Mou Ya-Ni, Fu Bo, Ren Kang, Ying Sheng-Hua, Feng Ming-Guang
MOE Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
MOE Laboratory of Biosystems Homeostasis and Protection, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China
mSystems. 2021 Mar 23;6(2):e00098-21. doi: 10.1128/mSystems.00098-21.
Small secreted proteins (SSPs), particularly cysteine-rich proteins secreted during fungal infection, comprise virulence effectors in plant-pathogenic fungi but remain unknown in insect-pathogenic fungi. We report here that only a small cysteine-free protein (CFP) is indispensable for insect pathogenicity of among 10 studied SSPs (99 to 274 amino acids [aa]), including seven hypothetical proteins containing 0 to 12 Cys residues. CFP (120 aa) features an N-terminal signal peptide (residues 1 to 17), a nuclear localization signal motif (residues 24 to 57), and no predictable domain. Its homologs exist exclusively in insect-pathogenic Cordycipitaceae and Clavicipitaceae. Fluorescence-tagged CFP fusion protein was localized in the nucleus but extracellularly undetectable, suggesting an inability for CFP to be secreted out. Disruption of resulted in abolished pathogenicity via normal cuticle infection, attenuated virulence via hemocoel injection, compromised conidiation capacity versus little growth defect, impaired conidial coat, blocked secretion of cuticle-degrading enzymes, impeded proliferation , disturbed cell cycle, reduced stress tolerance, and 1,818 dysregulated genes (genomic 17.54%). Hundreds of those genes correlated with phenotypic changes observed in the disruption mutant. Intriguingly, nearly 40% of those dysregulated genes encode hypothetical or unknown proteins, and another 13% encode transcription factors and enzymes or proteins collectively involved in genome-wide gene regulation. However, purified CFP showed no DNA-binding activity in an electrophoretic mobility shift assay. These findings unveil that CFP is a novel regulator of fungal insect-pathogenic life cycle and genomic expression and that cysteine richness is dispensable for distinguishing virulence effectors from putative SSPs in Small cysteine-rich proteins secreted during plant-pathogenic fungal infection comprise virulence effectors. Our study confirms that only a cysteine-free protein (CFP) is determinant to insect-pathogenic fungal virulence among 10 small putatively secreted proteins containing 0 to 12 Cys residues. Disruption of abolished insect pathogenicity and caused not only a series of compromised cellular events associated with host infection and disease development but also dysregulation of 1,818 genes, although no DNA-binding activity was detected in purified CFP samples. Nearly 13% of those genes encode transcription factors and enzymes or proteins collectively involved in transcriptional regulation. Altogether, CFP serves as a novel regulator of the fungal insect-pathogenic life cycle and genomic expression. Cysteine richness is dispensable for distinguishing virulence effectors from the fungal SSPs.
小分泌蛋白（SSPs），特别是真菌感染期间分泌的富含半胱氨酸的蛋白，是植物病原真菌中的毒力效应子，但在昆虫病原真菌中尚不清楚。我们在此报告，在所研究的10种SSPs（99至274个氨基酸[aa]）中，只有一种不含半胱氨酸的小蛋白（CFP）对昆虫致病性是不可或缺的，其中包括7种含有0至12个半胱氨酸残基的假定蛋白。CFP（120个氨基酸）具有一个N端信号肽（第1至17位氨基酸）、一个核定位信号基序（第24至57位氨基酸），且没有可预测的结构域。其同源物仅存在于昆虫病原麦角菌科和香柱菌科中。荧光标记的CFP融合蛋白定位于细胞核，但在细胞外无法检测到，这表明CFP无法分泌到细胞外。破坏该基因导致通过正常表皮感染的致病性丧失、通过血腔注射的毒力减弱、分生孢子形成能力受损而生长缺陷较小、分生孢子壁受损、角质层降解酶的分泌受阻、增殖受影响、细胞周期紊乱、应激耐受性降低以及1818个基因失调（占基因组的17.54%）。其中数百个基因与破坏突变体中观察到的表型变化相关。有趣的是，这些失调基因中近40%编码假定或未知蛋白，另外13%编码转录因子以及共同参与全基因组基因调控的酶或蛋白。然而，在电泳迁移率变动分析中，纯化的CFP未显示出DNA结合活性。这些发现揭示CFP是真菌昆虫致病生命周期和基因组表达的新型调节因子，并且富含半胱氨酸对于区分昆虫病原真菌中的毒力效应子与假定的SSPs并非必要。植物病原真菌感染期间分泌的富含半胱氨酸的小蛋白构成毒力效应子。我们的研究证实，在10种含有0至12个半胱氨酸残基的假定分泌小蛋白中，只有一种不含半胱氨酸的蛋白（CFP）是昆虫病原真菌毒力的决定因素。破坏该基因消除了昆虫致病性，不仅导致了一系列与宿主感染和疾病发展相关的细胞事件受损，还导致1818个基因失调，尽管在纯化的CFP样品中未检测到DNA结合活性。这些基因中近13%编码转录因子以及共同参与转录调控的酶或蛋白。总之，CFP作为真菌昆虫致病生命周期和基因组表达的新型调节因子。富含半胱氨酸对于区分真菌SSPs中的毒力效应子并非必要。
