Abstract: 【Objective】The objective of this study is to obtain the alpha-momorcharin (α-MC) and pokeweed antiviral protein (PAP) by cloning, observe the cellular localization of which in Nicotiana benthamiana by heterologous expression, and to evaluate the effects of α-MC on inhibiting Tobacco mosaic virus (TMV) and the resistance defense response of N. benthamiana.【Method】Based on the published sequences of α-MC and PAP, primer pairs for cloning PAP and α-MC were designed. RT-PCR and gene cloning were used to obtain the target genes α-MC and PAP from the leaves of Momordica charantia and Phytolacca acinosa, respectively. First, subcellular localization of the α-MC and PAP was predicted through Wolf PSORT, then the fusion protein vectors for verifying the subcellular localization were constructed by fusing the α-MC and PAP to the N-terminal of the GFP and DsRed2, respectively. The α-MC was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the α-MC expressed leaves were inoculated with TMV-GFP. The accumulation of the virions and viral RNA were detected by indirect ELISA and real-time quantitative PCR (qRT-PCR). In order to understand the antiviral mechanism of the α-MC, the expression of plant defense-related genes including non-expressor of pathogenesis-related genes (NPR1, PR1, PR2) were evaluated by qRT-PCR. 【Result】 The length of genes α-MC and PAP obtained by RT-PCR were 861 and 939 bp, respectively. The subcellular localization showed that the coding proteins of α-MC and PAP were predicted by Wolf PSORT to distribute on the plasma membrane. Under the confocal laser scanning microscope, the fusion proteins α-MC-GFP and PAP-DsRed2 also distributed on 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 PAP produced strong toxic effects on tobacco leaf cells, which led to necrosis of the cells, while the heterologously expressed α-MC showed no obvious toxic effect on tobacco leaf cells, which were kept intact. Additionally, following the heterologous expression of α-MC in N. benthamiana, TMV-GFP was inoculated. After 48 hours, there was no green fluorescence observed in the α-MC 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 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 was over 10 times more than the value of samples under treatment. qRT-PCR data showed that the expression level of TMV in control group was 149 times than that of the level in group after treatment. Those data indicate that α-MC has a significant impact on both replication and movement of TMV. The expression levels of several defense-related genes in N. benthamiana leaves expressing α-MC with or without TMV were tested through qRT-PCR. Data showed that NPR1 was induced in both cases while the expression level was 2.5 times in plant with TMV injection than the one without injection. As to PR1 and PR2, the expression of these two genes was 5-7 times higher in plants with TMV injection. Combining all those data together, it was 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. 【Conclusion】 The heterologously expressed α-MC significantly inhibited TMV, activated the plant defense response, enhanced the defense response of N. benthamiana, and produced few toxicity to the plant cells. Therefore, the results will provide a reference for the development of new products for the control of plant viruses based on heterologous expression of α-MC.

Key words: ribosome-inactivating proteins, α-momorcharin, Tobacco mosaic virus (TMV), heterologous expression, subcellular localization, defense-related genes