Department of Microbiology and Cell Science, University of Florida, Lake Alfred, FL, USA.
Mol Plant Microbe Interact. 2012 Jan;25(1):69-84. doi: 10.1094/MPMI-05-11-0121.
Citrus canker is caused by Xanthomonas citri subsp. citri and is one of the most devastating diseases on citrus plants. To investigate the virulence mechanism of this pathogen, a mutant library of strain 306 containing approximately 22,000 mutants was screened for virulence-deficient mutants in grapefruit (Citrus paradise). Eighty-two genes were identified that contribute to citrus canker symptom development caused by X. citri subsp. citri. Among the 82 identified genes, 23 genes were classified as essential genes, as mutation of these genes caused severe reduction of bacterial growth in M9 medium. The remaining 59 genes were classified as putative virulence-related genes that include 32 previously reported virulence-related genes and 27 novel genes. The 32 known virulence-related genes include genes that are involved in the type III secretion system (T3SS) and T3SS effectors, the quorum-sensing system, extracellular polysaccharide and lipopolysaccharide synthesis, and general metabolic pathways. The contribution to pathogenesis by nine genes (pthA4, trpG, trpC, purD, hrpM, peh-1, XAC1230, XAC1548, and XAC3049) was confirmed by complementation assays. We further validated the mutated genes and their phenotypes by analyzing the EZ-Tn5 insertion copy number using Southern blot analysis. In conclusion, we have significantly advanced our understanding of the putative genetic determinants of the virulence mechanism of X. citri subsp. citri by identifying 59 putative virulence-related genes, including 27 novel genes.
柑橘溃疡病由柑橘溃疡病菌（Xanthomonas citri subsp. citri）引起，是柑橘类植物上最具破坏性的病害之一。为了研究该病原体的毒力机制，我们对菌株 306 的突变体文库进行了筛选，以寻找在葡萄柚（Citrus paradise）中丧失毒力的突变体。共鉴定出 82 个基因参与了柑橘溃疡病菌引起的柑橘溃疡病症状的发展。在鉴定出的 82 个基因中，有 23 个被归类为必需基因，因为这些基因的突变导致细菌在 M9 培养基中的生长严重减少。其余 59 个基因被归类为假定的与毒力相关的基因，包括 32 个先前报道的与毒力相关的基因和 27 个新基因。32 个已知的与毒力相关的基因包括参与 III 型分泌系统（T3SS）和 T3SS 效应子、群体感应系统、胞外多糖和脂多糖合成以及一般代谢途径的基因。通过互补实验证实了 9 个基因（pthA4、trpG、trpC、purD、hrpM、peh-1、XAC1230、XAC1548 和 XAC3049）对致病性的贡献。我们进一步通过 Southern blot 分析分析 EZ-Tn5 插入拷贝数来验证突变基因及其表型。总之，我们通过鉴定 59 个假定的与毒力相关的基因，包括 27 个新基因，显著提高了对柑橘溃疡病菌毒力机制的假定遗传决定因素的认识。
