The Function Of Viral Suppressor P1/HC-Pro And P25 In Agrobacterium-mediated Transient Assay

Post-transcriptiona l gene silencing (PTGS) is a nucleotide sequence-specific process ofRNA degradation. It leads to the degradation of homologous RNA from among endogenousgenes, transgenes or RNA viruses. RNA silencing occurs in a wide variety of organism,including pla nts (cosuppression), anima ls (RNA interference, RNAi) and fungi (RNAquelling). RNA silencing represents an evolutionarily conserved defense mecha nism thatpla ys a key antiviral role in protecting pla nts against virus infection. Consistent with theantiviral nature of RNA silencing, ma ny viruses use a counterdefensive strategy, encodingproteins that block one or more steps in the RNA-silencing pathway.The viral suppressorproteinase1/helper component-proteinase (P1/HC-Pro) comprises the 5'-proxima l region ofthe tobacco etch virus (TEV) genomic RNA encoding P1, helper component proteinase (HCPro)and a sma ll part of P3, and is termed P1/ HC-Pro. Expression of the HC-Pro(encoded bypotato virus Y (PVY)﹑TEV and turnip mosa ic virus (TuMV))encoding sequence alone issufficient to suppress virus-ind uced gene silencing (VIGS). HC-Pro targets a ma intena ncestep of the RNA silencing pathway. P25 is a 25kD protein encoded by potato virus X (PVX).P25 targets the systemic signa ling of RNA silencing, which ma y include signa l production,signa l transport and signa l-med iated induction of RNA silencing. More tha n twenty genesilencing suppressors have been identified besides HC-Pro and P25 in recent years, whichhelp for the understa nding of eukaryote gene expression and affluence of gene engineeringtheory.Nontransgenic N. benthamiana,GFP-transgenic N. benthamiana(N. benthamiana (lin e16c))and HC-Pro-transgenic N. benthamiana(N. benthamiana (line ab34))were emloye das the pla nt materia ls. During this study the system of virus-ind uced post-transcriptiona l genesilencing and binary vector-based post-transcriptiona l gene silencing has been established andoptimized. We also studied the role of P1/HC-Pro and P25 in this system.1. The binary vector-based post-transcriptiona l gene silencing system has beenestablished. This system is fast and no limitation on the experimental material compared tovirus-ind uced gene silencing. This system is also cost effective to gene gun method. It is oftremendous potentia l for this system in identifica tion of gene silencing suppressor and genefunction.2. The GFP gene expression in Agrobacterium -mediated transient assay was enhanced bythe viral suppressor P1/HC-Pro. Expression together of the gene silencing suppressor and thetarget gene provided a new strategy of heterogeneous gene expressing in pla nts. This was of agreat potencia l for commercia l production of preteins to the market with pla nt bioreactor. 3. Polyclona l antibodies were generated in fema le BALB/c mice against Rubisco largeand small subunits. Polyclona l antibodies against Rubisco small subunit were applied indetection of rbcS silenced pla nts.In this study we developed a laboratory-orientated protocolto produce polyclona l antibodies, which has the character of saving time﹑low cost and hightitration.4. The virus vector induced post-transcriptiona l gene silencing system has beenestablished and optimized in which the seedlings at four-lea ve old and Agrobacteriumconcentration at OD600=0.6-1.5 showed the best results.5. P25 does not alter genomic DNA methylation pattern induced by GFP gene silencing.So P25 targets the systemic signa ling of RNA silencing which doesn't diffuse with the siRNAthat induces DNA methylation. We propose that P25 target a signa ling component of thesilencing signa l that doesn't pla y a role in the RNA-ind uced DNA methylation.6. P25 is distributed in the cytoplasm and nucleus in onion cells , which is consistent withthe transmission of the gene silencing signal between the cytoplasm and nucleus. This alsoproves that P25 targets the systemic signa ling of RNA silencing aside.Post-Transcriptiona l Gene Silencing(PTGS);RNA Silencing;Proteinase1/helper Component-Proteinase (P1/HC-Pro); P25; Methylation;Subcellula r Localization...