Screening Of Tobacco Interaction Protein For The Immune Activating Protein RhpPSP Of Rhodopseudomonas Palustris

As a kind of photosynthetic bacterium,Rhodopseudomonas palustrisis is rich in a variety of bioactive factors,such as riboflavin,5-aminolevulinic acid(ALA),extracellular polysaccharide(EPS),extracellular protein and acyl-serine lactone(AHL),which was used in fields of food,animal husbandry,environmental governance and agriculture.RhpPSP,as an extracellular protein of R.palustrisis,which has a direct inhibition effect on TMV virus and induces resistance to TMV in tobacco.For further research on RhpPSP induced resistant mechanism,this study used yeast two hybrid system to filtere the interaction protein with RhpPSP in N.benthamiana,and then the function of interaction proteins were verified by the technology of VIGS gene silencing.Finally,the interaction relationship of proteins was further verified by various means in vivo and in vitro,and the primary results were as follows:1.The result of yeast two-hybrid showed that the constructed yeast bait vector had no toxicity to yeast cells,and no self-activation activity.11 proteins interacting with RhpPSP were identified through hybridization with tobacco c DNA library,which involved in a various biological processes,such as chlorophyll binding,photosystem stability,vascular bundle development etc.The 11 proteins were verified by one-on-one yeast recovery with RhpPSP,respectively,and the NbGPI1 anchor protein,VAN3 binding protein and chlorophyll-binding protein A were determined interaction with RhpPSP,finally.2.In order to investigate the correlation between the three screened proteins interacting with RhpPSP and inducing plant resistance,NbGPI1,VAN3 and CAB10 A silenced plants were obtained by virus-mediated gene silencing.And then the gene silencing plants were treated with protein RhpPSP and TMV-GFP virus.Results showed that the TMV symptoms of NbGPI1-silenced plants were more serious,and the q PCR results showed that the TMV expression content was higher than the other experimental groups significantly,indicating that the resistance to TMV was weakened after treatment with RhpPSP in NbGPI1-silenced plant.3.The interaction between proteins RhpPSP and NbGPI1 was further verified through the methods of GST pull down,CO-IP and BIFC.In the experiment of GST pull down,the fusion protein His-NbGPI1 could be pulled down by GST-RhpPSP and detected by His antibody,indicating the protein RhpPSP was interacted with NbGPI1 protein.The CO-IP experiment result shown that the protein HA-NbGPI1 could be detected by HA antibody after eluted by GFP magnetic beads,and the interaction relationship between proteins RhpPSP and NbGPI1 was further demonstrated.In the BIFC experiment,the yellow fluorescence was observed on the cell membrane of tobacco,which proved that there was specific interaction between proteins RhpPSP and NbGPI1,and the interaction site was located on the cell membrane.