Sclerotinia sclerotiorum, the causal agent of white stem rot, is responsible for significant losses in crop yields around the globe. While our understanding of S. sclerotiorum infection is becoming clearer, genetic control of the pathogen has been elusive and effective control of pathogen colonization using traditional broad-spectrum agro-chemical protocols are less effective than desired. In the current study, we developed species-specific RNA interference-based control treatments capable of reducing fungal infection. Development of a target identification pipeline using global RNA sequencing data for selection and application of double stranded RNA (dsRNA) molecules identified single gene targets of the fungus. Using this approach, we demonstrate the utility of this technology through foliar applications of dsRNAs to the leaf surface that significantly decreased fungal infection and S. sclerotiorum disease symptoms. Select target gene homologs were also tested in the closely related species, Botrytis cinerea, reducing lesion size and providing compelling evidence of the adaptability and flexibility of this technology in protecting plants against devastating fungal pathogens.

Efforts to develop more environmentally friendly alternatives to traditional broad-spectrum pesticides in agriculture have recently turned to RNA interference (RNAi) technology. With the built-in, sequence-specific knockdown of gene targets following delivery of double-stranded RNA (dsRNA), RNAi offers the promise of controlling pests and pathogens without adversely affecting non-target species. Significant advances in the efficacy of this technology have been observed in a wide range of species, including many insect pests and fungal pathogens. Two different dsRNA application methods are being developed. First, host induced gene silencing (HIGS) harnesses dsRNA production through the thoughtful and precise engineering of transgenic plants and second, spray induced gene silencing (SIGS) that uses surface applications of a topically applied dsRNA molecule. Regardless of the dsRNA delivery method, one aspect that is critical to the success of RNAi is the ability of the target organism to internalize the dsRNA and take advantage of the host RNAi cellular machinery. The efficiency of dsRNA uptake mechanisms varies across species, and in some uptake is negligible, rendering them effectively resistant to this new generation of control technologies. If RNAi-based methods of control are to be used widely, it is critically important to understand the mechanisms underpinning dsRNA uptake. Understanding dsRNA uptake mechanisms will also provide insight into the design and formulation of dsRNAs for improved delivery and provide clues into the development of potential host resistance to these technologies.

RNA interference (RNAi) technologies have recently been developed to control a growing number of agronomically significant fungal phytopathogens, including the white mold pathogen,Sclerotinia sclerotiorum. Exposure of this fungus to exogenous double-stranded RNA (dsRNA) results in potent RNAi-mediated knockdown of target genes’ transcripts, but it is unclear how the dsRNA can enter the fungal cells. In nematodes, specialized dsRNA transport proteins such as SID-1 facilitate dsRNA uptake, but for many other eukaryotes in which the dsRNA uptake mechanisms have been examined, endocytosis appears to mediate the uptake process. In this study, using live cell imaging, transgenic fungal cultures and endocytic inhibitors, we determined that the uptake mechanism inS. sclerotiorumoccurs through clathrin-mediated endocytosis. RNAi-mediated knockdown of several clathrin-mediated endocytic genes’ transcripts confirmed the involvement of this cellular uptake process in facilitating RNAi in this fungus. Understanding the mode of dsRNA entry into the fungus will prove useful in designing and optimizing future dsRNA-based control methods and in anticipating possible mechanisms by which phytopathogens may develop resistance to this novel category of fungicides.

Current agricultural output is challenged by considerable losses in crop yield and post-harvest storage due to fungal infection. Traditional chemical fungicides used to treat these fungi can be ineffective and harmful to the environment if not used properly. With fungicide resistance increasing in fungal pathogens, new environmentally friendly and sustainable technologies are required to manage diseases on the world’s most important crops. RNA interference (RNAi) is an intrinsic cellular mechanism, mediated by double-stranded RNA (dsRNA), which can suppress protein expression through targeted destruction of mRNAs. With recent advances in dsRNA delivery or expression in plants, this mechanism has the potential to provide alternative disease management strategies. Examples of RNAi-based control to manage pathogenic fungal species are steadily increasing, and the technology offers new options to increase species-specificity and/or potency against fungi for which existing fungicides have been ineffective. RNAi technology can be adapted to provide either robust and multi-crop plant protection using topical sprays or can provide more durable resistance through transgene expression of dsRNAs within susceptible plant tissues. Using RNA sequencing to identify fungal gene targets, RNAi-based control technology continues to show promise as an alternative to traditional agrochemicals for crop protection.