The Inhibition Of Seneral Heterologous Expressed Ribosome-inactivating Proteins To TMV

Plant viruses,the important pathogens just after the fungus,are distributed in the worldand caused seriously increased lost of the important economic crops year by year.With the rapid development of genetic engineering,disease resistance breeding has becoming one of the effective methods for controlling plant viral diseases.Recently,ribosome-inactivating proteins(RIPs)genes have been widely used in pathogen-derived resistance.Transgenic plants expressing ribosome-inactivating protein genes have shown good resistance against plant viruses.In this study,we obtained four RIPs(the C-terminal deletion mutant of pkoweeed antiviral proteins PAP-c、alpha-momorcharin、momordica anti-HIV protein of 30kDa、luffin-a)from the plants and cellular localization of which in Nicotiana benthamiana(N.benthamiana)are investigated by heterologous expression.Indeed,we evaluated the effects the inhibition of RIPs to Tobacco mosaic virus(TMV)and the resistance defense response of N.benthamiana,and analysed the directly effect of different RIPs on plant virus and different RIPs induced plant resistance against virus.Based on the published sequences of PAP-c and α-MC,MAP30,Luffin-a,primer pairs for cloning RIPs genes were designed.RT-PCR and gene clone were used to obtain target genes PAP-c and α-MC,MAP30,Luffin-α from the leaves of the Momordica charantia and Phytolacca acinosa,Luffa cylindrica(L.)Roem respectively.First,subcellular localization of the PAP-c,α-MC,MAP30,and Luffin-a were predicted through Wolf PSORT,then the fusion protein vectors for verifying the subcellular localization were constructed by fused the RIPs to the N-terminal of the GFP,respectively.The RIPs were transiently expressed in the N.benthamiana leaves by agro-infiltration,and the RIPs expressed leaves were inoculated with TMV-GFP.The accumulation of the virions and viral RNA were detected by indirect ELISA real time quantitative RT-PCR(qRT-PCR).In order to understand the antiviral mechanism of the RIPs,the expression of plant defense-related genes including non-expressor of pathogenesis-related genes 1(NPR1,PR1,PR2)were evaluated by qRT-PCR.The length of genes α-MC,MAP30,Luffin-a and PAP-c obtained by RT-PCR with 861、861、831 and711 bp,respectively.The subcellular localization showed that the coding protein of RIPs were predicted by Wolf PSORT to distribute in the plasma membrane.Under the confocal laser scanning microscope,the fusion protein RIPs-GFP also distributed in the plasma membrane of the leaf epidermis cell of N.benthamiana,which is consistent with the predicted results.It was observed that the heterologously expressed RIPs showed no obvious toxic effect on tobacco leaf cells,which were kept intact.Additionally,following the heterologous expression of RIPs in N.benthamiana,TMV-GFP was inoculated.After 48 hours,there was no green fluorescence observed in the RIPs expressed leaves under UV light,but the green fluorescence could be observed in the control group.After 72 hours,sporadic green fluorescence was observed in the treatment group and the fluorescence in the control group started to spread.After 6 days,the green fluorescence spread to the spear leaf of the control group,while the no obvious change was found in that of the treatment group.ELISA assay results showed that after 6 days of TMV-GFP inoculation,the value of OD492 in the control group is over ten times more than the value of samples under treatment.qRT-PCR data showed that the expression level of TMV in control group is 149 times of the level in group after treatment.Those data indicate that RIPs has significant impact on both replication and movement of TMV.We also tested the expression level of several defense-related genes in N.benthamiana leaves expressing α-MC,MAP30,and Luffin-α with or without TMV expression through qRT-PCR.Data showed that NPR1 was induced in both cases while the expression level is 1.5-3 times in plant with TMV injection than the one without injection.As to PR1 and PR2,they express 5-7 times higher in plants with TMV injection.Combining all those data together,it suggested that the resistance effect of heterologously expressed α-MC on plant viruses could induce the expression of responsive defense-related genes including NPR1,PR1 and PR2,resulting in much stronger plant defense response.The heterologously expressed RIPs significantly inhibited TMV,activated the plant defense response,enhanced the defense response of N.benthamiana,and produced few toxic on the plant cells.Therefore,the results provided a reference for the development of new products to control plant viruses based on heterologous expression of RIPs.