Subcellular Localization Of Four Effectors From Meloidogyne Incognita And Screening Interaction Proteins In Plants Targeted By MiV901

Meloidogyne ineognita is an important plant-parasitic nematode and causes the serious damage to agricultural crops in China.Understanding the parasitism of root-knot nematodes in molecular level can provide a theoretical basis for new control strategies.In this paper,the genes MiV901 and MiV52 which encoding venom allergen-like protein(VAP),and genes Mi-CM1 and Mi-CM3 encoding chorismate mutase(CM)from M.incognita,were selected as the target genes and their subcellular localization in tobacco leaves were analyzed using the transient expressing mediated by Agrobact.erium tumefaciens.The effector MiV901 was selected for isolating the target protein in Arabidopsis thaliana using yeast two-hybrid,which may elucidate the possible function of MiV901 in the process of nematode parasitizingA.thaliana.The results are as followings:1.Subcellular localization of four effectors from M.incognitaFour fragments without the signal peptide of chorismate mutase Mi-CM1 and Mi-CM3,and the venom allergen-like protein MiV901 and MiV52,were amplified from cDNA template of M.incognita.The fragments were constructed into the plant expression vector pK7FWG2 using Gateway technique.The Mi-CM1?sp,Mi-CM3?sp,MiV901?sp and MiV52?sp were transiently expressed in tobacco leaves mediating by A.tumefaciens.The bands of four effector proteins detected by Western blot were consistent with the predicted molecular weight,indicating the fusion protein of 4 effectors and GFP could be expressed in tobacco leaves.The observation under cofocal microscopy revealed that Mi-CM1?sp and Mi-CM3?sp both were located at cytoplasm and nucleus of the plant cells,and no fluorescence signal was detected in the plastids.MiV52?sp was located at cytoplasm and nucleus of the plant cells.MiV901?sp was located at cytoplasm and no fluorescence signal was detect in the nucleus.2.Test of MiV901 regulating Cf-2/Rcr3pim-mediated defense response in tomatoIn order to investigate whether the MiV901 and the homologous protein Gr-VAP1 from Globodera rostochiensis have the similar mechanism in regulating immune system of plant,the Cf-2/Rcr3pim-mediated hypersensitive response(HR)in tomato induced by MiV901 was tested using agrobacterium-mediated transient expression,and the physical interaction between MiV901 and Rcr3pim was determined by yeast two-hybrid.The results showed that the transient expression of MiV901 could not trigger HR in Cf-2/Rcr3pim tomato.However,the tomato leaves injected with Gr-VAP1 and Avr2 agrobacterium transformants showing the necrotic symptom.In addition,yeast co-transformation assay showed that MiV901 failed to interact with Rcr3pim in tomato.The results demonstrated that the regulating mechanism of MIV901 in plant immune system was different from that of Gr-VAP1,which means MiV901 may play the function in nematode parasitism using other different targets or strategies.3.Screening the proteins in plants targeting by MiV901 using yeast two-hybridThe previous study has confirmed that MiV901 is closely related to nematode pathogenicity.In order to reveal the function of MiV901 in the process of parasitizing plants,the cDNA libraries of Solarium lycopersicum and Arabidopsis thaliana were screened by MiV901 bait vector using yeast two-hybrid technique.Five potential interaction proteins from S.lycopersicum library were targeted by MiV901,and finally confirmed to be false positive by cotransformation assay.Nine potential interaction targets were obtained from A.thaliana library,and only the colony At5-2 from partial sequences of cystine protease At4gl6190 was capable of activating the expression of X-?-Gal reporter gene and growing well on the QDO plate.The cotransformation assay revealed that the expression of pGADT7::At4gl6190 and pGBKT7::MiV901 failed in growing on the QDO plate.