病原真菌肠道感染昆虫的分子机制研究

Entomogenous fungus are widely used as biocontrol agents against insect pests, considering that a mass of insects displayed highly resistant to chemical insecticides. However, the applications of entomogenous fungus are limited by the notoriously slow killing action, for the reason that they infected hosts by means of cuticle instead of intestinal track in most cases. Previously, we tested Beauveria bassiana isolates for the capability to infect fifth-instar Bombyx. mori larvae by oral feeding. Survival assay revealed that a newly identified strain, B. bassiana Bb-bm01, is capable of killing B. mori larvae through intestinal infection. Meanwhile, Bb-bm01 displayed highly pathogenic to the silkworm by cuticle infection, and the virulence of which is significantly synergistically enhanced by cuticle and intestine coinfection. Therefore, firstly in our study, the process of the intestinal infection trigged by Bb-bm01 in insect host is determined by means of paraffin section, scanning electron microscopy visualization and fluorescence labeling. Secondly, genome-wild expression profiles of Bb-bm01 during different stages of intestinal infection process are investigated by RNA-seq, and the differentially expressed genes involved in intestinal infection of silkworm are selected by bioinformatics analysis. Subsequently, the candidate intestine-specific virulence factors were selected based on both RNA-seq and comparative genome analysis. Finally, the key intestine-specific virulence factors of Bb-bm01 are verified through genetic manipulation of filamentous fungi and the underlying mechanisms will be further studied. This study will reveal the molecular mechanism of intestinal infection trigged by entomogenous fungus, and provide new insights into the pathogenicity of entomogenous fungus.

昆虫病原真菌作为一种重要的生物杀虫剂，已被开发成多种制剂应用于农林害虫的防治。但是，鉴于病原真菌单一的体壁侵染途径，其防治效果受环境因子的制约，限制了真菌杀虫剂的推广应用。寻找具有新型感染途径的病原真菌将为真菌杀虫剂的开发提供新思路。我们前期通过口服饲喂的方法新发现了一株能够通过肠道感染家蚕的球孢白僵菌Bb-bm01；另外，体壁-肠道双途径感染有效提高了Bb-bm01的杀虫毒力。因此，本项目以球孢白僵菌Bb-bm01为研究材料，通过组织切片、扫描电镜检测以及荧光标记的方法确定Bb-bm01肠道感染家蚕的途径和过程；借助转录组测序分析Bb-bm01肠道感染家蚕不同阶段基因的表达差异，并结合比较基因组数据初步筛选Bb-bm01肠毒因子；进一步利用真菌分子遗传学分析和生物测定鉴定Bb-bm01关键肠毒因子的功能，揭示Bb-bm01肠道感染家蚕的分子机制，为拓展真菌杀虫剂的应用提供新途径和新思路。

昆虫病原真菌作为一种重要的生物杀虫剂，已被开发成多种制剂应用于农林害虫的防治。但是，鉴于病原真菌单一的体壁侵染途径，其防治效果易受环境因子制约，因而限制了真菌杀虫剂的推广应用。寻找具有新型感染途径的病原真菌将为真菌杀虫剂的开发提供新思路。我们前期通过口服饲喂的方法新发现了一株能够通过肠道感染家蚕的球孢白僵菌Bb-bm01，但是其肠道感染的分子机制尚不明确。本项目首先通过组织切片明确了Bb-bm01通过前肠感染家蚕的过程。利用比较基因组和转录组分析发现，Bb-bm01基因组中有12个株系特异的DNA区域（lineage-specific region, LS region）。最长的4号LS区域包含25个编码基因，其中19个是Bb-bm01菌株所特有的未知功能基因，这些基因大多数在Bb-bm01入侵家蚕体内阶段显著高表达。对9个Bb-bm01特有、并且在感染家蚕过程中上调表达的基因（命名为oral infection-related gene, OIG）分别进行敲除，突变株并没有影响真菌肠道感染毒力。鉴于家蚕前肠呈碱性，肠道感染可能需要真菌具有较强的耐碱性能力。在碱性胁迫条件下，Bb-bm01萌发速率显著快于不具有肠道感染的球孢白僵菌Bb-mo01。通过碱性诱导转录组，我们发现一个含有锌指结构域的转录因子bm01ZnF在碱性诱导下快速上调表达。敲除bm01znf后，Bb-bm01孢子萌发形成的菌丝生长受到碱性环境的显著抑制，肠道感染毒力显著降低。本项目研究揭示了球孢白僵菌Bb-bm01通过转录因子ZnF在碱性条件下的快速上调表达来调控耐碱性能力，从而能在昆虫碱性肠道中快速萌发和侵染的分子机制，为拓展真菌杀虫剂的应用提供新途径和新思路。项目分别在国际期刊Developmental and Comparative Immunology和Science Advances上各发表SCI论文1篇。培养博士研究生3名，其中2名博士生已顺利毕业。