Host-induced gene silencing of cytochrome P450 lanosterol C14α-demethylase–encoding genes confers strong resistance to Fusarium species

Abstract

Head blight, which is caused by mycotoxin-producing fungi of the genus Fusarium, is an economically important crop disease. We assessed the potential of host-induced gene silencing targeting the fungal cytochrome P450 lanosterol C-14α-demethylase (CYP51) genes, which are essential for ergosterol biosynthesis, to restrict fungal infection. In axenic cultures of Fusarium graminearum, in vitro feeding of CYP3RNA, a 791-nt double-stranded (ds)RNA complementary to CYP51A, CYP51B, and CYP51C, resulted in growth inhibition [half-maximum growth inhibition (IC 50 ) = 1.2 nM] as well as altered fungal morphology, similar to that observed after treatment with the azole fungicide tebuconazole, for which the CYP51 enzyme is a target. Expression of the same dsRNA in Arabidopsis and barley rendered susceptible plants highly resistant to fungal infection. Microscopic analysis revealed that mycelium formation on CYP3RNA-expressing leaves was restricted to the inoculation sites, and that inoculated barley caryopses were virtually free of fungal hyphae. This inhibition of fungal growth correlated with in planta production of siRNAs corresponding to the targeted CYP51 sequences, as well as highly efficient silencing of the fungal CYP51 genes. The high efficiency of fungal inhibition suggests that host-induced gene-silencing targeting of the CYP51 genes is an alternative to chemical treatments for the control of devastating fungal diseases.T he diseases Fusarium head blight (FHB) and root rot, caused by pathogenic ascomycete fungi of the genus Fusarium, such as Fusarium graminearum (Fg), are devastating diseases of cereal crops (1). Fusarium represents one of the most important cereal killers worldwide, exerting great economic and agronomic impact on global grain production and the grain industry. In addition to considerable yield losses, food quality is detrimentally affected by grain contamination with mycotoxins, which are produced by the fungi during plant infection (2-4). These contaminants represent a serious threat to human and animal health (5).Plant protection and toxin reduction strategies are presently mediated by chemical treatments, resistance breeding strategies, biological control, and genetic engineering. The latter relies on the use of antifungal transgenes, such as chitinase, defensins, polygalacturonase, and the use of mycotoxin detoxifying enzymes (6). However, the use of antifungal traits has not provided convincing practical solutions in terms of efficiency and reliability under agronomical practice.Currently, the application of systemic fungicides, such as sterol demethylation inhibitors (DMIs), is essential for controlling Fusarium diseases and thereby reaching the attainable production level of modern high-yield cultivars. DMI fungicides, such as tebuconazole, triadimefon, and prochloraz, act as ergosterol biosynthesis inhibitors because of cytochrome P450 lanosterol C-14α-demethylase (CYP51) binding, which subsequently disturbs fungal membrane integrity (7). Because of a shortage of alternative chemicals, D...

RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 α-Demethylase

“…A wide range of transgenic crops expressing dsRNAs that are subsequently processed into sRNAs targeting essential and/or pathogenicity genes are more resistant to viruses, viroids, bacteria, fungi, oomycetes, nematodes, and insects (Cai et al 2018). Several studies demonstrated that cereals can be protected from Fusarium species by expressing dsRNAs targeting essential fungal genes such as Chitin synthases, β-1,3-Glucan synthase, or the azole fungicide target Cytochrome P450 lanosterol C-14 α-demethylase (Koch et al 2013;Cheng et al 2015;Chen et al 2016). Moreover, banana could be protected from Fusarium wilt caused by the soilborne fungus Fusarium oxysporum f. sp.…”

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals

“…Silencing F. graminearum CYP51 genes in vitro , through the exogenous application of a 791‐nucleotide dsRNA complementary to each of the three CYP51 paralogs, inhibited fungal growth and caused the abnormal branching of developing hyphae. Moreover, detached leaves of both transgenic Arabidopsis and barley plants expressing the same dsRNA were more resistant to F. graminearum infection compared with wild‐type plants,36 demonstrating the capacity of HIGS to silence fungal genes and impede infection.…”

“…The majority of studies carried out with transgenic Bt maize demonstrated that these plants were also less contaminated with Fusarium mycotoxins, including fumonisin, DON and zearelone than non‐Bt maize 35. A more recent strategy includes direct RNAi approaches, such as HIGS and SIGS, that have successfully silenced essential fungal genes and/or essential biosynthetic pathways 33, 34, 36, 37. Although CYP51 is found in most eukaryotic organisms, the average nucleotide identity between CYP51 genes from different species is very low (25–30%).…”

“…This assisted in the design of HIGS constructs that could target just a single pathogen species. The use of HIGS to control F. graminearum was first demonstrated in 2013 under controlled environmental conditions, by silencing all three CYP51 genes, namely CYP51A, CYP51B and the Fusarium ‐specific CYP51C 36. CYP51 is the major target of azole fungicides, also known as DMIs (described in Section 2) 38.…”

“…Arabidopsis ) or tissues that do not represent natural F. graminearum floral infections (i.e. detached leaves) 33, 36. Nonetheless, the reduction of F. graminearum infection achieved through the silencing of CYP51 did provide novel mechanistic insights, while demonstrating that both the HIGS and SIGS can be used to silence F. graminearum genes which influence the outcome of infection.…”