Functional Analysis Of Colletotrichum Gloeosporiorum Specific Effector Genes CgSE1 And CgSE2

As an important industrial raw material and strategic resource,natural rubber is mainly derived from Hevea brasiliensis.Rubber tree anthracnose is one of the important factors affecting rubber production.Colletotrichum gloeosporioides and Colletotrichum acutatum can cause rubber tree anthracnose,and the pathogenicity of C.gloeosporioides on rubber trees is far stronger than that of C.acutatum.In the previous studies,the genome of C.gloeosporioides and C.acutatum were sequenced and C.gloeosporioides specific candidate effector encoding genes were predicted through the comparative genomics method.Two of these C.gloeosporioides specific effector genes were amplified and named as CgSE1 and CgSE2.were cloned and analyzed in this study.The functions of CgSE1 and CgSE2 were preliminarily investigated in this study.The main results were as follows.:1.C.gloeosporioides specific effector genes CgSE1 and CgSE2 were amplified by RT-PCR.According to the sequencing,CgSE1 contains an open reading frame of 639 bp,and CgSE2 contains a 435 bp open reading frame.2.CgSEl encodes a 212 amino acids peptide with a molecular weight of approximately 22.7 kDa.Bioinformatics analysis showed that the 1-16 aa of CgSE1 protein are typical signal peptide sequences.CgSE1 contains a HNHc conserved domain.Phylogenetic tree analysis showed that this protein belongs to the second subgroup of HNH protein family.According to the principle of homologous recombination,the knockout vector of CgSE1,pCB1532-CgSE1,was constructed and transferred into protoplast of C.gloeosporioides by PEG-mediated transformation method.The knockout mutant strain ?CgSE1 was obtained after chlorimuron-ethyl resistance selecting and molecular identification.?CgSE1 showed slower growth rate,decreased conidia production and weakened pathogenicity on rubber tree leaves than wild type stain.Further analysis revealed that the mechanical penetration ability of ACgSE1 mycelium was lost,and the resistance to oxidative stress was reduced.These may be the partial reason for its impaired virulence.At the same time,we transferred CgSE1 gene into the C.acutatum to analyze its effect on C.acutatum.The results above showed that CgSEl gene is not only participates in the regulation of the growth and development of C.gloeosporioides,but also plays an important role in the pathogenicity of C.gloeosporioides on rubber tree.3.CgSE2 encodes a peptide containing 144 amino acids and has a molecular weight of approximately 15 kDa.Bioinformatics analysis showed that CgSE2 protein contains a 16 amino acid signal peptide at N terminal of CgCE2.Similarly,the knockout mutant strain ?CgSE2 were constructed to study the function of the CgSE2.The results showed that the growth and conidia production of?CgSE2 were affected compared to the wild type(WT)of C.gloeosporioides,and the virulence of ?CgSE2 was also significantly reduced.At the same time,it was found that the mechanical penetration ability of ?CgSE2 mycelia was lost and the resistance to oxidative stress was reduced,suggesting that these contribute to impaired virulence of ?CgSE2 partially.The results above showed that CgSE2 was not only involved in the growth and development of C.gloeosporioides but also played important role in the pathogenicity on Hevea brasiliensis.