Virus-induced gene silencing in cultivated cotton (Gossypium spp.) using Tobacco rattle virus

Authors: MUSTAFA, ROMA - National Institute Of Biotechnology And Genetic Engineering (NIBGE); SHAFIQ, MUHAMMAD - National Institute Of Biotechnology And Genetic Engineering (NIBGE); MANSOOR, SHAHID - National Institute Of Biotechnology And Genetic Engineering (NIBGE); BRIDDON, ROB - National Institute Of Biotechnology And Genetic Engineering (NIBGE); Scheffler, Brian; Scheffler, Jodi; AMIN, IMRAN - National Institute Of Biotechnology And Genetic Engineering (NIBGE)

Submitted to: Molecular Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/3/2015
Publication Date: 11/24/2016
Publication URL: http://handle.nal.usda.gov/10113/62163
Citation: Mustafa, R., Shafiq, M., Mansoor, S., Briddon, R.W., Scheffler, B.E., Scheffler, J.A., Amin, I. 2016. Virus-induced gene silencing (VIGS) in cultivated cotton (Gossypium spp.) using Tobacco rattle virus (TRV). Molecular Biotechnology. 58:65–72. doi: 10.1007/s12033-015-9904-z.

Interpretive Summary: Cotton is a very difficult plant species to transform with gene constructs. The general technique is time and labor consuming. Transformed plants can be extremely useful for the study of genes to identify function and to characterize activity of various DNA alterations. Therefore cotton lags behind model species for gene characterization which in turn limits significant advancement in cotton traits for quality or in relation to abiotic or biotic stressors. An alternative method to study gene expression in cotton is via the use of a system called virus induced gene silencing (VIGS). A VIGS system does no result in stable transformants that are heritable, but rather VIGS produces somatic mutations through gene knockouts. VIGS have been utilized in some cotton research and it has been useful to study gene function more efficiently than by normal transformation methods. However, the VIGS systems tested in the past still have limited use. The research presented here investigated the use of a different VIGS system based off of tobacco rattle virus (TRV). This new VIGS system was then tested on three fiber producing cotton species. This included six varieties of upland cotton, the most common commercial cotton species in the U.S.A. Various parameters of the VIGS system were tested to identify optimal procedures for future studies. The study also identified response differences between the species and within upland cotton. This TRV based VIGS system should be a very useful tool for studying genes in cotton.

Technical Abstract: The study described here has optimized the conditions for virus induced gene silencing (VIGS) in three cultivated cotton species (Gossypium hirsutum, G. arboreum and G. herbaceum) using a Tobacco rattle virus (TRV) vector. The system was used to silence the homolog of the Arabidopsis thaliana chloroplastos alterados 1 (AtCLA1) gene, involved in chloroplast development, in G. herbaceum, G. arboreum and six commercial G. hirsutum cultivars. All plants inoculated with the TRV vector to silence CLA1 developed a typical albino phenotype indicative of silencing this gene. Although silencing in G. herbaceum and G. arboreum was complete, silencing efficiency differed for each G. hirsutum cultivar. Reverse transcriptase-PCR and real-time quantitative PCR showed a reduction in mRNA levels of the CLA1 homolog in all three species, with the highest efficiency (lowest CLA1 mRNA levels) in G. arboreum followed by G. herbaceum and G. hirsutum. The results indicate that TRV is a useful vector for VIGS in Gossypium species. However, selection of host cultivar is important. With the genome sequences of several cotton species recently becoming publicly available, this system has the potential to provide a very powerful tool for the rapid, large-scale reverse-genetic analysis of genes in several important Gossypium species.