Development of a VIGS vector based on SACMV for genomic studies in tobacco and cassava

Abstract

Cassava is a major food crop in sub-Saharan Africa where it is mostly cultivated by small scale farmers. Cassava mosaic disease (CMD) is a major cause of pathogen-associated yield loss in Africa and it is caused by cassava mosaic geminiviruses (CMGs). The genomic structure of CMGs consists of two genomic molecules, DNA-A and DNA-B, enclosed in a twinned icosahedral capsid. South African cassava mosaic virus (SACMV) is a CMG endemic to South Africa. SACMV infection in a plant triggers post transcriptional gene silencing (PTGS), a resistance mechanism intended to target viral mRNA post transcriptionally and to prevent viral proteins translation. SACMV and CMGs, in general, encode silencing suppressors that allow them to escape PTGS from the host. Using the ability of viruses to induce PTGS against their own mRNA, the virus-induced gene silencing (VIGS) technology was developed. It involves using viruses as silencing vectors for functional genomics. In this study, SACMV-A partial dimer infectious clones were constructed to contain one copy of the coat protein (AV1) and was used to construct a SACMV-A VIGS vector and used to silence NbNOA1 in N. benthamiana, the model plant system for geminivirus infection studies. SACMV-A VIGS vector was constructed by replacing a portion of AV1 from SACMV-A infectious partial dimer with a multiple cloning site (MCS). A 403 bp of N. tabacum su-gene was introduced to silence the su gene in N. benthamiana and cassava. Silencing of su-gene in N. benthamiana and cassava resulted in bleaching of leaves, a phenotype observed in su-gene null mutations. SACMV-A VIGS vector was also used to silence NbNOA1 in N. benthamiana, a gene involved in disease resistance in N. benthamiana against the fungi Colletotrichum lagenarium and whose homolog in Arabidopsis, AtNOA1, is involved in disease resistance against Pseudomonas aeruginosa. Attempts to amplify NbNOA1 homolog from cassava were unsuccessful, using AtNOA1 specific primers, and as a result NOA1 silencing experiments were not carried out in cassava. Positive silencing of NbNOA1 was quantified using quantitative real-time PCR at 14, 21 and 28 dpi. SACMV viral load in silenced plants was quantified at 14, 21 and 28 dpi using absolute quantitative PCR and was found to be elevated in plants infected with the silencing SACMV-A VIGS vector bearing the NbNOA1 than in plants infected with an empty VIGS vector. These results suggest a possible role of NbNOA1 in SACMV disease resistance in N. benthamiana.