2 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 CP Rp MP CP Rp MP protein-mediated resistance PMR RNA RNA mediated resistancerm

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1 2012 神奈川県農業技術センター研究報告第 155 号 ) Turnip mosaic virus TuMV resistance of transgenic Nicotiana benthamiana plants containing transgenes derived from TuMV genome Ken NOMURA, Hidetoshi UEKUSA and Nobuhiro KITA 1) TuMVCPNIb VPg TuMV-JO CP 864 bpcp bpcp3 NIb bpnib3 VPg 575 bpvpg Nicotiana benthamiana T 1 T 2 TuMV T 1 TuMV TuMV T 1 CP NIb3 7 VPg 3 CP3 2 T 2 CP TuMV sirna TuMV RNA TuMV,,, RNA Summary Key words: TuMV, virus resistance, transgenic, RNA silencing 1)

2 2 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 CP Rp MP CP Rp MP protein-mediated resistance PMR RNA RNA mediated resistancermrlomonossoff 1995 TMVRp 54K 54K 20 % PMR Carr et al 1992 AIMV Rp Palukaitis et al 1997TMV MP 30K WCIMV MP 13K cdna Cooper et al 1995, Beck et al 1994CMV Rp CPMV MP RMR Sijen et al 1996 Potyvirus Y PVY PSbMV NIb Audy et al 1994, Jones et al 1998 TEV VPg Swaney et al 1995 RMR Prins et al 2008, Fuchs and Gonsalves 2008 Potyvirus TuMV Tomlinson 1987 TuMV CP Arabidopsis thaliana TuMV Nomura et al 2004TuMV Nicotiana benthamiana TMV CMV PVY TuMV TuMV CP CP 3 NIb 3 VPg N.benthamiana TuMV TuMV CP NIb VPg TuMV-JO CP 864 bp Nomura et al 2004 CP bp CP3 NIb bp NIb3 VPg 575 bp VPg RT-PCR PCR CP3 CPF-2 GGATCCTGGAATTCCCGATCAA ACCG CPR-1GAGCTCTCATAACCCCTTAACG CCNIb3 NIbF1GGATCCTATTGCGAT

3 2012 神奈川県農業技術センター研究報告第 155 号 GCTGATGGCTC NIbR1 GAGCTCTTACTGGT GATAAACACAAGCTTVPg VPgF1GGATCCATGGCGAAAGGCAAGAGGCAAA G VPgR1GAGCTCTTACTCGTGGTCCACTG GGGACGA pbi121 BamHI SacI RT-PCR TuMV-JO RNA RNA LA PCR TaKaRaRT-PCR Nomura et al 2004 PCR pt7blue NovagenABI3130DNA BamHI SacI pbi121 GUS pbi-cp3 pbi-nib3 pbi-vpg 1 Ditta et al 1980 LBA4404 Nicotiana benthamiana N.benthamiana Horsch et al 1985N.bentahmiana 70 % 1cm MS-NB 1 µg/ml µg/ml MS-NB µg/ml 150 µg/ml MS-NB MS T µg/l MS T 1 TuMV-JO N.benthamiana T 1 T T 3 T 4 TuMV T 0 CP TuMV-CP PCR CP CPU1 5 -TTAACAGACGAGCAAAAGCA-3 CPL1 5 -TCCATCCAAGCCGAACAAAT-3 DNA PCR CP3 NIb3 VPg PCR T 1 MS

4 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 PCR 0.5 g DNA CTAB 0.11 g CTAB 15 ml 1/2 TE 15,000 g 10 15,000 g % 15,000 g 15 DNA 70 %10 RNase TE DNA Beckman DNA 10 µg DNA BamHI DNA 10 µl TE 1 % Bio-RadRoshe DNA DIG PCR Roshe 0.1 %SDS 2SSC 0.2SSC AP DIG Roche 5 15 RNA Easyprep RNA TaKaRa 0.5 g 5 µl RNA MOPS DIG PCR TuMV sirna RNA 0.5 g sirna RNA 50 µl DEPC 10 µl pt7blue CP DIG RNA TuMV-CP Nicotiana benthamiana TuMV N.benthamiana TuMV-CP CP3 20 DNA PCR T 0 T 1 TuMV TuMV-CP Nb-Tu-CP T 1 TuMV CP1CP2 A B C D 2 N.benthamiana (A) TuMV-CP (B~D) TuMV. A, BCD

5 2012 神奈川県農業技術センター研究報告第 155 号 TuMV-CPN.benthamiana T 1 T 2 TuMV CP9CP12CP13CP19 CP20 50% TuMV TuMV 1, 2DTuMV 2 C CP1CP2 CP12 TuMV CP2-R2CP2-R3 CP12-R1CP12-R2 CP1-R1CP1-R2CP1-R3CP2-R1CP12-R3 TuMV-CP3 Nb-Tu-CP3 TuMV Nb-Tu-CP CP3 1CP3 3CP3 4CP3 5CP3 6 CP3 9CP3 10CP3 12 CP T 1 CP3 3 CP3 9 2 CP3 3 CP3 10 CP3 3 T 1 5 T 2 CP3 3-R1R5Nb-Tu-CP TuMV CP3 3-R1 R3/CP3 3-R4, R5 T 1 T 2 TuMV NIb VPg Nicotiana benthamiana TuMV N.benthamiana TuMV NIb3 20

