Table_8.XLSX

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-Pseudomonas syringae (Pst) pathosystem with different Pst mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during Pst 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 Ca2+ 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, several potential PTI and ETI-specific markers are proposed. These results provide insights into the regulatory mechanisms underlying PTI and ETI in the tomato-Pst pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.

<p>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-Pseudomonas syringae (Pst) pathosystem with different Pst mutants that allow the dissection of PTI and ETI. A total of 225 proteins and 79 phosphopeptides differentially accumulated in tomato leaves during Pst 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-Pst pathosystem, and will promote future validation and application of the disease biomarkers in plant defense.</p>

Mitogen-activated protein kinases 3 and 6 (MPK3/MPK6) are central to pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in Arabidopsis, yet the involvement of long noncoding RNAs (lncRNAs, >200 nt) in these pathways is poorly understood. Here, transcriptomic analyses were performed to compare lncRNA and protein-coding gene (PCG) expression profiles in wild-type (WT) and MPK3/MPK6-deficient (MPK6SR) Arabidopsis plants. These plants were inoculated with either Pseudomonas syringae pv. tomato (Pst) DC3000, which elicits both PTI and ETI, or its type III secretion-deficient mutant, Pst DC3000 hrcC−, which induces only PTI. RNA sequencing (RNA-seq) analysis of 18 samples identified 1388 known and 70 novel lncRNAs, among which differentially expressed lncRNAs (DElncRNAs) involved in disease resistance were further identified. Using integrative analyses, including weighted gene co-expression network analysis (WGCNA), prediction of lncRNA cis-regulatory targets for PCGs, and validation via reverse transcription-quantitative PCR (RT-qPCR), three core lncRNA-mediated regulatory modules were identified: (i) MPK3/MPK6-dependent PTI and ETI, where lncRNAs amplify signals; (ii) MPK3/MPK6-dependent PTI, where lncRNAs fine-tune basal immunity; and (iii) MPK3/MPK6-independent PTI and ETI, where lncRNAs serve as a backup regulatory network. These modules form a multi-layered immune regulatory network via cis- and trans-regulation and further enable the identification of lncRNA-PCG pairs involved in both regulatory modes. This work enhances the understanding of the molecular mechanisms underlying plant innate immunity.

Plant cells possess a two-layered immune system consisting of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), mediated by cell surface pattern-recognition receptors and intracellular nucleotide-binding leucine-rich repeat receptors (NLRs), respectively. The CONSTITUTIVE EXPRESSION OF PR GENES 5 (CPR5) nuclear pore complex protein negatively regulates ETI, including ETI-associated hypersensitive response. Here, we show that CPR5 is essential for the activation of various PTI responses in Arabidopsis, such as resistance to the non-adapted bacterium Pseudomonas syringae pv. tomato DC3000 hrcC- . In a forward-genetic screen for suppressors of cpr5, we identified the mediator protein MED4. Mutation of MED4 in cpr5 greatly restored the defective PTI of cpr5. Our findings reveal that CPR5 plays opposite roles in regulating PTI and ETI, and genetically regulates PTI via MED4.

Introduction to a Virtual Special Issue on cell biology at the plant-microbe interface.

