Identification of barley stripe mosaic virus genes involved in viral RNA replication and systemic movement.

Abstract

Barley stripe mosaic hordeivirus (BSMV) has a tripartite positive‐sense RNA genome which encodes seven major polypeptides. Infectious in vitro transcripts derived from full‐length wild‐type and mutant cDNA clones have been used to investigate the contribution made by various BSMV gene products to viral RNA replication and systemic movement. We show that whereas all three of the BSMV RNA components are required for plant infection, RNAs alpha and gamma can replicate together in barley protoplasts, and therefore RNA beta must encode functions required for systemic invasion of plants. The alpha a and gamma a proteins, which contain helicase and RNA polymerase sequence motifs, together comprise the essential virus‐encoded components of BSMV RNA replicase. A second BSMV protein (beta b) which contains a helicase motif is not required for RNA replication. A small cysteine‐rich protein (gamma b) is dispensable for infection of plants, but in its absence the accumulation of viral coat (beta a) and beta b proteins is significantly reduced. In addition, mutations in both the gamma b and gamma a (replicase) proteins can affect the systemic movement phenotype.

RNA-binding activities of barley stripe mosaic virus b fusion proteins

“…Additional site-specific substitutions revealed that conserved amino acids within the two cysteine-rich clusters are dispensable for the in vitro RNA binding activity. We were unable to detect BSMVspecific RNA binding, but the results do show that the amino acid sequences affecting binding reside within one of the regions that we have previously shown to contribute to virulence Petty et al, 1990aPetty et al, , 1994.…”

“…BSMV-specific RNA probes were prepared from linearized transcription vector plasmids containing subcloned BSMV fragments. Plus-and minus-sense probes corresponding to the common 3' end (235nt) of BSMV RNAs were prepared from the vector pTZ18U and pTZI9U (Pharmacia) subclones constructed for hybridization analyses (Petty et al, 1990a). An RNAyspecific probe was obtained by subcloning a 210 bp EcoRV-KpnI 'blunt-ended' fragment containing the 7a-yb intergenic region of the genomic ND18 strain y42 cDNA clone (Petty et al, 1990b) into the HindIII site of pGEM-4Z (Promega), that bad been treated with Klenow polymerase.…”

“…Two proteins, ~a and 7a, constitute the BSMV-encoded replicase components (Petty et al, 1990a). Three genes organized in a' triple gene block' at the 3' end of RNAfl encode proteins that are required for cell-to-cell movement (Petty & Jackson, 1990;see Donald et al, 1995 for review).…”

“…Mutations within ~:b have been shown to affect pathogenesis and to have regulatory effects on gene expression (Petty et al, 1994;. The ~b protein is also dispensable for systemic invasion of barley in some BSMV strains, but not in others (Petty et al, 1990a).…”

“…Deletions and substitutions within the ~b gene that are designed to affect amino acids in the yb protein can have profound effects on the symptom phenotype Petty et al, 1994). Several mutations within ?b also affect accumulation of the yb protein in infected barley and modulate expression of RNAfl encoded proteins during infection (Petty et al, 1990a;. As with BSMV, the cysteine-rich proteins of TRV (Guilford et al, 1991) and BNYVV (Gilmer et al, 1992) are also dispensable for systemic infection.…”