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Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato

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Expression of artificial microRNA targeting ATP binding domain of AC1 in transgenic tomato confers resistance to Tomato leaf curl disease without impacting the yield of tomato.

Abstract

Tomato curl leaf disease caused by Tomato leaf curl virus (ToLCV) is a key constraint to tomato cultivation worldwide. Engineering transgenic plants expressing artificial microRNAs (amiRNAs) against the AC1 gene of Tomato leaf curl New Delhi virus (ToLCNDV), which is important for virus replication and pathogenicity, would consequently confer virus resistance and reduce crop loss in the economically important crops. This study relates to an amiRNA developed on the sequence of Arabidopsis miRNA319a, targeting the ATP/GTP binding domain of AC1 gene of ToLCNDV. The AC1-amiR was found to regulate the abundance of AC1, providing an excellent strategy in providing defense against ToLCNDV. Transgenic lines over-expressing AC1-amiR, when challenged with ToLCNDV, showed reduced disease symptoms and high percentage resistance ranging between ∼ 40 and 80%. The yield of transgenic plants was significantly higher upon ToLCNDV infection as compared to the non-transgenic plants. Although the natural resistance resources against ToLCNDV are not available, this work streamlines a novel amiRNA-based mechanism that may have the potential to develop viral resistance strategies in tomato, apart from its normal symptom development properties as it is targeting the conserved region against which higher accumulation of small interfering RNAs (siRNA) occurred in a naturally tolerant tomato cultivar.

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Acknowledgements

The authors’ work in the area of plant–virus interaction is supported by the JC Bose Fellowship (JCB/2018/00000l) from Science and Engineering Research Board (SERB), Govt. of India, India and core grant of National Institute of Plant Genome Research (NIPGR), New Delhi. The authors thank Dr. Muthamilarasan Mehanathan, School of Life Sciences, University of Hyderabad, India, for critically reading the manuscript. The authors are also thankful to DBT-eLibrary Consortium (DeLCON) for providing access to e-resources.

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  1. National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi, 110067, India

    Namisha Sharma & Manoj Prasad

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  1. Namisha Sharma
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  2. Manoj Prasad
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Contributions

MP and NS conceived and designed the experiments; NS conducted the experiments and analyzed data; NS and MP wrote the manuscript. All the authors have read and approved the manuscript.

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Correspondence to Manoj Prasad.

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Supplementary file1 (DOC 49 kb)

Supplementary file2 (DOC 29 kb)

299_2020_2584_MOESM3_ESM.tif

Supplementary file3 Supplementary Fig. S1. (A) Schematic representation of AC1 protein showing conserved motifs I–III required for dsDNA binding and the ATP-binding domain. (B) Multiple sequence alignment of amino acid (AA) sequence and (C) nucleotide sequence of AC1 from ToLCV strains causing the tomato leaf curl disease ToLCNDV-Mild (ToLCNDV-Mld; U15016), ToLCNDV-Lucknow (ToLCNDV-Luc; Y16421), ToLCNDV-Karnataka (ToLCNDV_Kar; NC003897), ToLCNDV-Jabalpur (ToLCNDV-Jb; AY260504), ToLCNDV- IARI (ToLCNDV-IARI; AF524893), ToLCNDV-Gujarat (ToLCNDV-Guj; AAM21569), ToLCNDV-Dharwad (ToLCNDV-Dh; AY260505), ToLCNDV-Bangladesh1 (ToLCNDV_Ban1; Z48182), ToLCNDV-Bangladesh (ToLCNDV_Ban; EF450316), ToLCBV-Bangladesh4 (ToLCBV-Ban4; AF165098), ToLCNDV-Bangladesh5 (ToLCBV-Ban5; AF295401), ToLCNDV-Israel (P27259). (D) Multiple sequence alignment of amino acid (AA) sequence and (E) nucleotide sequence of AC1 from different begomoviruses including Tomato Yellow Leaf Curl Virus-Almeria (TYLCV_Almeria; AJ489258), Tomato Yellow Leaf Curl Virus-Almeria (TYLCV; FJ956702), Tomato Golden Mosaic Virus (TGMV; NC001507), Squash leaf curl China virus (SLCCNV; KC222956) Tomato yellow leaf curl China virus (TYLCCNV; CAC85509), Tomato yellow leaf curl Sardinia virus (TYLSCV; AAA47955), African cassava mosaic virus (ACMV; AAD40938), Cotton leaf curl virus (CtLCV; KC412251), Pepper huasteco yellow vein virus (PHYVV; AAL02410), and Tomato mottle virus (ToMoV; AAC32414). The AA and nucleotide residues highlighted in black are non-conserved. The AA and nucleotide residues within the red box are the target site for AC1-amiR1 and within the blue box is the target site for AC1-amiR2. (TIF 15694 kb)

