Cloning And Functional Analyse Of CgSho1?CgCFEM1 And CgOPT2 In Colletotrichum Gloeosporioide

Anthracnose of rubber trees in the main planting areas in China, has been severely affecting the natural rubber production. Colletotrichum gloeosporioides (Colletotrichum gloeosporioides), a serious cause of leaf disease, will not only do harm to the growth of rubber tree, but also delay its tapping time. At present, the prevention and control of Anthracnose of Hevea brasiliensis is mainly based on the traditional chemical measure and agricultural measure. And few studies on the pathogenic mechanism of the pathogen lead to the lack of pertinence in prevention and cure measures. To study the pathogenic genes can help us to understand the pathogenesis so that early diagnosis,prevention scheme and the predicting model of the disease can be done on a scientific basis.Consulting relative literature and references,and referring to model fungal yeast and pathogenic gene significantly shaped among Magnaporthe grisea identified, three genes of Sho1, CFEM1, OPT2 has been selected in Colletotrichum gloeosporioides for cloning research. Following is the research content:1. HOG-MAPK (high osmolarity glycerol mitogen-activated protein kinase) signal pathway involving in response of osmotic pressure of MAPK pathway plays important role in growth and pathogenicity of plant pathogens. Shol (synthetic high osmolarity-sensitive protein 1), an important receptor in the upstream of HOG-MAPK signal pathway, often has different functions in different fungi. In this study, the homologous gene of Shol in Colletotrichum gloeosporioides was cloned and named as CgShol, which encodes a 291-amino acids protein, containing 4 transmembrane domains and a SH3 domain. The gene-knockout mutants of CgShol were obtained by homologous recombination.Comparing to wild type, the knockout mutants of CgShol showed slow growth, sparse aerial hyphae with hydrophobicity increasing, conidium production decreasing, and were so much more sensitive to oxidative stress and osmotic pressure that pathogenicity significantly decreased. The results also showed that the phenotype of the complementary strain was similar to that of the wild-type strain.2. A plant pathogenic fungus effector, a secreted protein or other small molecular substance, can alter the cell structure or function of the host plant, and plays an important role in the invasion, expansion and pathogenicity of pathogenic fungi. The unique CFEM(common in fungi several fungal extracellular membrane protein) protein of pathogenic fungi is indispensable for pathogenicity of pathogenic fungi, some of which secreted into the extracellular CFEM protein were proved to be pathogen effector. In this study,CFEM1 was cloned from Colletotrichum gloeosporioides in (hypothetical protein) effector homologous gene, named CgCFEMl, which encodes a protein of 384 amino acids, and contains a transmembrane domain and a CFEM domain. The gene knockout mutants obtained by homologous recombination, compared with wild-type knockout mutants,showed slow growth, sparse mycelia and hydrophobic enhanced sporulation decline, and were more sensitive to oxidative stress and osmotic pressure so that pathogenicity was weakened. These results indicate that CgCFEM1 is involved in the regulation of vegetative growth, conidial yield, oxidative stress response, osmotic stress response and pathogenicity of colletotrichum. In addition, the result shows that the phenotype of the complementary strain is similar to that of the wild-type strain.3. OPT (Oligopeptide transporter) is found in bacteria, fungi, plants and animals. In this study, the homologous gene of OPT1 in Colletotrichum gloeosporioides was cloned and named as CgOPT2, encoding a 982-amino acids protein and containing 15 transmembrane domains, both of which construct OPT domain and play an important role in growth and pathogenicity of pathogenic fungi. The gene-knockout mutants of CgOPT2 obtained by homologous recombination, compared to wild type, showed slow growth,sparse aerial hyphae with increasing hydrophobicity, and conidium production decline, and were more sensitive to oxidative stress and osmotic pressure so that pathogenicity decreased significantly. The result demonstrates that CgOPT2 is involved in regulation of vegetative growth, conidium production, oxidative stress response, osmotic pressure response and pathogenicity of C. gloeosporioides.