金龟子绿僵菌黏附素基因mad 1的敲除及功能分析

Knockout and functional analysis of the adhesin gene mad 1 in Metarhizium anisopliae

金龟子绿僵菌能够寄生昆虫,也能与植物共生。黏附素MAD1是绿僵菌与宿主互作初期的黏附因子。已知它在昆虫的侵染和致病中起重要作用,但与植物的互作机制报道甚少。为了研究MAD1在绿僵菌与植物共生中的作用,我们通过同源重组构建了金龟子绿僵菌mad 1敲除株,并检测了敲除株的生长、产孢、孢子萌发及毒力等生物学特性,进一步利用qRT-PCR分析了MAD1在调节花生免疫响应中的作用。结果显示,与野生型菌株相比,敲除株产孢量降低了43.67%,分生孢子萌发中时为27.69 h,显著长于野生型菌株的13.43 h。敲除株对家蚕的半致死时间LT 50为8.9 d,较野生型菌株的7.62 d显著延长。敲除株处理花生6 h后,花生免疫类基因CNGC 1、PCD 4,抗病基因SWEET 10及转录因子WRKY 41、MYB 86的转录水平出现显著上调,而钙调素CML 5、CML 19的转录表达受到明显抑制。本研究证明了mad 1是金龟子绿僵菌产孢、孢子萌发及毒力的正相关基因,并且MAD1在金龟子绿僵菌与植物相互作用初期,抑制植物抗性级联反应,减弱过敏反应,降低对微生物的抵御能力,同时增强共生信号的传导,这些作用有助于金龟子绿僵菌在花生根组织上定殖。

Metarhizium anisopliae is an entomopathogenic fungus and also an endophyte in plants.The adhesin MAD1 is the adhesion factor in the initial stage of interaction between M.anisopliae and insects or plant hosts.It is known that the adhesin is very important for infection and pathogenicity to insects,but its role in the interaction with plants is still unclear.Here,we aimed to explore the role of MAD1 in the symbiosis between M.anisopliae and plants.We constructed mad 1 knockout strain by homologous recombination and tested the biological characteristics such as growth,sporulation,spore germination and virulence of the mad 1 mutant.Furthermore,the effect of MAD1 on the peanut defense response was analyzed by qRT-PCR.The results showed that the spore production of the knockout strain reduced by 43.67%,and the spore semi-germination time was 27.69 h,which was significantly longer than 13.43 h of the wild type strain.The semi-lethal time LT 50 of the knockout strain against the Bombyx mori was 8.9 d,which was significantly longer than 7.62 d of the wild type strain.In addition,6 h after inoculating the knockout strain to peanut,the transcription levels of the immune genes CNGC 1 and PCD 4,disease resistance gene SWEET 10 and transcription factors WRKY 41 and MYB 86 were significantly up-regulated,while the transcriptional expression of calmodulin CML 5 and CML 19 were significantly inhibited.This study suggested that mad 1 was a positively correlated gene of sporulation,spore germination and virulence in M.anisopliae.Moreover,MAD1 inhibited the plant resistance cascade,weakened the allergic reactions and reduced the resistance to microorganisms,but synchronously enhanced the transduction of symbiotic signals at the initial stage of the interaction between M.anisopliae and plants.These effects were conducive to the colonization of M.anisopliae in peanut root tissues.