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Transmission of RNA silencing signal through grafting confers virus resistance from transgenically silenced tobacco rootstocks to non-transgenic tomato and tobacco scions

  • Md. Emran AliEmail author
  • Sumyya Waliullah
  • Kappei Kobayashi
  • Takashi Yaeno
  • Naoto Yamaoka
  • Masamichi NishiguchiEmail author
Original Article

Abstract

We examined the transmission of RNA silencing signal in non-transgenic tomato and tobacco scions grafted onto the tobacco Sd1 rootstocks, which is silenced in both NtTOM1 and NtTOM3 required for tobamovirus multiplication. When the non-transgenic tomato scions were grafted onto the Sd1 rootstocks, RT-PCR analysis of the scions showed the reduced level of mRNA compared with that before grafting in both LeTH3 and LeTH1, tomato homologs of NtTOM1 and NtTOM3, respectively. siRNAs from both genes were detected in the scions after grafting but not before grafting. Further tomato scions were inoculated with Tomato mosaic virus (ToMV) and used for virus infection. They showed very low level of virus accumulation. Necrotic responding tobacco to tobamovirus was grafted onto the rootstock of Sdl. RT-PCR analysis showed low level expression of both NtTOM1 and NtTOM3 in the scions but siRNA was detected after grafting. When the leaves of scions were inoculated with ToMV or Tobacco mosaic virus, they produced very few local necrotic lesions (LNLs) while the control scions did many LNLs. These results suggest that RNA silencing was transmitted to non-transgenic tomato and tobacco scions after grafting onto the Sd1 rootstocks and that virus resistance was induced in the scions.

Keywords

RNA silencing Graft-transmission Tobamovirus Virus resistance Tobacco Tomato TOM1/TOM3 

Abbreviations

ToMV

Tomato mosaic virus

TMV

Tobacco mosaic virus

LNLs

Local necrotic lesions

siRNA

Small interfering RNA

RISC

RNA-induced silencing complex

dsRNA

Double-stranded RNA

Notes

Acknowledgments

We are grateful to M. Ishikawa Y. Tamaki for providing Sd1 and Y. Sato for the tomato seeds, respectively. We also thank H. Takahashi and D. Murphy for the CMV-Y isolate and its cDNA clone and for checking the English in this manuscript, respectively. This work was supported by the Program for Promotion of Basic and Applied Research in Bio-oriented Industry (BRAIN), the Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry, the Ministry of Education, Culture, Sports and Technology of Japan [Grant-in-Aid for Scientific Research for Scientific Research (C), No. 24580065] to MN and the Japan Science and Technology Agency for the A-step (Adaptable & Seamless Technology Transfer Program through Target-driven R & D) to MN.

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

13562_2015_334_MOESM1_ESM.pptx (652 kb)
Fig. S1 RT-PCR and siRNA detection of grafted necrotic responding tobacco. I and II; RT-PCR and siRNA detection. A and D, Xanthi nc; B and E, Nicotiana glutinosa; C and F, Bright Yellow. CGS, grafted scion onto wild type rootstock; GS, grafted scion onto Sd1 rootstock; RS, Sd1 rootstock. Actin-F/ Actin-R were used to amplify the actin as an internal control. Eight weeks after grafting, total RNA was extracted from grafted scions and used for RT-PCR analysis and siRNA detection. The siRNA blots were hybridized with the [α-32P]dCTP-labeled cDNA probes specific for NtTOM1 (876 bp) or NtTOM3 (916 bp) for non-transgenic scions of necrotic responding tobacco varieties. (PPTX 652 kb)
13562_2015_334_MOESM2_ESM.pptx (1.1 mb)
Fig. S2 Grafted tobacco plants after TMV/ToMV inoculation. A and B, Xanthi nc (Xnc) grafted onto Sd1 and wild type rootstocks. C and D, Nicotiana glutinosa (NG) grafted onto Sd1 and wild type rootstocks. E and F, Bright Yellow (BY) grafted onto Sd1 and wild type rootstocks. Plants were inoculated 8 weeks after grafting and the photographs were taken 10 days after inoculation. (PPTX 1150 kb)
13562_2015_334_MOESM3_ESM.pptx (251 kb)
Fig. S3 ELISA and northern blot analysis of grafted tobacco. A, detection of CMV by ELISA. B, Northern blot analysis. Sample of scions from three individual grafted plants of each combination (Sd1/Sd1, Sam/Sd1 and Xnc/Sd1). Mock or M represents non-transgenic tobacco, Samsun, leaf grafted onto non-transgenic tobacco after inoculation only with buffer. Samples were taken 15 days after inoculation. Sd1, Sd1 line; Sam, Samsun; Xnc, Xanthi nc. (PPTX 251 kb)
13562_2015_334_MOESM4_ESM.pptx (380 kb)
Fig. S4 Nucleotide sequence comparison of NtTOM1, NtTOM3, LeTH1 and LeTH3. NtTOM1 (accession number AB193039), NtTOM3 (accession number AB193040), LeTH1 (accession number AB193041), LeTH3 (accession number AB193043). Gaps are designated by dots. Underlined nucleotides in lower case letters are used as primers for transgene constructs in Sd1 line (Asano et al. 2005). Only consensus nucleotides among four genes are written below in lower case letters. Nucleotides in red letters on blue back are different only in one and two genes from others. References Asano, M., R. Satoh, A. Mochizuki, S. Tsuda, T. Yamanaka, M. Nishiguchi, K. Hirai, T. Meshi, S. Naito, Ishikawa, M (2005) Tobamovirus-resistant tobacco generated by RNA interference directed against host genes. FEBS Lett 579: 4479–4484 Nozu, Y., Okada, Y (1968) Amino acid sequence of a common Japanese strain of tobacco mosaic virus. J Mol Biol 35(3): 643–646 Ohno, T., M. Aoyagi, Yamanashi, Y (1984) Nucleotide sequence of the tobacco mosaic virus (tomato strain) genome and comparison with the common strain genome. J Biochem. 96: 1915–1923. (PPTX 380 kb)
13562_2015_334_MOESM5_ESM.pptx (56 kb)
Fig. S5 Detection of Tobacco mosaic virus and Tomato mosaic virus in Xanthi nc and Bright yellow, respectively. A, detection of Tobacco mosaic virus (TMV) in inoculated Xanthi nc. TMV-OM (Nozu et al. 1968) was used as inoculum. B, detection of Tomato mosaic virus (ToMV-L) (Ohno et al. 1984) in Bright yellow. ToMV-L was used as inoculum. Detached leaves were inoculated with a suspension of virus (10 μg/ml) and the accumulation of virus was then tested 10 days after inoculation by ELISA. Mock represents non-transgenic leaf after inoculation only with buffer. Here, SB, scion (before grafting) and GS, grafted scion (after grafting). (PPTX 55 kb)
13562_2015_334_MOESM6_ESM.pptx (64 kb)
Table S1 (PPTX 64 kb)

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Copyright information

© Society for Plant Biochemistry and Biotechnology 2015

Authors and Affiliations

  • Md. Emran Ali
    • 1
    • 2
    Email author
  • Sumyya Waliullah
    • 1
  • Kappei Kobayashi
    • 1
  • Takashi Yaeno
    • 1
  • Naoto Yamaoka
    • 1
  • Masamichi Nishiguchi
    • 1
    Email author
  1. 1.Faculty of AgricultureEhime UniversityMatsuyamaJapan
  2. 2.Department of Plant PathologyThe Ohio State UniversityWoosterUSA

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