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.

Expression profiling of endo-xylanases during ripening of strawberry cultivars with contrasting softening rates. Influence of postharvest and hormonal treatments.

BACKGROUND: Softening is one of the main features that determine fruit quality during strawberry (Fragaria x ananassa, Duch.) ripening and storage. Being closely related to textural changes, the molecular and biochemical bases underlying strawberry cell-wall metabolism is a matter of interest. Here we investigated the abundance of transcripts encoding putative strawberry endo-xylanases in plant tissues, during fruit ripening and under postharvest and hormonal treatments. Total xylanase activity and expression of related genes in strawberry varieties with contrasting firmness were analyzed. RESULTS: FaXynA and FaXynC mRNA abundance was significantly higher than FaXynB in each plant tissue studied. Higher total xylanase activity was detected at the end of the ripening of the softer cultivar ('Toyonoka') in comparison with the firmer one ('Camarosa'), correlating with the abundance of FaXynA and FaXynC transcripts. Postharvest 1-methylcyclopropene treatment up-regulated FaXynA and FaXynC expressions. FaXynC mRNA abundance decreased with heat treatment but the opposite was observed for FaXynA. Calcium chloride treatment down-regulated FaXynA and FaXynC expression. Both genes responded differently to plant growth regulators' exposure. FaXynC expression was down-regulated by auxins and gibberellins treatment and up-regulated by abscisic acid. FaXynA was up-regulated by auxins, while no changes in mRNA levels were evident by abscisic acid and gibberellins treatment. Ethephon exposure did not change FaXynA and FaXynC expressions. CONCLUSION: New knowledge about the presence of xylanases in ripening strawberry fruit and their response to postharvest and hormonal treatments is provided. Our findings suggest a role for endo-xylanases in hemicelluloses depolymerization and possibly in strawberry fruit softening. © 2020 Society of Chemical Industry.

Molecular Characterization of Differences between the Tomato Immune Receptors Flagellin Sensing 3 and Flagellin Sensing 2.

Plants mount defense responses by recognizing indicators of pathogen invasion, including microbe-associated molecular patterns (MAMPs). Flagellin, from the bacterial pathogen Pseudomonas syringae pv. tomato (Pst), contains two MAMPs, flg22 and flgII-28, that are recognized by tomato (Solanum lycopersicum) receptors Flagellin sensing2 (Fls2) and Fls3, respectively, but to what degree each receptor contributes to immunity and whether they promote immune responses using the same molecular mechanisms are unknown. Here, we characterized CRISPR/Cas9-generated Fls2 and Fls3 tomato mutants and found that the two receptors contribute equally to disease resistance both on the leaf surface and in the apoplast. However, we observed striking differences in certain host responses mediated by the two receptors. Compared to Fls2, Fls3 mediated a more sustained production of reactive oxygen species and an increase in transcript abundance of 44 tomato genes, with two genes serving as specific reporters for the Fls3 pathway. Fls3 had greater in vitro kinase activity than Fls2 and could transphosphorylate a substrate. Using chimeric Fls2/Fls3 proteins, we found no evidence that a single receptor domain is responsible for the Fls3-sustained reactive oxygen species, suggesting involvement of multiple structural features or a nullified function of the chimeric construct. This work reveals differences in certain immunity outputs between Fls2 and Fls3, suggesting that they might use distinct molecular mechanisms to activate pattern-triggered immunity in response to flagellin-derived MAMPs.

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.

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.

Functional genomics of tomato for the study of plant immunity.

Tomato (Solanum lycopersicum), along with many other economically valuable species, belongs to the Solanaceae family. Understanding how plants in this family defend themselves against pathogens offers the opportunity of improving yield and quality of their edible products. The use of functional genomics has contributed to this purpose through both traditional and recently developed techniques that allow determination of changes in transcript abundance during pathogen attack. Such changes can implicate the affected gene as participating in plant defense. Testing the involvement of these candidate genes in defense has relied largely on posttranscriptional gene silencing, particularly virus-induced gene silencing. We discuss how functional genomics has played a key role in our current understanding of the defense response in tomato and related species and what are the challenges and opportunities for the future.

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.

Validation of reference transcripts in strawberry (Fragaria spp.).

Contemporary methods to assay gene expression depend on a stable set of reference transcripts for accurate quantitation. A lack of well-tested reference genes slows progress in characterizing gene expression in high-value specialty crops. In this study, a set of strawberry (Fragaria spp.) constitutively expressed reference genes has been identified by merging digital gene expression data with expression profiling. Constitutive reference candidates were validated using quantitative PCR and hybridization. Several transcripts have been identified that show improved stability across tissues relative to traditional reference transcripts. Results are similar between commercial octoploid strawberry and the diploid model. Our findings also show that while some never-before-used references are appropriate for most applications, even the most stable reference transcripts require careful assessment across the diverse tissues and fruit developmental states before being adopted as controls.

