Pseudomonas Syringae Infection Research Articles

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Pseudomonas syringae infectivity correlates to altered transcript and metabolite levels of Arabidopsis mediator mutants

Rapid metabolic responses to pathogens are essential for plant survival and depend on numerous transcription factors. Mediator is the major transcriptional co-regulator for integration and transmission of signals from transcriptional regulators to RNA polymerase II. Using four Arabidopsis Mediator mutants, med16, med18, med25 and cdk8, we studied how differences in regulation of their transcript and metabolite levels correlate to their responses to Pseudomonas syringae infection. We found that med16 and cdk8 were susceptible, while med25 showed increased resistance. Glucosinolate, phytoalexin and carbohydrate levels were reduced already before infection in med16 and cdk8, but increased in med25, which also displayed increased benzenoids levels. Early after infection, wild type plants showed reduced glucosinolate and nucleoside levels, but increases in amino acids, benzenoids, oxylipins and the phytoalexin camalexin. The Mediator mutants showed altered levels of these metabolites and in regulation of genes encoding key enzymes for their metabolism. At later stage, mutants displayed defective levels of specific amino acids, carbohydrates, lipids and jasmonates which correlated to their infection response phenotypes. Our results reveal that MED16, MED25 and CDK8 are required for a proper, coordinated transcriptional response of genes which encode enzymes involved in important metabolic pathways for Arabidopsis responses to Pseudomonas syringae infections.

The cytochrome P450 enzyme WsCYP71B35 from Withania somnifera has a role in withanolides biosynthesis and defence against bacteria

AbstractThe medicinal properties of Ashwagandha (Withania somnifera L. Dunal) are attributed to withanolides, which belong to the triterpenoid steroidal lactones class of compounds. Though it is proposed that intermediates of the universal phytosterol pathway are utilized by cytochrome P450 (CYP450) enzymes to form withanolides, studies on the functional characterization of these enzymes have been sparse. This study reports the functional characterization of a CYP450 candidate from W. somnifera, WsCYP71B35 that exhibited induced expression in response to methyl jasmonate treatment and showed higher expression in tissues that accumulate withanolides. Biochemical assay with yeast microsomal fraction expressing recombinant WsCYP71B35 indicated no activity when phytosterols and their intermediate 24‐methylene cholesterol were used as substrates. However, WsCYP71B35 catalyzed product formation with withaferin A, withanolide A, withanolide B, and withanoside IV among the tested substrates. Moreover, virus‐induced gene silencing (VIGS) and transient overexpression of WsCYP71B35 in W. somnifera leaves modulated the levels of withaferin A, withanolide A, and withanolide B, indicating the role of WsCYP71B35 in withanolides pathway. Furthermore, VIGS of WsCYP71B35 in W. somnifera reduced its tolerance to Pseudomonas syringae (DC3000) infection, whereas overexpression enhanced the tolerance to the bacterium in W. somnifera and transgenic tobacco. Overall, these results provide insights into the role of W. somnifera WsCYP71B35 in withanolides biosynthesis and bacterial defence.

Voltage-dependent anion channel 3 (VDAC3) mediates P. syringae induced ABA-SA signaling crosstalk in Arabidopsis thaliana

