Anti-Tobacco Mosaic Virus Effect And Action Mechanism Of Malformin A₁

Malformin A₁ is a natural cyclic pentapeptide produced by Aspergillus fungi,which has been reported to possess a variety of biological activities including plant growth stimulation.Previous studies have shown that malformin A₁ has significant anti-Tobacco mosaic virus?TMV?activity.In this paper,the effect and action mechanism of malformin A₁ inhibiting the multiplication of TMV in tobacco were studied using western-blot,green fluorescent protein labeling,fluorescence spectroscopy,high performance liquid chromatography and real-time fluorescent quantitative PCR,etc.The following results were obtained:The effect of malformin A₁ on the expression of TMV coat protein in Nicotiana tabacum cv.K₃₂₆ was detected by Western-blot.The results showed that malformin A₁ had a significant inhibitory effect on the expression of TMV-CP,and showed a significant dose-related relationship.Malformin A₁ at a concentration of 150?M almost completely inhibited the expression of TMV coat protein in host plants.Nicotiana benthamiana was injected by different concentrations of malformin A₁ with agrobacterium strain EHA 105 harbouring TMV based expression vector P35S:30B:GFP?Agro35S-30B:GFP?by green fluorescent protein labeling.Effects of malformin A₁ on the replication and spread of TMV in host plants was evaluated by observing the distribution of green fluorescence in plants.The results showed that 100?M malformin A₁ containing Agro35S-30B:GFP could significantly inhibit the proliferation and spread of TMV in host plants.During the experiment,when tobacco was treated with malformin A₁,blue fluorescence was observed around the treatment area of tobacco leaves under UV light?365 nm?.Based on this phenomenon,the following research was carried out:?1?Using thin layer chromatography combined with column chromatography including MCI column chromatography,RP-18 column chromatography,silica-gel column chromatography,etc.,separation and purification obtained blue fluorescent substance which induced by malformin A₁ in tobacco.The blue fluorescent substance was identified as scopolin by nuclear magnetic resonance spectroscopy(¹H and ¹³C NMR,DEPT,COSY,HMQC,HMBC and NOESY).?2?Tobacco was injected with different concentrations of malformin A₁.The fluorescence intensity and the content of blue fluorescence substance induced were determined by fluorescence spectroscopy and high performance liquid chromatography,respectively.The results showed that Nicotiana tabacum cv.K₃₂₆ and Nicotiana glutinosa were treated with malformin A₁,which can produce blue fluorescent substance scopolin,and the fluorescence intensity and content of scopolin were positively correlated with the concentration of malformin A₁.?3?Studies have shown that scopolin and its?-glycoside scopoletin which belong to coumarins,are important pytoalexin in tobacco.Both have no direct inhibitory effect on TMV,but when tobacco is stimulated by environmental stress or exogenous substances,and it is regulated by tobacco glucosyltransferase?TOGTs?to convert scopoletin into a water-soluble scopolin which transfered to the vacuole to exert an anti-TMV effect.In order to confirm whether malformin A₁ could induce plant disease resistance,this paper used real-time fluorescent quantitative PCR to detect the expression level of tobacco resistance-related genes PR1,NPR1 and JAZ3,and two key regulatory genes F6?H1 and F6?H2 in the biosynthesis pathway of phytoalexin.The results showed that malformin A₁?5?M?could induce high expression of tobacco pathogenesis-related protein gene PR1,while the expression levels of NPR1 and JAZ3 were significantly decreased,the expression level of F6?H1 was significantly increased,and the expression level of F6?H2 was not significantly changed.In summary,malformin A₁ can significantly inhibit the expression of TMV coat protein,affect the proliferation and spread of the TMV in the host plant,and the application of malformin A₁ could trigger the plant defense responses as indicated by the synthesis and accumulation of scopolin and the expression of pathogenesis-related protein,as well as the regulating of disease resistance-related genes.