Boosting baseline plant immunity using non-self RNAs

Plants and pathogens engage in a molecular arms race that determines infection outcomes. RNA interference is a defence mechanism used by both host plants and pathogens during infection and the subsequent disease response. Recent studies have also demonstrated that double-stranded RNAs (dsRNAs) play an important role in triggering defence responses to viral infection, by acting as Pathogen Associated Molecular Patterns (PAMPs) which are recognised by host plants. There is also evidence to suggest that non-self RNAs may also protect the plants against infection with fungal pathogens. One study observed that mutant Arabidopsis thaliana lines expressing dsRNA hairpins, targeting the Green Fluorescent Protein (GFP) gene, appeared to show enhanced resistance to infection with Verticillium dahliae. However, it was unclear why expression of the GFP hairpin in A. thaliana appeared to protect the mutant plant from infection, despite not targeting the genes of crucial virulence factors in the fungus.

In this project, we sought to validate the findings of these previous studies and investigate whether expression of non-self RNA hairpins enhances plants’ resistance to disease. We transiently expressed the hairpin in A. thaliana leaves and infected the transformed plants with the plant pathogen Pseudomonas syringae, to test of the introduction of the hairpin reduced the severity of disease symptoms in transformed plants. To further explore whether non-self RNA hairpins elicit the upregulation of defence pathways in host plants, we analysed RNASeq data from a study that used RNA hairpins against S. sclerotiorum in A. thaliana. This analysis revealed differentially expressed genes (DEGs) in hairpin-expressing versus wild-type plants prior to infection.

Although our disease assay was variable, when the controls succeeded, we demonstrated that the GFP hairpin did not reduce leaf damage after P. syringae infection in transformed plants. This contradicts previous observations of the GFP hairpin conferring a protective effect in A. thaliana. However, our analysis of RNASeq data revealed that the hairpin targeting the fungal Anhydrolase-3 gene enhanced baseline immunity in A. thaliana. At 0 d.p.i., the hairpin-expressing line showed an upregulation of baseline defence pathways compared to the wild-type, suggesting improved natural disease response to S. sclerotiorum. We hypothesised that, besides reducing S. sclerotiorum virulence by targeting , the hairpin also acted as a PAMP, leading to upregulation of defence pathways and enhancing the plant’s natural response to S. sclerotiorum.

Plant pathogens pose a significant threat to global food security. However, the use of commercial antimicrobial agents on crops is unsustainable due to the increasing difficulties associated with antimicrobial resistance, stricter legal regulations on chemical use, and the negative impacts many agricultural antimicrobials have on human health and the environment. Therefore, RNA technology is gaining traction in the agricultural sector as a means of developing chemical-free disease management strategies.