Serological Analysis of Barley Stripe Mosaic Virus-Encoded Proteins in Infected Barley

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Polyclonal antisera raised against proteins or peptides corresponding to barley stripe mosaic virus (BSMV) open reading frames were used in immunoblot experiments to investigate the in vivo expression of BSMV-encoded proteins during infection of barley. Six of the seven putative gene products, whose functional roles have been defined in previous genetic studies, were detected at one or more stages of infection in barley tissue sampled at 4, 7, 10, and 14 days postinoculation (DPI). The αa, βa, βb, βd, γa, and γb gene products were observed during the course of infection, but a protein corresponding to the βc gene could not be identified. The 130-kDa αa and the 74-kDa γa proteins, which comprise the essential BSMV-encoded replicase components, differed in the time course of their expression and in their subcellular distribution. The highest concentration of the αa protein coincided with the appearance of leaf symptoms at 4 DPI and declined gradually as infection progressed. The αa protein was found primarily in the soluble protein fraction but some of the protein was also found in the membrane and cell wall fractions. In contrast, the γa protein peaked in concentration later at 7 to 10 DPI and was more abundant in membranous fractions. The cysteine-rich 17-kDa γb protein was located predominantly in the soluble fraction and its concentration remained relatively constant during the course of infection. The 25-kDa capsid protein (βa) was present in the highest amounts in the soluble fraction at 4 DPI and it continued to increase in abundance throughout the sampling period. The transiently expressed 58-kDa βb protein, which like βc and βd is required for systemic infection, was most abundant at the onset of symptoms, but declined precipitously in concentration as the infection progressed. Although larger amounts of βb were present in the soluble fraction, the cell wall fraction contained a substantial portion estimated to be 30 to 40% of the protein. Affinity-purified antisera raised against a peptide corresponding to the hydrophobic 14-kDa βd ORF revealed the presence of a BSMV-specific protein of the predicted size in membrane and cell-wall fractions. Like the βb protein, the level of the βd protein decreased dramatically as infection progressed, suggesting that the synthesis of these two proteins is coordinately expressed from the RNAβ subgenomic RNA.