Pathogen severely affects plant mitochondrial processes including respiration, however, the roles and mechanism of mitochondrial protein during the immune response remain largely unexplored. The interplay of plant hormone signaling during defense is an outcome of plant pathogen interaction. We recently discovered that the Arabidopsis calcineurin B-like interacting protein kinase 9 (AtCIPK9) interacts with the voltage-dependent anion channel 3 (AtVDAC3) and inhibits MV-induced oxidative damage. Here we report the characterization of AtVDAC3 in an antagonistic interaction pathway between abscisic acid (ABA) and salicylic acid (SA) signaling in Pseudomonas syringae -Arabidopsis interaction. In this study, we observed that mutants of AtVDAC3 were highly susceptible to Pseudomonas syringae infection as compared to the wild type (WT) Arabidopsis plants. Transcripts of VDAC3 and CIPK9 were inducible upon ABA application. Following pathogen exposure, expression analyses of ABA and SA biosynthesis genes indicated that the function of VDAC3 is required for isochorisimate synthase 1 (ICS1) expression but not for Nine-cis-epoxycaotenoid dioxygenase 3 (NCED3) expression. Despite the fact that vdac3 mutants had increased NCED3 expression in response to pathogen challenge, transcripts of ABA sensitive genes such as AtRD22 and AtRAB18 were downregulated even after exogenous ABA application. VDAC3 is required for ABA responsive genes expression upon exogenous ABA application. We also found that Pseudomonas syringae-induced SA signaling is downregulated in vdac3 mutants since overexpression of VDAC3 resulted in hyperaccumulation of Pathogenesis related gene1 (PR1) transcript. Interestingly, ABA application prior to P. syringae inoculation resulted in the upregulation of ABA responsive genes like Responsive to ABA18 (RAB18) and Responsive to dehydration 22 (RD22). Intriguingly, in the absence of AtVDAC3, Pst challenge can dramatically increase ABA-induced RD22 and RAB18 expression. Altogether our results reveal a novel Pathogen-SA-ABA interaction pathway in plants. Our findings show that ABA plays a significant role in modifying plant-pathogen interactions, owing to cross-talk with the biotic stress signaling pathways of ABA and SA.

Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure.

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Expression of EPL1 from Trichoderma atroviride in Arabidopsis Confers Resistance to Bacterial and Fungal Pathogens.

During plant interaction with beneficial microorganisms, fungi secrete a battery of elicitors that trigger plant defenses against pathogenic microorganisms. Among the elicitor molecules secreted by Trichoderma are cerato-platanin proteins, such as EPL1, from Trichoderma atroviride. In this study, Arabidopsis thaliana plants that express the TaEPL1 gene were challenged with phytopathogens to evaluate whether expression of EPL1 confers increased resistance to the bacterial pathogen Pseudomonas syringae and the necrotrophic fungus Botrytis cinerea. Infection assays showed that Arabidopsis EPL1-2, EPL1-3, EPL1-4 expressing lines were more resistant to both pathogens in comparison to WT plants. After Pseudomonas syringae infection, there were reduced disease symptoms (e.g., small chlorotic spots) and low bacterial titers in the three 35S::TaEPL1 expression lines. Similarly; 35S::TaEPL1 expression lines were more resistant to Botrytis cinerea infection, showing smaller lesion size in comparison to WT. Interestingly, an increase in ROS levels was detected in 35S::TaEPL1 expression lines when compared to WT. A higher expression of SA- and JA-response genes occurred in the 35S::TaEPL1 lines, which could explain the resistance of these EPL1 expression lines to both pathogens. We propose that EPL1 is an excellent elicitor, which can be used to generate crops with improved resistance to broad-spectrum diseases.

Single-cell profiling of Arabidopsis leaves to Pseudomonas syringae infection

Plant response to pathogen infection varies within a leaf, yet this heterogeneity is not well resolved. We expose Arabidopsis to Pseudomonas syringae or mock treatment and profile >11,000 individual cells using single-cell RNA sequencing. Integrative analysis of cell populations from both treatments identifies distinct pathogen-responsive cell clusters exhibiting transcriptional responses ranging from immunity to susceptibility. Pseudotime analyses through pathogen infection reveals a continuum of disease progression from an immune to a susceptible state. Confocal imaging of promoter-reporter lines for transcripts enriched in immune cell clusters shows expression surrounding substomatal cavities colonized or in close proximity to bacterial colonies, suggesting that cells within immune clusters represent sites of early pathogen invasion. Susceptibility clusters exhibit more general localization and are highly induced at later stages of infection. Overall, our work shows cellular heterogeneity within an infected leaf and provides insight into plant differential response to infection at a single-cell level.

