| Glomerella leaf spot(GLS)caused by Colletotrichum fructicola is a new apple disease in China.This disease harms apple fruit and leaves,seriously affecting apple yield and quality.C.fructicola secretes effectors,which inhibit plant immunity and promote its infection.Clarifying the target of effectors will help to elucidate the mechanisms of pathogenicity and host defense.Previous studies have found that the effector CfEC92 is a key pathogenic factor of C.fructicola,but its infection mechanism is still unclear.This study screened the CfEC92 interaction target and validated its functionality,achieving the following main results:1.The candidate targets were screened using yeast hybridization and Bi FC technology to obtain the candidate interaction target Mal d 1j.Further in vitro and in vivo experiments ITC and Co-IP were conducted to demonstrate the direct interaction between CfEC92 and Mal d 1j.Instantaneous expression of CfEC92 and Mal d 1j in tobacco showed that both were co-localized in the cell membrane and nucleus.Inoculated the spore suspension of C.fructicola on apple leaves,and quantitative analysis showed that Mal d 1j began to be highly expressed at 8 h and reached its peak at 36 h,indicating that Mal d 1j was induced by pathogen infection.2.Phylogenetic analysis and homologous amino acid comparison of Mal d 1j revealed that Mal d 1j belonged to the PR10 family and was a member of the Mal d 1class of apple allergen proteins.Mal d 1j contains Bet v 1 domain and p-loop motif of GXGGXGXXK conserved sequence.3.Instantaneous overexpression of Mal d 1j in apples could enhance its resistance to C.fructicola.Overexpression of Mal d 1j in tobacco enhanced its resistance to Phytophthora nicotianae,after silencing the Mal d 1j homologous gene Nb PR10 in tobacco,the resistance was significantly reduced.Co-expression of CfEC92 and Mal d 1j in apple reduced resistance to C.fructicola.Co-expression of CfEC92 and Mal d 1j in tobacco reduced resistance to P.nicotianae.These results indicated that CfEC92 can inhibit Mal d 1j induced resistance in plants.4.In order to clarify the mechanism of CfEC92 affecting Mal d 1j,Mal d 1j was segmented and co-transferred into yeast with CfEC92.It was found that CfEC92 interacted with 100-159 aa of Mal d 1j,but did not interact with 34-66 aa containing p-loop motif,and p-loop motif was related to nuclease activity.This suggested that CfEC92 might not inhibit the immune function of Mal d 1j by affecting its nuclease activity.5.The antibacterial activity test in vitro of Mal d 1j showed that there was no direct antibacterial effect on C.fructicola and P.nicotianae.After overexpression of Mal d 1j in tobacco,quantitative analysis showed that disease resistance related genes Nb PR1 A,Nb PR2,Nb PR5,and Nb PDF were upregulated,histological observation showed a significant accumulation of callose,and co-expression of CfEC92 and Mal d1 j inhibited callose accumulation.These results indicated that Mal d 1j induced host resistance,while CfEC92 inhibited plant immunity through interaction with Mal d 1j. |
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