Michael G. K. Jones

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.

Healthy roots enable a plant to make full use of available water and nutrients. Plant parasitic nematodes are a neglected but economically group if plant pests that damage plant roots and cause annual crop losses estimated at US$120 billion p.a. New resources, in the form of the complete annotated genome of the model nematode, Caenorhabdhitis elegans, and more recently genomes of two root-knot nematode and one cyst nematode, provide new information to identify new target genes in plant parasitic nematodes. Using these resources, a range of transgenic dicotyledonous and monocotyledonous plants (wheat and sugarcane) have been generated containing synthetic resistance genes to confer resistance to different nematode parasites. Progress will be prevented on this research, including the results of nematode challenge experiments from two different approaches that show promise in conferring host plant resistance to economically important plant parasitic nematodes.

Many of the biggest threats to the biosecurity of Australia’s plant industries are bacterial, but difficulties in identification to pathovar level could seriously delay incursion management and affect market access. Pathovars are defined by host specificity so bioassays remain the definitive means of identification, but require physical containment and can be slow and subjective. Some pathovar‐specific serological and molecular tests are available but better diagnostic methods are often required. We have evaluated the use of proteomics and metabolomics, platforms that identify functional molecules potentially associated with plant‐pathogen interactions, to identify biomarkers that differentiate pathovars in Xanthomonas species. The proteomics component has focused on profiling membrane‐associated proteins extracted from selected bacterial isolates. Profiles show isolates of the same pathovar cluster together and proteins are differentially expressed between distinct pathovars. Differentially expressed proteins have been analysed by digestions and mass spectrometry and the genes that encode them identified by reference to genomic sequences. Based on this information, molecular tests to differentiate the pathovars are being designed and validated. The metabolomics component has analysed metabolite expression in selected bacterial pathovars. Results show separation between the different pathovars and differentially expressed metabolites are evident.

Root lesion nematodes (Pratylenchus spp., RLNs) are major pests of most crops, and reduce yields of wheat in Western Australia by up to 15%, with Australia-wide losses of more than $36 million per annum. The aim of this project is to investigate the use of RNA interference (RNAi) as an approach to confer resistance to RLNs. RNAi is a well established technology that can be used to silence specific genes in animals and plants. Exposure to artificially introduced double-stranded RNA (dsRNA) leads to the silencing of endogenous genes with homologous sequence. RNAi can silence genes in Caenorhabditis elegans, and some success has been reported in root-knot nematodes. There is no evidence yet that RNAi works for RLNs. RLNs are migratory endoparasitic nematodes, and so mobility is an important aspect of parasitism. In this study, we are investigating genes involved in locomotion in RLNs via RNAi. We have shown that P.thornei and P.zeae are indeed amenable to RNAi. Exposure to dsRNA for locomotion specific genes by 14 hours soaking in medium containing M9 buffer with 50 mM octopamine, 3 mM spermidine and 0.05% gelatin led to locomotion impairment in both of these species. In addition, dsRNA originating from P.thornei also led to abnormalities in the closely related species P.zeae and vice versa, indicating that inter-species gene knockdown is possible. The outcome of this study is economically significant as no reported natural resistance genes have broad effectiveness against RLNs. Bioengineered crops expressing dsRNA that silence essential target genes to interrupt the parasitic process represents a potential approach to develop novel, broadly applicable and durable RLN-resistance in crop plants .