果生刺盘孢G蛋白信号调控因子CfRGS4基因的生物学功能  

Biological functions of Colletotrichum fructicola G protein signaling regulator encoding gene CfRGS4

作　　者：胡秋月 曹凌雪 李河[1] HU Qiuyue;CAO Lingxue;LI He(Key Laboratory of National Forestry and Grassland Administration on Control of Artificial Forest Diseases and Pests in South China,Hunan Provincial Key Laboratory for Control of Forest Diseases and Pests,Central South University of Forestry and Technology,Changsha 410004,Hunan,China)

机构地区：[1]中南林业科技大学,南方人工林病虫害防控国家林业和草原局重点实验室,森林有害生物防控湖南省重点实验室,湖南长沙410004

出　　处：《菌物学报》2024年第8期40-53,共14页Mycosystema

基　　金：国家重点研发计划(2023YFD1401301)。

摘　　要：果生刺盘孢Colletotrichum fructicola是油茶炭疽病的优势病原菌。研究果生刺盘孢G蛋白信号调节因子CfRGS4基因的生物学功能,为揭示病菌致病机制以及防治油茶炭疽病提供理论依据。构建CfRGS4基因敲除载体片段,利用PEG介导的原生质体转化和抗性筛选及PCR电泳验证,获得果生刺盘孢CfRGS4基因敲除突变体及回补菌株。对野生型、△Cfrgs4和△Cfrgs4-C进行观察,结果表明:相较于野生型,突变体△Cfrgs4-1和△Cfrgs4-2在PDA培养基上的生长速率下降了24%和22%,在MM培养基上下降了22%和20%;分生孢子萌发率降低了65%和70%;附着孢形成率降低了24%和17%;在含有400μg/mL CR的细胞胁迫剂上,突变体△Cfrgs4-1和△Cfrgs4-2的生长抑制率为14%和11%;与野生型相比,突变体菌落表现出被水浸润的现象,荧光定量PCR结果显示,突变体△Cfrgs4中菌丝疏水相关基因CU1和MHP1基因均显著上调表达;在含有200μg/mL ABTs的PDA平板和PDB培养基中,突变体△Cfrgs4的胞外漆酶和过氧化物酶活性显著降低;突变体△Cfrgs4-1和△Cfrgs4-2在含有2.5 mol/L(30%和31%)和5 mol/L Cu^(2+)(73%和75%)的PDA培养基上的抑制率均显著高于野生型;在致病力测定中,突变体△Cfrgs4-1和△Cfrgs4-2在油茶叶片上造成的病斑面积减小了40%和48%,在苹果上造成的病斑面积减小了48%和55%。综上所述,G蛋白信号调控因子CfRGS4基因参与调控果生刺盘孢的生长发育、表面疏水性、附着胞形成、胞外漆酶和过氧化物酶活性、Cu^(2+)耐受性及致病性。Colletotrichum fructicola is the major pathogen causing anthracnose on tea-oil tree(Camellia oleifera).The biological function of a C.fructicola G protein signal regulator encoding gene,CfRGS4,was studied,to provide a new perspective for understanding the molecular basis of fungal pathogenicity and control of Ca.oleifera anthracnose.The CfRGS4 gene knockout vector was constructed,and the △Cfrgs4 and △Cfrgs4-C of C.fructicola were obtained by PEG-mediated protoplast transformation.Resistance screening and PCR electrophoresis were used for validation of mutant fungal strains.The results showed that compared with the wild type,the growth rates of mutants △Cfrgs4-1 and △Cfrgs4-2 on PDA medium decreased by 24%and 22%,and decreased by 22%and 20%on MM medium.Conidial germination rate decreased by 65%and 70%.The appressorium formation rate was reduced by 24%and 17%.The growth inhibition rates of the mutants △Cfrgs4-1 and △Cfrgs4-2 were 14%and 11%on the cell stress agent containing 400μg/mL CR.Compared with the wild type strain,the mutant colonies showed water saturated phenomenon.The expression of the CU1 and MHP1 genes was significantly up-regulated in the △Cfrgs4 mutant,as shown by the results of fluorescence quantitative PCR.The extracellular laccase and peroxidase activities of mutant △Cfrgs4 were significantly reduced in PDA plates and PDB media containing 200μg/mL ABTs.The inhibition rates of mutants △Cfrgs4-1 and △Cfrgs4-2 on PDA medium containing 2.5 mol/L(30%and 31%)and 5 mol/L Cu^(2+)(73%and 75%)were significantly higher than those of wild type strain.Determination of pathogenicity showed that the lesion area caused by △Cfrgs4-1 and △Cfrgs4-2 mutants on the leaves of Ca.oleifera decreased by 40%and 48%,and that on the apple decreased by 48%and 55%.In summary,CfRGS4 is involved in regulating the growth and development,surface hydrophobicity,appressorium formation,extracellular laccase and peroxidase activity,Cu^(2+)tolerance and pathogenicity of C.fructicola.

关 键 词：果生刺盘孢 油茶 CfRGS4基因 致病力 

分 类 号：S763.7[农业科学—森林保护学]