P3N-PIPO Is Involved In Synergism Between Potyviruses And Potato Virus X

Viruses of Potyvirus (family Potyviridae), the largest genus of plant infecting viruses, cause great threats to agriculture industry worldwide. Among the1000plant viruses reported, about30%of them belong to the genus Potyvirus. Recent studies show that a short frame-shift ORF, pipo, is embeded within the P3-coding region. The frame-shift protein was thought to be present in the form of P3N-PIPO and essential for potyvirus movement. To determine the form of PIPO produced in the Tobacco vein banding mosaic virus (TVBMV) infected plants, we modified pCamTVBMV-GFP, an infectious clone represents the TVBMV, to produce pCamTVBMV-6HISP3N-GFP which expressed a histidine tag-labeled P3. Symptom induced by pCamTVBMV-6HISP3N-GFP is similar to that induced by pCamTVBMV-GFP in Nicotiana benthamiana and N. tabacum plants. Western blot assay using an antibody against the histidine-tag showed that, two unique proteins of approximately40kDa and24kDa that are identical to the predicted size of TVBMV P3N-PIPO and P3had accumulated in the pCamTVBMV-6HISP3N-GFP infected N, benthamiana and N. tabacum plants by14days post agroinfiltration. This result indicates that both P3and P3N-PIPO fusion were expressed in the TVBMV infected plants.Synergism induced by mixed infection of potyviruses and Potato virus X (PVX) produces more severe symptom and increases plant damage. HC-Pro, a RNA silencing suppressor of potyviruses has been shown to involve in synergism between potyviruses and PVX. Here we showed a PVX-based vector encoding P3N-PIPOs of TVBMV and Potato virus Y (PVY) exacerbated the disease symptoms. The results of ELISA and Western blot assay showed the virus titer and CP expression level of PVX-P3N-PIPO was not enhanced. Further analysis showed the P3N domain of P3N-PIPO is responsible for this function. Nothern blot assay indicated neither of PIPO domain, P3N domain or whole P3N-PIPO enhanced the accumulation of PVX genomic and subgenomic RNA in N. benthamiana plants. No visible GFP fluorescence was observed in N. benthamiana leaves co-expressed with P3N-PIPO, GFP and hpGFP while strong GFP fluorescence appeared in positive control co-expressed with P3N-PIPO, GFP and hpGFP, which suggested P3N-PIPO has no RNA silencing suppression activity. These results suggest that potyviral P3N-PIPO is involved in synergism between potyviruses and PVX though a special mechanism distinct from surpression of RNA silencing.TVBMV is a distinct potyvirus infecting solanaceous crops including tobacco, potato and wild eggplant. The incidence of TVBMV increases drastically in China. We detected TVBMV on tomato samples collected from the city of Weifang and Linyi, Shandong Province. Here we showed20of32tomato species displayed light mosaic with GFP fluorescence under UV light in a TVBMV inoculation assay, suggesting TVBMV may creat potential threat to tomato production.