Complete genome sequence and integrated protein localization and interaction map for alfalfa dwarf virus, which combines properties of both cytoplasmic and nuclear plant rhabdoviruses

Abstract

We have determined the full-length 14,491-nucleotide genome sequence of a new plant rhabdovirus, alfalfa dwarf virus (ADV). Seven open reading frames (ORFs) were identified in the antigenomic orientation of the negative-sense, single-stranded viral RNA, in the order 3'-N-P-P3-M-G-P6-L-5'. The ORFs are separated by conserved intergenic regions and the genome coding region is flanked by complementary 3' leader and 5' trailer sequences. Phylogenetic analysis of the nucleoprotein amino acid sequence indicated that this alfalfa-infecting rhabdovirus is related to viruses in the genus Cytorhabdovirus. When transiently expressed as GFP fusions in Nicotiana benthamiana leaves, most ADV proteins accumulated in the cell periphery, but unexpectedly P protein was localized exclusively in the nucleus. ADV P protein was shown to have a homotypic, and heterotypic nuclear interactions with N, P3 and M proteins by bimolecular fluorescence complementation. ADV appears unique in that it combines properties of both cytoplasmic and nuclear plant rhabdoviruses.

Alfalfa dwarf cytorhabdovirus P protein is a local and systemic RNA silencing supressor which inhibits programmed RISC activity and prevents transitive amplification of RNA silencing

“…Gateway entry clones in pDONR221 for ADV N (used as negative control), P and P6 ORFs (Bejerman et al, 2015) (Life Technologies, California, USA) were recombined into plant expression destination vector pSITE-Flag-C1 (Chakrabarty et al, 2007) to yield N-terminal flag epitope-tagged proteins, following instructions described in the Gateway LR Clonase II Enzyme Mix kit (Life Technologies). For bimolecular fluorescence complementation (BiFC) protein-protein interaction assays, Gateway entry clones in pDONR221 for ADV P (Bejerman et al, 2015), AGO1b, AGO4a and RDR6 (kindly provided by Prof Peter Waterhouse, Queensland University of Technology; Nakasugi et al, 2013) and SGS3 were recombined into plant expression destination vectors pSITEBiFC-nEYFP-C1 and pSITE-BiFC-cEYFP-C1 to allow expression of these proteins as Cterminal fusions to the amino-or carboxy-terminal halves of yellow fluorescent protein (YFP), using Gateway LR Clonase II Enzyme Mix kit instructions (Life Technologies).…”

“…For bimolecular fluorescence complementation (BiFC) protein-protein interaction assays, Gateway entry clones in pDONR221 for ADV P (Bejerman et al, 2015), AGO1b, AGO4a and RDR6 (kindly provided by Prof Peter Waterhouse, Queensland University of Technology; Nakasugi et al, 2013) and SGS3 were recombined into plant expression destination vectors pSITEBiFC-nEYFP-C1 and pSITE-BiFC-cEYFP-C1 to allow expression of these proteins as Cterminal fusions to the amino-or carboxy-terminal halves of yellow fluorescent protein (YFP), using Gateway LR Clonase II Enzyme Mix kit instructions (Life Technologies). For co-localization studies ADV P was recombined into GFP-pSITE-C1, and AGO1b, AGO4a, RDR6 and SGS3 into RFP-pSITE-C1 (Chakrabarty et al, 2007).…”

“…At 2 dpi, confocal microscopy of agroinfiltrated N. benthamiana leaves showed that RFP-AGO1 fusions accumulated uniformly on the cell periphery and in the nucleus, while RFP-AGO4 was nuclear (Mann et al, 2016a). Next, we examined the localization of these RFP fusions when co-expressed with GFP-ADV P, which localizes to the nucleus (Bejerman et al, 2015). The partial cell periphery accumulation of RFP-AGO1 was no longer detectable when co-expressed with GFP-P, while the nuclear expression remained.…”

“…Targeting multiple components of the RNA silencing pathway LNYV P inhibited miRNA-guided AGO1 cleavage and translational repression, and RNA silencing amplification (Mann et al, 2016a). Given the importance of plant virus RSS function in the infection cycle and host interactions, we identified and examined the RSS of alfalfa dwarf virus (ADV), a virus that combines properties of both cytoplasmic and nuclear plant rhabdoviruses; ADV groups phylogenetically with cytorhabdoviruses, but its P protein accumulates in the cell nucleus (Bejerman et al, 2015). We also explored mechanisms of action of ADV RSS through study of targeted host components.…”

“…We also explored mechanisms of action of ADV RSS through study of targeted host components. The ADV genome consists of 14,491 nucleotides and encodes six proteins in the order 3'-N (nucleoprotein) -P (Bejerman et al, 2015;Mann et al, 2016b).…”