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Host RNAi-mediated silencing of Fusarium oxysporum f. sp. lycopersici specific-fasciclin-like protein genes provides improved resistance to Fusarium wilt in Solanum lycopersicum

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Main conclusion

Tomato transgenics expressing dsRNA against FoFLPs act as biofungicides and result in enhanced disease resistance upon Fol infection, by downregulating the endogenous gene expression levels of FoFLPs within Fol.

Abstract

Fusarium oxysporum f. sp. lycopersici (Fol) hijacks plant immunity by colonizing within the host and further instigating secondary infection causing vascular wilt disease in tomato that leads to significant yield loss. Here, RNA interference (RNAi) technology was used to determine its potential in enduring resistance against Fusarium wilt in tomato. To gain resistance against Fol infection, host-induced gene silencing (HIGS) of Fol-specific genes encoding for fasciclin-like proteins (FoFLPs) was done by generating tomato transgenics harbouring FoFLP1, FoFLP4 and FoFLP5 RNAi constructs confirmed by southern hybridizations. These tomato transgenics were screened for stable siRNA production in T0 and T1 lines using northern hybridizations. This confirmed stable dsRNAhp expression in tomato transgenics and suggested durable trait heritability in the subsequent progenies. FoFLP-specific siRNAs producing T1 tomato progenies were further selected to ascertain its disease resistance ability using seedling infection assays. We observed a significant reduction in FoFLP1, FoFLP4 and FoFLP5 transcript levels in Fol, upon infecting their respective RNAi tomato transgenic lines. Moreover, tomato transgenic lines, expressing intended siRNA molecules in the T1 generation, exhibit delayed disease onset with improved resistance. Furthermore, reduced fungal colonization was observed in the roots of Fol-infected T1 tomato progenies, without altering the plant photosynthetic efficiency of transgenic plants. These results substantiate the cross-kingdom dsRNA or siRNA delivery from transgenic tomato to Fol, leading to enhanced resistance against Fusarium wilt disease. The results also demonstrated that HIGS is a successful approach in rendering resistance to Fol infection in tomato plants.

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Data availability

All the data in the manuscript and supplementary information will be made available by the corresponding author upon request.

Abbreviations

DPI:

Days post-inoculation

Fol :

Fusarium oxysporum F. sp. lycopersici

HIGS:

Host-induced gene silencing

dsRNA:

Double-stranded RNA

siRNA:

Small interfering RNA

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Acknowledgements

We are grateful to Department of Biotechnology (Grant No. BT/PR0713/AGR/36/601/2008), New Delhi for financial assistance (to MVR). We are also grateful to the Department of Science and Technology, New Delhi for generous support in carrying out the RNAi work in the lab. MVR is grateful to the University Grants Commission (UGC) for BSR Faculty Fellowship. SC is thankful to the University of Delhi for UGC BSR-NET-JRF/SRF Fellowship. We also thank the UGC for SAP (DRS-III) programme, DST for FIST (Level 2) programme and DU-DST PURSE (Phase II) grant.

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Authors and Affiliations

  1. Department of Genetics, University of Delhi South Campus, Benito Juarez Marg, New Delhi, 110021, India

    Sambhavana Chauhan & Manchikatla Venkat Rajam

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  1. Sambhavana Chauhan
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Contributions

Conceived and designed the experiments: MVR SC. Performed the experiments: SC. Analyzed the data: SC MVR. Wrote the paper: SC. Corrected and edited the paper: MVR. Both the authors have approved the manuscript.

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Correspondence to Manchikatla Venkat Rajam.

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Fig. S1

FLP-RNAi constructs preparation and confirmation. A PCR amplification of off-target free FoFLP1 (sense- 201 bp (1) and antisense- 197 bp (2)), FoFLP4 (sense-156 bp (3) and antisense-152 bp (4)) and FoFLP5 (sense-145 bp (5) and antisense-141 bp (6)) using gene-specific primers (Table S1) at annealing temperatures 65 °C, 59 °C and 61 °C, respectively. B, C TA cloning of PCR amplified products of FoFLP1, FoFLP4 and FoFLP5 in pGEMT easy cloning vector and confirmation of positive transformations using EcoRI restriction enzyme digestion to release the desired fallout. Lane 1, 2 and 3 correspond to FoFLP sense and lane 4, 5 and 6 correspond to FoFLP antisense cloned in pGEMT vector. D, E Restriction enzyme digestions for confirmation of putative bacterial colonies harbouring pMVR-FLP-RNAi constructs.Lane 1 represents Uncut plasmid; lanes 2 and 3 represent vector and putative plasmid constructs restricted with AscI/XbaI; lanes 4 and 5 represent putative plasmid constructs restricted with AscI/SwaI and BamHI/XbaI for sense and antisense respectively; lanes 6 and 7 represent putative plasmid constructs restricted with AscI/BamHI and SwaI/XbaI for sense+intron and antisense+intron. F Plasmid PCR to amplify 35S promoter-sense-intron-antisense-OCS terminator for confirmation of pMVR-FLP-RNAi constructs after mobilising the confirmed construct from E. coli to A. tumefaciens strain LBA4404. B corresponds to vector control; lane 1 corresponds to empty vector control and lanes 2-15 correspond to pMVR-FLP-RNAi constructs. Supplementary file1 (TIF 1516 KB)

Fig. S2

T-DNA maps of FLP-RNAi constructs. A,B, C and D correspond to empty vector pMVR, FLP1, FLP4 and FLP5 RNAi constructs.. Supplementary file2 (TIF 545 KB)

Fig. S3

Agrobacterium-mediated tomato transformation with FLP-RNAi constructs. Cotyledonary leaves were independently transformed with FLP1, FLP4 and FLP5-RNAi constructs and maintained on 50 mg/L kanamycin concentration till hardening. SM, selection medium (MS + 3% maltose + 0.5mg/L IAA and 2.5 mg/L BAP); SRM, shoot regeneration medium (MS + 3% maltose + 0.5 mg/L BAP); RM, rooting medium (1/2 MS + 3% maltose).Supplementary file3 (TIF 3468 KB)

Fig. S4

Morphological characterization of T0 FLP-RNAi tomato transformants. T0 tomato transgenics of FLP1, FLP4 and FLP5 RNAi lines were compared with the non-transformant and empty vector (pMVRhp) controls for variations in plant morphology, flower and fruit.. Supplementary file4 (TIF 3803 KB)

Fig. S5

PCR screening of T0 FLP-RNAi tomato transformants to demonstrate the presence of transgenes. npt-II gene-specific primers were used to screen the putative FLP1, FLP4 and FLP5-RNAi transgenic lines for the presence of FLP1, FLP4 and FLP5-RNAi cassette, respectively. PCR product size of npt-II corresponds to 704 bp. Supplementary file5 (TIF 689 KB)

Fig. S6

Morphological characterization of T1 progenies of FLP-RNAi tomato transgenic lines. Line number 7.1, 7.4, 8.2 and 8.3 of FLP1, 8.17 and 8.31 of FLP4, 8.1 and 8.5 of FLP5, 8.15 of pMVR (empty vector control) and untransformed line showing morphology of plant, flowers and fruits.Supplementary file6 (TIF 9300 KB)

Supplementary file7 (DOCX 16 KB)

Supplementary file8 (DOCX 17 KB)

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Chauhan, S., Rajam, M.V. Host RNAi-mediated silencing of Fusarium oxysporum f. sp. lycopersici specific-fasciclin-like protein genes provides improved resistance to Fusarium wilt in Solanum lycopersicum. Planta 259, 79 (2024). https://doi.org/10.1007/s00425-024-04360-y

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