6 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 Nb-Tu-NIb3 T 0 T 1 TuMV 3 T 1 CP TuMV NIb3 3NIb3 7 NIb3 8NIb3 10NIb3 12NIb3 14 NIb ~90 % NIb3 3 NIb3 10 NIb3 3 NIb3 7 T 1 TuMV NIb3 3-R1 R3 NIb3 7-R1R3 T 1 T 2 TuMV VPg6VPg7 VPg9 3 T 1 TuMV TuMV-CP Nicotiana benthamiana Nb-Tu-CP T 1 CP1CP2CP9CP12CP13CP19 CP20 CP CP10 CP11CP14CP15CP16CP17 15 % CP TuMV VPg 20 T 0 Nb-Tu-VPgT 1 TuMV VPg1VPg2VPg6VPg7VPg8 VPg9VPg10 VPg19 8 4VPg3VPgVPg5 VPg % R R R R R R R S S S S S S S 3 TuMV-CP Nicotiana benthamiana T1. 1, CP1; 2, CP2; 3, CP9; 4, CP12; 5, CP13; 6, CP19; 7, CP20; 8, CP10; 9, CP11; 10, CP14; 11, CP15; 12, CP16; 13, CP17; 14, CP18. R S Nb-Tu-CP TuMV

7 2012 神奈川県農業技術センター研究報告第 155 号 RNA T 1 RNA CP1CP2CP9CP12CP13CP19 CP20 mrna CP10CP11CP14CP15 CP16CP17CP18 CP mrna NIb3 2NIb3 4NIb3 6 NIb TuMV-NIb R R R R S S S S TuMV-CP rrna R R R R R R R S S S S S S S rrna 4 TuMV-CP Nicotiana benthamiana T1. 1, CP1; 2, CP2; 3, CP9; 4, CP12; 5, CP13; 6, CP19; 7, CP20; 8, CP10; 9, CP11; 10, CP14; 11, CP15; 12, CP16; 13, CP17; 14, CP18. R S Nb-Tu-CP3 CP3 3-R1CP3 3-R2 CP3 3-R3 CP3 2 CP3 7 CP3 8 T 1 CP 5 6 TuMV-NIb3 Nicotiana benthamiana T1. 1, NIb3 3; 2, NIb3 7; 3, NIb3 8; 4, NIb3 10; 5, NIb3 2; 6, NIb3 4; 7, NIb3 6; 8, NIb3 11. Nb-Tu-VPg VPg1VPg10 T 0 VPg1VPg3 VPg4VPg5 mrna VPg6 VPg7VPg8 VPg10 VPg2VPg TuMV-VP mrna rrna R R R S S S TuMV-CP3 7 TuMV-VPg N.benthamiana T0. 1, VPg1; 2, VPg2; 3, VPg3; 4, VPg4; 5, VPg5; 6, VPg6; 7, VPg7; 8, VPg8; 9, VPg9; 10, VPg10. rrna 5 TuMV-CP3 N.benthamiana T1 T2. 1, CP3 3-R1; 2, CP3 3-R2; 3, CP3 -R3; 4, CP3 2; 5, CP3 7; 6, CP3 8. R S Nb-Tu-NIb T 1 NIb3 3 NIb3 7NIb3 8 NIb3 10 TuMV-CP Nicotiana benthamiana sirna Nb-Tu-CP sirna CP12-R1 T 1 TuMV sirna CP4 T 1 TuMV sirna sirna

8 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 CP10-S1 sirna sirna 9 TuMV-CP Nicotiana benthamiana TuMV sirna. 1, (CP10-S1); 2, CP4:TuMV ; 3, (CP4:TuMV ); 4, (CP12-R1: TuMV ). TuMV CP CP 3 CP3 NIb 3 NIb3 VPg N.benthamiana CP TuMV 7 T 1 N.benthamiana TuMV CP TuMV CP Potyvirus NIb VPg P1 HC-Pro Audy et al 1994, Jones et al 1998, Moreno et al 1998, Maki-Valkama et al 2000, Germundsson et al 2006 CP3 NIb3 VPg CP Nb-Tu-CP %Nb-Tu-NIb 7 35 % Nb-Tu-VPg 15 % TuMV CP mrna Nb-Tu-VPg mrna mrna TuMV mrna transcriptional gene silencing Vaucheret and Fagard 2001CP TuMV RNA sirna Hamilton and Baulcombe 1999 CP NIbVPg TuMV RNA TuMV TEV CP RNA TEV TEV TEV Lindbo et al 1993Dougherty et al 1994