A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research.

Nicotiana benthamiana is a widely used model plant species for the study of fundamental questions in molecular plant-microbe interactions and other areas of plant biology. This popularity derives from its well-characterized susceptibility to diverse pathogens and, especially, its amenability to virus-induced gene silencing and transient protein expression methods. Here, we report the generation of a 63-fold coverage draft genome sequence of N. benthamiana and its availability on the Sol Genomics Network for both BLAST searches and for downloading to local servers. The estimated genome size of N. benthamiana is 3 Gb (gigabases). The current assembly consists of approximately 141,000 scaffolds, spanning 2.6 Gb with 50% of the genome sequence contained within scaffolds >89 kilobases. Of the approximately 16,000 N. benthamiana unigenes available in GenBank, >90% are represented in the assembly. The usefulness of the sequence was demonstrated by the retrieval of N. benthamiana orthologs for 24 immunity-associated genes from other species including Ago2, Ago7, Bak1, Bik1, Crt1, Fls2, Pto, Prf, Rar1, and mitogen-activated protein kinases. The sequence will also be useful for comparative genomics in the Solanaceae family as shown here by the discovery of microsynteny between N. benthamiana and tomato in the region encompassing the Pto and Prf genes.

alpha-l-Arabinofuranosidase from strawberry fruit: cloning of three cDNAs, characterization of their expression and analysis of enzymatic activity in cultivars with contrasting firmness.

Softening of fleshy fruits during ripening is associated to catabolism of cell wall components. In strawberry, pectin degradation, as well as loss of neutral sugars (mainly arabinose), increases during ripening, and probably contributes to fruit softening. In this work, we report the activity of alpha-l-arabinofuranosidase (alpha-l-arafase) and the expression of related genes in strawberry. Activity of alpha-l-arafase was measured during ripening of cultivars with contrasting firmness. An important increment in the specific activity of alpha-l-arafase was detected during ripening in both cultivars. However, in the softest one (Toyonoka) the specific activities were higher than in the firmest (Camarosa). A combination of semi quantitative reverse transcriptase-PCR (RT-PCR) with degenerate primers and a screening of a cDNA library allowed the isolation and cloning of three cDNAs encoding putative alpha-l-arafases (FaAra1, FaAra2 and FaAra3). The deduced proteins revealed that FaAras belong to the glycoside hydrolase family 51 and not to glycoside hydrolase family 3. Expression studies, carried out by means of Northern-blot and semi quantitative RT-PCR, revealed that FaAras were predominantly expressed in fruit tissue and detected over the entire ripening process. Due to similarity of FaAras sequences, Northern-blot analysis probably grouped the expression of the three genes. The expression was high at small green stage, decreased at white stage and increased thereafter. The increment of the expression from white to 50% red stage was more evident in the softest cultivar (Toyonoka). Semi quantitative RT-PCR analysis allowed determining the expression of individual FaAras. The expression of the three genes was detected in all developmental and ripening stages. However, differences in expression levels could be detected between cultivars. In the softest cultivar, the expression of the three FaAras was higher at 50% and 75% red stages, and in the case of FaAra3 a higher expression was found also at 100% red stage. Overall, specific activity of alpha-l-arafase was higher in the softest cultivar; such activity reflects the expression of at least three putative FaAra genes.

ß-Xylosidase in strawberry fruit: Isolation of a full-length gene and analysis of its expression and enzymatic activity in cultivars with contrasting firmness.

Strawberry is a non-climateric fleshy fruit, which softens quickly and has short post-harvest life. Ripening is associated with an increment of pectin solubility and a reduction of the content of hemicelluloses. In this work, we have cloned the full-length cDNA encoding a ß-xylosidase (FaXyl1) from Fragaria×ananassa and we have characterized its expression in two strawberry cultivars with contrasting fruit firmness. The analysis of the predicted protein showed that FaXyl1 is closely related to other ß-xylosidases from higher plants. The recombinant protein obtained by over-expressing FaXyl1 in Escherichia coli had ß-xylosidase activity against the artificial substrate p-nitrophenyl ß-d-xilopyranoside. Differently from other bifunctional xylosidases, no α-l-arabinofuranosidase activity was detected in the recombinant enzyme. The expression of FaXyl1 gene was analyzed by northern-blot in Camarosa and Toyonaka strawberry cultivars, and compared with the corresponding protein data obtained by Western-blot and with the ß-xylosidase activity during ripening. The softest cultivar (Toyonaka) showed an early accumulation of FaXyl1 transcript and a higher expression of the corresponding protein during ripening, which correlates with a higher ß-xylosidase activity in all ripening stages analyzed.