The transcription factor LaMYC4 from lavender regulates volatile Terpenoid biosynthesis

BackgroundThe basic helix-loop-helix (bHLH) transcription factors (TFs), as one of the largest families of TFs, are essential regulators of plant terpenoid biosynthesis and response to stresses. Lavender has more than 75 volatile terpenoids, yet few TFs have been identified to be involved in the terpenoid biosynthesis.ResultsBased on RNA-Seq, reverse transcription-quantitative polymerase chain reaction, and transgenic technology, this study characterized the stress-responsive transcription factor LaMYC4 regulates terpenoid biosynthesis. Methyl jasmonate (MeJA) treatment increased volatile terpenoid emission, and the differentially expressed gene LaMYC4 was isolated. LaMYC4 expression level was higher in leaf than in other tissues. The expression of LaMYC4 decreased during flower development. The promoter of LaMYC4 contained hormone and stress-responsive regulatory elements and was responsive to various treatments, including UV, MeJA treatment, drought, low temperature, Pseudomonas syringae infection, and NaCl treatment. LaMYC4 overexpression increased the levels of sesquiterpenoids, including caryophyllenes, in Arabidopsis and tobacco plants. Furthermore, the expression of crucial node genes involved in terpenoid biosynthesis and glandular trichome number and size increased in transgenic tobacco.ConclusionsWe have shown that the stress-responsive MYC TF LaMYC4 from ‘Jingxun 2’ lavender regulates volatile terpenoid synthesis. This study is the first to describe the cloning of LaMYC4, and the results help understand the role of LaMYC4 in terpenoid biosynthesis.

Biorefinery of Tomato Leaves by Integrated Extraction and Membrane Processes to Obtain Fractions That Enhance Induced Resistance against Pseudomonas syringae Infection.

Tomato leaves have been shown to contain significant amounts of important metabolites involved in protection against abiotic and biotic stress and/or possessing important therapeutic properties. In this work, a systematic study was carried out to evaluate the potential of a sustainable process for the fractionation of major biomolecules from tomato leaves, by combining aqueous extraction and membrane processes. The extraction parameters (temperature, pH, and liquid/solid ratio (L/S)) were optimized to obtain high amounts of biomolecules (proteins, carbohydrates, biophenols). Subsequently, the aqueous extract was processed by membrane processes, using 30–50 kDa and 1–5 kDa membranes for the first and second stage, respectively. The permeate from the first stage, which was used to remove proteins from the aqueous extract, was further fractionated in the second stage, where the appropriate membrane material was also selected. Of all the membranes tested in the first stage, regenerated cellulose membranes (RC) showed the best performance in terms of higher rejection of proteins (85%) and lower fouling index (less than 15% compared to 80% of the other membranes tested), indicating that they are suitable for fractionation of proteins from biophenols and carbohydrates. In the second stage, the best results were obtained by using polyethersulfone (PES) membranes with an NMWCO of 5 kDa, since the greatest difference between the rejection coefficients of carbohydrates and phenolic compounds was obtained. In vivo bioactivity tests confirmed that fractions obtained with PES 5 kDa membranes were able to induce plant defense against P. syringae.

Silencing GhIAA43, a member of cotton AUX/IAA genes, enhances wilt resistance via activation of salicylic acid-mediated defenses

Auxin-mediated degradation of Aux/IAA proteins is a crucial step in auxin signaling. Recent researches indicate that Aux/IAA members also play a role in biotic and abiotic stresses. For example, Pseudomonas syringae infection causes Arabidopsis Aux/IAA protein (AXR2, AXR3) turnover. Here, by analyzing RNA-seq data we found that several cotton Aux/IAA genes are responsive to Verticillium dahliae infection, one of these named GhIAA43, was investigated for its role in cotton defense against V. dahliae infection. We demonstrate that the transcript levels of GhIAA43 were responsive to both V. dahliae infection and exogenous IAA application. By producing transgenic Arabidopsis plants overexpressing GhIAA43-GUS fusion, we show that IAA treatment and V. dahliae infection promoted GhIAA43 protein turnover. Silencing GhIAA43 in cotton enhanced wilt resistance, suggesting that GhIAA43 is a negative regulator in cotton defense against V. dahliae attack. By monitoring SA marker gene expression and measurement of SA content in GhIAA43-silenced cotton plants, we found that the enhanced resistance in GhIAA43-silenced cotton plants is due to the activation of SA-related defenses, and the activated defenses specifically occurred in the presence of V. dahliae. Furthermore, exogenous IAA application improve wilt resistance in cotton plants tested. Our results provide novel connection between auxin signaling and SA-related defenses in cotton upon V. dahliae attack.

