Steve Wylie

Plant RNA viruses are a very diverse group of viruses, capable of causing significant damage in crops, and potentially posing a threat to food security on local and global scales. Gene editing has enormous potential in the control of plant pathogens in agricultural crops. However, RNA viruses are not an obvious target for gene editing technologies because CRISPR/Cas9 targets DNA, not RNA. In this study we investigated novel strategies to control RNA viruses in plants using a highly flexible CRISPR/Cas9-based approach. The strategy depends on both specific and degenerate sgRNAs and their targeted delivery. Studies focused on design of the sgRNAs, their specific (species) and generalised (genera, family) targets, delivery to sites of action, and stability over time. Current studies will test the concept on one family of RNA viruses, the Potyviridae, but future studies will test the system against other groups of RNA viruses. The current study was done using Nicotiana tabacum L plants as a model system because of ease of transformation and large leaf surface area and biomass. Research in this model plant will provide proof of concept for applications to economically important crop species. Implications of a successful outcome of this research will be novel control strategies against, potentially, all plant RNA viruses.

The genetic diversity of Bean yellow mosaic virus (BYMV) was studied by comparing sequences of complete coat protein (CP) genes of 63 isolates. Phylogenetic analysis of CPs showed that isolates fell into eight distinct groups with an overall nucleotide diversity of 19%. The largest and most genetically diverse group was from natural infections in both wild and domesticated host plant species in both monocotyledonous and dicotyledonous plant families, indicating a generalized host strategy. The other seven groups had a narrower genetic base and in each case, isolates were from a single or limited number of domesticated species, indicating a specialized host strategy. We propose that the generalized group represents the ancestral type from which specialist groups evolved within domesticated plants after the advent of agriculture, since approximately ten millennia BP. The centre of origin of the ancestral group is unclear from its present distribution, but the specialized groups probably originated where their principal hosts were first domesticated, in most cases Eurasia. Specialist groups have adapted to domesticated hosts by often becoming seed-borne, facilitating their world-wide spread. Inter- and intra-specific recombination is a strategy used to increase genetic diversity in BYMV. Recombination was found in CPs and complete genomes and occurred between the generalized group and a specialized group, and between different specialized groups. Phylogenetic analysis of all genes within six complete BYMV genomes and 14VPg gene sequences showed that branch topologies were sometimes incongruous. This indicates that its different genes evolved independently.