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

Abstract

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.

Control of white mold (Sclerotinia sclerotiorum) through plant-mediated RNA interference

“…AT1703 plants demonstrate significant ABHYDROLASE-3 transcript reductions in S. sclerotiorum at 1 and 2 dpi, while reduced lesion size and fungal load are only identified at 3 dpi relative to wild-type Col-0 plants. Wytinck et al 16 showed similar results in transgenic B. napus, with significant reductions in ABHYDROLASE-3 transcript abundance and fungal load observed at 1 and 2 dpi respectively. In contrast, prior HIGS studies targeting S. sclerotiorum www.nature.com/scientificreports/ essential genes (TRX1, OA) detected both target transcript reduction and reduced lesion size at 2 dpi in host plants 33,41 , suggesting this delay to be specific to HIGS plants targeting ABHYDROLASE-3.…”

“…Limiting systemic infection has significant implications on reducing yield losses from S. sclerotiorum infection. This was demonstrated by Wytinck et al 16 , where transgenic B. napus lines expressing the same ABHYDROLASE-3 hpRNA construct against S. sclerotiorum show reduced vascular tissue colonization coupled with an increase in seed mass compared to untransformed B. napus post whole-plant S. sclerotiorum infection. As infection typically initiates on mature leaves before progressing into stem tissue through the vascular system 39,40 , it is critical to reduce vascular colonization directly at the site of infection, which can ultimately slow S. sclerotiorum progression throughout the host.…”

“…RNAi technology has the demonstrated ability to control pathogenic fungi in host plants through constitutively expressed dsRNA molecules in transgenic plants via host-induced gene silencing (HIGS). The constitutive expression of fungal-targeting dsRNAs allows for a durable means of protection against pathogen attack throughout the host plant lifecycle 16,33,34 . Protection of the plant relies on successful uptake and processing of dsRNA and processed siRNAs which can be generated in the host prior to uptake by the challenging pathogen 3 .…”

“…While the utility of HIGS in controlling S. sclerotiorum has been demonstrated in multiple host plants 16,33,34 , the underlying host response of these transgenic plants, as well as host-pathogen interactions in this system are less understood and dependent upon the specific gene being silenced. Here, global RNA sequencing and differential expression analyses provide insight into the response of AT1703 leaves to S. sclerotiorum directly at the site of infection.…”

“…Significant reductions in S. sclerotiorum lesion progression post application of ABHYDROLASE-3 targeting dsRNA molecules were correlated with reductions in target transcript levels as early as 24 h post infection. Subsequently, Wytinck et al 16 engineered B. napus to express hpRNAs targeting the S. sclerotiorum ABHYDROLASE-3 through HIGS and demonstrated reduced disease severity in inoculated stem tissues, coupled with significant ABHYDROLASE-3 transcript reduction. As S. sclerotiorum infection commonly initiates in leaves prior to progressing to stem tissues 17,18 , understanding how ABHYDROLASE-3 silencing influences the host response directly at the site of infection can provide…”