杨树腐烂病菌胞外分泌复合体亚基CcExo70的功能

Functional Analysis of the Exocyst Subunit CcExo70 in Cytospora chrysosperma

【目的】对杨树腐烂病菌金黄壳囊孢胞外分泌复合体亚基CcExo70进行功能分析,探究其在杨树腐烂病菌生长发育及致病过程中的生物学功能,为深入了解杨树腐烂病菌致病机制以及制定病害防控策略提供科学依据。【方法】1)利用PCR技术克隆得到CcExo70基因的DNA序列;2)通过生物信息软件对其编码的氨基酸序列进行特征分析并构建其同源基因的系统发育树;3)通过PEG介导的遗传转化法得到CcExo70基因的敲除突变体及回补菌株;4)通过PDA平板生长试验和PDB摇培试验,分析敲除CcExo70基因对该病菌生长发育、菌丝形态及H2O2胁迫响应的影响;通过对1年生欧美杨健康枝条烫伤接种的方法,测定CcExo70基因对该病菌致病性的影响;5)利用荧光增白剂(CFW)、二氨基联苯胺(DAB)和FM4-64染剂探究各菌株中几丁质沉积、活性氧积累及内吞作用情况;6)通过蛋白质组测序分析,筛选可能受CcExo70调控分泌的外泌蛋白。【结果】1)在杨树腐烂病菌中克隆得到了一个CcExo70基因,该基因全长1992 bp,包含1个内含子,编码636个氨基酸。2)系统发育和序列比对分析表明,CcExo70与其他病原真菌的同源基因在进化上是高度保守的。3)利用遗传转化法筛选得到2个敲除突变体(ΔCcExo70-8、ΔCcExo70-9),并获得了回补菌株(ΔCcExo70/C)。4)表型分析发现,与野生型和回补菌株相比,CcExo70敲除突变体在菌丝生长、形态发生、氧化应激反应和致病力等方面存在显著的缺陷。5)CFW染色观察显示,野生型和回补菌株菌丝尖端存在明显的几丁质沉积,而敲除突变体菌丝尖端未见明显的几丁质沉积但菌丝隔膜异常增加。此外,共聚焦显微镜观察发现,与野生型和回补菌株相比,CcExo70敲除突变体的内吞作用显著延迟。6)蛋白组测序分析发现,8个候选的外泌蛋白在CcExo70敲除突变体胞外培养滤液中的含量显著低于在野生型胞外培养滤液中的含量(降低了50%以上),其中有4个候选的外泌蛋白(CcSP2、CcSP3、CcSP6、CcSP7)在胞外培养滤液中的含量显著高于其对应菌株的胞内含量,其中包括1个候选的效应分子(CcSP2)和2个糖苷水解酶(CcSP6和CcSP7),表明这4个外泌蛋白的分泌受到了CcExo70的调控。【结论】胞外分泌复合体亚基CcExo70在杨树腐烂病菌的生长、胁迫响应、内吞作用和致病性等方面起着重要的调控作用。

【Objective】The purpose of this study is to analyze the functions of the exocyst subunit CcExo70 in poplar canker fungus,Cytospora chrysosperma,including its roles in fungal growth,development and pathogenicity.This study would provide a scientific basis for further understanding the pathogenic mechanism and establishing the disease control strategies.【Method】1)PCR amplification was used to clone the CcExo70 from the C.chrysosperma.2)The amino acid sequences were characterized by bioinformatics methods,and a phylogenetic tree of its homologous genes was constructed.3)The knockout mutants and complementary strains of CcExo70 were obtained by using the PEG-mediated transformation method.4)The effects of knockout of CcExo70 gene on growth and development,morphogenesis,and H2O2 stress response were analyzed on PDA plates or in liquid PDB,and the effect of CcExo70 gene on pathogenicity was tested with one-year-old Populus×euramericana.5)The calcofluor white(CFW),DAB(3,3’-diaminobenzidine),and FM4-64 were used to investigate the chitin deposition,reactive oxygen species accumulation and endocytosis of each strain.6)The comparative proteome analyses were performed to screen the putative secreted proteins regulated by CcExo70.【Result】1)The CcExo70 gene was cloned from the genomic DNA of C.chrysosperma.The full length of the gene was 1992 bp,including one intron encoding 636 amino acids.2)Phylogenetic analysis and sequence alignment showed that CcExo70 was highly conserved with Exo70 homologs from other pathogenic fungi.3)Two CcExo70 deletion mutants(ΔCcExo70-8 andΔCcExo70-9)were screened by using the split-marker method named,and the complemented strain(ΔCcExo70/C)was obtained.4)Phenotypic analyses showed that deletion mutants of CcExo70 displayed obvious defects in fungal growth,morphogenesis,oxidative stress response and pathogenicity compared to the wild-type and complemented strains.5)CFW staining showed that there was obvious chitin deposition at the mycelial tip of wild-type and compensatory strains,while there was no obvious chitin deposition at the mycelial tip of knockout mutants,but septa were increased.Additionally,confocal microscopy showed that endocytosis in CcExo70 deletion mutants was significantly delayed compared to the wild-type and complemented strains.6)Proteome sequencing analysis showed that the abundances of eight candidate secreted proteins were significantly reduced(over 50%)in culture filtrates of the CcExo70 deletion mutant compared to that in wild type by using proteomics analysis.Additionally,four of them(CcSP2k,CcSP3,CcSP6,CcSP7)were dramatically enriched in the culture filtrates compared to the corresponding intracellular protein profiles including 1 putative effector(CcSP2)and 2 glucoside hydrolases(CcSP6,CcSP7),indicating the secretion of these candidate proteins was regulated by CcExo70.【Conclusion】Collectively,these results suggest that the exocyst subunit gene CcExo70 is essential for the fungal growth,stress response,endocytosis and pathogenicity of C.chrysosperma.