The barley stripe mosaic virus gamma b gene encodes a multifunctional cysteine-rich protein that affects pathogenesis.

Abstract

Barley stripe mosaic virus contains seven genes, one of which specifies a 17-kD cysteine-rich protein, yb, that is known to affect virulence. To further characterize the role of yb in pathogenesis, we mutagenized sequences encoding amino acids within two clusters of cysteine and histidine residues in the cysteine-rich domain and a group of basic amino acids located between the clusters and determined the effects of these mutations on the symptom phenotype in barley. Three single amino acid substitutions in cluster 1 and two amino acid exchanges in the basic region caused bleached symptoms associated with pronounced elevations in accumulation of yb pmtein. In contrast, three single amino acid substitutions in cluster 2 and a mutation in the basic motif resulted in attenuated (Ynull") symptoms typical of those produced when the yb gene is deleted. Tissue infected with these "null" mutants accumulated slightly elevated amounts of the yb protein but significantly lower levels of coat protein and the putative movement protein pb. Genetic complementation tests revealed that cluster 1 mutations are dominant over the wild-type yb gene, whereas those in cluster 2 are recessive. These results highlight the pivotal role of yb in pathogenesis and suggest that the two cysteine-rich clusters are functionally distinct and that they affect different aspects of disease development.

The N-Terminal 85 Amino Acids of the Barley Stripe Mosaic Virus γb Pathogenesis Protein Contain Three Zinc-Binding Motifs

“…To engineer ␥b(Ϫ)C1C2, two blunt-ended fragments were amplified by PCR. The first reaction used the primers Ϫ1875 and C19/23R with the C1(9,10,19) mutant template (4), and the second reaction used the primers g2150F and BSMV3Ј with the C2(60,64) mutant template (4). These fragments were ligated, and the resulting product was amplified with the primers g1629F and C71/81R to produce intermediate F. The KpnI/MscI fragment of intermediate F was introduced into the corresponding sites of the C1(7,9,10) mutant (4).…”

“…The first reaction used the primers Ϫ1875 and C19/23R with the C1(9,10,19) mutant template (4), and the second reaction used the primers g2150F and BSMV3Ј with the C2(60,64) mutant template (4). These fragments were ligated, and the resulting product was amplified with the primers g1629F and C71/81R to produce intermediate F. The KpnI/MscI fragment of intermediate F was introduced into the corresponding sites of the C1(7,9,10) mutant (4). Next, the histidine to leucine mutation at position 85 was introduced by site-directed mutagenesis with the primers 85F and 85R with a QuikChange site-directed mutagenesis kit (catalog no.…”

“…Substitution of arginine and lysine residues within BM resulted in diverse disease phenotypes when the mutant RNA␥ derivatives were inoculated onto plants (4,5). Two BM mutants, BM26 (containing mutations R25Q and K26N) and BM29 (containing mutations R33G, K35N, and R36G) were also compromised in their ability to bind RNA.…”

“…Understanding the functions of these proteins during virus infections is of particular interest because, like ␥b, a number of them have been demonstrated to be viral pathogenicity determinants and are suggested to play regulatory roles during infection (4,7,12,13,18). Deletion of BSMV ␥b (5,21) and TRV p16 (18), both of which have RNA-binding activity, or PCV p15 (7) causes a dramatic decreases in viral RNA levels that can be rescued by supplying the appropriate CRP in trans.…”

“…Extensive analyses of mutations introduced into the C1, BM, and C2 regions of ␥b have demonstrated that these motifs each have important virulence functions (4). Subcellular fractionation of infected barley extracts has revealed that the site-specific mutations in these regions also have striking effects on the solubility of the protein and that mutant derivatives are often shifted from the soluble fraction to the cell wall and membrane-containing fractions (4). Furthermore, single-stranded RNA-binding activity has been demonstrated for a recombinant GST-␥b fusion protein in in vitro gel shift studies.…”