RNAi-mediated silencing of PEX6 and GAS1 g enes of Fusarium oxysporum f. sp. lycopersici confers resistance against Fusarium wilt in tomato

Abstract

In the present study, we have explored the potential of the RNAi mediated silencing of genes encoding peroxisomal biogenesis factor and β-1,3-glucanosyltransferase in Fusarium oxysporum f. sp. lycopersici (Fol) to confer resistance to Fusarium wilt in transgenic tomato plants. The partial gene fragments from these genes were utilized independently to generate hairpin RNAi constructs in appropriate silencing vectors and used for Agrobacterium-mediated transformation of tomato. The presence of gene-specific siRNAs was confirmed by stem-loop RT-PCR analysis of selected transgenic tomato lines. Transgenic lines expressing gene-specific dsRNA displayed enhanced resistance to Fol with delayed development of disease symptoms. The survival rate of transgenic tomato lines after fungal infection was higher as compared to that of the untransformed tomato plants.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-021-02973-8.

Keywords: Fungal resistance; Fusarium oxysporum; Gene silencing; RNA interference; Tomato.

Fig. 1

Semi-quantitative RT-PCR analysis for detection of FoPEX6-RNAi and FoGAS1-RNAi transcripts in transgenic tomato lines. Expression of transcripts was normalized with Actin gene from tomato. Semi-quantitative RT-PCR analysis was performed by utilizing total RNA from PCR positive transgenic tomato lines a FoPEX6-RNAi and b FoGAS1-RNAi, M—100 bp DNA Ladder, Lane 2—WT (wild-type) control, 3–8 RNAi tomato lines

Fig. 2

Detection of siRNAs in transgenic tomato lines a FoPEX6-RNAi and b FoGAS1-RNAi tomato lines showing siRNAs. Lane 1-ULM (Ultra low molecular weight markers), Lane 2-WT (Wild-type) control, Lanes 3–8 transgenic tomato lines

Fig.3

Fungal resistance assay with FoPEX6-RNAi and FoGAS1-RNAi transgenic tomato lines. Experiments were performed by inoculating the roots with fungal spores and disease severity index (DI) values were determined after 3 weeks for the transgenic lines relative to the wild-type control plants. DI values for wild-type control plants were recorded as 1.0, whereas transgenic lines have shown DI values ranging from 0.2 to 0.7; a lines 45 and 56 of FoPEX6RNAi with DI values ranging from 0.25 to 0.5 were 70–80% resistant. In contrast, line 46 was significantly less resistant with DI of 0.7 (30% resistant); b similarly, lines 7 and 15 of FoGAS1RNAi with DI values ranging from 0.3 to 0.5 were 70–80% resistant, while line 14 was 45–50% resistant with DI of 0.65; c Assessment of RNAi transgenic tomato lines for resistance against Fusarium wilt. Photographs were taken 3-week post-inoculation. Tomato seedlings (6 each from the wild-type and transgenic lines) were used for fungal resistance assays

Fig. 4

Invasive growth of F. oxysporum on transgenic tomato fruits. a Inoculation was performed by injecting 10³ microconidia/ml into wild-type and RNAi lines. The photographs were taken after incubation for 6 days at 26 °C and 90% RH. (i) H₂O Only, (ii) Wild-type control tomato fruits, (iii) and (iv) FoPEX6RNAi and FoGAS1RNAi tomato fruit infected with microconidia; b Relative lesion sizes of the infected transgenic tomato fruits were measured 6 day post-inoculation (dpi) using the ImageJ software. Error bars indicate the standard deviations (s.d.) of 12 samples