Anhui Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei, 230036, China.
School of Plant Protection, Anhui Agricultural University, Hefei, 230036, China.
BMC Microbiol. 2019 Jan 28;19(1):25. doi: 10.1186/s12866-019-1396-8.
Metarhizium robertsii, a widely distributed insect pathogen, is presently used as a natural alternative to chemical insecticides. Unfortunately, its worldwide commercial use has been restricted by a short shelf life and inconsistencies in virulence. In our previous study, a gene (GenBank accession number EFZ01626) was found to be significantly upregulated in heat-treated conidia. In the present study, this gene was characterized via gene disruption and complementation strategies.
The gene (amplified by rapid amplification of cDNA ends PCR) was 1219 bp long and contained an open reading frame (ORF) of 777 bp. It encoded a protein of 234 amino acid residues with a 26-residue signal peptide. Bioinformatics analyses did not identify conserved functional domains; therefore, it was assumed to be a secreted virulence-associated protein according to its signal peptide and bioassay results. We found that the conidial germination rate of the ΔMrSVP mutant fungi dramatically decreased after heat shock treatment in a thermotolerance test. In addition, transcription levels of all tested heat shock-related genes were significantly lower in the mutant than in the wild type. We also demonstrated that the mean lethal time to death (LT) of ΔMrSVP significantly increased relative to the wild type in insect bioassays (both topical inoculation and injection) involving Galleria mellonella. Moreover, similar rates of appressorium formation between ΔMrSVP and the wild type-and the significantly different expression of virulence-related genes such as acid trehalase and sucrose nonfermenting protein kinase in the haemocoel after injection-revealed that MrSVP is required for virulence in the insect haemocoel.
Overall, our data suggest that the Mrsvp gene contributes to thermotolerance and virulence of M. robertsii. Furthermore, this gene is deeply involved in the mycosis of insect cadavers and in immune escape rather than insect cuticle penetration during infection.
广泛分布的昆虫病原体玫烟色棒束孢目前被用作化学杀虫剂的天然替代品。不幸的是，由于保质期短和毒力不一致，其在全球范围内的商业应用受到限制。在我们之前的研究中，发现一种基因（GenBank 登录号 EFZ01626）在热处理分生孢子中显著上调。在本研究中，通过基因敲除和互补策略对该基因进行了特征分析。
该基因（通过快速扩增 cDNA 末端 PCR 扩增）长 1219bp，包含一个 777bp 的开放阅读框（ORF）。它编码一个由 234 个氨基酸残基组成的蛋白质，带有一个 26 个残基的信号肽。生物信息学分析未鉴定出保守的功能域；因此，根据其信号肽和生物测定结果，推测它是一种分泌的与毒力相关的蛋白质。我们发现，在耐热试验中，热休克处理后ΔMrSVP 突变真菌的分生孢子发芽率显著降低。此外，在突变体中，所有测试的热休克相关基因的转录水平均明显低于野生型。我们还证明，在涉及大蜡螟的昆虫生物测定（包括体表接种和注射）中，ΔMrSVP 的平均致死时间（LT）相对于野生型显著增加。此外，ΔMrSVP 与野生型之间的附着胞形成率相似，以及注射后血腔中与毒力相关的基因（如酸性海藻糖酶和蔗糖非发酵蛋白激酶）的表达显著不同，表明 MrSVP 是昆虫血腔中毒力所必需的。
总体而言，我们的数据表明 Mrsvp 基因有助于玫烟色棒束孢的耐热性和毒力。此外，该基因深度参与昆虫尸体的真菌病和免疫逃避，而不是感染过程中对昆虫表皮的穿透。