Pattern-recognition receptors (PRRs) located on the plant cell surface initiate immune responses by perceiving conserved pathogen molecules known as pathogen-associated molecular patterns (PAMPs). PRRs typically function in multiprotein complexes that include transmembrane and cytoplasmickinases and contribute to the initiation and signaling of pattern-triggered immunity (PTI). An important challenge is to identify molecular components of PRR complexes and downstream signaling pathways, and to understand the molecular mechanisms that mediate their function. In research activities supported by BARD-4931, we studied the role of the FLAGELLIN SENSING 3 (FLS3) PRR in the response of tomato leaves to flagellin-derivedPAMPs and PTI. In addition, we investigated molecular properties of the tomato brassinosteroid signaling kinase 830 (BSK830) that physically interacts with FLS3 and is a candidate for acting in the FLS3 signaling pathway. Our investigation refers to the proposal original objectives that were to: 1) Investigate the role of FLS3 and its interacting proteins in PTI; 2) Investigate the role of BSK830 in PTI; 3) Examine molecular and phosphorylation dynamics of the FLS3-BSK830 interaction; 4) Examine the possible interaction of FLS3 and BSK830 with Pstand Xcveffectors. We used CRISPR/Cas9 techniques to develop plants carrying single or combined mutations in the FLS3 gene and in the paralogsFLS2.1 and FLS2.2 genes, which encode the receptor FLAGELLIN SENSING2 (FLS2), and analyzed their function in PTI. Domain swapping analysis of the FLS2 and FLS3 receptors revealed domains of the proteins responsible for PAMP detection and for the different ROS response initiated by flgII-28/FLS3 as compared to flg22/FLS2. In addition, in vitro kinase assays and point mutations analysis identified FLS2 and FLS3 domains required for kinase activity and ATP binding. In research activities on tomato BSK830, we found that it interacts with PRRs and with the co-receptor SERK3A and PAMP treatment affects part of these interactions. CRISPR/Cas9 bsk830 mutant plants displayed enhanced pathogen susceptibility and reduced ROS production upon PAMP treatment. In addition, BSK830 interacted with 8 Xanthomonastype III secreted effectors. Follow up analysis revealed that among these effectors XopAE is part of an operon, is translocated into plant cells, and displays E3 ubiquitinligase activity. Our investigation was also extended to other Arabidopsis and tomato BSK family members. Arabidopsis BSK5 localized to the plant cell periphery, interacted with receptor-like kinases, and it was phosphorylatedin vitro by the PEPR1 and EFRPRRs. bsk5 mutant plants displayed enhanced susceptibility to pathogens and were impaired in several, but not all, PAMP-induced responses. Conversely, BSK5 overexpression conferred enhanced disease resistance and caused stronger PTI responses. Genetic complementation suggested that proper localization, kinase activity, and phosphorylation by PRRs are critical for BSK5 function. BSK7 and BSK8 specifically interacted with the FLS2 PRR, their respective mutant plants were more susceptible to B. cinereaand displayed reduced flg22-induced responses. The tomato BSK Mai1 was found to interact with the M3KMAPKKK, which is involved in activation of cell death associated with effector-triggered immunity. Silencing of Mai1 in N. benthamianaplants compromised cell death induced by a specific class of immune receptors. In addition, co-expression of Mai1 and M3Kin leaves enhanced MAPKphosphorylation and cell death, suggesting that Mai1 acts as a molecular link between pathogen recognition and MAPK signaling. Finally, We identified the PP2C phosphatase Pic1 that acts as a negative regulator of PTI by interacting with and dephosphorylating the receptor-like cytoplasmickinase Pti1, which is a positive regulator of plant immunity. The results of this investigation shed new light on the molecular characteristics and interactions of components of the immune system of crop plants providing new knowledge and tools for development of novel strategies for disease control.

