Early ETI and PTI responses in Arabidopsis thaliana camta3-D (sr1-4d) mutant
ABSTRACT: Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. In Arabidopsis, the CAMTA family of transcription factors plays a pivotal function in immunity. CAMTA binding motifs are highly enriched in the genes quickly induced during ETI and PTI. Using RNA-seq, we investigated the role of CAMTA TFs during the early ETI and PTI transcriptional responses.
Project description:Pattern recognition receptors (PRRs) and nucleotide-binding domain and leucine-rich repeat (LRR)-containing proteins (NLRs) initiate pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), respectively, each associated with the activation of an overlapping set of defence genes. The regulatory mechanism behind this convergence of PTI- and ETI-mediated defence gene induction remains elusive. We generated transgenic Arabidopsis plants that enable conditional NLR activation without pathogen infection to dissect NLR- and PRR-mediated transcriptional signals. A comparative analysis of over 40 transcriptome datasets linked calmodulin-binding transcription activators (CAMTAs) to the activation of overlapping defence genes in PTI and ETI. We used a dominant camta3 mutant (camta3-D) to assess CAMTA functions in the corresponding transcriptional regulation. Transcriptional regulation by NLRs, although highly similar to PTI responses, can be established independently of pathogen-associated molecular pattern (PAMP) perception, defence phytohormones and host cell death. Conditional expression of the N-terminal coiled-coil domain of the barley MLA (Mildew resistance locus A) NLR is sufficient to trigger similar transcriptional reprogramming as full-length NLRs. CAMTA-binding motifs are overrepresented in the 5' regulatory regions of the identified primary immune response genes, consistent with their altered expression and disease resistance responses in camta3-D plants. We propose that CAMTA-mediated transcriptional regulation defines an early convergence point in NLR- and PRR-mediated signalling.
Project description:The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)<sup>1-4</sup>. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades<sup>5,6</sup>. Here we show that Arabidopsis PRR and PRR co-receptor mutants-fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants-are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.
Project description:Plant innate immunity is composed of two layers. Basal immunity is triggered by pathogen-associated molecular patterns (PAMPs) such as the flagellin-peptide flg22 and is termed PAMP-triggered immunity (PTI). In addition, effector-triggered immunity (ETI) linked with programmed cell death and cytoskeletal reorganization can be induced by pathogen-derived factors, such as the Harpin proteins originating from phytopathogenic bacteria. To get insight into the link between cytoskeleton and PTI or ETI, this study followed the responses of actin filaments and microtubules to flg22 and HrpZ in vivo by spinning-disc confocal microscopy in GFP-tagged marker lines of tobacco BY-2. At a concentration that clearly impairs mitosis, flg22 can induce only subtle cytoskeletal responses. In contrast, HrpZ causes a rapid and massive bundling of actin microfilaments (completed in ~20 min, i.e. almost simultaneously with extracellular alkalinization), which is followed by progressive disintegration of actin cables and cytoplasmic microtubules, a loss of cytoplasmic structure, and vacuolar disintegration. Cytoskeletal disruption is proposed as an early event that discriminates HrpZ-triggered ETI-like defence from flg22-triggered PTI.
Project description:Plants can activate defence to pathogen attack by two layers of innate immunity: basal immunity triggered by pathogen-associated molecular pattern (PAMP) triggered immunity (PTI) and effector-triggered immunity (ETI) linked with programmed cell death. Flg22 and Harpin are evolutionary distinct bacterial PAMPs. We have previously shown that Harpin triggers hypersensitive cell death mimicking ETI in Vitis rupestris, but not in the Vitis vinifera cultivar 'Pinot Noir'. In contrast, the bacterial PAMP flg22 activating PTI does not trigger cell death. To get insight into the defence signalling triggered by flg22 and Harpin, we compared cellular responses upon flg22 and Harpin treatment in the two Vitis cell lines. We found that extracellular alkalinisation was blocked by inhibition of calcium influx, and modulated by pharmacological manipulation of the cytoskeleton and mitogen-activated protein kinase activity with quantitative differences between cell lines and type of PAMPs. In addition, an oxidative burst was detected that was much stronger and faster in response to Harpin as compared to flg22. In V. rupestris, both flg22 and Harpin induced transcripts of defence-related genes including stilbene synthase, microtubule disintegration and actin bundling in a similar way, whereas they differed in V. vinifera cv. 'Pinot Noir'. In contrast to Harpin, flg22 failed to trigger significant levels of the stilbene trans-resveratrol, and did not induce hypersensitive cell death even in the highly responsive V. rupestris. We discuss these data in a model, where flg22- and Harpin-triggered defence shares a part of early signal components, but differs in perception, oxidative burst, and integration into a qualitatively different stilbene output, such that for flg22 a basal PTI is elicited in both cell lines, while Harpin induces cell death mimicking an ETI-like pattern of defence.
