PLC对梨果皮蜡质疏水性诱导Alternaria alternata附着胞形成的调控机制研究

Fruit cuticular wax have important role in inducing infection structure formation of fungal pathogen. Our previous results indicated that there was significant positive correlation between pear cuticular wax hydrophobicity and appressorium formation rate of Alternaria alternata, and PLC involved the inductive process, however the detailed mechanism remains unclear. In this study, based on PLC gene cloning and its specific expression during appressorium formation of A. alternata, causal agents of pear black spot, induced by different hydrophobicity, the effect of PLC on morphological structure changes during appressorium forming process induce by pear wax hydrophibicity were studied by the means of microscopic observation. In addition, changes in cytosolic Ca2+ and differential genes expression of key regulatory proteins in calcium/ calmodulin-dependent signaling pathway during appressorium forming process were also analyzed by the biochemical and molecular techniques. Based on the above experiments, the regulatory mechanisms of PLC mediated calcium/calmodulin-dependent signaling pathway on appressorium formation induced by pear wax hydrophobicity will be systematically clarified. The results will provide a theoretical basis for further understanding mechanism of calcium/ calmodulin-dependent signaling pathway on appressorium formation of A. alternata and exploiting novel strategies for intervention of postharvest diseases in fruit and vegetables.

果实表皮蜡质对病原真菌侵染结构形成具有诱导作用。我们研究发现梨果皮蜡质疏水性与Alternaria alternata附着胞形成率呈显著正相关，且PLC参与其诱导过程，但具体调控机制不清。本项目在对梨果黑斑病菌A.alternataPLC基因克隆及其在蜡质疏水性诱导附着胞形成过程中特异性表达分析的基础上，通过PLC抑制剂或外源钙处理，采用显微技术和生物化学、分子生物学方法研究PLC对不同疏水性表面诱导A.alternata附着胞形成过程中形态结构变化的影响；分析比较A.alternata附着胞形成过程中胞内Ca2+的动态变化及钙信号途径关键调控蛋白基因表达差异，以揭示PLC介导的钙信号途径对梨果皮蜡质疏水性诱导A.alternata附着胞形成的调控机制，为进一步从分子水平上阐明钙离子信号通路对A.alternata附着胞形成的调控机制和开发梨果采后病害新的控制方法提供理论依据。

磷脂酶C（PLC）是钙信号途径中的关键酶，可通过调控胞内Ca2+浓度调节生物细胞发育等多种生理功能，在病原真菌响应外界刺激进而启动致病过程中具有重要的调控作用。项目以梨果黑斑病菌A. alternata为研究对象，在对AaPLCs基因克隆和生物信息学分析的基础上，通过药理学方法和分子生物学技术研究了AaPLCs的生物学功能及对梨果皮物化信号诱导A. alternata侵染结构形成的调控机制，同时研究了钙信号途径关键蛋白CaMK对A. alternata侵染结构分化的调控，结果表明：.1. 从A. alternata基因组中鉴定出了ORF全长为3315bp、2416bp和2010bp 的3个PLC基因，其分别编码1116、669和574个氨基酸。系统进化树分析表明AaPLC1与Stemphylium lycopersici同源性高达91%，AaPLC2和AaPLC3与Pyrenophora triticirepentis同源性高达84%和88%。.2. PLC抑制剂新霉素处理显著降低了梨果皮蜡质和疏水性诱导的A. alternata侵染结构形成；外源钙处理对新霉素处理引起附着胞形成的减少没有影响，但可部分恢复孢子萌发。.3. 与野生型相比，ΔAaPLC1突变株生长迟缓，AaPLC2缺失导致菌落形态发生变化，而ΔAaPLC3生长没有明显差异。AaPLCs对A. alternata响应外源胁迫的调控存在差异。此外ΔAaPLC1突变株致病性、毒素合成降低，黑色素含量增加，而AaPLC2和AaPLC3缺失对致病性影响不明显。.4. 通过体外及梨果皮试验发现果蜡涂膜的疏水表面能显著诱导野生型和ΔAaPLCs突变株侵染结构形成，而ΔAaPLCs对附着胞和侵染菌丝形成的影响存在差异，AaPLC1缺失导致附着胞和侵染菌丝形成显著低于野生型，而ΔAaPLC2和ΔAaPLC3均能正常形成侵染菌丝。.5. 对从A. alternata菌株中克隆得到的AaCaMK1、AaCaMK2和AaCaMK3基因敲除发现AaCaMKs单一缺失可显著降低A. alternata侵染结构形成和致病性。.可见AaPLCs在梨黑斑病菌A. alternata的生长、致病性和胁迫响应中具有重要的调控作用，且其介导的钙信号途径参与了A. alternata响应梨果皮蜡质物化信号进而启动侵染的调控。