In order to recognize a vast variety of attackers, plants possess a plethora of sophisticated detection systems. Perception of microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs) by the plant pattern recognition receptors (PRRs) leads to subsequent initiation of defense responses, a process collectively referred to as pattern-triggered immunity (PTI). PTI has been extensively studied in plant leaves, especially of the model organism Arabidopsis thaliana, whereas the mechanisms underlying PTI in roots so far attracted less attention. However, since a vast number of plant pests are soil-borne and attack roots in order to propagate and colonize whole plants, understanding the mechanisms underlying basic defense at the root level is of high interest for the development of new tools to combat root pathogens of crop plants. It has been demonstrated that recognition of flg22, the conserved epitope of the bacterial flagellin protein, leads to tissue-specific defense responses in roots. In order to investigate the cause for this tissue-specific induction of downstream responses, several approaches were employed during the course of this work. By studying the cellular localization of the PRR recognizing flg22, FLAGELLIN-SENSING 2 (FLS2), we were able to depict an expression map of FLS2 in wild-type Arabidopsis plants. Our study revealed that FLS2 was expressed in a highly tissue-specific manner in roots and shoots and that the FLS2 promoter activity was inducible upon environmental stimuli as well as during developmental processes, changing not only in intensity in expressing tissues but also in tissue-specificity. These results indicate an important role of the tissue-specific PRR localization in immunity mechanisms. In a parallel study, we expressed FLS2 under the control of several root tissue-specific promoters, which allowed us to analyze the competence of these tissues to detect flg22. Unexpectedly, all investigated root tissues were able to perceive externally applied flg22. In fact, PTI responses could be activated in intact roots as well as in dissected roots, suggesting that the peptide is able to penetrate through the different tissue layers. Remarkably, the expression level of the receptor was not the major parameter determining the magnitude of the immune response output. Thus, we postulated that perception of flg22 by certain tissues leads to stronger PTI responses potentially indicating why plants restrict immune receptor accumulation to tissue-specific locations possibly in order to balance the outcome of the defense activation. Due to the fact that many developmental or immunity processes in plants depend on systemic communication between different plant organs and that beneficial root microbes are known to prime and enhance resistance in aerial plant tissues, we hypothesized that MAMP perception by roots might induce a signaling event from roots to shoots. In order to address the potential existence of such systemic alarm signals, various methods were implemented. However, we encountered several technical limitations mainly concerning elicitor diffusion. Therefore, we focused on the development of an improved application method for studying systemic root-to-shoot signaling in Arabidopsis plants. Our system proved suitable to perform systemic signaling analysis and revealed that at the transcriptional level no systemically activated defense gene modifications were detectable in distal shoots of root-treated plants in our conditions. Like root pathogens, also viruses constitute a major threat in agro-economy and are responsible for immense crop losses. The basal defense response against viruses is thought to be mediated by RNA silencing, a process by which viral replication intermediates are cleaved and degraded by the plant silencing machinery through the recognition of virus-derived small RNAs. Intriguingly, a recent study conducted in our lab demonstrated a role of BRASSINOSTEROID INSENSITIVE1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1 (BAK1), a coreceptor of several PRRs involved in immunity and development, in antiviral defense. These results indicated that PTI may also contribute to antiviral resistance but the exact recognition process remained elusive. Because dsRNA produced during viral replication has been shown to act as a PAMP in animals, we decided to test whether dsRNA is perceived as a viral PAMP in planta as well. We found that natural as well as synthetic dsRNA is indeed perceived as a PAMP by Arabidopsis, leading to the activation of typical PTI responses. Remarkably, dsRNA application also promoted protection of Arabidopsis plants against viral infection. Taken together, this study provides new insights into the recognition mechanisms of bacteria- and virus-associated molecular patterns by different plant organs and contributes to elucidate the molecular defense strategy of plants against agriculturally important diseases.

ABSTRACTTo combat soilborne pathogens, roots activate pattern‐triggered immunity (PTI) through pattern‐recognition receptors (PRRs) that recognise microbe‐associated molecular patterns (MAMPs). Root PTI pathways can differ from their above‐ground counterparts and have been well‐characterised in the model plant Arabidopsis thaliana but are not well‐defined in crops. Gene repurposing coupled with differences in root tissues and root architecture in tomato species (Solanum lycopersicum and S. pimpinellifolium) led us to hypothesise that signalling pathways of Solanaceous‐specific PRRs diverge from canonical pathways. The objective of this study was to characterise PTI signalling pathways and responses (ROS, MAPK, gene expression, and growth inhibition) in roots of wild and domesticated tomatoes downstream of three immune receptors: the well‐conserved SlFLS2 and the Solanaeceous‐specific FLS3 and CORE. We find that Solanum root PTI responses are concentrated in early differentiating root regions compared to late differentiating regions or whole roots, and that FLS3 and CORE signalling pathways are overlapping but distinct from each other and from FLS2. Although the early differentiating root region had strong PTI responses across Solanum cultivars and species, different genetic backgrounds varied in their response dynamics. Our results underscore the complexity of PTI signalling across species and highlight the developmental‐stage specificity of tomato root immunity.

