稻瘟病菌热激蛋白(HSP)40编码基因MoMHF6的鉴定及功能研究
收稿日期: 2023-03-30
修回日期: 2023-04-20
网络出版日期: 2023-11-14
基金资助
国家自然科学基金资助项目(32070141)
Identification and Functional Characterization of the Heat Shock Protein (HSP) 40 Encoding Gene, MoMHF6, in Magnaporthe oryzae
Received date: 2023-03-30
Revised date: 2023-04-20
Online published: 2023-11-14
【目的】 探明稻瘟病菌热激蛋白（HSP）40在形态分化和致病过程中的作用。【方法】 利用DNA同源重组方法敲除了稻瘟病菌HSP40编码基因MoMHF6，获得ΔMomhf6突变体，并通过表型分析、基因回补、RNA-seq分析等对MoMHF6的生物学功能进行较系统的研究。【结果】 敲除MoMHF6基因导致稻瘟病菌气生菌丝生长、无性产孢、孢子萌发、子囊壳产生和附着胞形成均显著下降，但与野生菌株相比，ΔMomhf6突变体在CM培养基上的径向生长没有显著差异，在子囊壳中仍可形成子囊和子囊孢子。在1、2和3 mol/L甘油溶液处理条件下，ΔMomhf6突变体附着胞塌陷率显著升高，说明MoMHF6与附着胞膨压形成有关。ΔMomhf6突变体对洋葱内表皮的穿透能力和对感病寄主的致病性完全丧失，甚至在划伤大麦叶片表皮组织中的扩展也受明显限制。而且，ΔMomhf6突变体对氧化胁迫的敏感性增强，附着胞发育过程中糖原的转运和降解缓慢，说明MoMHF6参与氧化胁迫反应和附着胞的糖原代谢。将完整的MoMHF6基因重新导入ΔMomhf6突变体可回补突变体的所有表型缺陷。另外，RNA-seq分析显示，部分已知的稻瘟病菌致病相关基因表达可能受MoMHF6调控，如MoATG4、MoPL1、MoVPR和GAS1等。【结论】 综上所述，稻瘟病菌HSP40编码基因MoMHF6在产孢、附着胞形成、穿透寄主、氧化胁迫应答、致病等过程中起重要作用，研究结果对进一步阐明MoMHF6调控稻瘟病菌形态分化和致病过程的基因网络和分子机制具有重要意义。
童琪, 王春燕, 阙亚伟, 肖宇, 王政逸 . 稻瘟病菌热激蛋白(HSP)40编码基因MoMHF6的鉴定及功能研究[J]. 中国水稻科学, 2023 , 37(6) : 563 -576 . DOI: 10.16819/j.1001-7216.2023.230301
【Objective】 To explore the roles of chaperone HSP40 (heat shock protein 40) in morphological differentiation and pathogenesis in Magnaporthe oryzae. 【Method】 The HSP40 encoding gene MoMHF6 of M. oryzae was knocked out by DNA homologous recombination, and a deletion mutant ΔMomhf6 was generated. Biological function of the gene was systematically characterized via phenotypic analysis, gene complementation and RNA-seq analysis. 【Result】 Phenotypic analysis of the mutant revealed severe impairments in aerial hypha growth, asexual sporulation, conidial germination, perithecium production and appressorium formation. However, compared to the wild-type strain, the deletion of MoMHF6 did not affect mycelium radial growth on CM medium and the development of asci and ascospores in perithecia. Incipient cytorrhysis assays revealed that the appressorium collapse rate of the ΔMomhf6 mutant under treatment with 1, 2 or 3 mol/L glycerol was significantly increased compared to the wild-type strain, indicating that MoMHF6 is required for appressorium turgor generation. Also, we found that the ΔMomhf6 mutant was unable to penetrate into onion epidermis and completely nonpathogenic to susceptible hosts. Even in wounded barley leaves, infectious growth of the mutant was severely impaired. Moreover, the deletion of MoMHF6 resulted in significantly increased sensitivity to oxidative stress and delayed glycogen transportation and degradation during appressorium development, indicating that MoMHF6 is involved in oxidative stress response and glycogen metabolism of appressorium. All phenotypic defects of the ΔMomhf6 mutant could be restored by reintroducing the full-length MoMHF6 gene into the mutant. In addition, RNA-seq analysis of the ΔMomhf6 mutant revealed that several known pathogenicity-related genes in differentially expressed genes (DEGs) might be regulated by MoMHF6, such as MoATG4, MoPL1, MoVPR, and GAS1. 【Conclusion】 The HSP40 gene MoMHF6 plays an important role in asexual sporulation, appressorium formation, host penetration, oxidative stress response and pathogenicity in M. oryzae. These results are of great significance for further understanding the gene networks and molecular mechanisms in regulating morphological differentiation and pathogenesis governed by MoMHF6 in M. oryzae.
Key words： Magnaporthe oryzae; MoMHF6; heat shock protein 40; functional analysis; RNA-seq
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