| Tobacco mosaic virus disease is a plant disease widely distributed around the world,which seriously harms the yield and quality of tobacco.The protein complex in plant cells play a key role in disease-resistant signal transduction by interacting with TMV-encoded proteins.IP-L is a host protein screened from tobacco c DNA library that interacts with To MV CP,which participates in the long-distance movement of To MV.At the same time,it was found that the differentially expressed gene Nb CML30 can interact with IP-L in the silent IP-L transcriptome.Nb CML30 plays a positive regulatory role in the interaction between Nicotiana benthamiana and TMV.In this experiment,Nb CML30 and IP-L were transiently overexpressed in N.benthamiana to verify their role in the virus infection process,and combined with RNA-Seq to explore the mechanism of Nb CML30 inhibiting virus infection.Using LCI to further verify the interaction of IP-L and Nb CML30,and explore the effect of IP-L on the expression of Nb CML30.Analyze the RNA-Seq of N.benthamiana that silences Nb CML30,screen the differentially expressed genes and explore the role of HSP23,MBF1 c,and TLP1 in the process of virus infection through VIGS technology and transient overexpression.The homologous genes of Nb CML30 were searched in the whole tomato genome,and the function of SlCML30 in tomato response to TMV and Phytophthora capsici infection was studied by VIGS technology.Primers were designed according to the full-length sequence of Nb CML30,and plant expression vectors Nb CML30:mcherry and Nb CML30:Myc were constructed.Nb CML30:mcherry is transiently expressed in N.benthamiana.Observed by laser confocal microscope at 48-72 h,mcherry red fluorescence fused with Nb CML30 is localized in the nucleus and cytoplasm.It was found that the expression of Nb CML30 in N.benthamiana is tissue-specific,with the highest expression in N.benthamiana at the mature stage and the lowest expression in flowers.And it was also found that the relative expression level of Nb CML30 was the highest in N.benthamiana at the flowering stage,followed by the seedling stage,and the lowest at the maturity stage.After transient overexpression of IP-L and Nb CML30 in N.benthamiana,it was found that compared with the control treatment,overexpression of IP-L promoted the infection of the virus,and overexpression of Nb CML30 inhibited the infection of the virus.Combined with the analysis of RNA-Seq,it is found that silencing Nb CML30 can induce the up-regulation of RDR1 and promote the opening of stomata,but it does not affect the burst of ROS in N.benthamiana.We fused Nb CML30 with the C-terminal part of luciferase to construct the expression vector Nb CML30:c LUC,and IP-L:n LUC was constructed and stored by the laboratory.The LCI experiment further verified the interaction of Nb CML30 and IP-L in plants.The Agrobacterium-mediated method was used to transient express in N.benthamiana,and it was found that mcherry red fluorescence fused with Nb CML30 and e GFP fused with IP-L green fluorescence were both expressed in the cell membrane and nucleus of N.benthamiana.It showed that IP-L and Nb CML30 are co-localized on the plasma membrane and nuclear membrane.VIGS was used to co-silence IP-L and Nb CML30.The results showed that there was no significant difference in the relative expression of TMV-GFP between co-silenced plants and control plants at 4,6,and 8days after inoculation.It showed that co-silencing IP-L and Nb CML30 have no effect on the movement of the virus in N.benthamiana.Use the proteasome inhibitor MG132 to clarify the degradation pathway of Nb CML30.The results showed that the stability of Nb CML30 is regulated by the 26 S ubiquitin proteasome pathway,and IP-L can stabilize the expression of Nb CML30.Use Sol Genomic Network VIGS Tool to select non-conserved segments to construct TRV-mediated silencing vectors TRV:HSP23,TRV:MBF1c.The primers were designed according to the full-length sequence of the gene,and the expression vector TLP1:e GFP was constructed.Agrobacterium infiltration method was used for systemic silencing or transient expression in N.benthamiana.TMV-GFP was used for friction inoculation,and it was found that the expression of TMV-GFP in the silencing HSP23 and MBF1 c plants was less than that of the control plants,indicating that silencing HSP23 and MBF1 c was adverse to the replication and movement of TMV in N.benthamiana.The expression of TMV-GFP in the N.benthamiana that transiently overexpressed TLP1 was lower than that of the control plants,indicating that the overexpression of TLP1 inhibited the infection of TMV.The relationship between HSP23,MBF1 c,TLP1 and TMV helicase protein P50,movement protein,and coat protein were investigated by Y2 H.The results showed that HSP23,MBF1 c,TLP1 does not interact with TMV related proteins.In order to clarify whether HSP23,MBF1 c,TLP1 and Nb CML30,IP-L interact with each other,the Y2 H was used to find that the co-transformed yeasts of TLP1 and IP-L can grow normally on the auxotrophic medium.And under a confocal microscope,it was observed that p CV-n YFP:TLP1-ΔSP and p CV-c YFP:IP-L appeared fused yellow fluorescence in the chloroplast.It preliminarily showed that TLP1 interacts with IP-L.We observed the green fluorescence of e GFP fused with TLP1 is localized in the cytoplasm.Used the amino acid sequence of Nb CML30 to perform Blastp search in the whole tomato genome,the Nb CML30 homologous gene in Solanum lycopersicum was named SlCML30.RT-q PCR results showed that the expression of SlCML30 was the highest in tomato roots,followed by flowers,leaves and stems,and the lowest expression was in tomato fruits.And external application of Me SA,Me JA and ETH can reduce the expression of SlCML30 to varying degrees.We observed the green fluorescence of e GFP fused with SlCML30 is located on the plasma membrane and nucleus.Used VIGS to carry out the systematic silencing of SlCML30 in tomato,it was found that silencing SlCML30 enhanced the resistance of tomato plants to TMV and P.capsica.Preliminary experiments showed that SlCML30 plays a negative regulatory role in plant immunity. |