Plants rely on membrane-bound, pattern recognition receptors (PRRs) for extracellular recognition of pathogens as the first line of plant defense. This recognition of conserved microbial signatures, also called microbe-associated molecular patterns (MAMPs), initiates a broad-spectrum immune response known as pattern-triggered immunity (PTI). Overlapping mechanisms in PTI currently serve as the basis for some broad-spectrum resistance strategies; however, PRRs are known to be not only species-specific but also show tissue-specific expression patterns. Here, we identify the downstream PTI responses for three bacterial PRRs in roots of <i>Solanum lycopersicum</i> (tomato) and <i>Capsicum annuum</i> (pepper): FLAGELLIN SENSING 2 (FLS2) FLS3, and the cold-shock protein receptor CORE. Our results show that PTI responses in both tomato and pepper are PRR-specific. In tomato, the common readouts of PTI share overlapping, but distinct, responses to their cognate MAMP elicitor. We find that pepper and tomato share similarities in natural variation of PTI responses, though temporary root growth inhibition is a dominant phenotype for flg22-treated tomato but not flg22-treated pepper. Additionally, our results show that tomato root PTI responses are primarily located in developing tissues. We next shifted our focus from host PTI responses to pathogen-derived MAMPs. Exopolysaccharides (EPS) produced by the soil-borne pathogen <i>Ralstonia solanacearum</i> are required for virulence and have been shown to elicit responses in the host, though the exact mechanism remains unclear. We hypothesized that Ralstonia EPS would be recognized by plant roots and initiate PTI. Surprisingly, we found that crude EPS from two Ralstonia strains can serve as antioxidants for PTI-derived ROS burst in tomato roots. We show that crude EPS from both Ralstonia strains GMI1000 and K60 have different relative abundance in exopolysaccharide types. We show that loss of EPSI production significantly impacts the ability of GMI1000 to alleviate oxidative stress, while K60 relies on alternative mechanisms to mitigate oxidative damage. Together, this work highlights the differential sensitivity and signaling outputs to canonical MAMPs in the Solanaceae and, in turn, provides insight into how bacteria can evade these strategies in the Ralstonia-tomato pathosystem. Such findings will aid in advancing broad-spectrum engineering strategies using PRRs.

Plants rely on (in)direct detection of bacterial pathogens through plasma membrane-localized and intracellular receptor proteins. Surface pattern-recognition receptors (PRRs) participate in the detection of microbe-associated molecular patterns (MAMPs) and are required for the activation of pattern-triggered immunity (PTI). Pathogenic bacteria, such as Pseudomonas syringae pv. tomato (Pst) deploys ~ 30 effector proteins into the plant cell that contribute to pathogenicity. Resistant plants are capable of detecting the presence or activity of effectors and mount another response termed effector-triggered immunity (ETI). In order to investigate the involvement of tomato’s long non-coding RNAs (lncRNAs) in the immune response against Pst, we used RNA-seq data to predict and characterize those that are transcriptionally active in leaves challenged with a large set of treatments. Our prediction strategy was validated by sequence comparison with tomato lncRNAs described in previous works and by an alternative approach (RT-qPCR). Early PTI (30 min), late PTI (6 h) and ETI (6 h) differentially expressed (DE) lncRNAs were identified and used to perform a co-expression analysis including neighboring (± 100 kb) DE protein-coding genes. Some of the described networks could represent key regulatory mechanisms of photosynthesis, PRR abundance at the cell surface and mitigation of oxidative stress, associated to tomato-Pst pathosystem.

The innate immunity of plants is a dynamic, multilevel system traditionally divided into pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). Despite being activated by different types of receptors localized in different cell compartments, PTI and ETI are currently considered interdependent components of a single defense system. This view suggests that, due to various positive interactions between these two pathways, the innate immunity of plants is more than the sum of PTI andETI. Available data indicate that PTI and ETI enhance each other synergistically, increasing the concentration of signaling molecules, such as components of kinase cascades, reactive oxygen species, calcium ions, and phytohormones. This leads to the activation of defense genes, providing a local response to pathogens and the development of systemic plant resistance.

