Abstract
BackgroundThe genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses. Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Differential gene expression in Nicotiana benthamiana plants in response to infection with Bamboo mosaic virus (BaMV) was revealed using cDNA-amplified fragment length polymorphism (AFLP).ResultsFollowing inoculation with BaMV, N. benthamiana displayed differential gene expression in response to the infection. Isolation, cloning, and sequencing analysis using cDNA-AFLP furnished 90 cDNA fragments with eight pairs of selective primers. Fifteen randomly selected genes were used for a combined virus-induced gene silencing (VIGS) knockdown experiment, using BaMV infection to investigate the roles played by these genes during viral infection, specifically addressing the means by which these genes influence the accumulation of BaMV protein. Nine of the 15 genes showed either a positive or a negative influence on the accumulation of BaMV protein. Six knockdown plants showed an increase in the accumulation of BaMV, suggesting that they played a role in the resistance to viral infection, while three plants showed a reduction in coat protein, indicating a positive influence on the accumulation of BaMV in plants. An interesting observation was that eight of the nine plants showing an increase in BaMV coat protein were associated with cell rescue, defense, death, aging, signal transduction, and energy production.ConclusionsThis study reports an efficient and straightforward method for the identification of host genes involved in viral infection. We succeeded in establishing a cDNA-AFLP system to help track changes in gene expression patterns in N. benthamiana plants when infected with BaMV. The combination of both DNA-AFLP and VIGS methodologies made it possible to screen a large number of genes and identify those associated with infections of plant viruses. In this report, 9 of the 15 analyzed genes exhibited either a positive or a negative influence on the accumulation of BaMV in N. benthamiana plants.
Highlights
The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses
Screening of Bamboo mosaic virus (BaMV) infection-induced genes in N. benthamiana by cDNA-amplified fragment length polymorphism (AFLP) Total RNA was extracted from the mock- and BaMVinoculated leaves 1, 3, 5, and 7 days post inoculation, to identify differentially expressed genes in N. benthamiana plants following infection with BaMV
To avoid genomic contamination of the DNA and to enhance the efficiency of reverse transcription, we generated the cDNA from oligo-purified mRNAs and confirmed the efficiency of synthesizing cDNA on a 5% polyacrylamide gel before proceeding with the production of a standard cDNA-AFLP template [27]
Summary
The genes of plants can be up- or down-regulated during viral infection to influence the replication of viruses Identification of these differentially expressed genes could shed light on the defense systems employed by plants and the mechanisms involved in the adaption of viruses to plant cells. Identification of differentially expressed genes in plants during viral infection can help us to understand the defense systems employed by plants as well as the mechanisms behind the adaption of viruses to plant cells. Plant viruses use a variety of strategies to promote infection in susceptible hosts These strategies involve well-documented modifications to host cells such as the formation of replication complexes [4], the suppression of post-transcriptional gene silencing [5], alteration of cell-to-cell trafficking [6,7,8,9], and interference with the regulation of host cell cycle [10]
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