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Since its discovery in 2016, the Polerovirus Barley virus G has been reported in at least nine countries and multiple species of monocot plants. All of these reports have used PCR and/or sequencing based assays to identify BVG, however none have investigated the biology of BVG. In this study we detail the generation of the first infectious cDNA clone of BVG from archived RNA, thereby producing a valuable experimental tool and system for studying BVG biology. Using this system we identified two compatible aphid vectors and confirmed the susceptibility of several monocot plants, and the dicotyledonous plant host Nicotiana benthamiana , to BVG. • Construction of an infectious cDNA clone of the emergent Polerovirus Barley virus G. • Rhopalosiphom maidis and R. padi aphids are vectors of BVG. • R. maidis is a highly efficient BVG vector. • Multiple monocot crop plants are susceptible to BVG infection. [ABSTRACT FROM AUTHOR]

The protein P0 serves as the viral suppressor of RNA silencing (VSR) for poleroviruses, but elicits the hypersensitive response (HR) in specific Nicotiana species. We subjected P0 proteins from turnip yellows virus (P0Tu) and potato leafroll virus (P0PL) to serial deletion and performed extensive site-directed mutagenesis of P0Tu. Most deletions of the N-terminus and many substitution mutations disrupted both HR elicitation and VSR activity. Two conserved blocks of amino acid residues were found to be associated with HR. A double lysine to arginine substitution in HR-specific block 1 caused P0Tu to elicit a more robust HR. Conversely, deletion or mutation of block 2 in the C-terminus preserved VSR activity, but impaired HR elicitation, allowing virus escape from Nicotiana glutinosa resistance when expressed in the heterologous potato virus X vector. Our observations suggest that P0 residues responsible for suppressing RNA silencing and eliciting HR have overlapping, but distinct functions. • P0 proteins from Turnip yellows virus and Potato leafroll virus were studied by systematic mutagenesis. • P0 carboxy-termini were essential for hypersensitive response induction, but dispensible for RNA silencing suppression. • Two blocks of conserved amino acid sequence were associated with P0-induced hypersensitive response. • Amino acids responsible for distinct P0 protein functions are both separate and overlapping. [ABSTRACT FROM AUTHOR]

Pepper crops in Israel are infected by poleroviruses, Pepper vein yellows virus 2 (PeVYV-2) and Pepper whitefly-borne vein yellows virus (PeWBVYV). Herein we characterize the transmission of PeWBVYV and the aphid-transmitted PeVYV-2, and show that PeWBVYV is specifically transmitted by MEAM1 species of the whitefly Bemisia tabaci, with a minimum latency period of 120 h, and not by the Mediterranean (MED). PeWBVYV and PeVYV-2 were detected in the hemolymph of MED and MEAM1, respectively, however, amounts of PeWBVYV in the hemolymph of MED or PeVYV-2 in MEAM1 were much lower than PeWBVYV in hemolymph of MEAM1. Moreover, we show that PeWBVYV does not interact with the GroEL protein of the symbiont Hamiltonella and thus does not account for the non-transmissibility by MED. An insect glycoprotein, C1QBP, interacting in vitro with the capsid proteins of both PeWBVYV and PeVYV-2 is reported which suggests a putative functional role in polerovirus transmission. • Poleroviruses are strictly transmitted by aphids, yet a newly discovered poleroviruses has been associated with whiteflies. • The new polerovirus is transmitted by MEAM1 but not MED species of the whitefly Bemisia tabaci. • Transmission by MEAM1 but not MED is not associated with symbiotic bacteria. • The hemolymph plays a critical role in the inability of MED to transmit the new polerovirus. • The insect C1QBP protein interacts with the capsid proteins of both the aphid and whitefly transmitted poleroviruses. [ABSTRACT FROM AUTHOR]

Turnip yellows virus (TuYV), a phloem-limited virus, encodes a 74 kDa protein known as the readthrough protein (RT) involved in virus movement. We show here that a TuYV mutant deleted of the C-terminal part of the RT protein (TuYV-∆RT Cter ) was affected in long-distance trafficking in a host-specific manner. By using the C-terminal domain of the RT protein as a bait in a yeast two-hybrid screen of a phloem cDNA library from Arabidopsis thaliana we identified the calcineurin B-like protein-interacting protein kinase-7 (AtCIPK7). Transient expression of a GFP:CIPK7 fusion protein in virus-inoculated Nicotiana benthamiana leaves led to local increase of wild-type TuYV accumulation, but not that of TuYV-∆RT Cter . Surprisingly, elevated virus titer in inoculated leaves did not result in higher TuYV accumulation in systemic leaves, which indicates that virus long-distance movement was not affected. Since GFP:CIPK7 was localized in or near plasmodesmata, CIPK7 could negatively regulate TuYV export from infected cells. [ABSTRACT FROM AUTHOR]

Abstract: Potato leafroll virus (PLRV) is a positive-strand RNA virus that generates subgenomic RNAs (sgRNA) for expression of 3′ proximal genes. Small RNA (sRNA) sequencing and mapping of the PLRV-derived sRNAs revealed coverage of the entire viral genome with the exception of four distinctive gaps. Remarkably, these gaps mapped to areas of PLRV genome with extensive secondary structures, such as the internal ribosome entry site and 5′ transcriptional start site of sgRNA1 and sgRNA2. The last gap mapped to ∼500nt from the 3′ terminus of PLRV genome and suggested the possible presence of an additional sgRNA for PLRV. Quantitative real-time PCR and northern blot analysis confirmed the expression of sgRNA3 and subsequent analyses placed its 5′ transcriptional start site at position 5347 of PLRV genome. A regulatory role is proposed for the PLRV sgRNA3 as it encodes for an RNA-binding protein with specificity to the 5′ of PLRV genomic RNA. [Copyright &y& Elsevier]

Abstract: The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plant''s RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0PE, in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0PE has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0PE destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery. [Copyright &y& Elsevier]

Abstract: Poleroviruses are strictly transmitted by aphids. Glycosylation of Turnip yellows virus (TuYV) was previously reported and this modification was supposed to be required for aphid transmission. Using different approaches based on (i) a lectin-binding assay, (ii) use of specific complex glycan antibodies and (iii) mass spectrometry, we found no evidence that the structural proteins of TuYV and Cucurbit aphid-borne yellow virus (CABYV) carry glycan residues. Moreover, mutation of each of the four potential N-glycosylation sites of the structural protein sequences of CABYV indicated that, unless more than one site on the structural protein is glycosylated, N-glycosylation is not involved in aphid transmission. These results did not corroborate the previous hypothesis for the role of glycosylation in aphid transmission. They, however, revealed the presence of a glycosylated plant protein in purified polerovirus suspensions, whose function in aphid transmission should be further investigated. [Copyright &y& Elsevier]