Plant-pathogen interactions induce a signal transmission series that stimulates the plant's host defense system against pathogens and this, in turn, leads to disease resistance responses. Plant innate immunity mainly includes two lines of the defense system, called pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). There is extensive signal exchange and recognition in the process of triggering the plant immune signaling network. Plant messenger signaling molecules, such as calcium ions, reactive oxygen species, and nitric oxide, and plant hormone signaling molecules, such as salicylic acid, jasmonic acid, and ethylene, play key roles in inducing plant defense responses. In addition, heterotrimeric G proteins, the mitogen-activated protein kinase cascade, and non-coding RNAs (ncRNAs) play important roles in regulating disease resistance and the defense signal transduction network. This paper summarizes the status and progress in plant disease resistance and disease resistance signal transduction pathway research in recent years; discusses the complexities of, and interactions among, defense signal pathways; and forecasts future research prospects to provide new ideas for the prevention and control of plant diseases.

Arabidopsis LONG-CHAIN BASE KINASE 1 (LCBK1) interacts with MEDEA, a component of PCR2 complex that negatively regulates immunity. LCBK1 phosphorylates phytosphingosine and thereby promotes stomatal immunity against bacterial pathogens. Arabidopsis polycomb-group repressor complex2 (PRC2) protein MEDEA (MEA) suppresses both pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). MEA represses the expression of RPS2 and thereby attenuates AvrRpt2 effector-mediated ETI. However, the mechanism of MEA-mediated PTI diminution was not known. By screening the Arabidopsis cDNA library using yeast-2-hybrid interaction, we identified LONG-CHAIN BASE KINASE1 (LCBK1) as an MEA-interacting protein. We found that lcbk1 mutants are susceptible to virulent bacterial pathogens, such as Pseudomonas syringae pv maculicola (Psm) and P. syringae pv tomato (Pst) but not the avirulent strain of Pst that carries AvrRpt2 effector. Pathogen inoculation induces LCBK1 expression, especially in guard cells. We found that LCBK1 has a positive regulatory role in stomatal closure after pathogen inoculation. WT plants close stomata within an hour of Pst inoculation or flg22 (a 22 amino acid peptide from bacterial flagellin protein that activates PTI) treatment, but not lcbk1 mutants. LCBK1 phosphorylates phytosphingosine (PHS). Exogenous application of phosphorylated PHS (PHS-P) induces stomatal closure and rescues loss-of-PTI phenotype of lcbk1 mutant plants. MEA overexpressing (MEA-Oex) plants are defective, whereas loss-of-function mea-6 mutants are hyperactive in PTI-induced stomatal closure. Exogenous application of PHS-P rescues loss-of-PTI in MEA-Oex plants. Results altogether demonstrate that LCBK1 is an interactor of MEA that positively regulates PTI-induced stomatal closure in Arabidopsis.

Plants employ the innate immune system to discriminate between self and invaders through two types of immune receptors, one on the plasma membrane and the other in the intracellular space. The immune receptors on the plasma membrane are pattern recognition receptors (PRRs) that can perceive pathogen-associated molecular patterns (PAMPs) or host-derived damage-associated molecular patterns (DAMPs) leading to pattern-triggered immunity (PTI). Particular pathogens are capable of overcoming PTI by secreting specific effectors into plant cells to perturb different components of PTI signalling through various mechanisms. Most of the immune receptors from the intracellular space are the nucleotide-binding leucine-rich repeat receptors (NLRs), which specifically recognize pathogen-secreted effectors to mediate effector-triggered immunity (ETI). In this review, we will summarize recent progress in structural studies of PRRs, NLRs, and effectors, and discuss how these studies shed light on ligand recognition and activation mechanisms of the two types of immune receptors and the diversified mechanisms used by effectors to manipulate plant immune signalling.

Pattern‐triggered immunity (PTI) is typically initiated in plants by recognition of pathogen‐ or damage‐associated molecular patterns (PAMP/DAMPs) by cell surface‐localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid‐signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor‐like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence‐related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP‐binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor.