Loss-of-function mutations in WRKY22 and WRKY25 impair stomatal-mediated immunity and PTI and ETI responses against Pseudomonas syringae pv. tomato.

Plants defend themselves against pathogens using a two-layered immune system. The first response, pattern-triggered immunity (PTI), is activated upon recognition of microbe-associated molecular patterns (MAMPs). Virulent bacteria such as Pseudomonas syringae pv. tomato (Pst), deliver effector proteins into the plant cell to promote susceptibility. However, some plants possess resistance (R) proteins that recognize specific effectors leading to the activation of the second response, effector-triggered immunity (ETI). Resistant tomatoes such as Río Grande-PtoR recognize two Pst effectors (AvrPto and AvrPtoB) through the host Pto/Prf complex and activate ETI. We previously showed that the transcription factors (TF) WRKY22 and WRKY25 are positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens in Nicotiana benthamiana. Here, the CRISPR-Cas9 technique was used to develop three knockout tomato lines for either one or both TFs. The single and double mutants were all compromised in Pto/Prf-mediated ETI and had a weaker PTI response. The stomata apertures in all of the mutant lines did not respond to darkness or challenge with Pst DC3000. The WRKY22 and WRKY25 proteins both localize in the nucleus, but we found no evidence of a physical interaction between them. The WRKY22 TF was found to be involved in the transcriptional regulation of WRKY25, supporting the idea that they are not functionally redundant. Together, our results indicate that both WRKY TFs play a role in modulating stomata and are positive regulators of plant immunity in tomato.

CRISPR/Cas9-mediated phospholipase C 2 knock-out tomato plants are more resistant to Botrytis cinerea.

MAIN CONCLUSION: CRISPR/Cas9-mediated Phospholipase C2 knock-out tomato plants are more resistant to Botrytis cinerea than wild-type plants, with less ROS and an increase and reduction of (JA) and (SA)-response marker genes, respectively. Genome-editing technologies allow non-transgenic site-specific mutagenesis of crops, offering a viable alternative to traditional breeding methods. In this study we used CRISPR/Cas9 to inactivate the tomato Phospholipase C2 gene (SlPLC2). Plant PLC activation is one of the earliest responses triggered by different pathogens regulating plant responses that, depending on the plant-pathogen interaction, result in plant resistance or susceptibility. The tomato (Solanum lycopersicum) PLC gene family has six members, named from SlPLC1 to SlPLC6. We previously showed that SlPLC2 transcript levels increased upon xylanase infiltration (fungal elicitor) and that SlPLC2 participates in plant susceptibility to Botrytis cinerea. An efficient strategy to control diseases caused by pathogens is to disable susceptibility genes that facilitate infection. We obtained tomato SlPLC2-knock-out lines with decreased ROS production upon B. cinerea challenge. Since this fungus requires ROS-induced cell death to proliferate, SlPLC2-knock-out plants showed an enhanced resistance with smaller necrotic areas and reduced pathogen proliferation. Thus, we obtained SlPLC2 loss-of-function tomato lines more resistant to B. cinerea by means of CRISPR/Cas9 genome editing technology.

Inferring the Significance of the Polyamine Metabolism in the Phytopathogenic Bacteria Pseudomonas syringae: A Meta-Analysis Approach.

To succeed in plant invasion, phytopathogenic bacteria rely on virulence mechanisms to subvert plant immunity and create favorable conditions for growth. This process requires a precise regulation in the production of important proteins and metabolites. Among them, the family of compounds known as polyamines have attracted considerable attention as they are involved in important cellular processes, but it is not known yet how phytopathogenic bacteria regulate polyamine homeostasis in the plant environment. In the present study, we performed a meta-analysis of publicly available transcriptomic data from experiments conducted on bacteria to begin delving into this topic and better understand the regulation of polyamine metabolism and its links to pathogenicity. We focused our research on Pseudomonas syringae, an important phytopathogen that causes disease in many economically valuable plant species. Our analysis discovered that polyamine synthesis, as well as general gene expression activation and energy production are induced in the early stages of the disease. On the contrary, synthesis of these compounds is inhibited whereas its transport is upregulated later in the process, which correlates with the induction of virulence genes and the metabolism of nitrogen and carboxylic acids. We also found that activation of plant defense mechanisms affects bacterial polyamine synthesis to some extent, which could reduce bacterial cell fitness in the plant environment. Furthermore, data suggest that a proper bacterial response to oxidative conditions requires a decrease in polyamine production. The implications of these findings are discussed.

WRKY22 and WRKY25 transcription factors are positive regulators of defense responses in Nicotiana benthamiana.

KEY MESSAGE: NbWRKY22 and NbWRKY25 are required for full activation of bacteria-associated pattern- and effector-triggered immunity as well as for the response to other non-bacterial defense elicitors. Plants defend themselves against pathogens using a two-layered immune system. Pattern-triggered immunity (PTI) can be activated upon recognition of epitopes from flagellin including flg22. Pseudomonas syringae pv. tomato (Pst) delivers effector proteins into the plant cell to promote host susceptibility. However, some plants express resistance (R) proteins that recognize specific effectors leading to the activation of effector-triggered immunity (ETI). Resistant tomato lines such as Rio Grande-PtoR (RG-PtoR) recognize two Pst effectors, AvrPto and AvrPtoB, and activate ETI through the Pto/Prf protein complex. Using RNA-seq, we identified two tomato WRKY transcription factor genes, SlWRKY22 and SlWRKY25, whose expression is increased during Pst-induced ETI. Silencing of the WRKY25/22 orthologous genes in Nicotiana benthamiana led to a delay in programmed cell death normally associated with AvrPto recognition or several non-bacterial effector/R protein pairs. An increase in disease symptoms was observed in silenced plants infiltrated with Pseudomonas syringae pv. tabaci expressing AvrPto or HopQ1-1. Expression of both tomato WRKY genes is also induced upon treatment with flg22 and callose deposition and cell death suppression assays in WRKY25/22-silenced N. benthamiana plants supported their involvement in PTI. Our results reveal an important role for two WRKYs as positive regulators of plant immunity against bacterial and potentially non-bacterial pathogens.

Genome-wide analysis uncovers tomato leaf lncRNAs transcriptionally active upon Pseudomonas syringae pv. tomato challenge.

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.

Tomato Wall-Associated Kinase SlWak1 Depends on Fls2/Fls3 to Promote Apoplastic Immune Responses to Pseudomonas syringae.

Wall-associated kinases (Waks) are important components of plant immunity against various pathogens, including the bacterium Pseudomonas syringae pv. tomato (Pst). However, the molecular mechanisms of their role(s) in plant immunity are largely unknown. In tomato (Solanum lycopersicum), wall-associated kinase 1 (SlWak1), has been implicated in pattern recognition receptor (PRR)-triggered immunity (PTI) because its transcript abundance increases significantly after treatment with the flagellin-derived, microbe-associated molecular patterns flg22 and flgII-28, which activate the PRRs Fls2 and Fls3, respectively. We generated two SlWak1 tomato mutants (Δwak1) using CRISPR/Cas9 gene editing technology and investigated the role of SlWak1 in tomato-Pst interactions. Late PTI responses activated in the apoplast by flg22 or flgII-28 were compromised in Δwak1 plants, but PTI at the leaf surface was unaffected. The Δwak1 plants developed fewer callose deposits than wild-type plants, but retained early PTI responses such as generation of reactive oxygen species and activation of mitogen-activated protein kinases upon exposure to flg22 and flgII-28. Induction of Wak1 gene expression by flg22 and flgII-28 was greatly reduced in a tomato mutant lacking Fls2 and Fls3, but induction of Fls3 gene expression by flgII-28 was unaffected in Δwak1 plants. After Pst inoculation, Δwak1 plants developed disease symptoms more slowly than Δfls2.1/2.2/3 mutant plants, although ultimately, both plants were similarly susceptible. SlWak1 coimmunoprecipitated with both Fls2 and Fls3, independently of flg22/flgII-28 or of BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1. These observations suggest that SlWak1 acts in a complex with Fls2/Fls3 and is important at later stages of PTI in the apoplast.

Transcriptome-based identification and validation of reference genes for plant-bacteria interaction studies using Nicotiana benthamiana.

RT-qPCR is a widely used technique for the analysis of gene expression. Accurate estimation of transcript abundance relies strongly on a normalization that requires the use of reference genes that are stably expressed in the conditions analyzed. Initially, they were adopted from those used in Northern blot experiments, but an increasing number of publications highlight the need to find and validate alternative reference genes for the particular system under study. The development of high-throughput sequencing techniques has facilitated the identification of such stably expressed genes. Nicotiana benthamiana has been extensively used as a model in the plant research field. In spite of this, there is scarce information regarding suitable RT-qPCR reference genes for this species. Employing RNA-seq data previously generated from tomato plants, combined with newly generated data from N. benthamiana leaves infiltrated with Pseudomonas fluorescens, we identified and tested a set of 9 candidate reference genes. Using three different algorithms, we found that NbUbe35, NbNQO and NbErpA exhibit less variable gene expression in our pathosystem than previously used genes. Furthermore, the combined use of the first two is sufficient for robust gene expression analysis. We encourage employing these novel reference genes in future RT-qPCR experiments involving N. benthamiana and Pseudomonas spp.

The Tomato Kinase Pti1 Contributes to Production of Reactive Oxygen Species in Response to Two Flagellin-Derived Peptides and Promotes Resistance to Pseudomonas syringae Infection.

