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Transgenic rice plants expressing rice yellow mottle virus coat protein gene N. Kouassi, Plant Biology Division, International Laboratory for Tropical Agricultural Biotechnology, The Scripps Research Institute (ILTAB/ TSRI), MTC7, 10666 North Torrey Pine Road, La Jolla, CA 92037, USA; C. Brugidou, ILTAB Institut français de recherche scientifique pour le développement en coopération (ORSTOM); L. Chen, M. Ngon A. Yassi, and R. N. Beachy, ILTAB/TSRI; C. M. Fauquet, ILTAB-ORSTOM Coat protein-mediated resistance (CPMR) is used to induce resistance caused by the expression of a virus coat protein (CP) gene in transgenic plants (Powell et al 1986). We investigated the use of this strategy to produce transgenic plants resistant to rice yellow mottle virus (RYMV), an important viral disease in Africa. This virus was first reported in Kenya (Bakker 1970). The genome organization was achieved at ILTAB (Ngon A Yassi et al 1994). Transformation experiments using the biolistic method, which involves micro-projectile bombardment of embryogenic calli or immature embryos (Christou et al 1991; Li et al 1993; Sivamani et al 1996; Zhang et al 1996), followed. Four different constructs containing the cDNA sequence of the RYMV coat protein gene were inserted into the ubiquitin promoter-Nos terminator cassette and engineered in different ways to produce the CP (CP+), a truncated coat protein (CP NTS), a RNA sense (mRNA), and a RNA antisense (CP-). The biolistic method was used to introduce these into embryogenic calli of japonica rice variety Taipei 309. We obtained a relatively high transformation efficiency (see table). Cotransformation with various ratios (gene of selection/gene of interest) was achieved using the different CP chimeric plasmids and the plasmid pMON410 carrying the hygromycin resistance gene. After several rounds of selection on hygromycin, the plantlets were transferred to soil. Molecular analysis was performed using DNA extracted from leaves of putative R 0 transgenic plants following the method. We amplified by polymerase chain reaction (PCR) the hph gene and the entire cassette containing the gene of interest (ubiquitin- CPNos= 3000 bp). Southern blot experiments using a CP probe 32 P radiolabeled were also performed and confirmed the integration of the gene in R 0 plants (see figure). We tested 117 transgenic lines and all were found to carry the hph gene, while an average percentage (62%) of independent R 0 lines were positive by both PCR and Southern experiments for the ubiquitin-CP-Nos cassette (see table). Northern blot experiments showed different levels of accumulation of CP mRNA in the transgenic lines. Western

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Page 1: jameslitsinger.files.wordpress.com€¦  · Web viewN. Kouassi, Plant Biology Division, International Laboratory for Tropical Agricultural Biotechnology, ... Kofron M. 1991. Production

Transgenic rice plants expressing rice yellow mottle virus coat protein geneN. Kouassi, Plant Biology Division, International Laboratory for Tropical Agricultural Biotechnology,

The Scripps Research Institute (ILTAB/ TSRI), MTC7, 10666 North Torrey Pine Road, La Jolla, CA 92037, USA;

C. Brugidou, ILTAB Institut français de recherche scientifique pour le développement en coopération (ORSTOM);

L. Chen, M. Ngon A. Yassi, and R. N. Beachy, ILTAB/TSRI;C. M. Fauquet, ILTAB-ORSTOM

Coat protein-mediated resistance (CPMR) is used to induce resistance caused by the expression of a virus coat protein (CP) gene in transgenic plants (Powell et al 1986). We investigated the use of this strategy to produce transgenic plants resistant to rice yellow mottle virus (RYMV), an important viral disease in Africa.

This virus was first reported in Kenya (Bakker 1970). The genome organization was achieved at ILTAB (Ngon A Yassi et al 1994). Transformation experiments using the biolistic method, which involves micro-projectile bombardment of embryogenic calli or immature embryos (Christou et al 1991; Li et al 1993; Sivamani et al 1996; Zhang et al 1996), followed. Four different constructs containing the cDNA sequence of the RYMV coat protein gene were inserted into the ubiquitin promoter-Nos terminator cassette and engineered in different ways to produce the CP (CP+), a truncated coat protein (CP NTS), a RNA sense (mRNA), and a RNA antisense (CP-). The biolistic method was used to introduce these into embryogenic calli of japonica rice variety Taipei 309. We obtained a relatively high transformation efficiency (see table).

