In plants, a first layer of inducible immunity is conferred by pattern recognition receptors (PRRs) that bind microbe- and damage-associated molecular patterns to activate pattern-triggered immunity (PTI). PTI is strengthened or followed by another potent form of immunity when intracellular receptors recognize pathogen effectors, termed effector-triggered immunity. Immunity signaling regulators have been reported to influence abiotic stress responses as well, yet the governing principles and mechanisms remain ambiguous. Here, we report that PRRs of a leucine-rich repeat ectodomain also confer salt tolerance in Arabidopsis thaliana, following recognition of cognate ligands such as bacterial flagellin (flg22 epitope) and elongation factor Tu (elf18 epitope), and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires authentic PTI signaling components; namely, the PRR-associated kinases BAK1 and BIK1 and the NADPH oxidase RBOHD. Exposure to salt stress induces the release of Pep precursors, pointing to the involvement of the endogenous immunogenic peptides in developing plant tolerance to high salinity. Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following a preexposure to immunogenic patterns. In good accordance, plants challenged with nonpathogenic bacteria also acquired salt tolerance in a manner dependent on PRRs. Our findings provide insight into signaling plasticity underlying biotic or abiotic stress cross-tolerance in plants conferred by PRRs.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Pattern recognition receptors (PRRs) bind microbe- and damage-associated molecular patterns (MAMPs/DAMPs, respectively) to enhance host immunity in animals and plants. Here, we report that PRRs also confer salt tolerance in the model plant Arabidopsis thaliana following recognition of cognate ligands, such as bacterial flagellin and EF-Tu and the endogenous Pep peptides. Pattern-triggered salt tolerance (PTST) requires the PRR-associated kinases BAK1 and BIK1 , and the NADPH oxidase RBOHD . Transcriptome profiling reveals an inventory of PTST target genes, which increase or acquire salt responsiveness following an exposure to immunogenic patterns. In their regulatory DNA sequences, specific binding sites for a subset of WRKY transcription factors are over-represented. Accordingly, PTST requires WRKY40 and WRKY18 , which activate salt tolerance-related genes but attenuate pathogen defense-related genes, including the EDS1 immunity activator. PRR signaling leads to sustained WRKY40/WRKY18 accumulation under salt stress and utilizes both WRKY s for salt tolerance. The PRR-WRKY40/WRKY18 module also confers salt tolerance after challenge with non-pathogenic bacteria. Our findings give molecular insight into signaling plasticity underlying biotic-abiotic stress cross-tolerance in plants conferred by PRRs.

Small post-translationally modified peptides are important signalling components of plant defence responses against phytopathogens, acting as both positive and negative modulators. PAMP-INDUCED SECRETED PEPTIDE (PIP) 1 and 2 have been shown to amplify plant immunity. Here we investigate the role of the related peptide PIP3 in the regulation of immune response in Arabidopsis. Treatment with synthetic PIP peptides led to similar transcriptome reprogramming, indicating an effect on innate immunity-related processes and phytohormones, including jasmonic acid (JA) biosynthesis and signalling. PIP3 overexpressing (OX) plants showed enhanced growth inhibition in response to flg22 exposure. In addition, flg22-induced production of reactive oxygen species and callose deposition was significantly reduced in PIP3-OX plants. Interestingly, PIP3-OX plants showed increased susceptibility toward both Botrytis cinerea and the biotrophic pathogen Pseudomonas syringae. Expression of both JA and salicylic acid (SA) biosynthesis and signalling genes was more induced during B. cinerea infection in PIP3-OX plants compared with wild-type plants. Promoter and ChIP-seq analyses indicated that the transcription factors WRKY18, WRKY33, and WRKY40 cooperatively act as repressors for PIP3. The results point to a fine-tuning role for PIP3 in modulation of immunity through the regulation of SA and JA biosynthesis and signalling pathways in Arabidopsis.

