<b>Application of Exogenous Double-stranded RNA with Graphene Quantum Dot Nanocarriers to Target </b><b><i>Fusarium graminearum </i></b><b>Genes for Controlling Fusarium Head Blight in Wheat</b>
Creators: name: Gyawali, Binod
Publisher: name: Purdue University Graduate School
Year: 2024
Subjects: classificationCode: 300109
subject: Non-genetically modified uses of biotechnology

description: This research is about the innovative method for the control of the one of the fungal disease, Fusarium head blight, in wheat.<i>Fusarium graminearum</i> is a devastating fungus that causes Fusarium head blight (FHB) disease in cereal crops, including wheat, rice, barley, and oats, as well as ear rot and stalk rot in maize. Currently, application of synthetic agrochemical fungicides remains the major mitigation strategy for FHB disease control. However, fungicide treatment carries risks to human health and the environment. Spray-induced gene silencing (SIGS) is suggested as an effective, sustainable, and environmentally friendly alternative for the control of fungal diseases. SIGS uses double-stranded RNA (dsRNA) to induce RNAi against eukaryotic pathogens and pest’s genes. Unlike fungicides, dsRNA can be engineered and carefully designed to target specific fungal pathogens, essentially limiting off-target effects and reducing harm to non-target organisms, including beneficial species.In this study, we investigated the <i>in vitro</i> and <i>in vivo</i> effects of dsRNA application on growth and pathogenicity of <i>Fusarium graminearum</i> (<i>Fg</i>). We designed dsRNA against eight fungal genes <i>FgMGV1</i>, <i>FgRAS1</i>, <i>FgCOT1</i>, <i>FgPp2A</i>, <i>FgCAK1</i>, <i>FgTRI5</i>, <i>FgGMK1</i>, and <i>FgYCK1</i>, which have been previously reported to have a functional role in fungal growth and pathogenicity. I postulated that silencing the transcript expression of these selected fungal genes will lead to an attenuation of <i>F</i>. <i>graminearum</i> growth, development and virulence. For lab-scale <i>in vivo</i> dsRNA production, we first designed dsRNAs for each of the fungal genes using the pssRNAit webserver. These gene fragments were subsequently cloned into the L4440 plasmid and transformed into the RNAase III-deficient <i>E</i>. <i>coli</i> strain <i>HT115</i>(DE3). Induction of dsRNA production was mediated by the addition of Isopropyl-β-D-thiogalactoside (IPTG). Graphene Quantum Dots (GQDs) were used as nanocarrier, which were synthesized by pyrolyzing citric acid coupled with surface functionalization by using branched polyethyleneimine (bPEI).The performance of ds<i>TRI5</i>, ds<i>MGV1</i>, ds<i>YCK1</i> and ds<i>COT1</i> in reducing fungal biomass in SNA medium was better than dsRNA of other genes, as evidence by lowering fungal biomass by almost half, while ds<i>CAK1</i> and ds<i>GMK1</i> being the least effective in controlling biomass. The inhibitory effect of dsRNA on fungal growth in plate was also investigated. The mycelial growth of <i>F. graminearum</i> on plates was highly inhibited with distinct inhibition zones when plate media was made by mixing the dsRNAs. When wheat spikes of two varieties i.e., ‘AL105’ and ‘Gilat’ were point inoculated with <i>F</i>. <i>graminearum</i>, and after three days sprayed with dsRNAs, the percent symptomatic spikelets (PSS) significantly reduced to 20-25% in both varieties while non-treated control spikes showed 100% symptomatic spikelets. We also evaluated the effect of dsRNA on lowering the accumulation of mycotoxin deoxynivalenol (DON) post-infection followed by the analysis of transcript abundance for two genes <i>FgRAS1</i> and <i>FgPP2A</i> after the application of dsRNA in the liquid culture.In summary, we have provided the first example of utilizing graphene quantum dots, for the delivery of dsRNA in SIGS applications in wheat against <i>Fusarium graminearum</i>. In addition, exogenously applied dsRNA degrades naturally, leaving no harmful chemical residues on plants, soil, or water bodies, making it safer for human consumption and the environment. Further optimization of delivery systems for improving the uptake of pathogens efficiency should be done before commercial use of RNA-based disease management in fields.