Cotransformation with various ratios (gene of selection/gene of interest) was achieved using the different CP chimeric plasmids and the plasmid pMON410 carrying the hygromycin resistance gene. After several rounds of selection on hygromycin, the plantlets were transferred to soil. Molecular analysis was performed using DNA extracted from leaves of putative R 0 transgenic plants following the method. We amplified by polymerase chain reaction (PCR) the hph gene and the entire cassette containing the gene of interest (ubiquitin-CPNos= 3000 bp). Southern blot experiments using a CP probe 32 P radiolabeled were also performed and confirmed the integration of the gene in R 0 plants (see figure).

We tested 117 transgenic lines and all were found to carry the hph gene, while an average percentage (62%) of independent R 0 lines were positive by both PCR and Southern experiments for the ubiquitin-CP-Nos cassette (see table). Northern blot experiments showed different levels of accumulation of CP mRNA in the transgenic lines. Western blot analyses using the RYMV polyclonal antibodies revealed a weak accumulation of the coat protein in some appropriate transgenic plants (from CP+ lines). The level of accumulation of the coat protein and mRNA differed among lines.

Preliminary experiments were done to screen the transgenic plants against the virus. Based on observation of RYMV symptoms, different levels of tolerance for the RYMV virions have been recorded. Further experiments are in progress to quantify the virus replication in transgenic plants by enzyme-linked immunosorbent assay and to select the most resistant lines. In the meantime, variety BG 90-2 (widely used in West Africa and highly susceptible to RYMV) is being transformed for the same purpose with appropriate constructs. Other indica rice varieties cultivated in West Africa, such as Bouak 189 and Jaya, will be transformed with RYMV coat protein.

Cited referencesBakker W. 1970. Rice yellow mottle virus, a mechanically transmitted virus disease of rice in Kenya. Netherlands Journal of Plant Pathology Preliminary experiments were done 76:53-63.

Christou P, Ford TL, Kofron M. 1991. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9:957-962.

Ngon AYassi M, Ritzenthaler C, Brugidou C, Fauquet CM, Beachy RN. 1994. Nucleotide sequence and genome characterization of rice yellow mottle virus RNA. Journal of General Virology 75:249-257.

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Li L, Qu R, Kochko de A, Fauquet C, Beachy RN. 1993. An improved rice transformation system using the biolistic method. Plant Cell Reports 12:250-255.

Powell PA, Nelson RS, De B, Hoffman N, Rodgers SG, Fraley RT, Beachy RN. 1986. Delay of the disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-743.

Sivamani E, Shen P, Opalka N, Beachy RN, Fauquet CM. 1996. Selection of large quantities of embryogenic subcultured calli from Indica rice seeds for production of fertile transgenic plants using the biolistic method. Plant Cell Reports 15:322- 327.

Zhang S, Chen L, Qu R, Marmey P, Beachy RN, and Fauquet CM. 1996. Regeneration of fertile transgenic Indica (Group 1) rice plants following micropojectile transformation of embryogenic suspension culture cells. Plant Cell Reports 15:465-469.

Effi ciency of biolistic transformation and integration of the RYMV-CP gene in regenerated plants (T309).

Plasmid Rice tissue hph -resistant hph -resistant CP transgenic linesconstruct (no. of explants) calli (no.) plants (no.) (PCR, Southern) (%)

CP (+) Embryogenic 78 49 64calli (480)

CPΔNTS Embryogenic 28 19 64calli (100)

CP (-) Cell suspension 118 70 59rnRNA (360)

CP (+) Cell suspension 111 79 62mRNA (360) (360)

See figure below

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Page 4: jameslitsinger.files.wordpress.com€¦  · Web viewN. Kouassi, Plant Biology Division, International Laboratory for Tropical Agricultural Biotechnology, ... Kofron M. 1991. Production

Amplification and detection of the DNA fragment comprising the ubiquitin promoter, the RYMV-CP gene, and the Nos terminator cassette (Ubi-CP-Nos = 3 kbp). a) Agarose gel electrophoresis of DNA fragment amplified by PCR using total DNA extracted from hygromycin-resistant T309 rice leaves. Line 1 = 1 kb ladder used as MW marker; lines 2, 3, 4, 5, 6, 7, 8, 9, 11 = transgenic CP positive plants; line 10 = transgenic CP negative plant; line 12 = nontransgenic TP309 plant. b) Southern analyses of transformants (R 0 generation): detection by radioactive method using a CP fragment as a probe. Total DNA from transgenic plants (T309) were Hin dlll/ Afl lldigested, which releases the 3 kb Ubi-CP-Nos cassette. T = transgenic plants; NT = nontransgenic plant; P = CP chimeric plasmid used for the transformation.

Kouassi N, C Brugidou, L Chen, M Ngon A Yassi, RN Beachy, CM Fauquet. 1997. Transgenic rice plants expressing rice yellow mottle virus coat protein gene. International Rice Research Notes 22 (1) 14-15.