Host induced gene silencing of the Sclerotinia sclerotiorum ABHYDROLASE-3 gene reduces disease severity in Brassica napus.

Sclerotinia sclerotiorum is a pathogenic fungus that infects hundreds of crop species, causing extensive yield loss every year. Chemical fungicides are used to control this phytopathogen, but with concerns about increasing resistance and impacts on non-target species, there is a need to develop alternative control measures. In the present study, we engineered Brassica napus to constitutively express a hairpin (hp)RNA molecule to silence ABHYRDOLASE-3 in S. sclerotiorum. We demonstrate the potential for Host Induced Gene Silencing (HIGS) to protect B. napus from S. sclerotiorum using leaf, stem and whole plant infection assays. The interaction between the transgenic host plant and invading pathogen was further characterized at the molecular level using dual-RNA sequencing and at the anatomical level through microscopy to understand the processes and possible mechanisms leading to increased tolerance to this damaging necrotroph. We observed significant shifts in the expression of genes relating to plant defense as well as cellular differences in the form of structural barriers around the site of infection in the HIGS-protected plants. Our results provide proof-of-concept that HIGS is an effective means of limiting damage caused by S. sclerotiorum to the plant and demonstrates the utility of this biotechnology in the development of resistance against fungal pathogens.

dsRNA Uptake in Plant Pests and Pathogens: Insights into RNAi-Based Insect and Fungal Control Technology.

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.

Clathrin mediated endocytosis is involved in the uptake of exogenous double-stranded RNA in the white mold phytopathogen Sclerotinia sclerotiorum.

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 in S. sclerotiorum occurs 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.

Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea.

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.