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Gene expression of turnip crinkle virus and characterization of the two movement genes
Abstract
Turnip crinkle virus (TCV) encodes five open reading frames (ORFs) in a 4 kb plus-sense (+), single-stranded (ss) RNA genome. Previous mutagenesis studies resulted in preliminary assignment of functions to each of the ORFs (Hacker et al., 1992). In this current study, the translation products of the p28 and p88 replication associated genes was confirmed using in vitro translation, and through pulse labeling and immunoprecipitation of proteins synthesized in vivo. Western blot analysis of cellular fractions revealed possible membrane association of the p28 and p88 proteins. In vitro translation studies confirmed that the two putative movement genes, p8 and p9, could be translated from the 1.7 kb subgenomic RNA. Mutants of both genes were examined for cell-to-cell movement function by in situ hybridization of inoculated leaves. The results demonstrated that both p8 and p9 genes are necessary for cell-to-cell movement of the virus. The same assay was also used to demonstrate that the coat protein was not required for cell-to-cell movement in inoculated leaves of Arabidopsis. Co-inoculations of p8 and p9 mutants demonstrated that each would complement the other to promote cell-to-cell spread in inoculated leaves, but at a slower rate than wild type virus. Systemic invasion was slower in the complementation experiments and both of the defective mutants as well as recombinant wild-type virus was detected in systemic infections. Conclusive demonstration of the necessity for the p8 and p9 genes to complement each other to promote cell-to-cell movement function was obtained by expressing each transgenically in Arabidopsis. Transgenic plants expressing either gene separately or together developed normally without any obvious changes in cellular morphology, and the plants responded typically to TCV infection. Transformants expressing the corresponding gene(s) complemented the appropriate defective mutants in all cases. Antibodies generated from purified fusion proteins detected the p8 protein, but not the p9 protein, in Western blot analysis of the cellular fractions of both TCV infected plants and transgenic plants.
Subject Area
Plant pathology|Molecular biology|Genetics
Recommended Citation
Li, Wen-zhe, "Gene expression of turnip crinkle virus and characterization of the two movement genes" (1996). ETD collection for University of Nebraska-Lincoln. AAI9620343.
https://digitalcommons.unl.edu/dissertations/AAI9620343