FITNESS Acts as a Negative Regulator of Immunity and Influences the Plant Reproductive Output After Pseudomonas syringae Infection.

Plants, as sessile organisms, are continuously threatened by multiple factors and therefore their profitable production depends on how they can defend themselves. We have previously reported on the characterization of fitness mutants which are more tolerant to environmental stresses due to the activation of defense mechanisms. Here, we demonstrate that in fitness mutants, which accumulate moderate levels of salicylic acid (SA) and have SA signaling activated, pathogen infection is restricted. Also, we demonstrate that NPR1 is essential in fitness mutants for SA storage and defense activation but not for SA synthesis after Pseudomonas syringae (Pst) infection. Additionally, these mutants do not appear to be metabolically impared, resulting in a higher seed set even after pathogen attack. The FITNESS transcriptional network includes defense-related transcription factors (TFs) such as ANAC072, ORA59, and ERF1 as well as jasmonic acid (JA) related genes including LIPOXYGENASE2 (LOX2), CORONATINE INSENSITIVE1 (COI1), JASMONATE ZIM-domain3 (JAZ3) and JAZ10. Induction of FITNESS expression leads to COI1 downregulation, and to JAZ3 and JAZ10 upregulation. As COI1 is an essential component of the bioactive JA perception apparatus and is required for most JA-signaling processes, elevated FITNESS expression leads to modulated JA-related responses. Taken together, FITNESS plays a crucial role during pathogen attack and allows a cost-efficient way to prevent undesirable developmental effects.

Ath-miR164c influences plant responses to the combined stress of drought and bacterial infection by regulating proline metabolism

Plants under combined stresses exhibit a prominent shift in molecular responses compared with plants exposed to the same stresses independently. Profiling responses to individual and combined stressors at the gene expression level have identified several genes with intersecting responses to these stressors. However, the upstream regulators at the intersection of plant responses to individual and combined stresses are not known. Here, using the transcriptome of Arabidopsis thaliana under individual and combined drought and Pseudomonas syringae infection, we identified several genes whose expression overlaps between individual and combined stresses. To study the key regulator of such an overlapping gene, we predicted that the expression of 1-Pyrroline-5-carboxylate synthase 1 (AtP5CS1) is regulated by ath-miR164c at post-transcriptional level. Our results from the stem-loop RT-PCR based expression analysis revealed significant downregulation of ath-miR164c in response to P. syringae infection under both well-irrigated (pathogen only) and drought stress (combined stress) conditions. Furthermore, an Arabidopsis loss-of-function mutant of the miRNA ath-miR164c exhibited resistance to pathogen infection under combined stress, unlike the wild-type plants, implicating the role of ath-miR164c in regulating plant immunity. AtP5CS1 gene expression and proline accumulation were enhanced in the ath-miR164c mutant plants relative to the wild-type plants, demonstrating that ath-miR164c regulates AtP5CS1 of the proline biosynthesis pathway, which was also validated by 5’RLM-RACE results. This miRNA-mediated modulation of AtP5CS1 gene expression under combined stress fills crucial gaps in identifying the key convergent players in the current understanding of plant stress responses.

Engineering Bacillus velezensis with high production of acetoin primes strong induced systemic resistance in Arabidopsis thaliana

