半胱氨酸蛋白酶CfAtg4在油茶果生刺盘孢中的功能分析

doi:10.11707/j.1001-7488.LYKX20220819

摘要/Abstract

摘要：

目的: 揭示半胱氨酸蛋白酶CfAtg4在油茶果生刺盘孢中的生物学功能，为油茶炭疽病新型防控药剂的开发提供候选靶标位点。方法: 通过Overlap PCR构建CfATG4基因敲除片段，利用同源重组原理和PEG介导的原生质体转化法获得敲除突变体和回补菌株；测定野生型、突变体、回补菌株对自噬诱导剂雷帕霉素的敏感性，通过倒置荧光显微镜观察饥饿诱导前后自噬小体数量评价自噬发生程度，采用蛋白免疫印迹试验验证显微观察结果；测定野生型、突变体、回补菌株的生长速率、分生孢子产量、致病力、孢子萌发、附着胞形成以及对外界胁迫敏感性。结果: 1）果生刺盘孢CfATG4基因敲除突变体ΔCfatg4对雷帕霉素更敏感，自噬小体数量显著少于野生型，ΔCfatg4突变体饥饿诱导前后均不能降解自噬标记蛋白GFP-CfAtg8，自噬作用丧失。2）通过生物学表型分析发现，突变体ΔCfatg4在不同营养条件培养基上生长速率减缓，气生菌丝减少；ΔCfatg4突变体产孢量显著减少，在油茶叶片上致病性丧失，且无法穿透玻璃纸。3）突变体ΔCfatg4孢子萌发率和附着胞形成率显著降低。4）突变体ΔCfatg4对盐胁迫（NaCl、KCl）更耐受，对细胞壁完整性胁迫剂SDS更敏感，对CR更耐受；突变体ΔCfatg4在氧化环境（H₂O₂）下更敏感，在还原环境（DTT）下更耐受。结论: 半胱氨酸蛋白酶CfAtg4介导的自噬参与调控油茶果生刺盘孢生长发育、产孢、附着胞形成、外界胁迫应答和致病过程。

关键词: 油茶, 果生刺盘孢, 半胱氨酸蛋白酶Atg4, 致病力

Abstract:

Objective: This article aims to reveal the biological function of cysteine protease CfAtg4 in Colletotrichum fructicola on Camellia oleifera, so as to provide a candidate target site for the development of new prevention and control agents for anthracnose in C. oleifera. Method: The CfATG4 gene knockout fragment was constructed by Overlap PCR, and the knockout mutants and complemented strains were obtained by homologous recombination and PEG-mediated protoplast transformation. The sensitivity of wild-type, mutant and complemented strains to rapamycin was measured. The degree of autophagy was evaluated by observing the number of autophagosomes before and after starvation induction under an inverted fluorescent microscopy, and validating the microscopic observation results using the Western blot assays. The growth rate, conidia number, pathogenicity, spore germination rate, appressoria formation rate and response to external stresses were measured in the wild type, mutant and complemented strains. Result: 1) The CfATG4 knockout mutant, ΔCfatg4, was more sensitive to rapamycin and showed significantly fewer autophagosomes than wild-type CFLH16. The ΔCfatg4 mutant was not able to degrade the autophagy marker protein, GFP-CfAtg8, before and after starvation induction, exhibiting autophagy abolishment. 2) The biological phenotypic analysis revealed that ΔCfatg4 mutant grew slowly, and the aerial hyphae decreased on different nutrient media. ΔCfatg4 mutant significantly reduced conidiation, and lost pathogenicity on tea oil leave. 3) The spore germination rate and appressoria formation rate of ΔCfatg4 mutant were significantly reduced. 4) ΔCfatg4 mutant showed stronger tolerance to salt stresses (NaCl, KCl), and a cell wall integrity stress agent, Congo red (CR), but it was more sensitive to sodium dodecyl sulfate (SDS), another cell wall integrity stress agent. Furthermore, ΔCfatg4 mutant was more sensitive to oxidizing environment (H₂O₂), and however, more tolerant to reducing environment (dithiothreitol, DTT). Conclusion: The cysteine protease CfAtg4 is involved in regulating the growth and development, conidiation, appressoria formation, external stress responses, and pathogenicity through mediated autophagy. In this study, we have first elucidated that the CfAtg4-mediated autophagy regulates the pathogenicity of C. fructicola, which provides potential target sites for the development of novel fungicides.

Key words: Camellia oleifera, Colletotrichum fructicola, cysteine protease Atg4, pathogenicity

中图分类号:

S763.1

郭树峰,郭源,陈妍,李河,张盛培. 半胱氨酸蛋白酶CfAtg4在油茶果生刺盘孢中的功能分析[J]. 林业科学, 2024, 60(7): 65-72.

Shufeng Guo,Yuan Guo,Yan Chen,He Li,Shengpei Zhang. Functional Analysis of Cysteine Protease CfAtg4 in Colletotrichum fructicola on Camellia oleifera[J]. Scientia Silvae Sinicae, 2024, 60(7): 65-72.

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引物名称Primer	引物序列Sequence (5’-3’)
CfATG4-1F	TTCCGAAGCTAGGAAATCCA
CfATG4-2R	CGCGGATGCCATCGAGAGTA
CfATG4-3F	GGCGAGCAAGGCGCACAGGC
CfATG4-4R	GGTCGAAGTTCTCGAGTGTG
CfATG4-5F	GCGTGAACCACTGCTGGTAG
H855R	GCTGATCTGACCAGTTGC
CfATG4-7F	ATGGCTGCTGTGGATCTCGG
CfATG4-8R	TCAAACATCGAGGATAGTGT
CfATG4-ProF	ACTCACTATAGGGCGAATTGGGTACTCA AATTGGTT CATGGAGGTTTCGTTGTCGA
CfATG4-ProR	CCTCGCCCTTGCTCACCATCGCGGAT GCCATCGAGAGTA
GFPF	ATGGTGAGCAAGGGCGAGG
GFPR	CTTGTACAGCTCGTCCATGC
CfATG4F	GCATGGACGAGCTGTACAAGATGGC TGCTGTGGATCTCGG
CfATG4R	CACCACCCCGGTGAACAGCTCCTCGCCCTT GCTCACTCAAACATCGAGGATAGTGT
Hyg F	GGCTTGGCTCCAGCTAGTGGAGGT
Hyg R	CTCTATTCCTTTGCCCTCG

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