Analysis of the putative promoter of Indian Cassava Mosaic Virus, a Geminivirus

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Date
2017-07
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AAU, Jorhat
Abstract
Geminiviruses are single-stranded DNA viruses, considered as the largest group of plant pathogenic viruses having nine genera. Geminiviruses are considered as a rich source of promoter elements as the intergenic region (IR) of their genomes harbor a bi-directional promoter driving expression in the viral-sense and complementary-sense directions. Indian cassava mosaic virus (ICMV; genus: Begomoviridae) is a bipartite (having two circular genomes, DNA-A and DNA-B) geminivirus; and in this study, we tried to define and delineate its bi-directional promoter of the DNA-A. This promoter drives the expression of Coat Protein (CP) in the viral-sense and Replication associated protein (Rep) genes in complementary-sense direction. Four sequential deletion-constructs for each of these promoters were made, after a prior in silico analysis using plantCARE to ensure that no key regulatory motif such as TATA box get deleted, driving expression of Gus gene in pBI121. In transient expression assay in Agrobacterium, and tobacco, the deleted versions (del-1) showed higher expression than the full-length promoters of both CP and Rep. Transgenic Nicotiana tabacum plants were raised using the full-length CP, full-length Rep and their del-1 constructs and same observations were made. Besides, their phloem-specific activity of the CP promoter constructs was also observed. Subsequently, Arabidopsis transgenic plants were raised for all the constructs and a similar expression pattern was observed. However, visually higher Gus expression in Arabidopsis flowers was observed. In silico analyses showed that the transcription factor (TF), CDC5 (a known transcription enhancer), was over-represented in CP del-1 construct showing highest expression. Besides, another transcription factor, the MADS Box 13, was over-represented in the CP promoter constructs; this TF plays role in development of gametophytes and embryo. Copy number, as determined by quantitative PCR, was found to be 2, 1, 4 and 2 for CP, CP del-1, Rep and Rep del-1, respectively. The expression was also quantified, that showed a similar pattern. Based on the observations, putative positive and negative regulatory elements of the promoters were also identified. Two transcripts were mapped in the viral-sense direction; the longer starting at position 138, and the shorter at position 170; while the longer could express both the AV1 and AV2 ORFs, the shorter transcript could express only the AV1 ORF. It is the first report of a comparison of deletion constructs of viral-sense and complementary-sense cassava mosaic virus promoters and their phloem-limited expression.
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