Functional Analysis Of Candidate Genes CgNSPG1 And CgNSPG3 For The Specific Effect Protein Of Colletotrichum Gloeosporioides

Rubber tree(Hevea brasiliensis)is a tropical perennial crop and the main source of natural rubber.Natural rubber is an important industrial raw material and strategic resource in China.Colletotrichum gloeosporioides Hb and Colletotrichum acutatum Hb are the main pathogenic fungi of rubber anthracnose,which seriously affect the production of natural rubber.The pathogenicity of C.gloeosporioides Hb to rubber trees is much stronger than that of C.acutatum Hb.In the previous research,the laboratory had obtained two novel specific pathogenic gene of C.gloeosporioides(CgNSPG)by comparing the genome sequences of C.gloeosporioides Hb and C.acutatum Hb,and named them as CgNSPGl and CgNSPG3.The knockout mutants of CgNSPG1 and CgNSPG3(ΔCgNSPGl andΔCgNSPG3)were constructed for functional study.In this study,the complementary mutants of CgNSPGl and CgNSPG3 were generated based on the ΔCgNSPGl andΔCgNSPG3 for clarifying their function.In particular,transgenic CgNSPGl and CgNSPG3 mutants of C.acutatum Hb were constructed respectively to further explore their role in pathogenicity and host specialization.The research results are as follows:1.Functional Analysis of CgNSPG1(1)CgNSPGl contains an open reading frame of 435 bp,encoding 144 amino acids with a molecular weight of approximately 15.0 kDa.CgNSPGlprotein does not contain any conserved domains and transmembrane domains.According to the principle of gene complementation,the complementary mutant Res-ΔCgNSPG1 of CgNSPG1 was constructed based on the existing ΔCgNSPG1.(2)Through growth phenotype and pathogenicity analysis,it was found that compared with the phenotypes of ΔCgNSPG1 slowing down,conidia production ability decreased,and pathogenicity weakening,the gene complementary mutant Res-ΔCgNSPG1 recovered in the above aspects.Further analysis showed that the expression levels of fungal growth and pathogenic regulation-related factors CRZ1 and CMK1 in ΔCgNSPG1 were significantly down-regulated and up-regulated compared with Cg-WT,and there was no significant difference with the expression of Res-ΔCgNSPG1.The above results indicated that CgNSPG1 was involved in the growth and development and pathogenicity of C.gloeosporioides Hb,which may be related to the Ca2+ signaling pathway mediated by CRZ1 and the MAPK signaling pathway mediated by CMK1.(3)In order to analyze the possible role of CgNSPGl in host specialization,the constitutive heterologous expression of CgNSPGl in C.acutatum Hb(Ca-NSPG1)could significantly increase the growth and pathogenicity of C.acutatum Hb.The results of qRT-PCR analysis showed that the expression levels of CaCZRl and CaCMKl in Ca-NSPG1 were significantly increased compared with Ca-WT,suggesting that CgNSPG1 may promote the growth and pathogenicity of C.acutatum Hb relating to Ca2+ signaling pathway mediated by CRZ1 and the MAPK signaling pathway mediated by CMK1.(4)The DAB tissue staining method was used to analyze the generation and accumulation of ROS in the tissues of Cg-WT and ΔCgNSPG1 infected rubber tree leaves with C.gloeosporioides Hb.The results showed that the loss of CgNSPG1 gene led to a decrease in ROS production and accumulation,which indirectly proved that CgNSPG1 can enhance the level of reactive oxygen species in the leaves of rubber trees.(5)In order to further investigate whether CgNSPGl has elicitor function,the CgNSPG1 gene was transformed into the model plant Arabidopsis thaliana by Agrobacterium-mediated genetic transformation to obtain CgNSPG1 transgenic plants.The disease resistance test found that the resistance of CgNSPG1 transgenic plants to Botrytis cinerea was significantly enhanced.Based on the above experimental results,we believe that CgNSPGl as an effector protein not only participated in regulating the growth and pathogenicity of C.gloeosporioides Hb,but also was related to the host specificity of the pathogen;meanwhile,the expression of CgNSPGl in plants can improve disease resistance,suggesting that CgNSPGl has the function of an elicitor.2.Functional Analysis of CgNSPG3(1)CgNSPG3 contains an open reading frame of 639 bp,encoding 212 amino acids with molecular weight of approximately 23.0 kDa.CgNSPG3 contains an HNH conserved domain,and belongs to the HNH family members.Based on the principle of gene complementation,a CgNSPG3 gene complementary mutant Res-ΔCgNSPG3 was constructed.(2)The growth phenotype and pathogenicity analysis of of Cg-WT,ΔCgNSPG3 and Res-ΔCgNSPG3 on rubber tree leaves revealed that the gene deletion of CgNSPG3 caused growth of C.gloeosporioides Hb slightly slowed down,the conidia production ability reduced,the pathogenicity weakened,and it could be partially restored after gene supplementation,indicating that ΔCgNSPG3 is related to the growth and pathogenicity of C.gloeosporioides.(3)In order to analyze the possible role of CgNSPG3 in the host specialization,CgNSPG3 was transformed into C.acutatum Hb.The results of phenotypic analysis showed that the growth rate and pathogenicity of C.acutatum Hb expressing CgNSPG3 was significantly higher than that of wild-type C.acutatum Hb,suggesting that CgNSPG3 may be related to host specialization.(4)In order to further study the role of CgNSPG3 in the innate immunity of plants,the CgNSPG3 gene was transformed into Arabidopsis thaliana by Agrobacterium-mediated genetic transformation method.CgNSPG3 transgenic seedlings were obtained for further function study of CgNSPG3.As mentioned above,CgNSPG3 is not only involved in the growth and pathogenicity of mycelia of C.gloeosporioides Hb,but is also related to the host specialization.