External jasmonic acid isoleucine mediates amplification of plant elicitor peptide receptor (PEPR) and jasmonate-based immune signalling.

Jasmonic acid-isoleucine (JA-Ile) is a plant defence hormone whose cellular levels are elevated upon herbivory and regulate defence signalling. Despite their pivotal role, our understanding of the rapid cellular perception of bioactive JA-Ile is limited. This study identifies cell type-specific JA-Ile-induced Ca2+ signal and its role in self-amplification and plant elicitor peptide receptor (PEPR)-mediated signalling. Using the Ca2+ reporter, R-GECO1 in Arabidopsis, we have characterized a monophasic and sustained JA-Ile-dependent Ca2+ signature in leaf epidermal cells. The rapid Ca2+ signal is independent of positive feedback by the JA-Ile receptor, COI1 and the transporter, JAT1. Microarray analysis identified up-regulation of receptors, PEPR1 and PEPR2 upon JA-Ile treatment. The pepr1 pepr2 double mutant in R-GECO1 background exhibits impaired external JA-Ile induced Ca2+ cyt elevation and impacts the canonical JA-Ile responsive genes. JA responsive transcription factor, MYC2 binds to the G-Box motif of PEPR1 and PEPR2 promoter and activates their expression upon JA-Ile treatment and in myc2 mutant, this is reduced. External JA-Ile amplifies AtPep-PEPR pathway by increasing the AtPep precursor, PROPEP expression. Our work shows a previously unknown non-canonical PEPR-JA-Ile-Ca2+ -MYC2 signalling module through which plants sense JA-Ile rapidly to amplify both AtPep-PEPR and jasmonate signalling in undamaged cells.

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.

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.

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.

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.

The Arabidopsis thaliana At4g13040 gene, a unique member of the AP2/EREBP family, is a positive regulator for salicylic acid accumulation and basal defense against bacterial pathogens.

The Arabidopsis genome contains a large number of putative transcription factors, containing a DNA binding domain similar to APETALA2/ethylene response element binding protein (AP2/EREBP), for most of which a function is not known. Phylogenetic analysis divides the Apetala 2 (AP2) super-family into 5 major groups: AP2, RAV, ethylene response factor (ERF), dehydration response element binding protein (DREB) and At4g13040. Similar to ERF and DREB, the At4g13040 protein contains only one AP2 domain; however, its structural uniqueness places it into a distinct group. In this article, we report that At4g13040 (referred herein as Apetala 2 family protein involved in SA mediated disease defense 1 - APD1) is an important regulator for SA mediated plant defense. The APD1 gene is upregulated upon pathogen inoculation, exogenous SA application and in the mutant that constitutively activates SA signaling. The T-DNA insertion lines (inserted in the APD1 promoter), which fail to induce expression upon pathogen inoculation, are compromised for resistance against virulent bacterial pathogens and show reduced induction of pathogenesis related 1 gene. Our results suggest that APD1 functions downstream of PAD4 in Arabidopsis and promotes pathogen-induced SA accumulation. Exogenous SA application completely restores the loss-of-resistance phenotype of the apd1 mutant. Thus, APD1 is a positive regulator of disease defense that functions upstream of SA accumulation.