Investigation of the anti-virC2 transcript in Agrobacterium tumefaciens
Agrobacterium tumefaciens has long been an important tool for plant genetic transformation. Many important crop plants such as maize, soybean, wheat, and rice, as well as numerous other dicot plant species are able to be transformed using A. tumefaciens. While many potential regulatory RNA have been previously identified in A. tumefaciens, very few have been verified with a functional investigation. With the growing understanding of regulatory RNA, and its importance in gene regulation, it is essential to further classify regulatory RNA in the transformative tool A. tumefaciens. It is possible that regulatory RNA could be involved in virulence and their expression altered to increase transformation efficiency.
In this work, a particular candidate regulatory RNA, anti-virC2, was selected and investigated. While anti-virC2 was identified previous to this research, the function of anti-virC2 has never before been examined. The location of anti-virC2 is on the Ti-plasmid antisense to the virulence gene virC2 and it was initially hypothesized that anti-virC2 acted to regulate the virC2 gene in cis.
Through deletion of the promoter region, expression of the anti-virC2 transcript was reduced by ~39% in mutant strain C58 ∆Pavc2. Using this strain, changes in virC2 transcript and protein abundance were able to be observed and compared to wild type. In addition, other possible changes in virulence due to the drop in anti-virC2 expression were examined through Kalanchoe daigremontiana tumorigenesis assays and Arabidopsis thaliana infection experiments.
This work shows that anti-virC2 does not act to regulate the cis encoded virC2 gene but may have some impact on virulence. Additional anti-virC2 targets were also predicted and may be investigated in future studies. As well as the findings made regarding anti-virC2, this study provides an example for the functional study of additional regulatory RNA and may serve as a model for the investigation of other potential regulatory RNAs.