| As an important industrial raw material and strategic resource,natural rubber is mainly derived from Hevea brasiliensis.Anthracnose,mainly caused by Colletotrichum gloeosporioides,is one of the important fungal diseases of rubber tree leading to serious loss of rubber production.In the previous study,the genome of C.gloeosporioides of Hevea brasiliensis was sequenced and the genes encoding secretory proteins were predicted.Six of these candidate genes were cloned and their pathogenicity to the rubber tree was preliminarily investigated by constructing their gene knockout mutants respectively.Besides,further functional studies were performed on two pathogenic CFEM effectors of them.The specific results are as follows:1.The candidate effector encoding genes CgE4,CgE5,CgE6,CgE7,CgE8 and C.gE35 of C.gloeosporioides were amplified by RT-PCR.According to the principle of homologous recombination,the knockout mutant strains △CgE4,△CgE5,△CgE6,△CgE7,△CgE8 and △CgE35 were obtained.△CgE5,△CgE6,△CgE7 and △CgE8 showed significant weakened pathogenicity on rubber tree leaves than wild type stain,while there is no difference among △CgE4,△CgE35 and wild type stain in pathogenicity.These results indicated that C.gE5,CgE6,CgE7 and CgE8 were involved in the pathogenicity of C.gloeosporioides,but CgE4 and CgE35 had little relationship with the pathogenicity of C.gloeosporioides.Among them,both CgE6 and CgE8 contained only one CFEM conserved domain and were renamed as CgCFEM1 and CgCFEM2 respectively for further function research.2.Alignment analysis showed that both CgCFEM1 and CgCFEM2 contain eight conserved cysteine residues like other known CFEM effectors from different phytopathogenic fungi,indicating that CgCFEM1 and CgCFEM2 are CFEM effectors of C.gloeosporioides.The phylogenetic tree analysis showed that CgCFEM1 and CgCFEM2 belonged to different branches,indicating that CgCFEM1 and CgCFEM2 are far related and are from different resources.3.To further validate the role of the CgCFEMl and CgCFEM2 genes in the pathogenicity of C.gloeosporioides to the rubber tree,the complementary strains Res-△CgCFEM1 and Res-△CgCFEM2 were constructed.The pathogenicity analysis showed that there was no significant difference in the pathogenicity between the WT and the complementary strains on the leaves of the rubber tree,indicating CgCFEM1 and CgCFEM2 can restore the pathogenicity of △CgCFEM1 and △CgCFEM2.These data suggested that CgCFEM1 and CgCFEM2 play roles in the pathogenicity of C.gloeosporioides to the rubber tree.4.To explore the roles of CgCFEM1 and CgCFEM2 in the growth and development of C.gloeosporioides,we compared the growth speed and conidiation of wild-type strain,knockout mutants strains(△CgCFEM1 and △CgCFEM2),and complementary strains(Res-△CgCFEM1 and ReS-△CgCFEM2).The results showed that there was no significant difference between the wild type and the complementary strains in the growth rate of mycelium.The conidiation of ACgCFEMl and ACgCFEM2 were significantly lower than that of the wild type strain and their complement strains,but there was no significant difference between wild-type strains and their complementary strains.The above results indicated that CgCFEM1 and CgCFEM2 were related to the conidiation of C.gloeosporioides,but had little relationship with the growth of C.gloeosporioides,5.Iron plays an important role in the interaction between plants and pathogenic microorganisms.Previous studies have shown that some CFEM effectors from animal pathogenic fungi are involved in the absorption of iron.To investigate whether CgCFEM1 and CgCFEM2 are related to the iron uptake of C.gloeosporioides,we analyzed the expression of CgCFEM1 and CgCFEM1 under different iron ion conditions and the iron content in the mycelium of △CgCFEM1 and ACgCFEM2.Compared with the expression on normal conditions(Fe2+),Fe3+ could up-regulate the expression of CgCFEAM1 and CgCFEM2 genes.Under iron deficiency,the expression of CgCFEMl was up-regulated but CgCFEM2 was down-regulated.At the same time,under the normal condition(Fe2+),the iron content in the ACgCFEM1 mycelium was significantly lower than that of WT,and the △CgCFEM2 was significantly increased.Under the condition of Fe3+,there was no significant difference in the iron content of WT and the △CgCFEM1,but the iron content in the ACgCFEM2 decreased significantly compared to that of WT.It is indicated that CgCFEM1 may be associated with the uptake of Fe2+,while CgCFEM2 may be involved in the conversion of Fe3+.6.The changes of reactive oxygen species in the protoplasts of rubber tree expressing CgCFEM1 and CgCFEM2 after chitin treatment were analyzed by rubber tree mesophyll protoplasts transient expression system.It was found that both CgCFEM1 and CgCFEM2 could inhibit the PTI reaction induced by chitin.7.To study the subcellular location of CgCFEM1 and CgCFEM2 in rubber tree cells,the fusion protein CgCFEM1-GFP and CgCFEM2-GFP were expressed through the rubber tree mesophyll protoplasts transient expression system and the localization of the CgCFEM1 and CgCFEM2 were observed under fluorescent microscope.The results showed that CgCFEM1 and CgCFEM2 proteins were localized in the cytoplasm and cell membrane system respectively.Overall,in this study,we cloned 6 candidate effectors encoding genes of C.gloeosporioides and screened out 4 pathogenic related genes of them.Then,we focused on two pathogenic related CFEM type proteins,CgCFEM1 and CgCFEM2,for further function assay.The results proved that CgCFEM1 and CgCFEM2 are not only involved in the regulation of conidiation and iron absorption and transformation of C.gloeosporioides,but also play an important role in the pathogenesis of C.gloeosporioides on rubber trees through inhibiting the PTI reaction caused by chitin.This study provides new evidence for elucidating the pathogenesis of C.gloeosporioides. |
