Show me your secret(ed) weapons: a multifaceted approach reveals a wide arsenal of type III‐secreted effectors in the cucurbit pathogenic bacterium Acidovorax citrulli and novel effectors in the Acidovorax genus

Abstract

Summary The cucurbit pathogenic bacterium Acidovorax citrulli requires a functional type III secretion system (T3SS) for pathogenicity. In this bacterium, as with Xanthomonas and Ralstonia spp., an AraC‐type transcriptional regulator, HrpX, regulates expression of genes encoding T3SS components and type III‐secreted effectors (T3Es). The annotation of a sequenced A. citrulli strain revealed 11 T3E genes. Assuming that this could be an underestimation, we aimed to uncover the T3E arsenal of the A. citrulli model strain, M6. Thorough sequence analysis revealed 51 M6 genes whose products are similar to known T3Es. Furthermore, we combined machine learning and transcriptomics to identify novel T3Es. The machine‐learning approach ranked all A. citrulli M6 genes according to their propensity to encode T3Es. RNA‐Seq revealed differential gene expression between wild‐type M6 and a mutant defective in HrpX: 159 and 28 genes showed significantly reduced and increased expression in the mutant relative to wild‐type M6, respectively. Data combined from these approaches led to the identification of seven novel T3E candidates that were further validated using a T3SS‐dependent translocation assay. These T3E genes encode hypothetical proteins that seem to be restricted to plant pathogenic Acidovorax species. Transient expression in Nicotiana benthamiana revealed that two of these T3Es localize to the cell nucleus and one interacts with the endoplasmic reticulum. This study places A. citrulli among the ‘richest’ bacterial pathogens in terms of T3E cargo. It also revealed novel T3Es that appear to be involved in the pathoadaptive evolution of plant pathogenic Acidovorax species.

Identification and Functional Analysis of AopN, an Acidovorax Citrulli Effector that Induces Programmed Cell Death in Plants

“…are generally conserved, as determined by genome analysis [ 27 ]. Most of the predicted T3Es of A. citrulli shared similarities with those of Xanthomonas spp., as determined by bioinformatic analyses [ 21 ]. In this study, the homologous protein XopN in the A. citrulli Aac5 strain was identified by BLAST analysis and named AopN; the result is similar to that reported in the A. citrulli AAC00-1 strain and the M6 strain [ 21 ].…”

“…Most of the predicted T3Es of A. citrulli shared similarities with those of Xanthomonas spp., as determined by bioinformatic analyses [ 21 ]. In this study, the homologous protein XopN in the A. citrulli Aac5 strain was identified by BLAST analysis and named AopN; the result is similar to that reported in the A. citrulli AAC00-1 strain and the M6 strain [ 21 ]. Although AopN and XopN are homologous, they have just 26% similarity [ 27 ], and AopN was classified into subgroup III compared with Xanthomonas spp.…”

“…Through bioinformatic analysis, the AopN homologs were predicted to be a T3E in A. citrulli strain M6 [ 21 ]. It has been determined, by transcriptome data analysis that the expression of the AopN-coding gene was downregulated in hrpX mutant of M6 compared with that in the wild-type strain M6 [ 21 ].…”

“…Thus, identifying and analyzing functions of effector proteins of A. citrulli are essential in providing a foundation for studies on host resistance against BFB. In recent years, some progress has been made in A. citrulli effector proteins [ 21 ]. For example, A. citrulli group I and II strains differ in their effector compositions [ 22 ], and the presence or absence of these group-specific effectors may determine the preference of A. citrulli strains for different cucurbit hosts [ 23 ].…”

“…Using this hrpX mutant, we screened and identified an effector protein, AopP, which interfered with plant immune responses (unpublished). A recent study also used hrpX from A. citrulli group I strain M6 to screen for effector proteins using the transcriptome analysis [ 21 ]. However, only a few studies have been conducted to identify and characterize A. citrulli effector proteins.…”