Effect of wheat-streak-mosaic-virus infection on the activity of enzymes degrading starch and cell wall components in wheat

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Siddiq, Fauzia
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University of Guelph

Wheat streak mosaic virus infection reduces the starch content in infected mesophyll cells and causes the infected cells to collapse at a later stage of infection. In my research project, I investigated the hypothesis that these phenomena are due to a higher activity of degradative enzymes. Gel diffusion assays were developed which can distinguish between the different starch and cell wall component degrading enzymes. The activity of starch degrading enzymes (α- and β-amylase) was measured by gel diffusion assays and zymograms. Six different α-amylase isozymes and four major β-amylase isozymes were identified by isoelectric focusing zymogram and Western blot analysis. The cellular locations of the virus induced α-amylase isozymes were determined by isolating the protoplast. Two of the infection related isozymes were found to be localized within the cell. This study did not measure any change in β-amylase activity with infection which was supported by the similar β-amylase protein level observed in western analysis. A higher α-amylase activity was observed in infected cells (from 10 days post inoculation), which led me to propose that the lower starch content in wheat streak mosaic virus-infected cells is due to higher starch degrading enzyme activity. The cell wall components xylan, cellulose and 1,3;1,4-β-D-glucan might be degraded by the enzymes endo-xylanase, 1,4-β-D-glucanase, and 1,3-β-D-glucanase, 1,4-β-D-glucanase and 1,3;1,4-β-D-glucanase respectively. Xylanase activity was detected in extracts from healthy and infected leaves harvested at 2 to 8 days after inoculation. However, no difference between healthy and infected leaf tissue was observed, suggesting that the cell collapse in infected tissue was not due to xylan breakdown. Very little 1,4-β-D-glucanase activity was detected in healthy and infected leaf tissue. Thus cellulose degradation cannot be a factor in the cell collapse. 1,3;1,4-β-D-glucanase activity was detected at all times in the healthy leaf tissue, which supports the model of continuous synthesis and breakdown of the mixed glucan that has been proposed for grass cell walls. Infected tissue extracts had a higher activity at 2 to 8 days after infection. Also 1,3-β-D-glucanase activity was higher in infected tissue but only at a very late stage of infection. It was concluded therefore that the degradation of 1,3;1,4-β-D-glucan by an increased 1,3;1,4-β-D-glucanase activity contributed to the collapse of the cell walls in infected tissue.

wheat, wheat-streak-mosaic-virus infection, starch content, cell wall components, degradative enzymes