Functional Analysis Of Meloidogyne Incognita Effector MiV758 And Its Target Proteins In Host Plant

Root-knot nematodes(RKNs,Meloidogyne spp.)are obligate,sedentary endoparasites for plant hosts and cause serious damage to agricultural production and quality in worldwide.Exploring the function of effectors from RKNs and understanding their interaction with the target proteins in host plants is of great importance for making new strategies for the prevention and control of RKNs.The effector MiV758 is a kind of venom allergen-like protein secreted by M.incognita.The function of the MiV758 gene were analyzed by the in situ hybridization,qRT-PCR and RNAi in vivo techniques.The target proteins in tomato were screened and the key interaction domains were verified by the yeast two-hybrid(Y2H)with MiV758.The gene NbE3R14 from Nicotiana benthamiana,a homologous gene of the target tomato protein,was silenced to determine its effects on the base immunity of plant and the parasitism of M.incognita.The main results are as following:1.Expression pattern of effector MiV758 and effects of RNAi in vivo on parasitism of M.incognitaThe in situ hybridization confirmed that MiV758 gene was specifically expressed in the subventral esophageal gland cells of second stage juvenile(J2).The qRT-PCR detection revealed that the expression of MiV758 gene was highest in parasitic J2,which was 31.7 times higher than that in preparastic J2,while the expression in parasitic J3 or J4 was decreased,and in eggs,was similar to that in preparastic J2.The silencing of MiV758 gene in vivo was mediated by the Tobacco rattle virus(TRV).The qRT-PCR detection showed that the transcript level of MiV758 in nematodes was reduced 62.3%after 5 days post inoculation on TRV::V758 tomato line compared with that on wild type(WT)tomato line,which showing the significant difference(P<0.05).The numbers of galls,females and eggmasses produced on roots of TRV::V758 line were obviously reduced by 16.7%,17.8%and 16.8%,respectively,when compared with that on the WT line.The resultsdemonstrated that MiV758 could play an important role in the parasitism of M.incognita at the early stage.2.Screening the target proteins in tomato and the key interaction domain by Y2H with MiV758The yeast bait vector of MiV758 was constructed and the target proteins were screened from the yeast cDNA libraries of Solanum lycopersicum using the Y2H technique.Seven clones of the putative target proteins were obtained,and finally the protein encoded by the gene fragment of clone LOC101263135 was confirmed to be positively interacted with MiV758 by the yeast co-transformation assay.The full-length of the clone LOC101263135 was further cloned and named as the SlE3UP gene.The interaction of MiV758 with the E3 ubiquitin ligase enzyme SlE3UP was also confirmed using the Y2H.The analysis of amino acid sequences of protein encoded by the clone LOC101263135 revealed its containing part of the domains from protein SlE3UP.Seven mutant yeast vectors were constructed by deleting different domains of SlE3UP,and their interactions with MiV758 were verified using the Y2H.The results showed that the IBR2-C domain in SlE3UP was a key domain for its interaction with MiV758 in yeast.3.Effects of TRV-mediated silencing of the E3 ubiquitin ligase gene NbE3R14 in N.benthamiana on the plant based immune response and the parasitism of M.incognitaThe tobacco E3 ubiquitin ligase gene NbE3R14,a homologous gene of the target protein SlE3UP in tomato interacted with MiV758,was silenced mediating by TRV.The tobacco leaves in plant with NbE3R14 silenced were treated with peptide flg22,and the qRT-PCR detection revealed that the expression levels of PTI-related genes GRAS2,WRKY7 and WRKY8 in leaves were decreased by 41.3%,63.9%and 61.8%,respectively,when compared with that in the wild type(WT)tobacco,which showing the significant difference(P<0.05).After 36 hours inoculation with Phytophthora capsici,the diameter of lesions produced on the leaves of gene-silenced tobacco increased by 28.5%when compared with that on the WT tobacco,while 2 days after inoculation with Botrytis cinerea,the size of lesions produced on the leaves of gene-silenced tobacco also increased as well.The numbers of root knots,females and eggmasses produced by M.incognita after 8 weeks inoculation on roots of gene-silenced tobacco were increased significantly when compared with that on the WT tobacco.The results demonstrated that the silencing of NbE3R14 in tobacco plant reduced the level of plant based immunity,which resulted in the higher susceptibility to pathogens' infection.In conclusion,MiV758 could play an important role in the parasitism of M.incognita at the early stage and interact with the tomato target protein E3 ubiquitin ligase SlE3UP in the yeast.The domain IBR2-C in SlE3UP was a key domain for its interaction with MiV758.The E3 ubiquitin ligase may be involved in the positive regulation of plant immune responses,which deduced that the nematodes may inhibit the plant-based immune responses via interacting with SlE3UP,therefore can promote its infection to host plant.