During microbe-associated molecular pattern-triggered immunity more than 5000 Arabidopsis genes are significantly altered in their expression, and the question arises, how such an enormous reprogramming of the transcriptome can be regulated in a safe and robust manner? For the WRKY transcription factors (TFs), which are important regulators of numerous defense responses, it appears that they act in a complex regulatory sub-network rather than in a linear fashion, which would be much more vulnerable to gene function loss either by pathogen-derived effectors or by mutations. In this study we employed RNA-seq, mass spectrometry and chromatin immunoprecipitation-seq to find evidence for and uncover principles and characteristics of this network. Upon flg22-treatment, one can distinguish between two sets of WRKY genes: constitutively expressed and induced WRKY genes. Prior to elicitation the induced WRKY genes appear to be maintained in a repressed state mainly by the constitutively expressed WRKY factors, which themselves appear to be regulated by non-WRKY TFs. Upon elicitation, induced WRKYs rapidly bind to induced WRKY gene promoters and by auto- and cross-regulation build up the regulatory network. Maintenance of this flg22-induced network appears highly robust as removal of three key WRKY factors can be physically and functionally compensated for by other WRKY family members.

AbstractThe sections in this article areIntroductionDefense TranscriptomeRegulatory Sequences Mediating Defense Gene ExpressionTranscription Factors Involved in Defense Gene RegulationTranscriptional NetworksDefense Gene Suppression by PathogensSummary and OutlookAcknowledgments

Rapid and massive transcriptional reprogramming upon pathogen recognition is the decisive step in plant-phytopathogen interactions. Plant transcription factors (TFs) are key players in this process but they require a suite of other context-specific co-regulators to establish sensory transcription regulatory networks to bring about host immunity. Molecular, genetic and biochemical studies, particularly in the model plants Arabidopsis and rice, are continuously uncovering new components of the transcriptional machinery that can selectively impact host resistance toward a diverse range of pathogens. Moreover, detailed studies on key immune regulators, such as WRKY TFs and NPR1, are beginning to reveal the underlying mechanisms by which defense hormones influence the function of these factors. Here we provide a short update on such recent developments.

The large WRKY transcription factor family is mainly involved in regulating plant immune responses. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic processes towards Botrytis cinerea strain 2100 infection and is essential for resistance. In contrast to B. cinerea strain 2100, the strain B05.10 is virulent on wild-type (WT) Col-0 Arabidopsis plants highlighting the genetic diversity within this pathogen species. We analysed how early WRKY33-dependent responses are affected upon infection with strain B05.10 and found that most of these responses were strongly dampened during this interaction. Ectopic expression of WRKY33 resulted in complete resistance towards this strain indicating that virulence of B05.10, at least partly, depends on suppressing WRKY33 expression/protein accumulation. As a consequence, the expression levels of direct WRKY33 target genes, including those involved in the biosynthesis of camalexin, were also reduced upon infection. Concomitantly, elevated levels of the phytohormone abscisic acid (ABA) were observed. Molecular and genetic studies revealed that ABA negatively influences defence to B05.10 and effects jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) levels. Susceptibility/resistance was determined by the antagonistic effect of ABA on JA, and this crosstalk required suppressing WRKY33 functions at early infection stages. This indicates that B. cinerea B05.10 promotes disease by suppressing WRKY33-mediated host defences.

The WRKY proteins belong to a superfamily of TFs that play pivotal roles in responses to a wide range of biotic, abiotic, developmental and physiologic cues. Here, we assayed the accumulation of basal WRKY27 transcripts in diverse tissue including root, shoot, leaf and flowers. We demonstrated that plants over-expressing WRKY27 transcript levels exhibit growth aberrations and fertility defects. Scanning electron microscopic data suggest that WRKY27 overexpressor plants exhibit pollen dehiscence defects. Our fluorescein diacetate hydrolysis assay showed that flowers of plants overexpressing WRKY27 display significantly decreased pollen viability. These sterility-related phenotypes were not rescued by the exogenous applications of different phytohormones. Our results indicate the involvement of WRKY27 in particular for proper plant biomass accumulation and male fertility.

