Inhibition Of Plant Immunity And Interaction With Tomato Cysteine Protease Rcr3^pim By The Meloidogyne Incognita Effector MiV98

Root knot nematode Meloidogyne incognita is an important plant parasite that and causes great damage to the agricultural production.During its infection and parasitism process,nematode can secrete effector proteins to regulate plant immunity and promote its parasitism.The venom allergen-like protein(VAP)family is one of the important groups of effector proteins.In this study,the effector protein MiV98 from M incognita was obtained and its effects on inhibition of plant immunity as well as its interaction with the tomato cysteine protease Rcr3pim was explored.The main results are as the following:1.Sequence analysis and expression pattern of the effector MiV98 from M.incognitaThe MiV98 gene was cloned from the cDNA of M.incognita using the designed specific primers.The gene coding sequences(CDS)was 735 bp long,containing a secretion signal peptide with 21 amino acids.The bioelectrical point of MiV98 protein is 7.28,and the secondary structure prediction shows having 8 ?-helixs and 5 ?-sheets.The functional domain prediction of MiV98 reveals it containing a conserved domain of sperm coating-like protein(SCP),four glycosylation sites and 18 phosphorylation sites.Phylogenetic analysis based on amino acid sequences of VAP proteins revealed that MiV98 is closely related to HaVAP2 of Heterodera avenae.In situ hybridization revealed that the expression of MiV98 gene was localized in the subventral esophageal gland cells of M.incognita.The qRT-PCR detection revealed that the expression level of MiV98 gene was shown in the highest at the fourth stage juveniles of M.incognita.The results confirmed that MiV98 was secreted from the subventral esophageal gland cells and might play a role in the parasitism of M.incognita at the late stage.2.Effects of effector MiV98 on parasitism of M.incognita and plant immunityThe RNAi in vivo of MiV98 gene was mediated by the Tobacco rattle virus.The qRT-PCR detection showed that the transcription level of MiV98 gene in nematodes on TRV::MiV98 tomato line was reduced 65%compared with that on wild type(WT)tomato line.The number of females produced on roots of TRV::MiV98 line was reduced by around 20%,and the amount of eggs was decreased by about 22%,when compared with that on the WT line.The transient expression of MiV98 on leaves of Nicotiana benthamiana was performed using the Agrobacterium tumefaciens transformation technique.The results showed that MiV98 with the signal peptide specifically inhibited the programmed cell death(PCD)induced by the elicitor of R3a/Avr3a,and the expression levels of the PR-related genes including PR1,PR2,PR3,PR5 and Pti5 were also being inhibited.Moreover,MiV98 with the signal peptide significantly promoted the infection of Phytophthora capsici to tobacco leaves.3.Verification of the interaction of MiV98 with cysteine protease Rcr3pim and evaluation of the resistance of Cf-2/Rcr3pim tomato lines to M.incognitaThe interactions of protein MiV98 of Meloidogyne incognita with the tomato cysteine protease Rcr3pim as well as its functional domain Rcr3pimcd were verified using the techniques of the yeast two hybrid,bimolecular fluorescence complementation(BiFC)and the GST pull-down.The results showed that MiV98 interacted with Rcr3pim and Rcr3pimcd in yeast and tobacco systems.Meanwhile,MiV98 failed to interact with Rcr3pim in vitro,but succeeded in interacting with Rcr3pimcd weakly.The resistance of the Cf-2/Rcr3pim tomato lines to M.incognita was evaluated.The results showed that the Cf-0/Rcr3pim line was highly susceptible to M.incognita,while the Cf-2/Rcr3-3 line was moderately susceptible,and the Cf-2/Rcr3pim line was susceptible.Although the Cf-2/Rcr3pim genotype tomato line was less susceptible to the infection of M.incognita when compared with the Cf-0/Rcr3pim and Cf-2/Rcr3-3 lines,it failed to prevent the mass reproduction of nematodes on the roots,which indicating its unsuitable application in controlling root-knot nematodes.In conclusion,the protein MiV98 secreted from the subventral esophageal gland cells of M.incognita might play a role in the late stage of nematode parasitism The MiV98 protein can inhibit the PTI and ETI reactions in plant cells and promote the infection of Phytophthora capsici and M.incognita to plants.The MiV98 protein can interact with tomato cysteine protease Rcr3pim as well as its functional domain Rcr3pimcd,and the effectors of Cf-2/Rcr3pim can trigger the plant defense responses to prevent the infection of M.incognita.