Identification of CsLOB1 target genes that promote citrus canker disease

PthA4是柑橘溃疡病病原菌柑橘黄单胞菌的转录激活因子样效应物（TALE）。柑橘（Xcc），通过柑橘CsLOB1基因的转录激活来促进疾病，该基因编码侧器官边界（LBD）蛋白。由于LBD蛋白是转录因子，因此可以想象，不是CsLOB1本身，而是CsLOB1的靶基因，本文称为dtCsLOB1基因（CsLOB1的直接靶点），会诱导宿主细胞的生理变化，促进细菌性疾病。我们的目标是鉴定dtCsLOB1基因，并打算阐明它们的表达如何转化为促进细菌性疾病的生理变化。CsLOB1靶启动子将通过转录组分析（RNA-Seq）和染色质免疫沉淀（ChIP-Seq）的组合来鉴定。通过将疾病受损的Xcc pthA4突变株（不能激活CsLOB1）与特异性诱导单个dtCsLOB1基因表达的dTALEs进行功能互补，将研究鉴定出的dtCsLOB1基因可能对疾病的贡献。当Xcc效应物在宿主细胞内起作用时，病原体在外质体中繁殖，这表明tale诱导的CsLOB1表达应引起外质体液体的变化。因此，我们还将通过基于质谱的代谢物/蛋白质组分析来研究CsLOB1表达对柑橘叶片外胞液组成的影响，以确定影响Xcc生长的变化。结合基于基因/转录的方法，代谢物/蛋白质组学分析将提供dtCsLOB1基因的表达如何转化为促进Xcc胞外生长的生理反应的指示。我们设想我们的研究有可能揭示新的微生物入侵策略，这些策略可能在系统发育不同的致病和/或共生植物相关微生物中保守。

PthA4, a transcription activator-like effector (TALE) from the citrus canker pathogen Xanthomonas citri pv. citri (Xcc), promotes disease via transcriptional activation of the citrus CsLOB1 gene that encodes a lateral organ boundary (LBD) protein. Since LBD proteins are transcription factors its conceivable that not CsLOB1 itself but rather target genes of CsLOB1, herein designated as dtCsLOB1 genes (direct targets of CsLOB1), will induce physiological changes in host cells that promote bacterial disease. We aim to identify dtCsLOB1 genes and intend to clarify how their expression translates into physiological changes that promote bacterial disease. CsLOB1 target promoters will be identified by a combination of transcriptome profiling (RNA-Seq) and chromatin immunoprecipitation (ChIP-Seq). The possible disease contribution of identified dtCsLOB1 genes will be studied by functional complementation of disease-compromised Xcc pthA4 mutant strains (incapable of activating CsLOB1) with dTALEs that specifically induce expression of individual dtCsLOB1 genes. While Xcc effectors act inside host cells, the pathogen multiplies in the apoplast, which implies that TALE-induced CsLOB1 expression should cause changes in apoplastic fluids. Accordingly, we will also study the impact of CsLOB1 expression on the composition of apoplastic fluids from citrus leaves via mass-spectrometry-based metabolite/proteome profiling to identify changes that affect Xcc growth. Metabolite/proteome-profiling in conjunction with the gene/transcript-based approaches should provide indications of how expression of dtCsLOB1 genes translates into physiological responses that promote apoplastic growth of Xcc. We envision that our studies have the potential to uncover novel microbial invasion strategies that are possibly conserved across phylogenetically diverse pathogenic and/or symbiotic plant-associated microbes.