The soil-borne pathogen Verticillium dahliae has a worldwide distribution and a plethora of hosts of agronomic value. Molecular analysis of virulence processes can identify targets for disease control. In this work, we compared the global gene transcription profile of random T-DNA insertion mutant strain D-10-8F, which exhibits reduced virulence and alterations in microsclerotium formation and polar growth, with that of the wild-type strain. Three genes identified as differentially expressed were selected for functional characterization. To produce deletion mutants, we developed an updated version of one-step construction of Agrobacterium-recombination-ready plasmids (OSCAR) that included the negative selection marker HSVtk (herpes simplex virus thymidine kinase gene) to prevent ectopic integration of the deletion constructs. Deletion of VdRGS1 (VDAG_00683), encoding a regulator of G protein signaling (RGS) protein and highly upregulated in the wild type versus D-10-8F, resulted in phenotypic alterations in development and virulence that were indistinguishable from those of the random T-DNA insertion mutant. In contrast, deletion of the other two genes selected, vrg1 (VDAG_07039) and vvs1 (VDAG_01858), showed that they do not play major roles in morphogenesis or virulence in V. dahliae. Taken together the results presented here on the transcriptomic analysis and phenotypic characterization of D-10-8F and ∆VdRGS1 strains provide evidence that variations in G protein signaling control the progression of the disease cycle in V. dahliae. We propose that G protein-mediated signals induce the expression of multiple virulence factors during biotrophic growth, whereas massive production of microsclerotia at late stages of infection requires repression of G protein signaling via upregulation of VdRGS1 activity.

中文翻译：

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通过正向遗传和转录组学表征控制大黄萎病黄萎病病害周期的分子机制。

土壤传播的病原体黄萎病菌在世界范围内分布，并具有大量具有农艺价值的寄主。毒力过程的分子分析可以确定疾病控制的目标。在这项工作中，我们比较了随机T-DNA插入突变菌株D-10-8F的全局基因转录谱，该菌株与野生型菌株相比，其毒力降低，微菌核形成和极性生长的改变。选择鉴定为差异表达的三个基因进行功能表征。为了产生缺失突变体，我们开发了农杆菌重组就绪质粒（OSCAR）的一步构建的更新版本，该质粒包括阴性选择标记HSVtk（单纯疱疹病毒胸苷激酶基因），以防止缺失构建体的异位整合。与D-10-8F相比，VdRGS1（VDAG_00683）的编码G蛋白信号（RGS）蛋白的调节子的缺失，并且在野生型中的表达上调与D-10-8F高度相关，导致发育和毒力的表型改变与随机T- DNA插入突变体。相反，删除选定的其他两个基因，vrg1（VDAG_07039）和vvs1（VDAG_01858），表明它们在大麦弧菌的形态发生或毒力中不发挥主要作用。综上所述，D-10-8F和∆VdRGS1菌株的转录组分析和表型表征结果均提供了证据，表明G蛋白信号的变化控制了大丽花病的病程。我们建议G蛋白介导的信号在生物营养生长过程中诱导多种毒力因子的表达，