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.

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.

JMJ14 encoded H3K4 demethylase modulates immune responses by regulating defence gene expression and pipecolic acid levels.

Epigenetic modifications have emerged as an important mechanism underlying plant defence against pathogens. We examined the role of JMJ14, a Jumonji (JMJ) domain-containing H3K4 demethylase, in local and systemic plant immune responses in Arabidopsis. The function of JMJ14 in local or systemic defence response was investigated by pathogen growth assays and by analysing expression and H3K4me3 enrichments of key defence genes using qPCR and ChIP-qPCR. Salicylic acid (SA) and pipecolic acid (Pip) levels were quantified and function of JMJ14 in SA- and Pip-mediated defences was analysed in Col-0 and jmj14 plants. jmj14 mutants were compromised in both local and systemic defences. JMJ14 positively regulates pathogen-induced H3K4me3 enrichment and expression of defence genes involved in SA- and Pip-mediated defence pathways. Consequently, loss of JMJ14 results in attenuated defence gene expression and reduced Pip accumulation during establishment of systemic acquired resistance (SAR). Exogenous Pip partially restored SAR in jmj14 plants, suggesting that JMJ14 regulated Pip biosynthesis and other downstream factors regulate SAR in jmj14 plants. JMJ14 positively modulates defence gene expressions and Pip levels in Arabidopsis.

GBF1 differentially regulates CAT2 and PAD4 transcription to promote pathogen defense in Arabidopsis thaliana.

G-BOX BINDING FACTOR 1 (GBF1) influences light-regulated seedling development in Arabidopsis, and inhibits CATALASE 2 (CAT2) expression during senescence. CAT2 functions as a scavenger of hydrogen peroxide. The role of GBF1 in the defense response is not known. We report here that GBF1 positively influences the defense against virulent and avirulent strains of Pseudomonas syringae. The gbf1 mutants are susceptible, whereas GBF1 over-expresser transgenic plants are resistant to bacterial pathogens. GBF1 negatively regulates pathogen-induced CAT2 expression and thereby positively regulates the hypersensitive response. In addition to CAT2 promoter, GBF1 binds to the G-box-like element present in the intron of PHYTOALEXIN DEFICIENT 4 (PAD4). This association of GBF1 with PAD4 intron is enhanced upon pathogenesis. GBF1 positively regulates PAD4 transcription in an intron-dependent manner. GBF1-mediated positive regulation of PAD4 expression is also evident in gbf1 mutant and GBF1 over-expression lines. Similar to pad4 mutants, pathogen-induced camalexin and salicylic acid (SA) accumulation, and expression of SA-inducible PATHOGENESIS RELATED1 (PR1) gene are compromised in the gbf1 mutant. Exogenous application of SA rescues the loss-of-defense phenotypes of gbf1 mutant. Thus, altogether, our results demonstrate that GBF1 is an important component of the plant defense response that functions upstream of SA accumulation and, by oppositely regulating CAT2 and PAD4, promotes disease resistance 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.

Arabidopsis flowering locus D influences systemic-acquired-resistance- induced expression and histone modifications of WRKY genes.

A plant that is in part infected by a pathogen is more resistant throughout its whole body to subsequent infections--a phenomenon known as systemic acquired resistance (SAR). Mobile signals are synthesized at the site of infection and distributed throughout the plant through vascular tissues. Mechanism of SAR development subsequent to reaching the mobile signal in the distal tissue is largely unknown. Recently we showed that flowering locus D (FLD) gene of Arabidopsis thaliana is required in the distal tissue to activate SAR. FLD codes for a homologue of human-lysine-specific histone demethylase. Here we show that FLD function is required for priming (SAR induced elevated expression during challenge inoculation) of WRKY29 and WRKY6 genes. FLD also differentially influences basal and SAR-induced expression of WRKY38, WRKY65 and WRKY53 genes. In addition, we also show that FLD partly localizes in nucleus and influences histone modifications at the promoters of WRKY29 and WRKY6 genes. The results altogether indicate to the possibility of FLD's involvement in epigenetic regulation of SAR.