基于RNA高通量测序分析灰葡萄孢侵染垫形成相关基因

High-throughput RNA sequencing-based analysis of differentially expressed genes associated with infection cushion formation in Botrytis cinerea

灰葡萄孢产生功能上类似附着胞的侵染垫来侵染寄主植物,但目前对于侵染垫形成的分子机制尚不明确。本文利用高通量测序技术对菌株CanBC-1(弱毒,不形成侵染垫)和CanBC-1c-66(强毒,正常形成侵染垫)进行了转录水平比较。结果表明:在菌株CanBC-1中共检测到2 333个差异表达基因(与菌株CanBC-1c-66相比较),其中1 425个基因表达上调,908个基因表达下调。对差异表达基因进行功能注释(GO)分析发现,在细胞组分(Cellular component)中细胞(Cell)和细胞部分(Cell part)这2个亚类所占比例较大,在分子功能(Molecular function)中结合活性(Binding)和催化活性(Catalytic)这2个亚类所占比例较大。这些结果暗示差异基因主要参与细胞、代谢和发育等生物学过程。筛选得到12个与真菌致病相关的基因,其中BC1G_03994和BC1G_01012与稻瘟病菌侵染相关的classⅡ疏水蛋白基因Mhp1同源。RT-PCR检测发现这2个基因在弱毒菌株CanBC-1中表达下调,同时该菌株疏水性降低,推测它们可能参与灰葡萄孢侵染垫形成。这些差异基因的获得为揭示灰葡萄孢侵染形成的分子机制奠定了基础。

Botrytis cinerea usually infect plant tissues through infection cushions （ICs） or complex appressoria. However, little is known so far about the molecular mechanisms involved in formation of infection cushions by this fungus. In this study, high-throughput RNA sequencing was performed to investigate the global gene expres- sion profiles in B. cinerea strain CanBC-1 （avirulent, deficient in formation of ICs） and its single-conidium progeny CanBC-1c-66 （virulent, normal formation of ICs）. Results showed that a total of 2 333 genes were dif- ferentially expressed in CanBC-1 compared with that in CanBC-1c-66. Of these genes, 1 425 genes were up-regulated, whereas 908 genes were down-regulated. Gene ontology analysis revealed that most of the differentially expressed genes （DEGs） in the Cellular component category involved in Cell and Cell part. Most of the DEGs in the Molecular function category were predicted to have the binding and catalytic activities, which may involve in various biological processes such as cellular metabolism and development. Further screening confirmed 12 DEGs as the pathogenicity-related factors in B. cinerea or other plant pathogens. Among them, two genes, namely BC1G03994 and BC1GO1012, were found to be highly homologous to the class II hydrophobin gene Mhpl in Magnaporthe grisea. RT-PCR test showed that these two genes were down-regulated in strain CanBC-1, which was reduced hydrophobicity in mycelia and conidia in compared with strain CanBC-1c-66. Therefore, they might involve in infection cushion formation in B. cinerea. The finding will be helpful for understanding of the molecu- lar mechanisms for infection by B. cinerea.