants are resistant to virulent pathogens only, MYC2 over-expressing plants are hyper-resistant to multiple virulent and avirulent strains of bacterial pathogens. MYC2 promotes pathogen-induced callose deposition, SA biosynthesis, expression of PR1 gene, and SA-responsiveness. Using bacterially produced MYC2 protein in electrophoretic mobility shift assay (EMSA), we have shown that MYC2 binds to the promoter of several important defense regulators, including PEPR1, MKK4, RIN4, and the second intron of ICS1. MYC2 positively regulates the expression of RIN4, MKK4, and ICS1; however, it negatively regulates the expression of PEPR1. Pathogen inoculation enhances MYC2 association at ICS1 intron and RIN4 promoter. Mutations of MYC2 binding site at ICS1 intron or RIN4 promoter abolish the associated GUS reporter expression. Hyper-resistance of MYC2 over-expressing plants is largely light-dependent, which is in agreement with the role of MYC2 in SA biosynthesis. The results altogether demonstrate that MYC2 possesses dual regulatory roles in SA biosynthesis, SA signaling, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) in Arabidopsis.

Recent advances in plant thermomemory.

KEY MESSAGE: This review summarizes the process of thermal acquired tolerance in plants and the knowledge gap compared to systemic acquired resistance that a plant shows after pathogen inoculation. Plants are continuously challenged by several biotic stresses such as pests and pathogens, or abiotic stresses like high light, UV radiation, drought, salt, and very high or low temperature. Interestingly, for most stresses, prior exposure makes plants more tolerant during the subsequent exposures, which is often referred to as acclimatization. Research of the last two decades reveals that the memory of most of the stresses is associated with epigenetic changes. Heat stress causes damage to membrane proteins, denaturation and inactivation of various enzymes, and accumulation of reactive oxygen species leading to cell injury and death. Plants are equipped with thermosensors that can recognize certain specific changes and activate protection machinery. Phytochrome and calcium signaling play critical roles in sensing sudden changes in temperature and activate cascades of signaling, leading to the production of heat shock proteins (HSPs) that keep protein-unfolding under control. Heat shock factors (HSFs) are the transcription factors that read the activation of thermosensors and induce the expression of HSPs. Epigenetic modifications of HSFs are likely to be the key component of thermal acquired tolerance (TAT). Despite the advances in understanding the process of thermomemory generation, it is not known whether plants are equipped with systemic activation thermal protection, as happens in the form of systemic acquired resistance (SAR) upon pathogen infection. This review describes the recent advances in the understanding of thermomemory development in plants and the knowledge gap in comparison with SAR.

Systemic acquired resistance specific proteome of Arabidopsis thaliana.

KEY MESSAGE: A comparative proteomic study between WT and SAR-compromised rsi1/fld mutant reveals a set of proteins having possible roles in the SAR development. A partly infected plant shows enhanced resistance during subsequent infection through the development of systemic acquired resistance (SAR). Mobile signals generated at the site of primary infection travel across the plant for the activation of SAR. These mobile signals are likely to cause changes in the expression of a set of proteins in the distal tissue, which contributes to the SAR development. However, SAR-specific proteome is not revealed for any plant. The reduced systemic immunity 1 (rsi1)/(allelic to flowering locus D; fld) mutant of Arabidopsis is compromised for SAR but shows normal local resistance. Here we report the SAR-specific proteome of Arabidopsis by comparing differentially abundant proteins (DAPs) between WT and fld mutant. Plants were either mock-treated or SAR-induced by primary pathogen inoculation. For proteomic analysis, samples were collected from the systemic tissues before and after the secondary inoculation. Protein identification was carried out by using two-dimensional gel electrophoresis (2-DE) followed by tandem mass spectrometry. Our work identified a total of 94 DAPs between mock and pathogen treatment in WT and fld mutant. The DAPs were categorized into different functional groups along with their subcellular localization. The majority of DAPs are involved in metabolic processes and stress response. Among the subcellular compartments, plastids contained the highest number of DAPs, suggesting the importance of plastidic proteins in SAR activation. The findings of this study would provide resources to engineer efficient SAR activation traits in Arabidopsis and other plants.

DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2 of Arabidopsis thaliana is a negative regulator of local and systemic acquired resistance.