9 2012 神奈川県農業技術センター研究報告第 155 号 TuMV RNA Pang 1997 Tomato spotted wilt virustswv N N.benthamiana bp TSWV bp 236~387 bp GFP TuMV-CP T 2 CP 864 bp 35 % CP3 430 bp CP 864 bp Nb-Tu-CP T 1 TuMV TuMV-CP CP3 NIb3 VPg N.benthamiana 2 TuMV RNA Audy, P., Palukaitis, P., Slack, S. A., and Zaitlin, M Replicase-mediated resistance to potato virus Y in transgenic tobacco plants. Mol. Plant-Microbe Interact. 7: Beck, D. L., van Dolleweerd, C. J., Lough, T. J., Balmori, E., Voot, D. M., Andersen, M. T., O Brien, I. E. W., and Forster, R. L. S Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block. Proc. Natl. Acad. Sci. USA 91: Carr, J. P., Marsh, L. E., Lomonossoff, G. P., Sekiya, M. E., and Zaitlin, M Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein. Mol. Plant-Microbe Interact. 5: Cooper, B., Lapidot, M., Heick, J. A., Dodds, J. A., and Beachy, R. N A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility. Virology 206: Ditta, G., Stanfield, S., Corbin, D., and Helinski, D. R Broad host range DNA cloning system for gram-negative bacteria: Construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: Dougherty, W. G., Lindbo, J.A., Smith, H. A., Parks, T. D., Swaney, S., and Proebsting, W. M RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation. Mol. Plant-Microbe Interact. 7: Fuchs, M., and Gonsalves, D Safety of virus-resistant transgenic plants two decades after their introduction: lessons from realistic field risk assessment studies. Annu. Rev. Phytopathol 45:

10 10 カブモザイクウイルス (TuMV) ゲノム由来の遺伝子を導入した Nicotiana benthamiana における TuMV 抵抗性 Germundsson, A., and Valkonen, J. P. T P1- and VPg-transgenic plants show similar resistance to Potato virus A and may compromise long distance movement of the virus in plant sections expressing RNA silencing-based resistance. Virus Research 116: Hamilton, A. J., and Baulcombe, D. C A species of small antisense RNA in posttranscriptional gene silencing in plants. Science 286: Horsch, R. B., Fry, J. E., Hoffman, N. L., Eichholtz, D., Rogers, S. G., and Fraley, R. T A simple and general method for transferring genes into plants. Science 227: Jones, A. L., Johansen, I. E., Bean, S. J., Bach, I., and Maule, A. J Specificity of resistance to pea seed-borne mosaic potyvirus in transgenic peas expressing the viral replicase (NIb) gene. J. Gen. Virol. 79: Lindbo, J. A., and Dougherty, W. G Plant pathology and RNAi: a brief history. Annu. Rev. Phytopathol. 43: Lomonossoff, G. P Pathogen-derived resistance to plant viruses. Annu. Rev. Phytopathol. 33: Maki-Valkama, T., Valkonen, J. P. T., Kreuze, J. F., and Pehu, E Transgenic resistance to PVY O associated with post-transcriptional silencing of P1 transgene is overcome by PVY N strains that carry highly homologous P1 sequences and recover transgene expression at infection. Mol. Plant-Microbe Interact. 13: Moreno, M., Bernal, J. J., Jimenez, I., and Rodriguez-Cerezo, E Resistance in plants transformed with the P1 or P3 gene of tobacco vein mottling potyvirus. J. Gen. Virol. 79: Nomura, K., Ohshima, K., Anai, T., Uekusa, H., and Kita, N RNA-silencing of the introduced coat protein gene of Turnip mosaic virus confers the broad spectrum resistance in transgenic Arabidopsis. Phytopathology 94: Palukaitis, P., and Zaitlin, M Replicase-mediated resistance to plant virus disease. Adv. Virus Res. 48: Pang, S-Z., Jan, F-J., and Gonsalves, D Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proc. Natl. Acad. Sci. USA 94: Prins, M., Laimer, M., Noris, E., Schubert, J., Wassenegger, M., Tepfer, M Strategies for antiviral resistance in transgenic plants. Mol. Plant. Pathol 9: Sijen, T., Wellink, J., Hiriart, J. B., and van Kammen, A RNA-mediated virus resistance: Role of repeated transgenes and delineation of targeted regions. Plant Cell 8: Swaney, S., Powers, H., Goodwin, J., Silva Rosales, L., and Dougherty, W. G RNA-mediated resistance with nonstructural genes from the tobacco etch virus genome. Mol. Plant-Microbe Interact. 8: Tomlinson, J. A Epidemiology and control of virus diseases of vegetables. Ann. Appl. Biol. 110: Vaucheret, H., and Fagard, M Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17:29-35.