Molecular Mechanism Of Inhibition Of Plant Immunity By Effector Sntf2 From Colletotrichum Gloeosporioides

Glomerella leaf spot of apple（GLSA）,caused by Colletotrichum gloeosporioides,is a devastating disease that leads to severe defoliation and fruit spots and threatens industry development and security.Effectors are the vital pathogenic factor of plant pathogens and play a role in manipulating host immune responses and facilitating pathogen colonization.The identification and functional study of pathogenic effector,so as to understand the pathogenic mechanism and host defense mechanism,is of great significance for developing disease resistance breeding and innovating new disease control strategies.In C.gloeosporioides,many studies focus on pathogens,occurrence and control of the disease,and identification of pathogenicity-related genes.A few study the interaction mechanisms between C.gloeosporioides and host plant.The molecular mechanism of effectors regulating host immune response is not fully understood.In this study,transcriptome sequencing was performed on tissues of C.gloeosporioides appressorium stage and mycelium stage.We systematically analyzed and screened the candidate effector genes,and further investigated the functions of the effector Sntf2.The study clarified the mechanism of inhibition of plant immunity by the effector Sntf2 from C.gloeosporioides.The main results of this study are as follows:1.The differentially expressed genes between appressorium and mycelium of C.gloeosporioides were analyzed based on transcriptome sequencing.39 candidate effector genes that up-regulated at appressorium formation phase were screened through further bioinformatics analysis.Effector Sntf2inhibiting BAX-induced programmed cell death（PCD）was clarified by transient expression of Agrobacterium tumefaciens in tobacco leaves.2.Sntf2 is a virulence effector of C.gloeosporioides and the deletion of SNTF2 significantly reduced the pathogenicity.Sntf2 contained a signal peptide（SP）,and its secretion activity was demonstrated by Yeast Signal Sequence Trap.Expectedly,the complementation strain without SP could not restore the pathogenicity of the deletion mutantΔsntf2-1.q RT-PCR analysis identified that SNTF2 is up-regulated during the appressorium-mediated penetration and the formation of infection hyphae.The deletion mutantΔsntf2-1 increased H₂O₂ accumulation and callose deposition in the host during the infection.Consistent with this,Sntf2 inhibits BAX-induced cell death.The results indicated that Sntf2promotes the biotrophic phase infection of pathogenic by suppressing the host immune response.3.The localization of Sntf2-e GFP fusion protein during the infection was observed by using a confocal microscope.The results showed that Sntf2 is secreted and transferred into plant cells where it performs functions.Sntf2-e GFP fusion protein was also detected in apple chloroplasts.Transient expression of Sntf2^Δsp-e GFP fusion protein was detected in tobacco leaves infected by Agrobacterium tumefaciens.The result also confirmed that Sntf2 is localized to chloroplasts.4.Potential interaction proteins of Sntf2 were obtained by a yeast two-hybrid system.Mdycf39was confirmed to interact with Sntf2 in chloroplasts by subcellular co-localization analysis,Bi FC analysis,and pull-down analysis.5.The key region analysis of Sntf2 interaction with Mdycf39 showed that the C-terminal of Sntf2is essential for its interaction with Mdycf39.The pathogenicity test of mutants with different structural regions of Sntf2 showed that the C-terminal region of Sntf2 was necessary for Sntf2 to play its pathogenicity.6.Mdycf39 is homologous to Arabidopsis thaliana HCF244 protein（a photosystem II assembly factor）with identify 80.76%.The transgenic silencing lines of Mdycf39 did not grow normally with pale-white leaves.Mdycf39 interacts with the light-harvesting complex-like protein Md OHP2 in chloroplasts.The transgenic overexpression lines（OE-ycf39）of Mdycf39 in GL-3 showed the susceptibility to C.gloeosporioides is increased.The H₂O₂accumulation is reduced in OE-ycf39 lines during the infection.Overall,these results showed that the effector Sntf2 of C.gloeosporioides perturbs the function of chloroplast by targeting a photosystem II assembly factor Mdycf39,indicating that the Sntf2 inhibits apple immune response and facilitates fungal colonization in plant.