Genetic Engineering Research Center, College of Bioengineering, Chongqing University, Chongqing, P R China.
BMC Microbiol. 2012 Aug 1;12:163. doi: 10.1186/1471-2180-12-163.
The efficacy of entomopathogenic fungi in pest control is mainly affected by various adverse environmental factors, such as heat shock and UV-B radiation, and by responses of the host insect, such as oxidative stress, osmotic stress and fever. In this study, an adenylate cyclase gene (MaAC) was cloned from the locust-specific entomopathogenic fungus, Metarhizium acridum, which is homologous to various fungal adenylate cyclase genes. RNA silencing was adapted to analyze the role of MaAC in virulence and tolerance to adverse environmental and host insect factors.
Compared with the wild type, the vegetative growth of the RNAi mutant was decreased in PD (potato dextrose medium), Czapek-dox and PDA plates, respectively, demonstrating that MaAC affected vegetative growth. The cAMP levels were also reduced in PD liquid culture, and exogenous cAMP restored the growth of RNAi mutants. These findings suggested that MaAC is involved in cAMP synthesis. The knockdown of MaAC by RNAi led to a reduction in virulence after injection or topical inoculation. Furthermore, the RNAi mutant grew much slower than the wild type in the haemolymph of locust in vitro and in vivo, thus demonstrating that MaAC affects the virulence of M. acridum via fungal growth inside the host locust. A plate assay indicated that the tolerances of the MaAC RNAi mutant under oxidative stress, osmotic stress, heat shock and UV-B radiation was decreased compared with the wild type.
MaAC is required for virulence and tolerance to oxidative stress, osmotic stress, heat shock and UV-B radiation. MaAC affects fungal virulence via vegetative growth inside the insect and tolerance against oxidative stress, osmotic stress and locust fever.
昆虫病原真菌在害虫防治中的功效主要受到各种不利环境因素的影响，如热冲击和 UV-B 辐射，以及宿主昆虫的反应，如氧化应激、渗透应激和发热。在这项研究中，从蝗虫专性昆虫病原真菌绿僵菌中克隆了一个腺苷酸环化酶基因（MaAC），该基因与各种真菌的腺苷酸环化酶基因同源。采用 RNA 干扰技术分析 MaAC 在真菌毒力和对不利环境及宿主昆虫因子的耐受中的作用。
与野生型相比，RNAi 突变体在 PD（土豆葡萄糖培养基）、Czapek-dox 和 PDA 平板上的营养生长分别降低，表明 MaAC 影响营养生长。cAMP 水平在 PD 液体培养中也降低，外源性 cAMP 恢复了 RNAi 突变体的生长。这些发现表明 MaAC 参与 cAMP 的合成。MaAC 的 RNAi 敲低导致注射或局部接种后毒力降低。此外，RNAi 突变体在体外和体内蝗虫血液中的生长速度明显慢于野生型，表明 MaAC 通过真菌在宿主蝗虫体内的生长影响绿僵菌的毒力。平板测定表明，与野生型相比，MaAC RNAi 突变体对氧化应激、渗透应激、热冲击和 UV-B 辐射的耐受能力降低。
MaAC 是毒力和对氧化应激、渗透应激、热冲击和 UV-B 辐射耐受所必需的。MaAC 通过昆虫体内营养生长和对氧化应激、渗透应激和蝗虫发热的耐受影响真菌的毒力。