Many plant growth promoting rhizobacteria such as Bacillus velezensis GJ11 can produce acetoin to trigger induced systemic resistance (ISR) in plants. For improving acetoin production, the mutant strains were respectively constructed by knockout of the gene of bdh (2,3-butanediol dehydrogenase) and gdh (glycerol dehydrogenase) in GJ11, but only GJ11Δbdh produced a high level of acetoin triggering strong ISR against Pseudomonas syringae infection in plants. GJ11Δbdh could induce H2O2 accumulation in plants by producing a high level of acetoin. H2O2 was necessary for triggering ISR against the pathogen infection because after scavenging H2O2 with ascorbic acid or catalase, the inhibition role to pathogen infection induced by acetoin almost disappeared in plants. Further investigation found the plants treated with GJ11Δbdh in an obvious “priming” state, in which the mild immune response was observed such as a slight increase of H2O2 production, callose deposition, and enzymes activity related with defence response (e.g. POD, PAL and PPO). The plants in “priming” could rapidly respond to the pathogen infection accompanying with a significant increase of H2O2 production, callose deposition, and enzymes activity. Collectively, this study provides new insight into the role of acetoin as a strong elicitor of defense response, and ascribes a new approach to construct the mutant strains with high production of acetoin for triggering stronger ISR against pathogens infection in plants.

Ammonium mediated changes in carbon and nitrogen metabolisms induce resistance against Pseudomonas syringae in tomato plants

Predominant NH4+ nutrition causes an “ammonium syndrome” that induces metabolic changes and thereby provides resistance against Pseudomonas syringae infection through the activation of systemic acquired acclimation (SAA). Hence, to elucidate the mechanisms underlying NH4+-mediated SAA, the changes in nutrient balance and C and N skeletons were studied in NH4+-treated plants upon infection by P. syringae. A general decrease in cation and an increase in anion levels was observed in roots and leaves of NH4+-treated plants. Upon NH4+-based nutrition and infection, tomato leaves showed an accumulation of S, P, Zn, and of Mn. Mn accumulation might be required for ROS detoxification since it acts as a cofactor of superoxide dismutase (SOD). Primary metabolism was modified in both tissues of NH4+-fed plants to counteract NH4+ toxicity by decreasing TCA intermediates. A significant increase in Arg, Gln, Asn, Lys, Tyr, His and Leu was observed in leaves of NH4+-treated plants. The high level of the putrescine precursor Arg hints towards the importance of the Glu pathway as a key metabolic check-point in NH4+-treated and infected plants. Taken together, NH4+-fed plants displayed a high level of basal responses allowing them to activate SAA and to trigger defense responses against P. syringae through nutrient imbalances and changes in primary metabolism.

Characterization of squalene-induced PgCYP736B involved in salt tolerance by modulating key genes of abscisic acid biosynthesis

Cytochrome P450 genes as the one of the largest superfamily genes mediate a wide range of plant biochemical pathways. In this study, a full-length cytochrome P450 monooxygenase (CYP736B) cDNA was isolated and characterized from Panax ginseng. It was revealed that the deduced amino acid of PgCYP736B shares a high degree of sequence homology with CYP736A12 encoded by P. ginseng. Expression of PgCYP736B was differentially induced not only during a Pseudomonas syringae infection (7.7-fold) and wounding (47.3-fold) but also after exposure to salt (7.4-fold), cold (8.3-fold), and drought stress (3.24-fold). The gene transcription was highly affected by methyl jasmonate (476-fold) in the ginseng, suggesting that PgCYP736B was elicitor-responsive. Furthermore, we overexpressed the PgCYP736B gene in Arabidopsis and found that PgCYP736B is a transmembrane protein. Overexpression of PgCYP736B in Arabidopsis conferred enhanced resistance to salt stress via decreased H2O2 accumulation, increased carotenoid levels, and through abscisic acid biosynthesis gene expression. Our results suggest that the induction of ginsenoside biosynthetic pathway genes along with PgCYP736B by an exogenous supply of 10–100 μM of squalene most likely affects the metabolite profile of ginsenoside triterpenoid. Overall, our findings indicate that PgCYP736B protects ginseng from salt stress and may contribute to triterpenoid biosynthesis.

Arabidopsis RETICULON-LIKE3 (RTNLB3) and RTNLB8 Participate in Agrobacterium-Mediated Plant Transformation.