To fine tune defense response output, plants recruit both positive and negative regulators. Here we report Arabidopsis DORMANCY/AUXIN ASSOCIATED FAMILY PROTEIN 2(DAP2) gene as a negative regulator of basal defense against virulent bacterial pathogens. Expression of DAP2 enhances upon pathogen inoculation. Our experiments show that DAP2 suppressed resistance against virulent strains of bacterial pathogens, pathogen-induced callose deposition, and ROS accumulation; however, it did not influence effector-triggered immunity. In addition, DAP2 negatively regulated systemic acquired resistance (SAR). DAP2 expression was enhanced in the pathogen-free systemic tissues of SAR-induced plants. Previously, Arabidopsis Flowering locus D (FLD) gene has been shown to be essential for SAR but not for local resistance. We show here that FLD function is necessary for SAR-induced expression of DAP2, suggesting DAP2 as a target of FLD for activation of SAR in Arabidopsis.

MEDEA-interacting protein LONG-CHAIN BASE KINASE 1 promotes pattern-triggered immunity in Arabidopsis thaliana.

KEY MESSAGE: Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

CaMPK9 increases the stability of CaWRKY40 transcription factor which triggers defense response in chickpea upon Fusarium oxysporum f. sp. ciceri Race1 infection.

KEY MESSAGE: Physical interaction and phosphorylation by CaMPK9 protects the degradation of CaWRKY40 that induces resistance response in chickpea to Fusarium wilt disease by modulating the transcription of defense responsive genes. WRKY transcription factors (TFs) are the global regulators of plant defense signaling that modulate immune responses in host plants by regulating transcription of downstream target genes upon challenged by pathogens. However, very little is known about immune responsive role of Cicer arietinum L. (Ca) WRKY TFs particularly. Using two contrasting chickpea genotypes with respect to resistance against Fusarium oxysporum f. sp. ciceri Race1 (Foc1), we demonstrate transcript accumulation of different CaWRKYs under multiple stresses and establish that CaWRKY40 triggers defense. CaWRKY40 overexpressing chickpea mounts resistance to Foc1 by positively modulating the defense related gene expression. EMSA, ChIP assay and real-time PCR analyses suggest CaWRKY40 binds at the promoters and positively regulates transcription of CaDefensin and CaWRKY33. Further studies revealed that mitogen Activated Protein Kinase9 (CaMPK9) phosphorylates CaWRKY40 by directly interacting with its two canonical serine residues. Interestingly, CaMPK9 is unable to interact with CaWRKY40 when the relevant two serine residues were replaced by alanine. Overexpression of serine mutated WRKY40 isoform in chickpea fails to provide resistance against Foc1. Mutated WRKY40Ser.224/225 to AA overexpressing chickpea resumes its ability to confer resistance against Foc1 after application of 26S proteasomal inhibitor MG132, suggests that phosphorylation is essential to protect CaWRKY40 from proteasomal degradation. CaMPK9 silencing also led to susceptibility in chickpea to Foc1. Altogether, our results elucidate positive regulatory roles of CaMPK9 and CaWRKY40 in modulating defense response in chickpea upon Foc1 infection.

APD1, the unique member of Arabidopsis AP2 family influences systemic acquired resistance and ethylene-jasmonic acid signaling.

Arabidopsis AP2 FAMILY PROTEIN INVOLVED IN DISEASE DEFENSE (APD1) is a member of AP2/EREBP super-family that positively regulates SA biosynthesis and defense against virulent bacterial pathogens. Here we report additional roles of APD1 in plant defense and development. We show that APD1 function is required for light-mediated defense against bacterial pathogens and systemic acquired resistance (SAR). We demonstrate that APD1 function is not required for generating SAR mobile signal at the site of primary inoculation but is required at the distal end for SAR manifestation. In addition, the APD1 function is required for PTI-induced callose deposition, defense against necrotrophic pathogen Botrytis cinerea and Alternaria alternata, which are ethylene (ET) or ethylene-Jasmonate (JA) dependent responses. Development of seedling under dark and ET is partly dependent on APD1. The mutant apd1 plants are non-responsive towards exogenous ACC application regarding apical hook formation and hypocotyl shortening, however, possess WT-like ET-mediated root growth inhibition. JA-mediated root growth inhibition is also impaired in apd1 seedlings. Altogether our results suggest that APD1 impacts multiple aspects of plant growth and development.