Project description:Innate immune responses of plant cells confer the first line of defence against pathogens. Signals generated by activated receptors are integrated inside the cell and converge on transcriptional programmes in the nucleus. The Arabidopsis Toll-related intracellular receptor RPS4 operates inside nuclei to trigger resistance and defence gene reprogramming through the stress response regulator, EDS1. In order to test a role for RPS4 at the chromatin level during Effector-Triggered Immunity (ETI), we undertook a ChIP-seq approach.
Project description:The soybean cyst nematode (SCN), Heterodera glycines, is one of the most destructive pathogens of soybeans. SCN is an obligate and sedentary parasite that transforms host plant root cells into an elaborate permanent feeding site, a syncytium. Formation and maintenance of a viable syncytium is an absolute requirement for nematode growth and reproduction. In turn, sensing pathogen attack, plants activate defence responses and may trigger programmed cell death at the sites of infection. For successful parasitism, H. glycines must suppress these host defence responses to establish and maintain viable syncytia. Similar to other pathogens, H. glycines engages in these molecular interactions with its host via effector proteins. The goal of this study was to conduct a comprehensive screen to identify H. glycines effectors that interfere with plant immune responses. We used Nicotiana benthamiana plants infected by Pseudomonas syringae and Pseudomonas fluorescens strains. Using these pathosystems, we screened 51 H. glycines effectors to identify candidates that could inhibit effector-triggered immunity (ETI) and/or pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). We identified three effectors as ETI suppressors and seven effectors as PTI suppressors. We also assessed expression modulation of plant immune marker genes as a function of these suppressors.
Project description:Plants have evolved a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI and ETI are functionally linked, but also have distinct characteristics. Unraveling how these immune systems coordinate plant responses against pathogens is crucial for understanding the regulatory mechanisms underlying plant defense. Here we report integrative proteomic and phosphoproteomic analyses of the tomato-<i>Pseudomonas syringae (Pst)</i> pathosystem with different <i>Pst</i> mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during <i>Pst</i> infection. The abundances of many proteins and phosphoproteins changed during PTI or ETI, and some responses were triggered by both PTI and ETI. For most proteins, the ETI response was more robust than the PTI response. The patterns of protein abundance and phosphorylation changes revealed key regulators involved in Ca<sup>2+</sup> signaling, mitogen-activated protein kinase cascades, reversible protein phosphorylation, reactive oxygen species (ROS) and redox homeostasis, transcription and protein turnover, transport and trafficking, cell wall remodeling, hormone biosynthesis and signaling, suggesting their common or specific roles in PTI and/or ETI. A NAC (NAM, ATAF, and CUC family) domain protein and lipid particle serine esterase, two PTI-specific genes identified from previous transcriptomic work, were not detected as differentially regulated at the protein level and were not induced by PTI. Based on integrative transcriptomics and proteomics data, as well as qRT-PCR analysis, several potential PTI and ETI-specific markers are proposed. These results provide insights into the regulatory mechanisms underlying PTI and ETI in the tomato-<i>Pst</i> pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.
Project description:Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.
Project description:We observed inhibition of the hypersensitive response (a typical ETI response) by PTI signaling in an Arabidopsis quadruple mutant dde2 ein2 pad4 sid2 (quad). Thus, we designed an experiment to see the interaction between ETI and PTI signaling at the transcriptome level. The Arabidopsis line dde2 ein2 pad4 sid2 Ed-AvrRpt2 (quadAvrRpt2) was used (Ed-AvrRpt2, estradiol-inducible AvrRpt2 transgene). In this plant line, ETI can be elicited by estradiol (Ed) treatment via in planta expression of AvrRpt2. Transcriptome responses to PTI only, ETI only, and PTI+ETI together were recorded.
Project description:Plant immunity consists of two arms: pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI), induced by surface-localized receptors, and effector-triggered immunity (ETI), induced by intracellular receptors. Despite the little structural similarity, both receptor types activate similar responses with different dynamics. To better understand phosphorylation events during ETI, we employed a phosphoproteomic screen using an inducible expression system of the bacterial effector avrRpt2 in Arabidopsis thaliana, and identified 109 differentially phosphorylated residues of membrane-associated proteins on activation of the intracellular RPS2 receptor. Interestingly, several RPS2-regulated phosphosites overlap with sites that are regulated during PTI, suggesting that these phosphosites may be convergent points of both signaling arms. Moreover, some of these sites are residues of important defense components, including the NADPH oxidase RBOHD, ABC-transporter PEN3, calcium-ATPase ACA8, noncanonical G? protein XLG2 and H+ -ATPases. In particular, we found that S343 and S347 of RBOHD are common phosphorylation targets during PTI and ETI. Our mutational analyses showed that these sites are required for the production of reactive oxygen species during both PTI and ETI, and immunity against avirulent bacteria and a virulent necrotrophic fungus. We provide, for the first time, large-scale phosphoproteomic data of ETI, thereby suggesting crucial roles of common phosphosites in plant immunity.