Hu Yang, Duan Shuo, Zhang Yunzeng, Shantharaj Deepak, Jones Jeffrey B, Wang Nian
First, fourth, and fifth authors: Department of Plant Pathology, University of Florida, Gainesville 32611; and second, third, and sixth authors: Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, 700 Experiment Station Road, Lake Alfred 33850.
Phytopathology. 2016 May;106(5):442-51. doi: 10.1094/PHYTO-09-15-0201-R. Epub 2016 Mar 31.
Citrus canker, caused by Xanthomonas citri subsp. citri, is a devastating disease of most commercial citrus varieties. In our previous study, we analyzed the transcriptional response of 'Valencia' sweet orange to X. citri subsp. citri wild-type and pthA4 mutant infection at 48 h postinoculation (hpi). Using microarray analysis, two PthA4 targets, CsLOB1 and CsSWEET1, were identified. We have shown that PthA4 binds to the effector binding element (EBE) of CsLOB1 and activates gene expression of this susceptibility gene. However, how PthA4 modulates host genes at different stages of infection remains to be determined. In this study, we compared the transcriptional profiles between citrus leaf tissue inoculated with Xcc306 and those inoculated with a pthA4-deletion mutant strain (Xcc306∆pthA4) at 6, 48, and 120 hpi. At both 48 and 120 hpi, the PthA4-mediated infection significantly upregulated expression of a variety of genes involved in cell-wall degradation and modification, DNA packaging, G-protein, protein synthesis, sucrose metabolism, and cell division functions, while the downregulated genes were mainly enriched in photosynthesis, transport, secondary metabolism, cytochrome P450, and various plant defense-associated mechanisms. To validate microarray results, gene expression of 26 genes representing genes associated with cell-wall-associated, immunity system, and carbohydrate metabolism was confirmed using quantitative reverse-transcription polymerase chain reaction. Expression patterns of these genes at 48 and 120 hpi were consistent with the microarray results. We also identified putative EBE for PthA4 (EBEPthA4) in the promoter regions of multiple genes upregulated by PthA4, to which PthA4 might bind directly to control their gene expression. Our study provided a dynamic picture of citrus genes regulated by PthA4 during the X. citri subsp. citri infection of citrus leaves at different stages. This study will be useful in further understanding the virulence mechanism of X. citri subsp. citri and identifying potential targets of PthA4.
柑橘溃疡病由柑橘黄龙病菌引起，是大多数商业柑橘品种的毁灭性病害。在我们之前的研究中，我们分析了‘伏令夏橙’甜橙在接种后48小时对柑橘黄龙病菌野生型和pthA4突变体感染的转录反应。通过微阵列分析，鉴定出两个PthA4靶标，即CsLOB1和CsSWEET1。我们已经表明，PthA4与CsLOB1的效应子结合元件（EBE）结合并激活这个感病基因的基因表达。然而，PthA4在感染的不同阶段如何调节宿主基因仍有待确定。在本研究中，我们比较了接种Xcc306和接种pthA4缺失突变菌株（Xcc306∆pthA4）的柑橘叶片组织在接种后6、48和120小时的转录谱。在48和120小时时，PthA4介导的感染显著上调了参与细胞壁降解和修饰、DNA包装、G蛋白、蛋白质合成、蔗糖代谢和细胞分裂功能的多种基因的表达，而下调的基因主要富集在光合作用、运输、次生代谢、细胞色素P450和各种植物防御相关机制中。为了验证微阵列结果，使用定量逆转录聚合酶链反应确认了代表与细胞壁相关、免疫系统和碳水化合物代谢相关基因的26个基因的表达。这些基因在48和120小时的表达模式与微阵列结果一致。我们还在多个被PthA4上调的基因的启动子区域中鉴定出PthA4的推定EBE（EBEPthA4），PthA4可能直接与其结合以控制它们的基因表达。我们的研究提供了柑橘黄龙病菌感染柑橘叶片不同阶段PthA4调控柑橘基因的动态图景。这项研究将有助于进一步了解柑橘黄龙病菌的致病机制并确定PthA4的潜在靶标。
