Down-regulation of Fusarium oxysporum endogenous genes by Host-Delivered RNA interference enhances disease resistance

Abstract

Fusarium oxysporum is a devastating pathogen causing extensive yield losses in a variety of crops and development of sustainable, environmentally friendly methods to improve crop resistance is crucial. We have used Host-Delivered RNA interference (HD-RNAi) technology to partially silence three different genes (FOW2, FRP1, and OPR) in the hemi-biotrophic fungus F. oxysporum f. sp. conglutinans. Expression of double stranded RNA (dsRNA) molecules targeting fungal pathogen genes was achieved in a number of transgenic Arabidopsis lines. F. oxysporum infecting the transgenic lines displayed substantially reduced mRNA levels on all three targeted genes, with an average of 75, 83, and 72% reduction for FOW2, FRP1, and OPR, respectively. The silencing of pathogen genes had a clear positive effect on the ability of the transgenic lines to fight infection. All transgenic lines displayed enhanced resistance to F. oxysporum with delayed disease symptom development, especially FRP1 and OPR lines. Survival rates after fungal infection were higher in the transgenic lines compared to control wild type plants which consistently showed survival rates of 10%, with FOW2 lines showing 25% survival; FRP1 lines 30-50% survival and OPR between 45 and 70% survival. The down-regulation effect was specific for the targeted genes without unintended effects in related genes. In addition to producing resistant crops, HD-RNAi can provide a useful tool to rapidly screen candidate fungal pathogenicity genes without the need to produce fungal knockout mutants.

Keywords: Fusarium oxysporum; Host-Delivered RNAi; disease control; disease resistance; host-induced gene silencing; plant fungal pathogens.

Figure 1

Quantification of Fusarium oxysporum mRNA levels. Wild type and T3 homozygous transgenic lines expressing the HD-RNAi constructs for (A) FOW2, (B) FRP1, and (C) OPR were infected with F. oxysporum f. sp. conglutinans. Total RNA extracted from above-ground tissues of 3-week-old transgenic lines at 7 days post inoculation was used (n = 20) for cDNA synthesis as a template for quantitative real time PCR with pathogen gene-specific primers. F. oxysporum actin levels (GenBank JQ965663.1) were used for normalization purposes. For each gene, the relative mRNA level measured in F. oxysporum infecting wild type plants was given the arbitrary value of one and the remaining mRNA levels referred to it. Values shown are means ± SE of three biological replicates. ^*p ≤ 0.05; ^**p ≤ 0.005.

Figure 2

Disease progression of transgenic FOW2-RNAi lines. Two independent T3 homozygous Arabidopsis transgenic lines carrying an HD-RNAi construct targeting the F. oxysporum FOW2 gene were infected with F. oxysporum. (A) Disease progression was monitored by quantifying the number of yellow leaves. Values shown are means ± SE (n = 20). The experiment was repeated three times. (B) Survival was assessed 2–4 weeks after infection. Plants surviving after 2 weeks would grow to maturity and set seeds. Wild type Col-0 plants were used as control. A representative of three experiments is shown. ^*p ≤ 0.05; ^***p ≤ 0.0005.

Figure 3

Disease progression of transgenic FRP1-RNAi lines. Two independent T3 homozygous Arabidopsis transgenic lines carrying an HD-RNAi construct targeting the F. oxysporum FRP1 gene were infected with F. oxysporum. (A) Disease progression was monitored by quantifying the number of yellow leaves. Values shown are means ± SE (n = 20). The experiment was repeated three times. (B) Survival was assessed 2–4 weeks after infection. Plants surviving after 2 weeks would grow to maturity and set seeds. Wild type Col-0 plants were used as control. A representative of three experiments is shown. ^***p ≤ 0.0005; ^****p ≤ 0.0001.

Figure 4

Disease progression of transgenic OPR-RNAi lines. Three independent T3 homozygous Arabidopsis transgenic lines carrying an HD-RNAi construct targeting the F. oxysporum OPR gene were infected with F. oxysporum. (A) Disease progression was monitored by quantifying the number of yellow leaves. Values shown are means ± SE (n = 20). The experiment was repeated three times. (B) Survival was assessed 2–4 weeks after infection. Plants surviving after 2 weeks would grow to maturity and set seeds. Wild type Col-0 plants were used as control. A representative of three experiments is shown. ^****p ≤ 0.0001.

Figure 5

Time-lapse images and disease progression in transgenic OPR-RNAi and FRP1-RNAi lines. (A) Three independent T3 homozygous Arabidopsis OPR transgenic lines (#7, #8, and #9) carrying an HD-RNAi construct targeting the F. oxysporum OPR gene were infected with F. oxysporum. Disease progression was photographed 7, 9, 11, 15, 18, and 20 days after inoculation. Plants surviving after 2 weeks would grow to maturity and set seeds. Wild type Col-0 plants were used as control. A representative of three experiments is shown. (B) Two independent T3 homozygous Arabidopsis FRP1 transgenic lines (#24 and #40) carrying an HD-RNAi construct targeting the F. oxysporum FRP1 gene were infected with F. oxysporum. Disease progression was photographed 9 days after inoculation.