蛋白质-酪氨酸磷酸酶在调控稻瘟菌致病与抗氧化过程中的分子机理

The balances between phosphorylation-dephosphorylation in proteins of organisms are dynamic, which is controlled by both protein kinases and protein phosphatases. Protein kinases play important roles in regulating pathogenicity of fungal pathogens through phosphorylation signaling, however, roles of protein phosphatases in fungal pathogens are less known. Several mutants, which are sensitive to the oxidative stressor H2O2, were screened from T-DNA library of Magnaporthe oryzae; among these mutants, the R28515 strain was genetically and biologically identified and characterized. In R28515 strain, a putative protein-tyrosine phosphate phosphatase gene (PTP1) was disrupted by the T-DNA insertion at the upstream of no translation region. PTP1 deletion mutant was more sensitive to H2O2 in comparison with the wild type and lost pathogenicity. In order to clarify the function and regulation mechanisms of PTP1, this project is going to focus on the following four points. (1) Biochemical analysis for PTP1, which involves phosphatase activity detection, and catalytic active sites confirmation through site-directed mutation; (2) proteomics study on PTP1 substrates, to obtain substrate protein candidates and predict the corresponding genes, Pull down assisted with Phosphate-tyrosine antibody /Phos-tag SDS-PAGE and MS/MS MALDI TOF technologies are concerned; (3) Biological analysis of PTP1 substrate genes through constructing deletion mutant strain and complemented strains, pathogenicity tests are particularly performed in this point; (4) To clarify the relationship among PTP members, the other 7 predicted PTP homologs will be selected for further biological analysis; to obtain multiple genes’ disruption, CRISP-Cas9 approach will be used. This project will verify the dephosphorylation role of PTP1 to specific phosphate-proteins as substrates; and reveal pathogenicity regulation mechanisms of M. oryzae protein-tyrosine phosphatase family in response to ROS stress. The results of this project will provide potential molecular targets for designing novel fungicide.

蛋白磷酸化水平是蛋白激酶与磷酸酶共同作用的结果。已有研究表明，蛋白激酶在调控病原菌致病过程中扮演重要角色，但磷酸酶在致病中的作用却仍不明确。通过筛选过氧化氢敏感突变体，我们初步鉴定了一个稻瘟菌蛋白质-酪氨酸磷酸酶基因（PTP1），敲除PTP1，突变体对氧化更加敏感、且丧失致病力。为探明PTP1在调控致病和抗氧化过程中的分子机理，本项目拟开展：⑴ PTP1生化功能研究，测定磷酸酶活性，确定活性位点； ⑵ PTP1底物的蛋白组学研究，利用Phos-tag电泳与质谱分析技术，推断磷酸酶底物及其编码基因；⑶ PTP1底物的验证与生物功能分析，通过底物磷酸化水平和致病性鉴定，确定PTP1底物及其致病功能； ⑷ PTPs成员的关联分析，通过构建PTPs多位点突变体，进行生物功能比较。预期结果将明确PTP1的功能和催化底物，丰富蛋白激酶信号途径的内容，为设计靶向PTP1途径的新型抗菌药物提供参考。

蛋白磷酸化水平是蛋白激酶与磷酸酶共同作用的结果。蛋白激酶在调控病原菌致病过程中扮演着重要角色，但磷酸酶在病原真菌致病中的作用却仍不明确。本项目通过鉴定一个稻瘟菌PTP1同源基因，揭示了PTP1参与稻瘟菌致病调控的分子机理。我们发现MoPTEN基因发生了可变剪接，其两种转录形式；稻瘟菌MoPTEN两个可变剪接变体（MoPTEN-1/2）具有双磷酸酶（dual-lipid and protein）活性，但底物不同。通过单个变体互补证明，两个变体分别在侵染前和侵染后高效表达，并以接力形式贡献稻瘟病菌的致病性发育。MoPTEN基因的可变剪接受到稻瘟菌SMN/MOS因子调控，而MoPTEN-2与推定的激酶MoRANX互作，并最终影响稻瘟病菌的生长、发育和致病。本项目明确了MoPTEN的生物功能和生化特性，丰富了蛋白激酶信号途径的内容，为设计靶向PTP1途径的新型抗菌药物提供参考。