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

Abstract

BackgroundPlants 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.ResultsWe 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.ConclusionsUsing 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.

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

“…As observed for the PTI reporter gene, the activation of the ETI marker gene was over-estimated. Additionally, the gene expression reduction between 6 and 12 h previously reported18, could not be observed when using GAPDH normalization. Again, the combined use of ARD2 / VIN3 leads to a drastic reduction in standard deviation values.…”

“…To validate the selection of reference genes, we measured the expression of a PTI- and an ETI-specific gene that were previously reported18. As recommended51, we estimated the relative expression using the normalization factor NF calculated as the geometric mean of Cq values obtained for ARD2 and VIN3 , the most stable reference genes.…”

Defining essential processes in plant pathogenesis with Pseudomonas syringae pv. tomato DC3000 disarmed polymutants and a subset of key type III effectors

“…A VIGS forward screen in N. benthamiana for PTI genes revealed surprisingly few essential genes and no silver-bullet candidates for the PTI-protected state (Chakravarthy et al, 2010). However, RNA-seq transcription profiling aimed at distinguishing PTI and ETI responses of tomato to DC3000 derivatives revealed enrichment for genes associated with the phenylpropanoid pathway in the PTI-specific set (Pombo et al, 2014). Phenylpropanoid pathway genes were similarly found in a transcriptome study of PTI genes in tobacco (Szatmari et al, 2014), and the N. benthamiana VIGS screen identified the CA4H gene which plays a role in phenylpropanoid biosynthesis (Chakravarthy et al, 2010).…”

Expression of tomato reference genes using established primer sets: Stability across experimental set‐ups

“…NormFinder uses a model-based approach allowing intra-and intergroup comparisons of the obtained Ct values, and it provides a ranking of the candidate reference genes based on the estimated standard deviations. Recent studies have proposed different reference genes for expression studies during tomato (Solanum lycopersicum) development (Expósito-Rodríguez, Borges, Borges-Pérez, & Pérez, 2008) and tomato-pathogen interactions (Fradin et al, 2009;Müller et al, 2015;Pombo et al, 2014). Primer pairs for ten of these genes-four classical housekeeping genes, such as elongation factor EF1-α and ATPase (Expósito-Rodríguez et al, 2008;Pombo et al, 2014), and six newly suggested ones (Müller et al, 2015;Pombo et al, 2014)-were selected and evaluated in tomato-grey mould interaction.…”

“…tomato interaction. The pathogen-leaf infiltrations were performed on whole plants from which leaf samples were harvested at 3, 6, 9 and 12 hrs postinoculation (hpi) (Pombo et al, 2014). Primers amplifying importin β (primer set IMPβ), PHD finger family protein (PHD), cytochrome c oxidase subunit (COX), polyribonucleotide 5′-hydroxyl-kinase (CLP1) and U6 snR-NA-associated Sm-like protein LSm7 (LSM7) were used in a tomato (cv.…”