Screening And Preliminary Identification Of RNA Silencing Suppressor Candidates In Avian Viruses

RNA silencing is an anti-viral defense mechanism involved in degradation of exogenous nucleic acid via sequence-specific interaction. To counteract the host defense mechanism, many viruses have evolved suppressor proteins to overcome the resistance of the corresponding RNA silencing. Since the identification of gene silencing suppressors encoded by the plant viruses in 1998, many suppressor proteins have been found and studied in a variety of plant and animal viruses. At present, gene silencing suppressor identification and functional mechanism analysis have become the research hot spot of virology. In this study, we selected several pathogenic proteins of Avian virus as RNA silencing suppressor candidates. And these candidates were identified using the agrobacterium-mediated transient expression system in GFP transgenic N.benthamiana(16c),Then the inhibitory mechanism of the identified candidate was discussed. The results are as follows:1. F gene of Newcastle disease virus(NDV), NS1 gene of Avian Influenza virus(AIV) and HVT gene of Herpes virus of turkey(HVT) were cloned, and inserted between the 35S promoter and nos terminator sequences of the binary vector pBI121respectively, named as 35S-F, 35S-HVT and 35S-NS1. These recombinant vectors were successfully transformed into Agrobacterium tumefaciens strain GV3101.2. P19, GFP and P25 gene fragments were cloned respectively from the pBIN61-P19 plasmid, total DNA of GFP transgenic N.benthamiana and the reverse transcripts of total RNA in Potato virus X(PVX). The plant binary expression vector (35S-P19, 35S-GFP and 35S-P25) were constructed and then transfered to Agrobacterium tumefaciens strain GV3101.3. Using the agrobacterium-mediated transient expression system in GFP transgenic N.benthamiana(16c), the above silencing suppressor candidates were identified. The 35S-GFP + 35S-empty vector was designed as negative control, while 35S-GFP + 35S-P19 and 35S-GFP + 35S-P25 were as positive control. The results showed that F gene was invalid in regulating GFP expression in GFP transgenic N.benthamiana. The HVT protein seems to accelerate the GFP silencing. The NS1 was a functional suppressor.4. Monitoring GFP expression in upper noninfiltrated leaves to determine NS1 could interfere with systemic RNA silencing.Systemic RNA silencing was observed in plants infiltrated with 35S-GFP plus 35S-NS1 at 9 dpi. By 32dpi, although the spread of systemic silencing was completed in plants, while the infiltrated patches still exhibited bright GFP fluorescence due to local silencing suppression activity of NS1.Therefore, these results demonstrated that NS1 can efficiently suppress local silencing, it is not able to block the spread of the systemic silencing.5. The E.coli expression vector PET-GFP was constructed and transformed into BL21(DE3)plysS. The inducing proteins were injected into mice to prepare antiserum after being purified. The titer of antibody was detected as 1:1500.6. Western bloting analysis revealed that at 3 dpi the steady-state levels of GFP proteins were very low in leaf patches expressing 35S-GFP plus empty vector.In contrast,high GFP expression was detected in the two combinations expressing P19 and NS1,with similar efficiencies,there is no GFP expression detected in leaves infiltrated with 35S-GFP plus the empty control at 7 dpi.confirming the silencing suppression activity of NS1.7. Northern bloting analysis revealed that at 3 dpi the levels of GFP mRNA were very low in leaf patches expressing 35S-GFP plus empty vector.In contrast,high GFP mRNA accumulation was detected in the two combinations expressing P19 and NS1,with similar efficiencies,the drop in GFP mRNA levels detected in leaves infiltrated with 35S-GFP plus the empty control at 7 dpi.confirming the silencing suppression activity of NS1.