Agrobacterium tumefaciens can genetically transform various eukaryotic cells because of the presence of a resident tumor-inducing (Ti) plasmid. During infection, a defined region of the Ti plasmid, transfer DNA (T-DNA), is transferred from bacteria into plant cells and causes plant cells to abnormally synthesize auxin and cytokinin, which results in crown gall disease. T-DNA and several virulence (Vir) proteins are secreted through a type IV secretion system (T4SS) composed of T-pilus and a transmembrane protein complex. Three members of Arabidopsis reticulon-like B (RTNLB) proteins, RTNLB1, 2, and 4, interact with VirB2, the major component of T-pilus. Here, we have identified that other RTNLB proteins, RTNLB3 and 8, interact with VirB2 in vitro. Root-based A. tumefaciens transformation assays with Arabidopsis rtnlb3, or rtnlb5-10 single mutants showed that the rtnlb8 mutant was resistant to A. tumefaciens infection. In addition, rtnlb3 and rtnlb8 mutants showed reduced transient transformation efficiency in seedlings. RTNLB3- or 8 overexpression transgenic plants showed increased susceptibility to A. tumefaciens and Pseudomonas syringae infection. RTNLB1-4 and 8 transcript levels differed in roots, rosette leaves, cauline leaves, inflorescence, flowers, and siliques of wild-type plants. Taken together, RTNLB3 and 8 may participate in A. tumefaciens infection but may have different roles in plants.

A Localized Pseudomonas syringae Infection Triggers Systemic Clock Responses in Arabidopsis

The circadian clock drives daily rhythms of many plantphysiological responses, providing a competitive advantage that improves plant fitness and survival rates [1-5]. Whereas multiple environmental cues are predicted to regulate the plant clock function, most studies focused on understanding the effects of light and temperature [5-8]. Increasing evidence indicates a significant role of plant-pathogen interactions on clock regulation [9, 10], but the underlying mechanisms remain elusive. In Arabidopsis, the clock function largely relies on a transcriptional feedback loopbetween morning (CCA1 and LHY)- and evening (TOC1)-expressed transcription factors [6-8]. Here, we focused on these core components to investigate the Arabidopsis clock regulation using a unique biotic stress approach. We found that a single-leaf Pseudomonas syringae infection systemically lengthened theperiod and reduced the amplitude of circadian rhythms in distal uninfected tissues. Remarkably, the low-amplitude phenotype observed upon infection was recapitulated by a transient treatment with the defense-related phytohormone salicylic acid (SA), which also triggered a significant clock phase delay. Strikingly, despite SA-modulated circadian rhythms, we revealed that the master regulator of SA signaling, NPR1 [11, 12], antagonized clock responses triggered by both SA treatment and P.syringae. In contrast, weuncovered that the NADPH oxidase RBOHD [13] largely mediated the aforementioned clock responses after either SA treatment or the bacterial infection. Altogether, we demonstrated novel and unexpected roles for SA, NPR1, and redox signaling in clock regulation by P.syringae and revealed a previously unrecognized layer of systemic clock regulation by locally perceived environmental cues.

Nonredundant functions of Arabidopsis LecRK-V.2 and LecRK-VII.1 in controlling stomatal immunity and jasmonate-mediated stomatal closure.

Stomatal immunity restricts bacterial entry to leaves through the recognition of microbe-associated molecular patterns (MAMPs) by pattern-recognition receptors (PRRs) and downstream abscisic acid and salicylic acid signaling. Through a reverse genetics approach, we characterized the function of the L-type lectin receptor kinase-V.2 (LecRK-V.2) and -VII.1 (LecRK-VII.1). Analyses of interactions with the PRR FLAGELLIN SENSING2 (FLS2) were performed by co-immunoprecipitation and bimolecular fluorescence complementation and whole-cell patch-clamp analyses were used to evaluate guard cell Ca2+ -permeable cation channels. The Arabidopsis thaliana LecRK-V.2 and LecRK-VII.1 and notably their kinase activities were required for full activation of stomatal immunity. Knockout lecrk-V.2 and lecrk-VII.1 mutants were hyper-susceptible to Pseudomonas syringae infection and showed defective stomatal closure in response to bacteria or to the MAMPs flagellin and EF-Tu. By contrast, Arabidopsis over-expressing LecRK-V.2 or LecRK-VII.1 demonstrated a potentiated stomatal immunity. LecRK-V.2 and LecRK-VII.1 are shown to be part of the FLS2 PRR complex. In addition, LecRK-V.2 and LecRK-VII.1 were critical for methyl jasmonate (MeJA)-mediated stomatal closure, notably for MeJA-induced activation of guard cell Ca2+ -permeable cation channels. This study highlights the role of LecRK-V.2 and LecRK-VII.1 in stomatal immunity at the FLS2 PRR complex and in MeJA-mediated stomatal closure.

