| Root-knot nematodes(RKN,Meloidogyne spp.)are plant sedentary endoparasites,and cause serious damage to agricultural production all over the world.RKNs can secrete effectors to modulate host physiological metabolism and immune response,thus to establish feeding sites for completing their life cycle.It is critical important to elucidate the fuction of these effectors from RKNs in order to understand the mechanism of their pathogenecity.The effector MiV901 from M.incognita was focused on this study.The transgenic Arabidopsis thaliana lines overexpressing MiV901 were cultivated,and the effects of overexpression MiV901 gene on plant disease resistance and PTI responses were determined.The various techniques including pull-down,luciferase complementation assay,Bi FC assay and subcellular localization were further used to validate the interaction between MiV901 and Arabidopsis cysteine protease RD21A.The main results are as follows:1.Heterologous expression of MiV901 in A.thaliana and determination of resistance phenotypeThe transgenic A.thaliana lines of MiV901 gene was cultivated using Agrobacterium mediated floral dip method.The T2 lines were screened by Basta herbicide and RT-PCR detection,then the seeds of T2 lines were sowed on the MS medium.Two weeks later,the root length and plant growth of MiV901 overexpressing lines were observed,and there is no significantly difference between trangenic and wild-type(WT)lines.The results of inoculation with Botrytis cinerea showed that the wilting and yellowing symtem on leaves of MiV901-OE-1 and MiV901-OE-2 lines were more severe than that on WT lines.After inoculation with Pst-DC3000,the population of DC3000 in WT leaves was 6.22 CFU/cm~2,and in MiV901-OE-1 and MiV901-OE-2 lines leaves was 7.01 CFU/cm~2and 6.80 CFU/cm~2,when compared with WT leaves,showing great significant difference(P<0.01).After 45days of M.incognita inoculation,the number of galls on overexpressing lines was 86.0%higher than that on the WT lines,showing significant difference(P<0.05).These results indicated that the overexpression of MiV901 gene in Arabidopsis could promote the infection of fungi,bacteria as well as root-knot nematodes.The leaves of MiV901 overexpressing Arabidopsis lines were treated with flg22,and PTI responses in leaves were detected.The q RT-PCR detection showed that the expression levels of PTI-related genes CBP60G,NHL10,WRKY30,FRK1 and WRKY33 in overexpression lines were decreased by 50.0%,83.0%,85.0%,22.0%and 29.0%,respectively when compared with the WT lines,which showing significant difference(P<0.05),while the expression levels of CYP81 and WRKY53 were not signifancant changed.The aniline blue staining showed that the callose deposition in the MiV901overexpression lines responded to flg22 were less than that in WT lines.Meanwhile,the luminoluminescence assay showed that ROS production in leaves of the MiV901overexpression lines was significantly inhibited when compared with that in the WT lines.The above results indicated that the overexpression of MiV901 gene could inhibit the PTI responses in plant.2.Interaction between effector MiV901 of M.incognita and cysteine protease RD21A of A.thalianaBased on the results from previous yeast two-hybrid experiments,the subcellular localization assay was futher carried out and the results showed that MiV901 of M.incognita,the intermediate i RD21A and the mature m RD21A of A.thaliana were distributed in plant vesicles.The co-location of MiV901 with i RD21A and with m RD21A were observed in vesicles.The detection of luciferase complementation assay revealed that MiV901 interacted with the intermediate i RD21A and with the mature m RD21A in tobacco cells,while the Bi FC assay indicated that MiV901 interacted weakly with i RD21A,but strongly with m RD21A.The pull-down assay validated MiV901 interacting with mature m RD21A.These results indicated that the effector MiV901 of M.incognita can interact either in vitro or in vivo with cysteine protease RD21A of A.thaliana. |
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