The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense.

Plants recruit positive and negative regulators for fine tuning the balance between growth and development. Negative regulators of pathogen defense generally modulate defense hormone biosynthesis and signaling. Here, we report a mechanism for attenuation of the defense response in Arabidopsis (Arabidopsis thaliana), which is mediated by the polycomb-group repressor MEDEA (MEA). Our results showed that pathogen inoculation or exogenous application of salicylic acid, methyl jasmonate, or the bacterial 22-amino acid domain of flagellin peptide induces the expression of MEAMEA expression was higher when plants were inoculated with the avirulent strain of Pseudomonas syringae pv. tomato (Pst) carrying the AvrRpt2 effector (Pst-AvrRpt2) compared to the virulent Pst strain. MEA remains suppressed during the vegetative phase via DNA and histone (H3K27) methylation, and only the maternal copy is expressed in the female gametophyte and endosperm via histone and DNA demethylation. In contrast, Pst-AvrRpt2 induces high levels of MEA expression via hyper-accumulation of H3K4me3 at the MEA locus. MEA-overexpressing transgenic plants are susceptible to the fungal pathogen Botrytis cinerea and bacterial pathogens Pst and Pst-AvrRpt2, whereas mea mutant plants are more resistant to bacterial pathogens. AvrRpt2-mediated immunity requires the function of RESISTANCE TO P. SYRINGAE2 (RPS2) in Arabidopsis. Using transcriptional analysis and chromatin immunoprecipitation, we established that MEA directly targets RPS2 and suppresses its transcription. We screened an Arabidopsis cDNA library using MEA as the bait in a yeast two-hybrid assay and identified DROUGHT-INDUCED19, a transcription factor that interacts with MEA and recruits it at the RPS2 promoter. The results identified a previously unknown mechanism of defense response attenuation in plants.

AtOZF1 Positively Regulates Defense Against Bacterial Pathogens and NPR1-Independent Salicylic Acid Signaling.

Plant hormone salicylic acid (SA) plays critical roles in defense signaling against biotrophic pathogens. Pathogen inoculation leads to SA accumulation in plants. SA activates a transactivator protein NPR1, which, in turn, transcriptionally activates many defense response genes. Reports also suggest the presence of NPR1-independent pathways for SA signaling in Arabidopsis. Here, we report the characterization of a zinc-finger protein-coding gene AtOZF1 that positively influences NPR1-independent SA signaling. Mutants of AtOZF1 are compromised, whereas AtOZF1-overexpressing plants are hyperactive for defense against virulent and avirulent pathogens. AtOZF1 expression is SA-inducible. AtOZF1 function is not required for pathogenesis-associated biosynthesis and accumulation of SA. However, it is required for SA responsiveness. By generating atozf1npr1 double mutant, we show that contributions of these two genes are additive in terms of defense. We identified AtOZF1-interacting proteins by a yeast-two-hybrid screening of an Arabidopsis cDNA library. VDAC2 and NHL3 are two AtOZF1-interacting proteins, which are positive regulators of basal defense. AtOZF1 interacts with NHL3 and VDAC2 in plasma membrane and mitochondria, respectively. Our results demonstrate that AtOZF1 coordinates multiple steps of plant-pathogen interaction.

Arabidopsis thaliana GLUTATHIONE-S-TRANSFERASE THETA 2 interacts with RSI1/FLD to activate systemic acquired resistance.

A partly infected plant develops systemic acquired resistance (SAR) and shows heightened resistance during subsequent infections. The infected parts generate certain mobile signals that travel to the distal tissues and help to activate SAR. SAR is associated with epigenetic modifications of several defence-related genes. However, the mechanisms by which mobile signals contribute to epigenetic changes are little known. Previously, we have shown that the Arabidopsis REDUCED SYSTEMIC IMMUNITY 1 (RSI1, alias FLOWERING LOCUS D; FLD), which codes for a putative histone demethylase, is required for the activation of SAR. Here, we report the identification of GLUTATHIONE-S-TRANSFERASE THETA 2 (GSTT2) as an interacting factor of FLD. GSTT2 expression increases in pathogen-inoculated as well as pathogen-free distal tissues. The loss-of-function mutant of GSTT2 is compromised for SAR, but activates normal local resistance. Complementation lines of GSTT2 support its role in SAR activation. The distal tissues of gstt2 mutant plants accumulate significantly less salicylic acid (SA) and express a reduced level of the SA biosynthetic gene PAL1. In agreement with the established histone modification activity of FLD, gstt2 mutant plants accumulate an enhanced level of methylated and acetylated histones in the promoters of WRKY6 and WRKY29 genes. Together, these results demonstrate that GSTT2 is an interactor of FLD, which is required for SAR and SAR-associated epigenetic modifications.

