| Root-knot nematode Meloidogyne incognita is one of the most important parasitic-nematodes causing serious damage to crops worldwide.During the parasitic process on host plant,the nematode needs to secrete a large number of effectors via its esophageal gland cells and to inject them into the plant cells.Among these effectors,the venom allergen-like proteins play an important role in the early infection stage of M.incognita.The transcriptomic for avirulent population and virulent population of M.incognita after invading into the Mi-1 tomato was analyzed using RNAseq technique.The gene MiV901 encoding the venom allergen-like protein which expression was significantly up-regulated in the virulent population of M.incognita,was selected for the transient expression in tobacco leaves and over-expression in Arabidopsis thaliana,which may help us understanding the biological functions of MiV901 in nematode parasitism.The main results are as following:1.Differentially expressed genes in virulent versus avirulent population of M.incognita after infection to Mi-1 tomatoIn order to investigate the differentially expressed genes during the compatible and incompatible interaction between M.incognita and Mi-1 tomato,the transcriptomic for the avirulent population MIJS8-AV and virulent population MIJS8-VI after 3 days inoculation to Mi-1 tomato 'Tanaki' was analyzed using RNAseq technique.The results showed that there were 93 differentially expressed genes(DEGs)between the library AVTL and VITL which were constructed from 'Tanaki' root tips infected with MIJS8-AV and MIJS8-VI,respectively.Compared with the AVTL library,69 DEGs were up-regulated and 24 DEGs were down-regulated in the VITL library.Twelve DEGs were selected for RT-qPCR validation,and the transcriptional levels of 9 of 12 DEGs were consistent with those revealed by the transcriptome analysis,which indicating the transcriptomic data can be used for further analysis.The functional annotation by GO terms revealed that the DEGs maybe involved in five different biological functions,including transcription factor regulation activity,structural molecular activity,growth,reproduction and movement.The metabolic pathway annotated by KEGG revealed that the DEGs mainly involved in the pathway of'Translation of the ribosomes' and 'Signal transduction'.2.Analyzing the inhibition of plant defense response induced by MiV901 from M.incognitaIn order to investigate whether the gene MiV901 differentially expressed in virulence and avirulent populations of M.incognita regulate the plant immunity,the fragments with and without the signal peptide of MiV901 gene were amplified and their plant expression vectors PK7FWG2-901sp and PK7FWG2-901?sp were constructed.The programmed cell death(PCD)induced by proteins BAX,INF1,Avr4/Cf4 and Avr9/Cf9 were detected by Agrobacterium-mediated transient expression of MiV901 gene in tobacco leaves.The transcription levels of PTI related genes Acre31,Grans2 and Pti5,the salicylic acid pathway related genes PAL and PR1,as well as the jasmonic acid pathway related gene LOX,were detected in tobacco leaves by RT-qPCR after treating with flg22.The results showed that the transient expression of MiV901sp and MiV901?sp in tobacco leaves could not inhibit the PCD induced by BAX,INF1,Avr4/Cf4 and Avr9/Cf9.The RT-qPCR revealed that the transient expression of MiV901?sp decreased the transcription levels of Grans2 and Pti5 in tobacco leaves triggered by flg22,while the expression of MiV901sp did not affect the transcription levels of Acre31,Grans2 and Pti5.In addition,the transient expression of MiV901?sp and MiV901sp reduced the transcription levels of PAL,PR1 and LOX significantly.3.Effects of heterologous expression of MiV901 on immunity of A.thalianaIn order to understand the biological function of MiV901 in the process of M.incognita parasitizing the plants,the plant expression vectors containing the fragments of target genes were constructed,and transformed into A.thaliana using inflorescence impregnation in Agrobacterium tumefaciens.The positive transformants were screened,and the growth of seedlings and the sensitivity to infection of several pathogens were evaluated.The positive transformants of Arabidopsis containing the signal peptide MiV901sp were screened using antibiotic plates.The phenotype of T3 transgenic plants were observed,and revealed the expression of MiV901 prompting the growth of Arabidopsis root.The A.thaliana seedlings over-expressed MiV901 gene showed the increase of its sensitivity to M.incognita infection.Compared with the control,the number of nematodes invaded into the transgenic line 901-12 increased by 94.9%.The necrotic lesions produced on the seedlings of transgenic lines 901-8 and 901-12 after inoculation with Botrytis cinerea were significantly larger than that produced on the wild type line.These results indicated that heterologous expression of MiV901 gene can affect the growth of plants,and also inhibit the plant immune response,thereby promoting the infection of M.incognita and B.cinerea. |
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