Identification of CsLOB1 target genes that promote citrus canker disease

PthA4，柑橘溃烂病病原菌Xanthomonas Citri PV的转录激活因子样效应子(TALL)。Citri(Xcc)，通过转录激活编码横向器官边界(LBD)蛋白的柑橘CsLOB1基因而促进疾病。由于LBD蛋白是转录因子，可想而知，不是CsLOB1本身，而是CsLOB1的靶基因，在这里被称为dtCsLOB1基因(CsLOB1的直接靶基因)，将在宿主细胞中诱导促进细菌疾病的生理变化。我们的目标是识别dtCsLOB1基因，并打算澄清它们的表达如何转化为促进细菌疾病的生理变化。CsLOB1靶向启动子将通过转录组分析(RNA-Seq)和染色质免疫沉淀(ChIP-Seq)相结合的方法进行鉴定。已识别的dtCsLOB1基因可能的疾病贡献将通过疾病受损的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的功能互补来研究。当XCC效应器在宿主细胞内作用时，病原体在质外体中繁殖，这意味着TALE诱导的CsLOB1表达应该引起质外体液体的变化。因此，我们还将通过基于质谱学的代谢物/蛋白质组图谱来研究CsLOB1表达对柑橘叶片质外体液组分的影响，以确定影响Xcc生长的变化。代谢物/蛋白质组谱结合基于基因/转录本的方法应该提供dtCsLOB1基因的表达如何转化为促进Xcc质外体生长的生理反应的迹象。我们设想，我们的研究有可能发现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微生物和/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.