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Silencing of the Ornithine Decarboxylase Gene of Fusarium oxysporum f. sp. lycopersici by Host-Induced RNAi Confers Resistance to Fusarium Wilt in Tomato

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Abstract

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici, a hemibiotrophic filamentous fungal pathogen is one of the important diseases of tomato. Recently, RNA interference (RNAi) has emerged as a novel alternative strategy for the control of plant diseases caused by fungal pathogens. Therefore, we tested the potential of RNAi for the control of Fusarium wilt in tomato by targeting a key polyamine (PA) biosynthesis gene, ornithine decarboxylase (ODC) of the pathogen as PAs (putrescine, spermidine, and spermine) are absolutely essential for normal growth and development. The target fungal ODC gene fragment was cloned in a hairpin RNA construct, which was used to develop several transgenic tomato plants. These RNAi transgenic lines expressed the intended small interfering RNAs (siRNAs) and exhibited moderate to high resistance when challenged with the spores of F. oxysporum. Interestingly, the silencing effect was confined only to the fungal pathogen and had no influence on the plant ODC gene expression, polyamine levels, and morphology. These results confirm that the ODC gene is vital for fungal growth and is a suitable target for disease control through host-induced gene silencing (HIGS) and also implicates the transfer of dsRNA/siRNAs from host plant cells to the fungal cells. To further validate the uptake of plant-derived siRNAs by fungal cells in a visual manner, silencing of the GFP transgene was observed in F. oxysporum GFP transformants upon infecting the dsGFP-expressing transgenic tomato plants. These experiments demonstrate the applicability of RNAi-based approach for crop protection from the filamentous fungal pathogens.

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Acknowledgments

NS acknowledges the Council of Scientific and Industrial Research for senior research fellowship. We also thank the University Grants Commission, New Delhi, for special assistance program (DRS-III); Department of Science and Technology (DST), New Delhi, for FIST program (Level 2); and DU-DST PURSE grant.

Funding

We are grateful to the Department of Biotechnology (Grant No. BT/PR 10713/AGR/36/601/2008), New Delhi, for the financial assistance (to MVR).

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Authors and Affiliations

  1. Plant Polyamine, Transgenic and RNAi Lab, Department of Genetics, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India

    Neeru Singh & Manchikatla Venkat Rajam

  2. Division of Plant Pathology, ICAR- Indian Agricultural Research Institute, New Delhi, 110012, India

    Sunil Kumar Mukherjee

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  1. Neeru Singh
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MVR has conceived the concept and designed the experiments. NS performed the experiments, generated and analyzed the data, and written the manuscript. MVR and SKM revised the manuscript and interpreted the data. All the authors approved the final manuscripts.

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Correspondence to Manchikatla Venkat Rajam.

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Key Message

• Silenced ornithine decarboxylase (ODC) gene of Fusarium oxysporum f. sp. lycopersici by host-induced RNAi

• Tomato RNAi lines have exhibited Fusarium wilt resistance.

• Validated uptake of plant-derived siRNAs by fungi using GFP gene

• Demonstrated that ODC gene is vital for fungal development and good target for plant fungal infections through RNAi

Electronic Supplementary Material

ESM. 1

(DOCX 12 kb)

Fig. S1

Pathway for biosynthesis of major plant and fungal polyamines. Initial step in the pathway involves formation of diamine, putrescine from ornithine decrboxylation by ODC enzyme activity in all the organisms including, fungi. α-Difluoromethylornithine (DFMO) irreversibly inhibits ODC activity their by blocking putrescine synthesis. Alternatively, putrescine is also produced from arginine by the activity of ADC enzyme in plants, bacteria and some fungi. Higher polyamines, triamine spermidine and tetraamine spermine are synthesized from putrescine by the catalytic activity of enzymes spermidine synthase (SPDSYN) and spermine synthase (SPMSYN), respectively, by sequential additions of aminopropyl groups donated by decarboxylated S- adenosylmethionine (dcSAM), which is in turn synthesized from SAM by SAM decarboxylase. (JPG 41 kb)

Fig. S2

Diagrammatic representation of pFGC5941 RNAi vector. It has basta resistance (bar) gene for plant selection, CaMV35S promoter to drive the dsRNA expression, ChsA intron to stabilize the dsRNA molecules hairpin formation. The 541 bp ODC gene sequence was inserted in the vector in sense and antisense orientation with AscI, SwaI and BamHI, XbaI restriction sites, respectively (a) and this complete expression cassette was cleaved with EcoRI and PstI restriction enzymes and inserted into pCambia 2300 binary vector, which has NPT-II as the selection marker (b). (JPG 47 kb)

Fig. S3

Transgene integration in ODC RNAi tomato plants. The presence of transgenes was checked by PCR analysis using both ODC and NPT-II gene primers, Lane 1 (of a, b, c, d) is marker (M, 1 kb DNA ladder) and lane 2 is +ve control (plasmid DNA), Lane 3–11 (a), 2–9 (b) are the transgenic lines showing ODC gene integration and Lane 2, 3 (c) are the untransformed control and + ve control respectively, lanes 4–11 (c); lane 2–13 (d) are the transgenic lines showing NPT-II gene integration. (JPG 61 kb)

Fig. S4

The expression of native ODC and ADC genes in ODC hp. tomato transgenic plants by RT- PCR with tomato ODC and ADC primers. Lane 1 (of a, b, c, d) is the marker (1 kb DNA ladder); Lane 2 (a, b, c, d) are the wild-type control and Lanes 3–10 (a, ODC), 3–7 (c, ADC) represent the transgenic lines. The β- actin RT-PCR product was taken as the internal control in this experiment (b, d). This experiment was repeated thrice. (JPG 58 kb)

Fig. S5

(a) The diagrammatic representation of pCAMBIA 1302 vector carrying sGFP gene used for F. oxysporum transformation; (b) Diagrammatic representation of GFP hp. construct used for tomato transformation (JPG 43 kb)

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Singh, N., Mukherjee, S.K. & Rajam, M.V. Silencing of the Ornithine Decarboxylase Gene of Fusarium oxysporum f. sp. lycopersici by Host-Induced RNAi Confers Resistance to Fusarium Wilt in Tomato. Plant Mol Biol Rep 38, 419–429 (2020). https://doi.org/10.1007/s11105-020-01205-2

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