Functional Study And Host Target Identification Of An Effector Protein Vm4797 From Valsa Mali

Apple(Malus domestica Borkh.)has wild planting areas,and its output and sales rank first in China.However,a fungal disease caused by Valsa mali is threatening the sustainability of the China’s apple industry.To date,the key virulence factors and pathogenic process are obscure.To study the pathogenic mechanism of Valsa mali at molecular level can provide a theoretical basis for further understanding and prevention of the disease.Effector proteins secreted by fungi can regulate host defense response and promote pathogens infection and colonization.Based on 12 effector proteins obtained,we identified an effector protein Vm4797 which could promote the infection of Phytophthora capsici and suppress programmed cell death in Nicotiana benthamiana.Furthermore,we analyzed the function of Vm4797 in plant immune response,screened and analyzed function of its target in apple.The main results are as follows:1.Identification and functional analysis of Vm4797 in plant immune responseThe effector protein Vm4797 promoted Phytophthora capsici infection,suppressed PCD induced by INF1 and callose deposition induced by PAMP in Nicotiana benthamiana.Yeast secretion system showed that Vm4797 signal peptide has secretory functions.The expression of Vm4797 was upregulated in different periods of apple leaves infected by V.mali and the expression level was high in the later period.The deletion of Vm4797 led to significant reductions in virulence on apple twigs and leaves compared with wild type.Vm4797 over-expressing apple callus markedly promoted V.mali infection.These results indicated that Vm4797 was involved in the pathogenic process of V.mali.2.Regulation analysis of Vm4797 on apple transcriptome in V.mali infectionRNA-seq was carried out using wild-type(WT)and Vm4797 overexpressed apple callus.The results showed that Vm4797 over-expressing apple callus up-regulated 1,955 genes and down-regulated 1574 genes compared with WT.Analyses of GO function and KEGG pathway of DEGs indicated that oxidoreductases activity and phenylpropanoid biosynthesis were influenced and enriched differentially in transgenic lines.After inoculated V.mali,a total of 1900 genes were altered in transgenic lines,among which1269 genes were up-regulated and 631 genes were down-regulated compared with WT.Analyses of GO function and KEGG pathway showed that transcription factor activity and plant hormone signal transduction were affected and enriched in overexpressing Vm4797 lines.The transcription levels of SA and JA pathway related genes in transgenic lines were lower than WT when infected V.mali.The results indicated that Vm4797 may promote the infection of V.mali by regulating secondary metabolites,SA and JA signaling pathways.3.Identification and function analysis of apple target of Vm4797MdUBOX62 protein was shown the virulence target of Vm4797 using a yeast two-hybrid screening.we confirmed Vm4797 interacts with Md UBOX62 by bimolecular fluorescence(Bi FC).The Md UBOX62 over-expressing transgenic apple callus enhanced resistance to V.mali,and the transcription levels of SA and JA pathway related genes were higher than WT during the infection.The results suggested that Md UBOX62 might increase resistance to V.mail through SA and JA signaling pathways.