Epigenetic regulation of the expression of WRKY75 transcription factor in response to biotic and abiotic stresses in Solanaceae plants.

SlyWRKY75: gene expression was induced in response to biotic stresses, especially in Botrytis cinerea-infected tomato plants, in which Sly-miR1127-3p is a putative SlyWRKY75 regulator and epigenetic marks were detected. WRKY75 transcription factor involved in Pi homeostasis was recently found also induced in defense against necrotrophic pathogens. In this study, we analyzed by RT-qPCR the expression of SlyWRKY75 gene in tomato plants in response to abiotic stresses (drought or heat) and biotic stresses (Colorado potato beetle larvae infestation, Pseudomonas syringae or Botrytis cinerea infection) being only differentially expressed following biotic stresses, especially upon B. cinerea infection (55-fold induction). JA and JA-Ile levels were significantly increased in tomato plants under biotic stresses compared with control plants, indicating that SlyWRKY75 might be a transcriptional regulator of the JA pathway. The contribution of miRNAs and epigenetic molecular mechanisms to the regulation of this gene in B. cinerea-infected tomato plants was explored. We identified a putative Sly-miR1127-3p miRNA predicted to bind the intronic region of the SlyWRKY75 genomic sequence. Sly-miR1127-3p miRNA was repressed in infected plants (0.4-fold) supporting that it might act as an epigenetic regulation factor of SlyWRKY75 gene expression rather than via the post-transcriptional mechanisms of canonical miRNAs. It has been proposed that certain miRNAs can mediate DNA methylation in the plant nucleus broadening miRNA functions with transcriptional gene silencing by targeting intron-containing pre-mRNAs. Histone modifications analysis by chromatin immunoprecipitation (ChIP) demonstrated the presence of the activator histone modification H3K4me3 on SlyWRKY75 transcription start site and gene body. The induction of this gene in response to B. cinerea correlates with the presence of an activator mark. Thus, miRNAs and chromatin modifications might cooperate as epigenetic factors to modulate SlyWRKY75 gene expression.

Cyclic Dipeptides from Bacillus vallismortis BS07 Require Key Components of Plant Immunity to Induce Disease Resistance in Arabidopsis against Pseudomonas Infection.

Cyclic dipeptides (CDPs) are one of the simplest compounds produced by living organisms. Plant-growth promoting rhizobacteria (PGPRs) also produce CDPs that can induce disease resistance. Bacillus vallismortis strain BS07 producing various CDPs has been evaluated as a potential biocontrol agent against multiple plant pathogens in chili pepper. However, plant signal pathway triggered by CDPs has not been fully elucidated yet. Here we introduce four CDPs, cyclo(Gly-L-Pro) previously identified from Aspergillus sp., and cyclo(L-Ala-L-Ile), cyclo(L-Ala-L-Leu), and cyclo(LLeu-L-Pro) identified from B. vallismortis BS07, which induce disease resistance in Arabidopsis against Pseudomonas syringae infection. The CDPs do not directly inhibit fungal and oomycete growth in vitro. These CDPs require PHYTOALEXIN DEFICIENT4, SALICYLIC ACID INDUCTION DEFICIENT2, and NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 important for salicylic acid-dependent defense to induce resistance. On the other hand, regulators involved in jasmonate-dependent event, such as ETHYLENE RECEPTOR1, JASMONATE RESPONSE1, and JASMONATE INSENSITIVE1, are necessary to the CDP-induced resistance. Furthermore, treatment of these CDPs primes Arabidopsis plants to rapidly express PATHOGENESIS-RELATED PROTEIN4 at early infection phase. Taken together, we propose that these CDPs from PGPR strains accelerate activation of jasmonate-related signaling pathway during infection.