A viral noncoding RNA complements a weakened viral RNA silencing suppressor and promotes efficient systemic host infection

Systemic movement of beet necrotic yellow vein virus (BNYVV) in Beta macrocarpa depends on viral RNA3, whereas in Nicotiana benthamiana this RNA is dispensable. RNA3 contains a coremin motif of 20 nucleotides essential for the stabilization of noncoding RNA3 (ncRNA3) and for long\u2010distance movement in Beta species. Coremin mutants that are unable to accumulate ncRNA3 also do not achieve systemic movement in Beta species. A mutant virus carrying a mutation in the p14 viral suppressor of RNA silencing (VSR), unable to move long distances, can be complemented with the ncRNA3 in the lesion phenotype, viral RNA accumulation, and systemic spread. Analyses of the BNYVV VSR mechanism of action led to the identification of the RNA\u2010dependent RNA polymerase 6 (RDR6) pathway as a target of the virus VSR and the assignment of a VSR function to the ncRNA3. \ua9 2016 by the authors

Synthesis and recombination of infectious in vitro transcripts from Beet soil-borne mosaic virus and Beet necrotic yellow vein virus cDNA clones for a preliminary analysis of plant response

Studio della sintesi e della ricombinazione di cloni cDNA di Beet soil-borne mosaic virus e Beet necrotic yellow vein virus. PS 12-60

Beet necrotic yellow vein virus RNA-1 and 2 replicate and encapsidate Beet soil- borne mosaic virus RNA-3 in planta

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) are members of Benyvirus genus. BSBMV has been reported only in the United States while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae, possess similar host ranges, particles number and morphology. Both viruses are not serologically related but have similar genomic organizations. Field isolates consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles on plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed BSBMV and BNYVV are different enough to be classified in two different species. Considering RNA-3 involvement on symptoms development, we synthesised biologically active cDNA clones from which complete copies of the BSBMV genomic RNA-3 are transcribed. Sequence and structure similarities were found in BNYVV and BSBMV RNA-3 UTR domains. Recombination experiments showed capability of BNYVV RNA-1 and 2 to replicate and encapsidate BSBMV RNA-3 in planta and allow us to demonstrate that BSBMV RNA-3 support long-distance movement of BNYVV RNA-1, -2 when inoculated on Beta macrocarpa plants. However, competition occurred when BSBMV RNA-3 derived replicons were used together with BNYVV derived RNA-3 but not when RNA-5-derived component was used. Our data demonstrate the close relation between BSBMV and BNYVV RNA-3 even if the BSBMV RNA-3 encoded p29 is much closer to RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Molecular interactions between sugar beets and Benyviruses will be better-investigated exploiting similarities and divergences between BSBMV and BNYVV

Beet soil-borne mosaic virus RNA-3 is replicated and encapsidated in the presence of BNYVV RNA-1 and -2 and allows long distance movement in Beta macrocarpa.

Beet soil-borne mosaic virus (BSBMV) and Beet necrotic yellow vein virus (BNYVV) belong to the Benyvirus genus. BSBMV has been reported only in the United States, while BNYVV has a worldwide distribution. Both viruses are vectored by Polymyxa betae and possess similar host ranges, particle number and morphology. BNYVV and BSBMV are not serologically related but they have similar genomic organizations. Field isolates usually consist of four RNA species but some BNYVV isolates contain a fifth RNA. RNAs 1 and 2 are essential for infection and replication while RNAs 3 and 4 play important roles in plant and vector interactions, respectively. Nucleotide and amino acid analyses revealed that BSBMV and BNYVV are sufficiently different to be classified as two species. Complementary base changes found within the BSBMV RNA-3 5′ UTR made it resemble to BNYVV 5′ RNA-3 structure whereas the 3′ UTRs of both species were more conserved. cDNA clones were obtained, and allowed complete copies of BSBMV RNA-3 to be trans-replicated, trans-encapsidated by the BNYVV viral machinery. Long-distance movement was observed indicating that BSBMV RNA-3 could substitute BNYVV RNA-3 for systemic spread, even though the p29 encoded by BSBMV RNA-3 is much closer to the RNA-5-encoded p26 than to BNYVV RNA-3-encoded p25. Competition occurred when BSBMV RNA-3-derived replicons were used together with BNYVV-derived RNA-3 but not when the RNA-5-derived component was used. Exploitation of the similarities and divergences between BSBMV and BNYVV should lead to a better understanding of molecular interactions between Benyviruses and their hosts

