Molecular Mechanism Analysis On RNA Silencing Enhancement And Virus Resistance Induced By Meq Gene

The Meq gene is the major oncogenic gene of Marek’s disease virus (MDV), which plays an important role in the multiplication and pathogenicity of MDV. The N-terminal and the C-terminal of MEQ protein are the basic leucine zipper domain and prolines-rich domain respectively. The Agrobacterium transient coinfiltration assays shows that MEQ can significantly enhance the transient coinfiltration-mediated RNA silencing which is the main defense strategy against virus infection. The molecular mechanism analysis on the silencing enhancement of MEQ would help to understand RNA silencing better, as well as provide a new way to acquire the broad-spectrum viral resistance. In order to reveal the key amino acids and functional domain of MEQ involved in RNA silencing enhancement, the deletion mutations and point mutations of Meq were constructed for functional analysis. To further explore the possible mode by which Meq gene participates in the silencing pathway, the real-time fluorescence quantitative PCR was performed to test the expression of the genes which are related to silencing pathway in the case of Meq gene expressing. In additional, the virus resistance of the Meq transgenic tobacco and Arabidopsis were analyzed through virus artificial inoculation. The main results are as follows:Meq gene could prominently enhance both local and systemic RNA silencing induced by transient coinfiltration. The Meq gene was constructed into binary plant expression vector pBI121and transformed into Agrobacterium tumefaciens strain GV3101. RNA silencing enhancement activity of Meq gene was identified in green fluorescent protein (GFP) transgenic Nicotiana benthamiana line16c using Agrobacterium-mediated transient expression system and the expression of green fluorescent protein was observed persistently under a long wave UV lamp. The results indicated that the green fluorescent couldn’t be seen in the patches coinfiltrated with35S-MEQ and35S-GFP at3days post infiltration (dpi), while slight GFP fluorescent was observed in35S-GFP alone infiltration. Northern blot analysis indicated that the accumulation of GFP mRNA levels was much higher in the35S-GFP expressing tissues than that of35S-GFP plus35S-MEQ coinfiltration. While the accumulation of GFP siRNA in the patches coinfiltrated with35S-MEQ and35S-GFP was higher than that of35S-GFP infiltration. At15dpi,35S-MEQ and35S-GFP coinfiltrated plants were observed much more red new emerging leaves than that of35S-GFP infiltrated plants under UV light. It is suggested that Meq gene also accelerated the systemic RNA silencing.The MEQ protein comprised of399amino acids. The first1-117amino acids are the DNA-binding domain and the118-399amino acids are transcriptional activation domain. Among the first117amino acids, the1-28Pro/Gln-rich region can interact with the transcriptional corepressor CtBp; the28-78region contains two positively charged amino acid-rich DNA-binding domains and the nuclear localization signal; the79-117amino acids are the leucine zipper. The mutation analysis of MEQ suggested that the A1-28mutation could enhance RNA silencing but the A1-78mutation not, suggesting the DNA-binding region between28-78amino acids were necessary to silencing enhancement. A118-399mutation lost silencing enhancement activity while A175-399and A302-399mutations had no effect on the enhancement activity, which showed that118-174were critical for enhancement function of MEQ. The alanine substitution of two or three leucines (L92AL99A and L92AL99AL106A) weakened the silencing enhancement activity of MEQ while one site substitution (L92A) did not affect the enhancement activity, which demonstrated that the leucine zipper region was the key functional domain of MEQ on RNA silencing enhancement. The sub-cellular localization analysis turned out that the MEQ-GFP fusion protein mainly distributed in the nucleus, and the enhancement activity needed the DNA-binding domain and transcriptional activation domain to work together. Therefor it is presumable that MEQ acts as a transcription factor to enhance RNA silencing by regulating the expressing of other genes.In order to further understand the mechanisms on MEQ involved in silencing pathway, twenty genes that were proved to relat to RNA silencing pathway in N. benthamiana were selected to test their expression levels in the case of Meq expressing using real-time fluorescence quantitative PCR. The results showed seven genes had a significant increase in the expression level, especially the AGO1and HEN1genes, MET1, NRPD2a, SGS3, AGO1a and DRM3genes were next. The five genes HEN1、MET1、NRPD2a、SGS and DRM3were related to DNA methylation and the remaining two genes belonged to the AGO gene family which were the important proteins participating in RNA silencing. It’s speculate that the Meq gene might involve in RNA silencing pathway by increasing the expressing of AGO genes. And whether there was a link between RNA silencing enhancement and DNA methylation need to be further studied.To analyze the virus resistance of Meq transgenic tobacco, four transgenic lines were obtanied using Agrobacterium-mediated method which exhibited kanamycin resistance and positive results in PCR tests. Cucumber mosaic virus (CMV)、Potato virus X (PVX) and Potato virus Y (PVY) were inoculated in the transgenic plants respectively, and the ELISA results and Northern blot analysis revealed that the transgenic lines presented significant resistance in CMV but no obvious resistance to PVX and PVY. Moreover, there was no significant correlation between the CMV resistance and the expression level of Meq gene. And the virus resistance analysis on transgenic Arabidopsis is going on.