Disease resistance of plants involves two distinct forms of chemical communication with the pathogen: recognition and defense. Both are essential components of a highly complex, multifaceted defense response, which begins with non-self recognition through the perception of pathogen-derived signal molecules and results in the production, inter alia, of antibiotically active compounds (phytoalexins) and cell wall-reinforcing material around the infection site. To elucidate the molecular details and the genomic basis of the underlying chains of events, we used two different experimental systems: suspension-cultured cells of Petroselinum crispum (parsley) and wildtype as well as mutant plants of Arabidopsis thaliana. Particular emphasis was placed on the structural and functional identification of signal and defense molecules, and on the mechanisms of signal perception, intracellular signal transduction and transcriptional reprogramming, including the structural and functional characterization of the responsible cis-acting gene promoter elements and transacting regulatory proteins. Comparing P. crispum and A. thaliana allows us to distinguish species-specific defense mechanisms from more universal responses, and furthermore provides general insights into the nature of the interactions. Despite the complexity of the pathogen defense response, it is experimentally tractable, and knowledge gained so far has opened up a new realm of gene technologyassisted strategies for resistance breeding of crop plants.

BACKGROUND:The interaction of Pseudomonas syringae with Arabidopsis is one of the most commonly used systems to study various bacterial-host interrelationships. Currently, most studies are based on the growth quantification of the pathogen to characterize resistance or virulence targets. However, the standard available method for determining bacterial proliferation in planta is laborious and has several limitations.RESULTS:Here we present an alternative robust approach, which is based on the quantification of bacterial DNA by real-time PCR. We directly compared this assay with the routinely used plate counting method to access bacterial titers in a number of well described Arabidopsis mutants.CONCLUSIONS:These studies showed that the DNA-based technique is highly reliable and comparable. Moreover, the technique is easily applicable, robust, and ideal for routine experiments or for larger scale analyses.

During microbial-associated molecular pattern-triggered immunity (MTI), molecules derived from microbes are perceived by cell surface receptors and upon signaling to the nucleus initiate a massive transcriptional reprogramming critical to mount an appropriate host defense response. WRKY transcription factors play an important role in regulating these transcriptional processes. Here, we determined on a genome-wide scale the flg22-induced in vivo DNA binding dynamics of three of the most prominent WRKY factors, WRKY18, WRKY40, and WRKY33. The three WRKY factors each bound to more than 1000 gene loci predominantly at W-box elements, the known WRKY binding motif. Binding occurred mainly in the 500-bp promoter regions of these genes. Many of the targeted genes are involved in signal perception and transduction not only during MTI but also upon damage-associated molecular pattern-triggered immunity, providing a mechanistic link between these functionally interconnected basal defense pathways. Among the additional targets were genes involved in the production of indolic secondary metabolites and in modulating distinct plant hormone pathways. Importantly, among the targeted genes were numerous transcription factors, encoding predominantly ethylene response factors, active during early MTI, and WRKY factors, supporting the previously hypothesized existence of a WRKY subregulatory network. Transcriptional analysis revealed that WRKY18 and WRKY40 function redundantly as negative regulators of flg22-induced genes often to prevent exaggerated defense responses.

The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity.

Arabidopsis immunity towards B. cinerea 2100 2 3 Shouan Liu, Barbara Kracher, Joerg Ziegler, Rainer P. Birkenbihl and Imre 4 E. Somssich* 5 Department of Plant Microbe Interactions, Max Planck Institute for Plant 6 Breeding Research, Carl-von-Linné Weg 10, 50829 Cologne, Germany 7 Department of Molecular Signal Processing, Leibnitz Institute of Plant 8 Biochemistry, Weinberg 3, 06120 Halle (Saale), Germany 9 10 11 12 *Corresponding author: 13 Imre E. Somssich 14 +49-221-5062-310 15 Email: somssich@mpipz.mpg.de 16 17 18 19 20

Next to numerous abiotic stresses, plants are constantly exposed to a variety of pathogens within their environment. Thus, their ability to survive and prosper during the course of evolution was strongly dependent on adapting efficient strategies to perceive and to respond to such potential threats. It is therefore not surprising that modern plants have a highly sophisticated immune repertoire consisting of diverse signal perception and intracellular signaling pathways. This signaling network is intricate and deeply interconnected, probably reflecting the diverse lifestyles and infection strategies used by the multitude of invading phytopathogens. Moreover it allows signal communication between developmental and defense programs thereby ensuring that plant growth and fitness are not significantly retarded. How plants integrate and prioritize the incoming signals and how this information is transduced to enable appropriate immune responses is currently a major research area. An important finding has been that pathogen-triggered cellular responses involve massive transcriptional reprogramming within the host. Additional key observations emerging from such studies are that transcription factors (TFs) are often sites of signal convergence and that signal-regulated TFs act in concert with other context-specific TFs and transcriptional co-regulators to establish sensory transcription regulatory networks required for plant immunity.