CBL-interacting protein kinase 6 negatively regulates immune response to Pseudomonas syringae in Arabidopsis.

Cytosolic calcium ion (Ca2+) is an essential mediator of the plant innate immune response. Here, we report that a calcium-regulated protein kinase Calcineurin B-like protein (CBL)-interacting protein kinase 6 (CIPK6) functions as a negative regulator of immunity against the bacterial pathogen Pseudomonas syringae in Arabidopsis thaliana. Arabidopsis lines with compromised expression of CIPK6 exhibited enhanced disease resistance to the bacterial pathogen and to P. syringae harboring certain but not all avirulent effectors, while restoration of CIPK6 expression resulted in abolition of resistance. Plants overexpressing CIPK6 were more susceptible to P. syringae. Enhanced resistance in the absence of CIPK6 was accompanied by increased accumulation of salicylic acid and elevated expression of defense marker genes. Salicylic acid accumulation was essential for improved immunity in the absence of CIPK6. CIPK6 negatively regulated the oxidative burst associated with perception of pathogen-associated microbial patterns (PAMPs) and bacterial effectors. Accelerated and enhanced activation of the mitogen-activated protein kinase cascade in response to bacterial and fungal elicitors was observed in the absence of CIPK6. The results of this study suggested that CIPK6 negatively regulates effector-triggered and PAMP-triggered immunity in Arabidopsis.

Arabidopsis thaliana methionine sulfoxide reductase B8 influences stress-induced cell death and effector-triggered immunity.

KEY MESSAGE: Reactive oxygen species (ROS) oxidize methionine to methionine sulfoxide (MetSO) and thereby inactivate proteins. Methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Our results show that Arabidopsis thaliana MSR enzyme coding gene MSRB8 is required for effector-triggered immunity and containment of stress-induced cell death in Arabidopsis. Plants activate pattern-triggered immunity (PTI), a basal defense, upon recognition of evolutionary conserved molecular patterns present in the pathogens. Pathogens release effector molecules to suppress PTI. Recognition of certain effector molecules activates a strong defense, known as effector-triggered immunity (ETI). ETI induces high-level accumulation of reactive oxygen species (ROS) and hypersensitive response (HR), a rapid programmed death of infected cells. ROS oxidize methionine to methionine sulfoxide (MetSO), rendering several proteins nonfunctional. The methionine sulfoxide reductase (MSR) enzyme converts MetSO back to the reduced form and thereby detoxifies the effect of ROS. Though a few plant MSR genes are known to provide tolerance against oxidative stress, their role in plant-pathogen interaction is not known. We report here that activation of cell death by avirulent pathogen or UV treatment induces expression of MSRB7 and MSRB8 genes. The T-DNA insertion mutant of MSRB8 exaggerates HR-associated and UV-induced cell death and accumulates a higher level of ROS than wild-type plants. The negative regulatory role of MSRB8 in HR is further supported by amiRNA and overexpression lines. Mutants and overexpression lines of MSRB8 are susceptible and resistant respectively, compared to the wild-type plants, against avirulent strains of Pseudomonas syringae pv. tomato DC3000 (Pst) carrying AvrRpt2, AvrB, or AvrPphB genes. However, the MSRB8 gene does not influence resistance against virulent Pst or P. syringae pv. maculicola (Psm) pathogens. Our results altogether suggest that MSRB8 function is required for ETI and containment of stress-induced cell death in Arabidopsis.

Arabidopsis thaliana serpins AtSRP4 and AtSRP5 negatively regulate stress-induced cell death and effector-triggered immunity induced by bacterial effector AvrRpt2.