Properties of post-transcriptional gene silencing suppression proteins of Benyviruses

Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) belong to the Benyvirus genus, possess a multipartite genome formed by four ssRNAs(+) and are both transmitted by the plasmodiophorid Polymyxa betae. BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. The innate mechanism that plants use to protect themselves against viral infections is called Post Transcriptional Gene Silencing (PTGS). PTGS is triggered by the presence of aberrant RNA or dsRNA generated during the replication of viral genomes and leads to their degradation. To counteract this innate mechanism, viruses co-evolved with their hosts and express viral suppressors of RNA silencing (VSR) that inhibit the PTGS. In this work we investigated properties of the Benyvirus VSRs. Such VSR consist of cysteine-rich proteins (CRP) of 14kDa expressed from RNA2 of both BNYVV and BSBMV species used in this study. We demonstrated that P14s have a zinc-finger domain (Znf) able to bind nucleic acids. Agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins action, without interfering with the transitivity. Sequence motifs essential for the nucleolus targeting of the protein (NoLS) and cysteine residues essential to the Znf structure folding, have been also identified. Both p14s localize in the nucleolus, form homodimers and bind the \u201ccoremin\u201d sequence, a stretch of 20 nucleotides present in the RNAs-3 sequence of Benyviruses and required for their systemic spread in the plant. Moreover, the coremin sequence is able to complement defective BNYVV P14 mutants in long distance movement illustrating an obvious link between P14, suppression of RNA silencing activity, RNAs-3 coremin sequence and long distance movement

Post-transcriptional gene silencing suppression study of Beet soil-borne mosaic virus: characterization of p14 and production of chimeric isolates of Benyviruses

Beet soil-borne mosaic virus (BSBMV) belongs to the Benyvirus genus, together with Beet necrotic yellow vein virus (BNYVV). Both viruses possess a multipartite genome formed by four ssRNAs(+). BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. Recent studies demonstrated a possible amplification and transmission of BSBMV RNAs by BNYVV. In the United States of America, both benyviruses are frequently present in the same cultivated field, infecting the same plant but no chimeric forms have been described from field isolates so far. Chenopodium quinoa infection has been carried out using in vitro infectious transcripts of both BNYVV and BSBMV RNA-1 and -2 and the behavior of BSBMV/BNYVV combinations and wild type isolates has been compared. In parallel, the properties of the BSBMV VSR, a cysteine-rich protein (CRP) of 14 kDa expressed by RNA-2, have been investigated and compared to the BNYVV p14 RNA silencing suppressor. P14 has a zinc-finger domain able to bind nucleic acids and agroinfection of Nicotiana benthamiana plants demonstrated that p14 is able to suppress the PTGS downstream of the Dicer proteins, without interfering with the transitivity. Moreover, both p14 are localized in the nucleolus, forms homodimers and binds the \u201ccoremin\u201d sequence, a stretch of 20 nucleotides present in the RNA-3 of Benyviruses and necessary for the systemic spread of viruses in the plant. Experiments performed to investigate relationships between BSBMV/BNYVV p14s and VSR activity, \u201ccoremin\u201d sequence, long distance movement and absence of natural chimeras of Benyviruses will be presented

Phloem specific virus-induced gene-silencing using recombinant poleroviruses

National audienc

CHARACTERIZATION OF POST TRANSCRIPTIONAL GENE SILENCING SUPPRESSOR PROTEINS OF BENYVIRUS

An innate mechanism that plants use to protect themselves against viral infections is called Post Transcriptional Gene Silencing (PTGS). PTGS is triggered by the presence of aberrant RNA or dsRNA generated during the replication of viral genomes and leads to their degradation. To counteract this innate mechanism, viruses co-evolved with their hosts and express viral suppressors of RNA silencing (VSR) that inhibit the PTGS. . The properties of the Benyvirus VSRs have been investigated. Such VSR consist of cysteine-rich proteins (CRP) of 14kDa expressed from RNA2 of both benyvirus species used in this study. The CRPs have a zinc-finger domain able to bind nucleic acids. Agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins, without interfering with the transitivity. Both p14s are localized in the nucleolus and in the cytoplasm in and out of the viral context. Moreover yeast two-hybrid and three-hybrid tests proved that these proteins form homodimers and bind the “coremin” sequence, a stretch of 20 nucleotides present in the RNA-3 of both viruses and necessary for the systemic spread of the virus in the plant. Some of the investigation performed to elucidate the existing connection between the p14s and VSR activity, the “coremin” sequence and the long distance movement of Benyviruses will be presented