Trichome patterning in Arabidopsis thaliana is regulated by a regulatory feedback loop of the trichome promoting factors TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA3 (GL3)/ENHANCER OF GL3 (EGL3), and GL1 and a group of homologous R3MYB proteins that act as their inhibitors. Together, they regulate the temporal and spatial expression of GL2 and TTG2, which are considered to control trichome cell differentiation. In this work, we show that TTG2 is a specific activator of TRY (but not CPC or GL2). The WRKY protein TTG2 binds to W-boxes in a minimal promoter fragment of TRY, and these W-boxes are essential for rescue of the try mutant phenotype. We further show that TTG2 alone is not able to activate TRY expression, but rather drastically enhances the activation by TTG1 and GL3. As TTG2 physically interacts with TTG1 and because TTG2 can associate with GL3 through its interaction with TTG1, we propose that TTG2 enhances the activity of TTG1 and GL3 by forming a protein complex.

The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues. DOI: http://dx.doi.org/10.7554/eLife.00983.001.

BACKGROUND:The identification of endogenous cis-regulatory DNA elements (CREs) responsive to endogenous and environmental cues is important for studying gene regulation and for biotechnological applications but is labor and time intensive. Alternatively, by taking a synthetic biology approach small specific DNA binding sites tailored to the needs of the scientist can be generated and rapidly identified.RESULTS:Here we report a novel approach to identify stimulus-responsive synthetic CREs (SynCREs) from an unbiased random synthetic element (SynE) library. Functional SynCREs were isolated by screening the SynE libray for elements mediating transcriptional activity in plant protoplasts. Responsive elements were chromatin immunoprecipitated by targeting the active Ser-5 phosphorylated RNA polymerase II CTD (Pol II ChIP). Using sequential enrichment, deep sequencing and a bioinformatics pipeline, candidate responsive SynCREs were identified within a pool of constitutively active DNA elements and further validated. These included bonafide biotic/abiotic stress-responsive motifs along with novel SynCREs. We tested several SynCREs in Arabidopsis and confirmed their response to biotic stimuli.CONCLUSIONS:Successful isolation of synthetic stress-responsive elements from our screen illustrates the power of the described methodology. This approach can be applied to any transfectable eukaryotic system since it exploits a universal feature of the eukaryotic Pol II.

Summary In Arabidopsis thaliana , small peptides ( A t P eps) encoded by PROPEP genes act as damage‐associated molecular patterns ( DAMP s) that are perceived by two leucine‐rich repeat receptor kinases, PEPR 1 and PEPR 2, to amplify defense responses. In particular, expression of PROPEP2 and PROPEP3 is strongly and rapidly induced by A t Peps, in response to bacterial, oomycete, and fungal pathogens, and microbe‐associated molecular patterns ( MAMP s). The cis ‐regulatory modules ( CRM s) within the PROPEP2 and PROPEP3 promoters that mediate MAMP responsiveness were delineated, employing parsley ( Petroselinum crispum ) protoplasts and transgenic A. thaliana plants harboring promoter‐reporter constructs. By chromatin immunoprecipitation in vivo , DNA interactions with a specific transcription factor were detected. Furthermore, the phast C ons program was used to identify conserved regions of the PROPEP3 locus in different B rassicaceae species. The major MAMP ‐responsive CRM within the PROPEP2 promoter is composed of several W boxes and an as1 / OCS ( activation sequence‐1/octopine synthase ) enhancer element, while in the PROPEP3 promoter the CRM is comprised of six W boxes. The WRKY 33 transcription factor binds in vivo to these promoter regions in a MAMP ‐dependent manner. Both the position and orientation of the six W boxes are conserved within the PROPEP3 promoters of four other B rassicaceae family members. WRKY factors are the major regulators of MAMP ‐induced PROPEP2 and PROPEP3 expression.