摘要
液泡在调控细胞的生长发育方面具有多重作用。真菌的液泡和哺乳动物中的溶酶体以及植物中的液泡有类似的功能,包括离子的代谢和储存、pH和渗透压调控、营养运输以及细胞凋亡等。液泡是真菌生理活动中的关键细胞器,直接参与菌丝细胞内的长距离营养运输,调控菌丝的扩展和分支的形成；通过对自噬的调控间接参与真菌某些关键结构的形成,如附着胞的形成和假菌丝的生长等。稻瘟病菌(Magnaporthe oryzae)是一种严重危害水稻产量的植物病原丝状真菌；同时稻瘟病菌也是研究病原真菌与寄主植物互作的模式生物之一。了解其致病过程和机理对于水稻稻瘟病的防治有重要作用。
本论文主要研究和阐明液泡运输与稻瘟病菌生长、发育和致病性之间的关系。获得的主要研究结果如下：
1)在稻瘟病菌附着胞的差减文库中,找到了细胞自噬相关基因的EST (expressed sequence tags),进而找到基因,定名为MoYpt7,通过保守结构域分析该基因编码Rab GTPase。对MoYpt7进行细胞定位,该基因在菌丝和分生孢子的液泡膜上强烈表达；通过构建MoYpt7基因置换载体,得到基因MoYpt7缺失突变体AMoYpt7。
2) AMoYpt7突变体在CM培养基上的菌落形态发生变化,菌丝稀疏；几乎不产孢,少数孢子可以萌发,但不形成附着胞；在完整的水稻或者大麦叶片上不能形成可见病斑,对水稻根部也不能完成侵染,致病性丧失；突变子在OMA培养基上与2539菌株交配,不能产生子囊壳,育性受到影响；基因MoYpt7缺失,细胞自噬过程中断,菌丝中大量小液泡聚集。
3)通过同源序列比对,PCR的方法,得到液泡运输相关基因MoMonl;将稻瘟病菌MoMonl基因,互补到酵母Monl基因缺失突变体中,能够恢复酵母的液泡形态；通过构建MoMonl基因置换载体,得到基因MoMonl缺失突变体ΔMoMonl。
4)基因缺失突变体MoMonl,液泡融合受阻,细胞自噬过程中断；菌落形态改变,菌丝稀疏；孢子产量显著下降,不能形成附着胞；与2539菌株的交配,不能形成子囊壳；在大麦和水稻上的致病性丧失。
液泡广泛存在于真核生物中,液泡参与细胞内营养物质的运输等多个生命过程,与液泡相关的运输过程在植物病原真菌中的研究很少,结合细胞自噬在稻瘟病菌中研究液泡的运输国内尚属首次；同时,研究过程中采用的方法对其它病原真菌的研究起到借鉴作用。
In eukaryotic cells, the vacuole/lysosome is a complex organelle with a range of various functions, such as nutrient transport, the storage of Ca2+, ion homeostasis and degradation of macromolecules. In filamentous fungi, the inheritance and morphology of vacuole are vital for fungal growth, differentiation and symbiosis and/or pathogenesis. Magnaporthe oryzae is a filamentous ascomycete fungus that has been served as an excellent model organism for studying plant-pathogen interactions. A deep understanding of the molecular background of this fungus is beneficial for the control of the rice blast.
This work is aimed to investigate the relationship between vacuole trafficking and cell development, differentiation and pathogenicity in M. oryzae. We isolated and cloned two vacuole fusion-related genes, MoYpt7and MoMonl, which is a homolog of gene Ypt7or Monl respectively in Saccharomyces cerevisiae. The functional analyses of MoYpt7and MoMonl during the infection process of rice blast were discussed.
An EST (expressed sequence tags) for MoYpt7was found in the appressorium of the rice blast fungus. MoYpt7is a Rab GTPase, conserved from yeast to mammalian. The ΔMoYpt7mutant, in which the MoYpt7gene has been deleted, exhibits defective in mycelial growth and conidial production. Among the few conidia generated, conidia are malformed and defective in appressorial formation. Consequently, the ΔMoYpt7mutant fails to cause diseases on rice CO-39and barley ZJ-8. The cellular localization assay shows that the MoYpt7is mainly localized to vacuolar membranes. Furthermore, the ΔMoYpt7mutant shows lots of small and fragmented vacuoles in hypha, blocked in autophagy process, a breach in cell wall integrity and more sensitive to ions stress.
Similarly, a sequence homologue was isolated by using the amino acid sequence of Monl searching against M. oryzae genome databases, assigned MoMonl. A targeted gene deletion strategy was adopted to determine the role of MoMonl in fungal development and pathogenicity at the molecular level. The ΔMoMonl mutant showed accumulation of small punctuate vacuoles in the hypha and hypersensitive to monensin, an antibiotic that can block intracellular protein transport. Secondly, the ΔMoMonl mutant exhibited reduced in hyphal development, conidiogenesis and failed to produce the appressorium. Additionally, the ΔMoMonl mutant revealed blocked in autophagy and a defect in pathogenicity.
In summary, MoYpt7and MoMonl also have essential function in vacuolar assembly and growth, conidiation, appressorium formation and pathogenicity in M. oryzae.
引文
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