RNA interference in Fusarium graminearum – opportunities and challenges as a biological toolkit
RNA interference (RNAi) mediated by small RNA (sRNAs) is a conserved eukaryotic molecular mechanism involved in a plethora of cellular processes, ranging from developmental control to immune responses. Recent studies highlight the fact that the sRNA-orchestrated gene expression regulation is playing a considerable role in specific plant-pathogen interactions. For instance, Botrytis cinerea was demonstrated to secrete sRNAs hijacking the plant immune system, and B. cinerea spores were sensitive to exogenously applied inhibitory sRNAs (exoRNAi). Similarly, Fusarium graminearum was demonstrated to be susceptible to exoRNAi targeting CYP51 genes. These reports rise the question whether exoRNAi presents a conserved molecular toolkit applicable for gene function explorations and fungal control, or whether it confines a peculiar signaling situation within a specific environmental situation.
We report an assessment of exoRNAi for gene silencing in F. graminearum. Novel Fusarium lines expressing a destabilized nanoLuciferase (nanoLuc) enzyme were generated, allowing the ultrasensitive quantification of silencing of the nanoLuc reporter gene in vivo. Using these reporter lines, the silencing potency of stably expressed hairpin sRNA constructs was validated and compared to exoRNAi triggered by treatment of axenic cultures with different sRNA triggers. Systematic approaches to evaluate the potential and tentatively improve exoRNAi efficiency in F. graminearum were undertaken, i) the screening of a large sRNA library targeting genes essential for growth, ii) the genetic engineering of lines with increased sRNA uptake and iii) of lines overexpressing components of the RNAi machinery. Collectively, our data point towards a transient and limited exoRNAi phenotype in F. graminearum in vitro. In view of the previously reported in planta RNAi efficacy against selected plant pathogens, our findings advocate a contrasting in vitro situation, suggesting distinct sRNA uptake, processing and amplification mechanisms during plant-pathogen interactions.