致病疫霉PiSAK1的生物学功能分析

Biological roles of Phytophthora infestans PiSAK1

摘要:

【背景】致病疫霉是引起世界范围内马铃薯晚疫病的重要病原菌。Stress-activated protein kinases (SAPKs)是一类胁迫激活的mitogen-activated protein kinases (MAPKs)，研究表明真菌SAPKs在调控细胞应答外界胁迫等方面有重要作用。致病疫霉中存在一个SAPK，即PiSAK1，其生物学功能并不明确。【目的】探究PiSAK1在致病疫霉生长发育、抵抗外界胁迫及侵染马铃薯过程中发挥的生物学功能。【方法】利用生物信息学手段分析PiSAK1的特性，通过RT-qPCR分析明确致病疫霉PiSAK1在不同发育阶段及侵染马铃薯不同时期的表达量，最后构建PiSAK1沉默、过表达菌株并测定其各项生物学表型。【结果】PiSAK1具有丝裂原活化蛋白激酶典型的Ser/Thr蛋白激酶催化结构域，并且与其他卵菌的SAPKs同属一个进化分支。致病疫霉PiSAK1分别在休止孢阶段、侵染马铃薯48 h时表达量最高，且0.3 mol/L NaCl及3 mmol/L H₂O₂胁迫刺激0.5 h后PiSAK1的表达量均显著升高。构建PiSAK1沉默、过表达菌株并测定其表型，结果表明基因沉默菌株在菌落生长、孢子囊产量、抵御NaCl及H₂O₂胁迫、清除活性氧、致病性方面均比野生型菌株显著减弱，而基因过表达菌株除菌落生长与野生型菌株无显著差异外，其他表型相较野生型菌株均显著增强。【结论】致病疫霉PiSAK1在生长发育、渗透压调节、氧化胁迫应答和侵染马铃薯的过程中发挥重要作用。该结果为后续研究新的杀菌剂作用靶标、选育马铃薯抗病新品种提供了理论依据。

Abstract:

[Background] Phytophthora infestans is a major pathogen causing potato late blight. Stress-activated protein kinases (SAPKs), a class of mitogen-activated protein kinases (MAPKs), play a role in regulating cell responses of fungi to external stress factors. The biological functions of a SAPK in P. infestans, i.e., PiSAK1, remain unknown. [Objective] To explore the biological roles of PiSAK1 in the growth and development, the responses to external stress factors, and the infection in potato. [Methods] The bioinformatics tools were used to characterize PiSAK1. The expression levels of PiSAK1 at different development and infection stages were determined by RT-qPCR. Finally, the PiSAK1-silenced and-overexpressed strains were constructed and characterized. [Results] PiSAK1 had a typical Ser/Thr protein kinase catalytic domain of MAPKs and shared the same evolutionary branch with the SAPKs of other oomycetes. The expression level of PiSAK1 was the highest at the stage of cysts or 48 h after infecting potato. Moreover, it was significantly up-regulated after exposure to 0.3 mol/L NaCl or 3 mmol/L H₂O₂ for 0.5 h. The results showed that the PiSAK1-silenced strains were significantly weaker than the wild type in terms of colony growth, sporangium production, resistance to NaCl and H₂O₂, scavenging of reactive oxygen species, and pathogenicity. However, the PiSAK1-overexpressed strains showed significantly enhanced phenotypic characteristics compared with the wild type, except for no significant difference in colony growth. [Conclusion] PiSAK1 plays a role in the growth and development, osmotic regulation, oxidative stress response, and infection in potato. Our results provide a theoretical basis for further research on new targets of fungicides and breeding of resistant potato varieties.