The Pti1 kinase was identified from a reverse genetic screen as contributing to pattern-triggered immunity (PTI) against Pseudomonas syringae pv. tomato (Pst). The tomato genome has two Pti1 genes, referred to as Pti1a and Pti1b. A hairpin-Pti1 (hpPti1) construct was developed and was used to generate two independent stable transgenic tomato lines that had reduced transcript abundance of both genes. In response to P. syringae pv. tomato inoculation, these hpPti1 plants developed more severe disease symptoms, supported higher bacterial populations, and had reduced transcript accumulation of PTI-associated genes, as compared with wild-type plants. In response to two flagellin-derived peptides, the hpPti1 plants produced lesser amounts of reactive oxygen species (ROS) but showed no difference in mitogen-activated protein kinase (MAPK). Synthetic Pti1a and Pti1b genes designed to avoid silencing were transiently expressed in the hpPti1 plants and restored the ability of the plants to produce wild-type levels of ROS. Our results identify a new component of PTI in tomato that, because it affects ROS production but not MAPK signaling, appears to act early in the immune response.

Use of RNA-seq data to identify and validate RT-qPCR reference genes for studying the tomato-Pseudomonas pathosystem.

The agronomical relevant tomato-Pseudomonas syringae pv. tomato pathosystem is widely used to explore and understand the underlying mechanisms of the plant immune response. Transcript abundance estimation, mainly through reverse transcription-quantitative PCR (RT-qPCR), is a common approach employed to investigate the possible role of a candidate gene in certain biological process under study. The accuracy of this technique relies heavily on the selection of adequate reference genes. Initially, genes derived from other techniques (such as Northern blots) were used as reference genes in RT-qPCR experiments, but recent studies in different systems suggest that many of these genes are not stably expressed. The development of high throughput transcriptomic techniques, such as RNA-seq, provides an opportunity for the identification of transcriptionally stable genes that can be adopted as novel and robust reference genes. Here we take advantage of a large set of RNA-seq data originating from tomato leaves infiltrated with different immunity inducers and bacterial strains. We assessed and validated 9 genes that are much more stable than two traditional reference genes. Specifically, ARD2 and VIN3 were the most stably expressed genes and consequently we propose they be adopted for RT-qPCR experiments involving this pathosystem.

A novel method of transcriptome interpretation reveals a quantitative suppressive effect on tomato immune signaling by two domains in a single pathogen effector protein.

BACKGROUND: Effector proteins are translocated into host cells by plant-pathogens to undermine pattern-triggered immunity (PTI), the plant response to microbe-associated molecular patterns that interferes with the infection process. Individual effectors are found in variable repertoires where some constituents target the same pathways. The effector protein AvrPto from Pseudomonas syringae has a core domain (CD) and C-terminal domain (CTD) that each promotes bacterial growth and virulence in tomato. The individual contributions of each domain and whether they act redundantly is unknown. RESULTS: We use RNA-Seq to elucidate the contribution of the CD and CTD to the suppression of PTI in tomato leaves 6 h after inoculation. Unexpectedly, each domain alters transcript levels of essentially the same genes but to a different degree. This difference, when quantified, reveals that although targeting the same host genes, the two domains act synergistically. AvrPto has a relatively greater effect on genes whose expression is suppressed during PTI, and the effect on these genes appears to be diminished by saturation. CONCLUSIONS: RNA-Seq profiles can be used to observe relative contributions of effector subdomains to PTI suppression. Our analysis shows the CD and CTD multiplicatively affect the same gene transcript levels with a greater relative impact on genes whose expression is suppressed during PTI. The higher degree of up-regulation versus down-regulation during PTI is plausibly an evolutionary adaptation against effectors that target immune signaling.

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins.

BACKGROUND: Plants have two related immune systems to defend themselves against pathogen attack. Initially,pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses. RESULTS: We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling. CONCLUSIONS: Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Transcriptomics-based screen for genes induced by flagellin and repressed by pathogen effectors identifies a cell wall-associated kinase involved in plant immunity.

BACKGROUND: Microbe-associated molecular patterns, such as those present in bacterial flagellin, are powerful inducers of the innate immune response in plants. Successful pathogens deliver virulence proteins, termed effectors, into the plant cell where they can interfere with the immune response and promote disease. Engineering the plant immune system to enhance disease resistance requires a thorough understanding of its components. RESULTS: We describe a high-throughput screen, using RNA sequencing and virus-induced gene silencing, to identify tomato genes whose expression is enhanced by the flagellin microbe-associated molecular pattern flgII-28, but reduced by activities of the Pseudomonas syringae pv. tomato (Pst) type III effectors AvrPto and AvrPtoB. Gene ontology terms for this category of Flagellin-induced repressed by effectors (FIRE) genes showed enrichment for genes encoding certain subfamilies of protein kinases and transcription factors. At least 25 of the FIRE genes have been implicated previously in plant immunity. Of the 92 protein kinase-encoding FIRE genes, 33 were subjected to virus-induced gene silencing and their involvement in pattern-triggered immunity was tested with a leaf-based assay. Silencing of one FIRE gene, which encodes the cell wall-associated kinase SlWAK1, compromised the plant immune response resulting in increased growth of Pst and enhanced disease symptoms. CONCLUSIONS: Our transcriptomic approach identifies FIRE genes that represent a pathogen-defined core set of immune-related genes. The analysis of this set of candidate genes led to the discovery of a cell wall-associated kinase that participates in plant defense. The FIRE genes will be useful for further elucidation of the plant immune system.