Generation Of CgLysM RNAi Line Of Colletotrichum Gloeosporioides And Its Protein Subcellular Localization

Anthracnose of rubber tree was mainly caused by Colletotrichum gloeosporioides on the blade and petiole and leads to serious economic losses in rubber planting area in China.Effectors are important pathogenic factors for fungal pathogen. LysMs (Lysin Motifs) type effectors, named for the LysM conserved domain in the protein, are apoplast effector and widely existed in diverse fungi. In the previous study, genome of C. gloeosporioidase was sequenced and the genes encoding secreted proteins were predicted. One of the genes was named as CgLysM (LysM of C. gloeosporioide). The ORF (open reading frame) of CgLysM contains486base pairs, encoding a peptide with161amino acids. Protein structure analysis shows that CgLysM contains a signal peptide comprised of the first18amino acids and two conserved LysM domains locating at46to87and115to158amino acid residue, respectively.Gene knockout or gene silencing mutants are important to study the gene function. Thus, the C. gloeosporioides protoplasts transformation system was modified and established by PEG-mediated method. GFP (green fluorescent protein)-marked transformant of C. gloeosporioidase was generated and GFP can be observed clearly under the fluorescence microscope. Further experiment showed that there is no significant difference between GFP transformant with wild-type in the phenotypes of mycelia and its pathogenicity. These results indicated that the GFP transformant can be used as control in the subsequent research.To investigate the function of CgLysM, the RNAi vector pSilient-CgLysM was constructed and transformed into wild type C. gloeosporioidase to get CgLysM RNAi mutants. Semi quantitative RT-PCR demonstrated that expressions of CgLysM in RNAi transformants were decreased at different levels compared with that in the wild type. Further analysis found that the hyphal and connidial morphology and the pathogenicity of CgLysM of RNAi transformants showed no significant difference with in wild type.The recombination vector pCB1532-ToxA-CgLysM-GFP was constructed to generate CgLysM-GFP transformants for the localization of CgLysM in C. gloeosporioidase. Then, the location of CgLysM-GFP in mycelium, spores and the process of spore germination were observed. Results showed that the fluorescence was distributed in the whole cell of the mycelium and spore in transformant with GFP only. However, Fluorescence in mycelium of CgLysM-GFP transformants was mainly distributed in the peripheral position near the cell membrane. In the CgLysM-GFP conidia, the fluorescence was concentrated at one point. During conidium germination, CgLysM-GFP appeared on the top of the germ tube or in appresorium.One of the most important functions of effectors is PTI interference in plant. In this study, the effect of CgLysM on PTI was analyzed through Arabidopsis protoplast transient expression system. The results showed that CgLysM effector did not significantly affect the PTI in plant cell.