In vivo trans-specific gene silencing in fungal cells by in planta expression of a double-stranded RNA

Abstract

Background: Self-complementary RNA transcripts form a double-stranded RNA (dsRNA) that triggers a sequence-specific mRNA degradation, in a process known as RNA interference (RNAi), leading to gene silencing. In vascular plants, RNAi molecules trafficking occur between cells and systemically throughout the plant. RNAi signals can spread systemically throughout a plant, even across graft junctions from transgenic to non-transgenic stocks. There is also a great interest in applying RNAi to pathogenic fungi. Specific inhibition of gene expression by RNAi has been shown to be suitable for a multitude of phytopathogenic filamentous fungi. However, double-stranded (ds)RNA/small interfering (si)RNA silencing effect has not been observed in vivo.

Results: This study demonstrates for the first time the in vivo interference phenomenon in the pathogenic fungus Fusarium verticillioides, in which expression of an individual fungal transgene was specifically abolished by inoculating mycelial cells in transgenic tobacco plants engineered to express siRNAs from a dsRNA corresponding to the particular transgene.

Conclusion: The results provide a powerful tool for further studies on molecular plant-microbe and symbiotic interactions. From a biotechnological perspective, silencing of fungal genes by generating siRNAs in the host provides a novel strategy for the development of broad fungi-resistance strategies in plants and other organisms.

Figure 1

Tobacco GUS-expressing line was re-transformed to express a double-stranded RNA for silencing the gus gene. (A) General scheme of the intron-spliced hairpin (hp)RNA vector (pC1302GUSi) constructed to promote gus gene silencing in GUS⁺ transformed tobacco lines. In order to generate the gus inferring cassette (hpGUS) a 627-bp fragment from gus gene (gus frag) was directionally cloned in order to generate sense and antisense arms, flanking the malate synthase gene intron 3 from Arabidopsis thaliana (ms-i3). (B) A GUS expressing tobacco line (GUS) was re-transformed with pC1302GUSi and regenerated plants (GUS-RNAi lines) did not show observable gus gene expression. (C) transgenic plants were analysed by polymerase chain reaction n order to detect both gus transgene in transformed plants (GUS) and the gus inferring cassette (hpGUS) in the GUS-RNAi lines. (D) Detection of small interfering RNA in a GUS expressing plants, GUS-RNAi line and control. Ethidium bromide-stained RNA serves as the loading control. Control in C and D is a non-transformed plant. Molecular size markers are indicated on the left in C and D.

Figure 2

Inoculations of GUS-RNAi tobacco lines with GUS expressing Fusarium verticillioides. (A) Mycelium cells exhibiting stable and high gus gene expression were used for the inoculation of plant leaves. (B) GUS assay carried out with GUS-RNAi tobacco leaves inoculated with the GUS⁺ fungus. Fungal mycelia interacting with the re-transformed plant did not show GUS expression; only a few germinating spores presented GUS expression. (C) GUS assay with non-transformed tobacco leaves inoculated with GUS⁺ fungus. Fungal mycelia showed high GUS expression level. (D, E) After GUS assay, leaf surfaces (presented in B and C) were observed under a scanning electron microscope in order to observe spores germinating and penetrating into stomata.

Figure 3

Isolation of Fusarium verticillioides colonies from both re-transformed and non-transformed tobacco lines. (A) The gus gene expression was quantified in fungal colonies isolated from non-transgenic (NT) and transgenic (RT1 and RT2) lines. (B) The two colonies isolated from GUS-RNA interference plant lines presented a reduction of the gus gene expression and were analysed for GUS expression over eight passages.

Figure 4

Presence and expression of the gus gene in transformed Fusarium verticillioides isolated from small interfering (si)RNA-expressing tobacco plants. (A) Polymerase chain reaction (PCR) analysis for the presence of the gus gene in F. verticillioides isolates that were not inoculated in tobacco plants (N), which were isolated from inoculated GUS-RNA interference plants and exhibited a reduction of approximately 62% (RT1) and 96% (RT2) in the gus gene expression and RT2 isolate after reversion to the normal GUS⁺ phenotype after the 7^th passage (R). (B) Reverse transcriptase (RT)-PCR analysis for the presence of transcripts from the endogenous gus gene in fungal cells. (C) RT-PCR analysis for the presence of transcripts from the fungal 5.8S rRNA housekeeping gene (internal control). (D) Detection of siRNA isolated from fungi isolated from transgenic (RT1, RT2 and R) and non-transgenic plants (N). The position corresponding to 18 and 24 nucleotides is indicated. C+: 100 pg of a gus gene-derived oligomer. Ethidium bromide-stained RNA serves as the loading control.

Figure 5

Southern analysis of genomic DNA to detect the foreign gus gene in Fusarium verticillioides isolates. C = non-transformed; N = transgenic isolate that was not inoculated in tobacco plants; RT1 and RT2 = isolated from inoculated GUS-RNA interference plants; R = RT2 isolate after reversion to the normal GUS⁺ phenotype.