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.
Highlights
The genus Fusarium includes several species of fungi that are broadly spread in soil and organic substrates worldwide
FRP1 is necessary for F. oxysporum f. sp. lycopercisi pathogenicity and, as with F. oxysporum Wilt 2 (FOW2), FRP1-deficient mutants were still able to grow in artificial media but did not colonize host plants (Duyvesteijn et al, 2005)
In this work, we have shown that expression of double stranded RNA (dsRNA) molecules targeting pathogen genes in transgenic Arabidopsis plants can effectively reduce the mRNA levels of three different genes in F. oxysporum infecting the transgenic lines
Summary
The genus Fusarium includes several species of fungi that are broadly spread in soil and organic substrates worldwide. Fusarium oxysporum is one of the most relevant species of this genus and is the causal agent of root rots, damping-off and wilt diseases in more than 100 plants species, including a wide range of economically important horticultural crops, flowers, trees, and a number of field crops such as cabbage, banana, and cotton (Michielse and Rep, 2009; Takken and Rep, 2010; Dean et al, 2012; Swarupa et al, 2014). Colonization and toxin production by the pathogen results in blockage of the host vascular system (Michielse and Rep, 2009), causing characteristic disease symptoms including vasculature yellowing, vein clearing, chlorosis, and necrosis in leaf veins and leaves, leaf detachment and wilting (Di Pietro et al, 2003; Czymmek et al, 2007). F. oxysporum is most prevalent in tropical and subtropical regions and it is expected that its geographical range will extend due to climate change (Okubara and Paulitz, 2005)
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