299_2020_2584_MOESM4_ESM.tif

Supplementary file4 Supplementary Fig. S2. Stem-loop structure of precursor ath-miR219, AC1-amiR1 and AC1-amiR2 predicted by Mfold software. The highlighted part indicates the mature sequence of ath-miRNA319 and amiRNAs. (TIF 1403 kb)

299_2020_2584_MOESM5_ESM.tif

Supplementary file5 Supplementary Fig. S3. PCR amplification profile using vector specific primers of kanamycin gene (448bp) from genomic DNA of transgenic lines (L1-L7), Plasmid control (PC) and non-transformed control (NTC) plants. (TIF 695 kb)

299_2020_2584_MOESM6_ESM.tif

Supplementary file6 Supplementary Fig. S4. Southern blot analysis for copy number of transgene in transgenic lines. Genomic DNA was isolated from leaves of non-transformed control (NTC) and transgenic plants (L1-L6) plants, and digested with EcoRI, which cut T-DNA at single site. Digested genomic DNA was separated on 0.8%agarose gel. (A) Blot developed by using 448 bp kanamycin amplicon (NPT-II) for probing the digested genomic DNA. (B) The EtBR stained agarose gel depicting equal loading of digested DNA from different tomato samples. (TIF 1993 kb)

299_2020_2584_MOESM7_ESM.tif

Supplementary file7 Supplementary Fig. S5. Expression analysis of AC1-amiR in transgenic lines (T2:L1- L6) and non-transformed control plants by Northern blot. Antisense of mature sequence of AC1-amiR1 was used as probe. EtBr-stained rRNA was used as loading control. (TIF 255 kb)

299_2020_2584_MOESM8_ESM.tif

Supplementary file8 Supplementary Fig. S6. Southern blot analysis for virus accumulation (A) the total DNA of transgenic (T2: L1_T, L4_T and L6_T), non-transformed control (NTC_T) and cultivar H-88-78-1 (H_T) tomato plants after 21dpi digested with EcoRV. (B) Undigested total DNA of transgenic (T1: L1_T, L4_T and L6_T), non-transformed control (NTC_T) and cultivar H-88-78-1 (H_T) tomato plants after 21dpi. Different forms of ToLCNDV genome is shown as open circular (OC), linear (Lin), supercoiled (SC) and single strand (SS). Equivalent loading of DNA was shown in the Ethidium bromide stained DNA. (TIF 2653 kb)

299_2020_2584_MOESM9_ESM.tif

Supplementary file9 Supplementary Fig. S7. Expression analysis of AC1 in virus infected transgenic lines (L1, L4 and L6), H-88-78-1 and non-transformed control plants after 21dpi (A) T2 and (B) T1. EtBr-stained rRNA is shown as the equivalent loading control for the experiment. (TIF 731 kb)

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Sharma, N., Prasad, M. Silencing AC1 of Tomato leaf curl virus using artificial microRNA confers resistance to leaf curl disease in transgenic tomato. Plant Cell Rep 39, 1565–1579 (2020). https://doi.org/10.1007/s00299-020-02584-2

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