pHANNIBAL vector

Viruses cause many serious plant diseases, leading to huge losses in crop yields worldwide. Therefore, the development of new methods to control plant viruses is crucial to meet the needs of a growing world population. In recent years, RNA interference (RNAi) has been widely used to develop virus-resistant plants. Once genome replication and assembly of virions is completed within the host plant, mature virions or sometimes naked viral genomes spread between cells via plasmodesmata by interacting with virus-encoded motor proteins (MPs). Researchers used an RNAi approach to inhibit MP gene expression, which in turn prevented potato leafroll virus (PLRV) systemic infection in Solanum tuberosum cv. Khufri Ashoka. Potato plants Agrobacterium infiltrated with the MP siRNA construct showed no rolling symptoms after PLRV infection, indicating that silencing of MP gene expression is an effective method to generate PLRV-resistant potato plants.

In this study, specific primers were designed for amplification of MP gene sequences in sense and antisense directions. Use these primers to perform RT-PCR on total RNA extracted from infected potato leaves and amplify the desired sequence. PCR amplification of an oligo-dT-primed first-strand cDNA template prepared from mRNA extracted from infected leaves produced 471 bp bands in both sense and antisense orientations (Figure 1A). These fragments were cloned into the pHANNIBAL vector and digested with the appropriate enzyme set (Figure 1B). The MP gene in pHANNIBAL was carefully analyzed in sense orientation (pHANNIBAL-sense MP) and antisense orientation (pHANNIBAL-antisense MP) clones using different sets of restriction enzymes (Figure 1D). Additionally, the gene orientation was confirmed by sequencing (IDT). Another round of sequencing was also performed in a cloning vector carrying sense and antisense MP (pHANNIBAL-MP). The resulting siRNA construct was further digested with NotI and introduced into the digested linear binary vector pART27 to generate the pART27-MP-siRNA construct (Figure 1C-F). The accuracy of the siRNA constructs in pHANNIBAL and pART27 binary vectors was further confirmed by restriction digestion analysis (Figure 1D).

Figure 1. RT-PCR was performed to amplify sense and antisense PLRV_MP sequences from the leaves of infected potato plants. (Kumari P, et al. 2020)