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.

中文翻译：

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向我展示您的秘密武器：多方面的方法揭示了葫芦科病原菌Acidovorax citrulli中广泛分泌III型效应子的库，以及Acidovorax属中的新型效应器。

瓜类致病性细菌Acidovorax citrulli需要功能性III型分泌系统（T3SS）才能致病。在这种细菌中，如黄单胞菌和Ralstonia spp 。AraC型转录调节子HrpX调节编码T3SS成分和III型分泌效应子（T3Es）的基因的表达。测序的柑橘曲霉菌株的注释揭示了11个T3E基因。假设这可能被低估了，我们的目标是发现柑桔曲霉的T3E库。模型应变，M6。彻底的序列分析揭示了51个M6基因，其产物与已知的T3E相似。此外，我们将机器学习与转录组学相结合，以鉴定出新颖的T3E。机器学习方法根据所有A. citrulli M6基因对它们编码T3E的倾向进行排名。RNA‐Seq揭示了野生型M6和突变型HrpX中的缺陷之间的差异基因表达：159和28个基因分别相对于野生型M6显着降低和增加了该突变体中的表达。这些方法结合的数据导致鉴定了7种新的T3E候选物，这些候选物已使用依赖T3SS的易位测定法进一步验证。这些T3E基因编码的假设蛋白似乎仅限于植物致病性嗜酸性种类。在本氏烟草中的瞬时表达表明，这些T3E中有两个位于细胞核中，一个与内质网相互作用。根据T3E货物，这项研究将柑桔曲霉列为“最丰富的”细菌病原体。它还揭示了新型T3E，似乎参与了植物病原性嗜酸性空肠物种的病理适应性进化。