柑橘响应溃疡菌入侵的生长素作用机制研究

Citrus canker is a quarantine disease worldwide caused by Xanthomonas axonopodis pv. citri (Xac). Auxin plays an important role in plant-pathogen interaction. To clarify the role of auxin in response to Xac infection in citrus, the transgenic citrus plants harboring the auxin reporter gene DR5::GUS-YFP will be used in our research. Local auxin accumulation at the innoculated sites of citrus host will be analyzed at first via GUS assay; then the gene expression profiles of the hosts will be compared by RNA-seq technology after Xac inoculation on the leaves pretreated by sterile water, auxin and auxin synthesis inhibits respectively.The differentially expressed genes will be screened and the expression patterns of the key genes selected from expression profiling data will be analyzed by qRT-PCR. Physiologycal measurement will also be done on the metabolic pathways involved. Furthermore, the effect of local auxin accumulation on tissue structure, stomata opening at the innoculation sites will be observed; the accumulation of reactive oxygen species (ROS) and salicylic acid (SA) will be detected. Finally, the relationship of auxin on ROS and SA signaling pathways will also be clarified. Based on the above mentioned works we will elucidate the role of auxin in citrus-Xac interaction. The work following the former research will deepen the understanding of the role of auxin in pathogenesis of citrus canker. The result is expected to contribute improvement of canker resistance of citrus with important theoretical and potential application value.

柑橘溃疡病是受溃疡菌侵染引起的一种世界检疫性病害。生长素在植物-病原菌互作中发挥重要作用。为解析生长素在柑橘响应溃疡菌入侵中的作用机制，以转生长素报告体系基因DR5::GUS-YFP柑橘为材料，首先通过GUS组织染色法直观分析溃疡菌接种对柑橘侵染部位生长素局部积累的影响规律。对无菌水、生长素及生长素合成抑制剂预处理的柑橘寄主的侵染部位组织进行基因表达谱测序，鉴定差异表达的基因和代谢途径。通过相关的形态和生理指标的测定，分析生长素对接种部位组织的细胞结构、气孔开闭、活性氧及水杨酸积累的影响，探讨生长素与活性氧及水杨酸介导的抗病信号途径之间的关系，最终阐明生长素在柑橘响应溃疡菌入侵中的作用机制。本项目从生长素角度对柑橘受溃疡菌入侵的应答机制进行探讨，将进一步深化对柑橘溃疡病致病机理的认识，为柑橘抗病育种提供新的思路，因而具有重要的理论意义和潜在应用价值。

生长素在植物—病原菌互作中具有重要作用。柑橘溃疡病是世界检疫性病害；目前，有关柑橘抗病遗传改良的研究已开展较多，而柑橘与溃疡菌互作部位生长素应答相关研究开展极少。本申请项目首先通过发根农杆菌介导的遗传转化方法获得转生长素报告基因DR5::GUS-YFP柑橘材料枳再生植株，在此基础上以该转基因枳为主要材料，开展了柑橘响应溃疡菌入侵的生长素应答机制研究。结果表明：（1）柑橘溃疡菌侵染后可导致转基因枳叶片侵染部位GUS组织化学染色显色更深，生长素报告基因YFP、生长素响应基因GH3.1、GH3.9等上调表达，表明侵染部位存在生长素积累；这与酶联免疫检测的结果一致。（2）生长素预处理可促进溃疡菌接种后枳叶片柑橘溃疡病发生，表现在病斑显著增大；（3）活性氧染色结果表明，柑橘溃疡菌侵染导致的生长素积累对活性氧积累没有抑制作用；气孔观察表明，生长素浸泡处理也不促进叶片气孔的开放；（4）q-PCR分析表明，柑橘溃疡菌接种下调水杨酸信号途径ICS1、NPR1基因表达，但PAL1、PR2基因却呈上调表达；生长素浸泡处理则总体上调水杨酸信号途径关键基因表达。（5）转录组测序及q-PCR分析结果均表明柑橘溃疡菌接种可导致枳叶片与细胞壁延展有关的细胞壁重塑相关蛋白如β-1,3内切葡聚糖酶（B1,3G）、β-1,4内切葡聚糖酶（B1,4G）、果胶酯酶（PEC等基因显著上调；此外，未接种条件下生长素溶液浸泡处理也导致上述枳叶片细胞壁重塑相关蛋白基因的上调表达。. 综上可知，柑橘溃疡菌侵染导致柑橘寄主叶片组织局部生长素积累，进一步通过上调细胞壁重塑相关蛋白基因的表达促进溃疡菌的侵染。本申请项目深化了柑橘与溃疡菌互作过程生长素应答机制的认识，为通过调控柑橘内源生长素手段开展抗病遗传改良奠定了理论基础。