Epsilon Archive for Student Projects

Abstract

Begomoviruses are whitefly-transmitted viruses, which can induce severe diseases in various vegetable, fiber and cereal crops around the world. Begomoviruses have a circular single-stranded (ss) DNA genome, and they often infect plants together with other circular ssDNA molecules, which are the so-called alphasatellites and betasatellites. Alpha-Rep is a replication initiator protein encoded by the genome of alphasatellites. RNA silencing is an important part of plants’ natural defense against viruses. Plant viruses and their associated satellites adopt different strategies to overcome this defense mechanism and promote viral infection. The aim of this project was to test whether the Alpha-Rep proteins of two alphasatellites (Okra yellow crinkle Cameroon alphasatellite, OYCrCMA; Ageratum leaf curl Cameroon alphasatellite, ALCCMA) can suppress RNA silencing. Gene cloning technology was used to express these two Alpha-Rep proteins in RNA silencing-induced plants. The βC1 protein specified by betasatellites has been reported to have strong suppression activity of RNA silencing. The suppressor activity of βC1 encoded by Cotton leaf curl Gezira betasatellite (CLCuGB) was investigated mostly as a positive control. The Alpha-Rep genes of OYCrCMA and ALCCMA and the βC1 gene of CLCuGB were first cloned into the intermediate pJET1.2 vector, and then transferred to the binary vector PLH7000. To test the ability of the satellite proteins to suppress RNA silencing, a PA-GFP construct (to express the GFP transgene) and an RNAi-GFP construct (to induce RNA silencing) were infiltrated into whole leaves of wildtype Nicotiana benthamiana plants using Agrobacterium tumefaciens at the 4 leaf stage. At 4 dpi (days post inoculation), GFP-silenced leaves were inoculated with Agrobacterium containing the cloned satellite genes. Visual detection of green fluorescence in the N. benthamiana plants infiltrated with distinct constructs was performed using ultraviolet (UV) illumination. The results showed that both of the Alpha-Rep proteins can suppress RNA silencing, and that the Alpha-Rep protein of ALCCMA has much stronger suppression activity than that of OYCrCMA.