摘要
稻瘟病菌(Magnaporthe grisea)能引起水稻上的一种重要病害-稻瘟病，了解其致病过程和机理对于防治稻瘟病菌有重要作用。近年研究表明细胞自噬可能参与了稻瘟病菌侵染水稻过程中分生孢子体死亡的过程。细胞自噬是细胞中一种物质降解方式，在一些特定情况下，自噬被诱导，一些长寿蛋白质和细胞器被运送到液泡／溶酶体中被降解，从而能量和物质得以重新利用。此过程在许多生物体中是保守的，一些参与过程的基因也是保守的。稻瘟病菌中存在与啤酒酵母自噬相关基因ScATG1和ScATG8同源的两个基因MgATG1和MgATG8，它们的缺失突变子表型与野生型存在很大差异。本文则分离和克隆了ScATG9在稻瘟病菌中的同源基因MgATG9，通过基因敲除的方法初步分析了MgATG9在致病及相关过程中的作用。研究结果如下：
1．利用ScATG9基因预测的氨基酸序列在稻瘟病菌基因组数据库中检索序列相似的基因，定名为MgATG9。
2．PCR扩增获得cDNA克隆。测序分析CDS长2754nt，编码918aa。
3．构建MgATG9基因置换载体，得到基因MgATG9缺失突变体⊿mgatg9。
4．基因MgATG9缺失后，细胞自噬过程中断，在液泡中未见自噬泡的积累。
5．突变子在CM培养基上生长时菌丝稀疏，产孢量显著下降。
6．突变子分生孢子发育受到影响。
7．突变子附着胞膨压显著下降。
8．突变子致病性几乎完全丧失，在水稻和大麦叶片上不能形成可见病斑；在有伤口的大麦叶片上只能产生微弱的病斑。
9．突变子在OMA培养基上与2539菌株交配，产生子囊壳时间推迟并且数量明显减少，育性受到影响。
Magnaporthe grisea(Hebert)Barr,is well-known as the efficient and devastatingagent of rice blast.A deep understanding of the molecular background of this fungusis beneficial for the control of rice blast.Ever more powerful molecular geneticanalysis of the rice blast fungus brings to a comprehensive understanding of themolecular basis of host-pathogen interactions in rice blast.Blast is thus developed as amodel system for the study of interactions between an economically important cropplant and a damaging fungal pathogen.
Recent research demonstrated the involvement of autophagy within the processof the cell death of the conidium during the appressorium development.Autophagy isa ubiquitous degradation process.Only under some instances,autophagy is induced.Long-lived proteins and organelles are sequestered within double-membrane vesiclesthat deliver the contents to the lysosome/vacuole for degradation and recycling of theresulting macromolecules.Autophagy is a conserved process among many species,andsome genes related to autophagy could be conserved either.Here,in this paper,weisolated and cloned MgATG9 gene,which is a homolog of ATG9 gene inSaccharomyces cerevisiae.The functional analysis of MgATG9 gene in the infectionprocess of rice blast were discussed.
A sequence homologue was isolated by using the amino acid sequence ofScATG9 searching against M.grisea genome databases,assigned MgATG9.The fulllength of MgATG9 ORF and cDNA clones were obtained by PCR strategy.Nucleotidesequence analysis revealed a coding sequence of 2754 nt,putatively coding a 918amino acid peptide,containing a conserved apg9 domain.
Autophagy was blocked in the△mgatg9 mutant,in which MgATG9 gene wasdeleted by targeted gene replacement;the mutant△mgatg9 also showed delayedgermination of conidia,lower turgor pressure of the appressorium.The△mgatg9 lostits ability to penetrate and infect the two tested host,namely rice and barley.Theseproperties were restored when re-introduced MgATG9 coding sequence into themutants.
In summary,MgATG9 plays a significant role in autophagy of M.grisea,which isnecessary for normal development of conidia and appressoria and pathogenicity of M.grisea.
引文
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