Expression, subcellular localization and estudies on silencing suppression of a new member of the potyviridae family in Brazil

Abstract

Soybean yellow shoot virus (SoyYSV) has a high potential for severe crop destruction and is a threat to soybean production in Brazil. Recently, SoyYSV was sequenced and molecular analyzes indicated that it is a new species belonging to the Potyviridae family. Therefore, it is important to understand the subcellular location of the viral proteins in the host cell and its possible silencing suppression activity to understand the molecular mechanisms involved in viral infection processes. For this, the genes coding for the SoyYSV viral proteins named P1, HCPro, P3, PIPO, 6K1, NIa, NIb and CP, were cloned into the entry clone plasmid of the Gateway system (PDONR221). After cloning, it was subcloned into the pSITE expression vector fused to the GFP fluorescence protein and expressed by Agrobacterium tumefasciens, in two lines of Nicotiana benthamiana transgenic plants expressing the RFP fluorescent protein, one of these fused to histone 2b, inducing fluorescence in the nucleus, and the other one with protein expressed in the endoplasmic reticulum. The analysis of the infiltrated plants, coupled with observations of confocal microscopy, showed that the proteins P1, HCPro and Nia were located in the nucleus and cytoplasm; P3 and Nib accumulated in the cytoplasm, nucleus and nucleolus, whereas the PIPO and 6K1 proteins were located in the cell membranes in the cytoplasm. The CP protein accumulated exclusively in the nuclear periphery. On the other hand, in the second experiment, the objective was to investigate the possible silencing suppression activity presented by some proteins of Soybean yellow shoot virus (SoyYSV), Turnip mosaic virus (TUMV), Chinese yan necrotic mosaic virus (CYNMV), Blackberry virus Y (BlVY) Arepa palm necrotic ringspot virus (APNRV) and Brome streak mosaic virus (BSTMV), belonging to the Potyviridae family. For this, an infectious clone of Plum pox virus (PPV) was used, inserting the genes encoding the proteins of interest. The constructs were expressed in Nicotiana benthamiana plants by Agrobacterium tumefasciens. The images were obtained by epifluorescence microscopy and fluorescence quantification using fluorometry. The results obtained in this work did not show silencing suppression activity, but allowed to obtain information that contributed to a better understanding of the silencing mechanisms of SoyYSV in the infection process. Further studies should be done by knowing the role of viral proteins when they are associated with virus proteins or when interacting specifically with host proteins.