Protease inhibitors and their cognate proteases regulate growth, development and defense. Serine protease inhibitors (serpins) constitute a large family of genes in most metazoans and plants. Drosophila NECROTIC (NEC) gene and its homologues in the mammalian system are well-characterized serpins, which play a role in regulating proteases that participate in cell death pathways. Although the Arabidopsis genome contains several serpin homologs, biological function is not known for most of them. Here we show that two Arabidopsis serpins, AtSRP4 and AtSRP5, are closest sequence homologue of Drosophila NEC protein, and are involved in stress-induced cell death and defense. Expression of both AtSRP4 and AtSRP5 genes induced upon ultra-violet (UV)-treatment and inoculation with avirulent pathogens. The knockout mutants and amiRNA lines of AtSRP4 and AtSRP5 exaggerated UV- and hypersensitive response (HR)-induced cell death. Over-expression of AtSRP4 reduced UV- and HR-induced cell death. Mutants of AtSRP4 and AtSRP5 suppressed whereas over-expression of AtSRP4 supported the growth of bacterial pathogen Pseudomonas syringae pv. tomato DC3000 carrying the AvrRpt2 effector, but not other avirulent or virulent pathogens. Results altogether identified AtSRP4 and AtSRP5 as negative regulators of stress-induced cell death and AvrRpt2-triggered immunity; however, the influence of AtSRP4 was more prominent than AtSRP5.

Rice MYC2 (OsMYC2) modulates light-dependent seedling phenotype, disease defence but not ABA signalling.

Arabidopsis MYC2 (AtMYC2) is a bHLH class transcription factor that mediates light-dependent seedling development, disease defence, JA and ABA signalling. AtMYC2 gene modulates hypocotyl elongation and expression of chlorophyll A/B binding protein 1 (CAB1) and rubisco small subunit protein1 (RBCS1) under blue light. The atmyc2 mutants are resistant against virulent bacterial pathogens. MYC2 orthologues from several crop plants have been characterized. The rice gene Os10g42430 has been referred earlier as OsMYC2 and has been shown to promote expression of JA-inducible genes. However, the role of OsMYC2 in seedling development under ABA, dark or light of specific wavelengths was not known. It was also not known whether OsMYC2 complements AtMYC2 function in Arabidopsis. We show here that expression of OsMYC2 in the atmyc2 mutant of Arabidopsis complements the blue-light-mediated defects in hypocotyl elongation and expression of CAB1 and RBCS1. We generated multiple transgenic rice lines for over-expression and RNAi-mediated suppression of OsMYC2. In agreement with AtMYC2 function, OsMYC2 over-expression and RNAi lines showed enhanced and suppressed seedling growth compared to WT plants respectively under blue light, and showed little effect under white light or dark. In agreement with the negative regulatory role of AtMYC2 in disease defence, the RNAi lines showed enhanced resistance against bacterial pathogen Xanthomonas oryzae pv oryzae. However, in contrast to AtMYC2 function, OsMYC2 influences seedling development under red light and show no effect in ABA-mediated seed germination. Thus, the results suggest evolutionarily conserved as well as the distinct role of OsMYC2 in comparison with AtMYC2.

The rice OsSAG12-2 gene codes for a functional protease that negatively regulates stress-induced cell death.

Senescence is the final stage of plant development. Although expression of most of the genes is suppressed during senescence, a set of genes referred as senescence-associated genes (SAGs) is induced. Arabidopsis thaliana SAG12 (AtSAG12) is one such gene that has been mostly studied for its strict association with senescence. AtSAG12 encodes a papain-like cysteine protease, expressed predominantly in senescence-associated vacuoles. Rice genome contains multiple AtSAG12 homologues (OsSAGs). OsSAG12-1, the closest structural homologue of AtSAG12, is a negative regulator of developmental and stress-induced cell death. Proteolytic activity has not been established for any SAG12 homologues in vitro. Here, we report that OsSAG12-2, the second structural homologue of AtSAG12 from rice, codes for a functional proteolytic enzyme. The recombinant OsSAG12-2 protein produced in Escherichia coli undergoes autolysis to generate a functional protease. The matured OsSAG12-2 protein shows 27 percent trypsin-equivalent proteolytic activity on azocasein substrate. Dark-induced senescence activates OsSAG12-2 expression. Down-regulation of OsSAG12-2 in the transgenic artificial miRNA lines results in enhanced salt- and UV-induced cell death, even though it does not affect cell viability in the stress-free condition. Our results show that OsSAG12-2 codes for a functional protease that negatively regulates stress-induced cell death in rice.