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 (IC50) = 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.

Keywords: Fusarium head blight; HIGS; RNA interference; crop protection; small interfering RNA.

Fig. 1.

Infection symptoms on Arabidopsis leaves following inoculation with F. graminearum. (A) Detached leaves of 5-wk-old plants were treated with 5 × 10⁴ macroconidia mL⁻¹ and evaluated for necrotic lesions at 3 dpi. (i) wild-type (Col-0), (ii) Col-0 ev control, (iii) Col-0 expressing CYP3RNA (representative line L8), and (iv) wild-type treated with Tween water (mock). (B) Quantification of infected leaf area at 3 dpi; typical infection symptoms are recorded as a percent of the total leaf area. Bars represent mean values ± SDs of three independent experiments, each using 20 leaves collected from 15 different plants of each transgenic line, as well as wild-type and Col-0 ev plants. The reduction in infection symptoms on CYP3RNA-expressing leaves compared with the wild-type and Col-0 ev control was statistically significant (***P < 0.0001; Student´s t test). (C) Fg-inoculated Arabidopsis leaves at 5 dpi. (i) The Col-0 ev leaf is heavily infected with Fg; (ii) Col-0 expressing CYP3RNA does not show infection symptoms.

Fig. 2.

Abundance of CYP51 gene transcripts and siRNAs in Fg-infected Arabidopsis leaves. (A–C) Gene-specific analysis of CYP51 transcripts 3 dpi by quantitative RT-PCR using fungal β-tubulin as the reference gene. (A) CYP51A, (B) CYP51B, and (C) CYP51C. cDNA was generated from total RNA isolated from Fg-inoculated leaves collected during the detached leaf assay. Bars represent mean values ± SDs of three independent sample collections. The reduction in CYP51 gene expression in the Fg-inoculated Col-0-CYP3RNA leaves compared with the ev control was statistically significant (**P < 0.001, ***P < 0.0001; Student´s t test) (D). Detection of low molecular mass siRNA complementary to the CYP51 genes in pooled leaf tissue from uninfected and Fg-infected Arabidopsis plants at 3 dpi. Northern blot analysis using ³²P-labeled CYP3RNA was followed by autoradiography. No signal was detected in the control samples from wild-type and Col-0 ev plants. Ethidium bromide-stained rRNA in the Lower panel served as the loading control.

Fig. 3.

Quantification of fungal CYP51 transcripts by quantitative RT-PCR in Fg-inoculated barley leaves and roots. (A) Transcript abundance of detached leaves at 5 dpi: (i) CYP51A, (ii) CYP51B, (iii) CYP51C. Bars represent mean values ± SDs of two independent experiments. (B) Transcript abundance at 3 dpi in roots generated by the STARTS method (41): (i) CYP51A, (ii) CYP51B, (iii) CYP51C. Bars represent mean values ± SDs of three independent experiments. The reduction in CYP51 gene expression in Fg-inoculated Col-0-CYP3RNA roots compared with the ev control was statistically significant (***P < 0.0001; Student´s t test).

Fig. 4.

Infection symptoms on barley leaves and caryopses following inoculation with F. graminearum. (A) Detached second leaves of 2-wk-old plants were treated with 5 × 10⁴ macroconidia mL⁻¹ and evaluated for necrotic lesions at 5 dpi. Wt, wild-type Golden Promise; ev, Golden Promise empty vector control; L2-L9, L14, L42, Golden Promise lines expressing CYP3RNA. (B) Caryopses were treated with 1.2 × 10⁴ macroconidia per mL⁻¹ and evaluated for Fg infection at 4 dpi. CYP3RNA +Fg, Golden Promise (L2) expressing CYP3RNA; CYP3RNA –Fg, mock-inoculated (Tween water) Golden Promise expressing CYP3RNA. (C) Microscopy of Fg infection in the epicarp and inner tissue of barley caryopses at 4 dpi: (i and ii) cross-sections of caryopses from ev plants inoculated with Fg (i) and with a GFP-expressing Fg strain (ii); (iii and iv) cross-sections of caryopses collected from CYP3RNA expressing plants treated with Fg (iii) and with GFP-expressing Fg (iv). a, aleurone; c, cross cells; cl, cell layers; e, endosperm; m, macroconidia; p, pericarp; t, testa; arrow heads (black and white), infection hyphae.