Functional Analysis Of Pathogenic LysM-type Effectors In Colletotrichum Gloeosporioides

As an important strategic resource and industrial raw material,natural rubber is mainly produced from the Brazilian rubber tree(Hevea brasiliensis).The rubber tree anthracnose caused by filamentous fungus Colletotrichum gloeosporioides results in serious loss of latex production worldwide.Lys M-type effector is a class of secreted effector containing Lys M conserved domain,which function to prevent chitin recognition by host immune receptors and play an important role in the interactions between fungi and plant.However,the molecular basis for Lys M-type effecctors of C.gloeosporioides remained unclear.In previous research,10 genes encoding Lys M-type proteins were predicted in the genome of the C.gloeosporioides.In this study,these genes were cloned and their roles in pathogenicity to the rubber tree were preliminarily investigated by constructing their gene knockout mutants respectively.Further functional studies were performed on two of them,CgLysM2 and CgLysM3,which were relating to pathogenicity.The results were as follows:1.The results from bioinformatics analysis showed that the size of these Lys M-type effectors,named as Cg E11-20,ranges between 162 and 785 amino acid residues(Cg E17 and Cg E19,respectively)and harbored from one to six Lys M domains.Of them,the full-length coding sequences of seven,Cg E12-18,were successfully amplified by RT-PCR.Based on the principle of homologous recombination,the knockout mutant strains of Cg E12-14 and Cg E16-18 were constructed.Virulence analysis showed that Cg E11,Cg E12 and Cg E13 were involved in the pathogenicity of C.gloeosporioides to rubber tree,Cg E14,Cg E16,Cg E17 and Cg E18 had no significant correlation with the pathogenicity of C.gloeosporioides.Among them,Cg E12 and Cg E13 with full length of encoding sequence were renamed as CgLysM2 and CgLysM3 respectively.CgLysM3 contains a 486 bp open reading frame(ORF)encoding 156 amino acids that consists of the two conserved Lys M domains.CgLysM2 contains a 1983 bp ORF encoding a peptide with 661 amino acids that contains four Lys M domains.2.The chitin-binding activity assay showed that CgLysM3 could bind to chitin directly in vitro,while chitin-binding activity of CgLysM2 was not detected.The subcellular localization of the CgLysM2 and CgLysM3 showed that both CgLysM2 and CgLysM3 proteins were located in the cell membrane and cytoplasm,but the specific location in cytoplasm still need further to be confirmed.3.The complementary strain of CgLysM2 and CgLysM3(Res-ΔCgLysM3 and Res-ΔCgLysM2)were constructed to further confirm their functions.The pathogenicity analysis showed that CgLysM3 and CgLysM2 restored the pathogenicity of ?CgLysM3 and?CgLysM2,suggesting that CgLysM3 and CgLysM2 did play roles in the pathogenicity of C.gloeosporioides to the rubber tree.4.ΔCgLysM2 and ΔCgLysM3 showed significantly weaker mechanical penetration capacity and antioxidant stress ability than wild-type strains,but no significant difference between ΔCgLysM2 and wild-type strains in the conidia production and growth speed,suggesting CgLysM2 was not related to the sporulation capacity and the growth of C.gloeosporioides.However,the conidia production of ΔCgLysM3 were obviously lower than that of WT strains,indicating that CgLysM3 affected the conidia production of C.gloeosporioides,but did not affect the growth of C.gloeosporioides.The melanin content inΔCgLysM2 was significantly lower than that in WT and Res-ΔCgLysM2 strains.Furthermore,the expression levels of some melanin biosynthesis related genes such as alb1,abr1,arp1,arp2,ayg1 and cmr1 in ΔCgLysM2 were significantly lower than WT and Res-ΔCgLysM2,suggesting that CgLysM2 gene had effects on melanin biosynthesis in C.gloeosporioides by regulating the expression of these key genes in the melanin pathway.5.The effect of CgLysM3 and CgLysM2 on reactive oxygen species(ROS)in rubber tree cells were analyzed using the transient expression system of rubber mesophyll protoplast.It was found that CgLysM3 and CgLysM2 can inhibit the accumulation of ROS in rubber tree mesophyll protoplasts.The expression patterns of Hb PR1 and Hb PR5 were analyzed by q RT-PCR and the results showed that CgLysM3 and CgLysM2 inhibited expression levels of these genes,suggesting that CgLysM3 and CgLysM2 could prevent PTI responses trigged by chitin in H.brasiliensis.6.Transgenic Arabidopsis thaliana plants CgLysM3 and CgLysM2 were generated by Agrobacterium-mediated flower dip method.It was found that CgLysM3 and CgLysM2 could enhance the susceptibility of A.thaliana to Botrytis cinerea by inhibiting accumulation of ROS in leaves and decreasing expression level of key genes in SA and ET/JA signaling pathway(At PR1,At SID2,At ACS6,At LOX1、At JAZ1,etc.).In addition,compared with Col-0,the susceptibility of transgenic plant CgLysM3 to Pst.DC3000 was enhanced,while the susceptibility of transgenic plant CgLysM2 to Pst.DC3000 had no significant change,and both of these two transgenic plants had no significant difference to Alternaria altermata after infection compared with Col-0.7.The interaction protein of CgLysM2,named as S-adenosine methiothiocyanine synthase1-like(Hb Sas1-like),was screened by yeast two-hybrid in rubber trees.The sequence of Hb Sas1 contains a 1182 bp ORF encoding 384 amino acids.The specific function of Hb Sas1-like needs further study.