1.12.23
THE POSSIBLE MECHANISM OF RESISTANCE IN A TRANSGENIC TOBACCO PLANT EXPRESSING A VIRAL REPLICON

M SUZUKI1 , C MASUTA2, Y TAKANAMI3, S KUWATA4 and T HI BI1

1Department of Agricultural and Environmental Biology, University of Tokyo, Tokyo113-8657, Japan; 2Department of Agrobiology and Bioresources, Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan; 3Faculty of Agriculture, Kyushu University, Fukuoka 812-81, Japan; 4Plant Breeding and Genetics Research Laboratory, Japan Tobacco Inc., Iwata-gun, Shizuoka 438, Japan

Background and objectives
Cucumber mosaic virus (CMV) possesses a genome consisting of three plus-sense, single-stranded RNA molecules, designated RNA1, RNA2 and RNA3 in decreasing order of molecular weight. The 1a and 2a proteins encoded by RNA1 and RNA2, respectively, are necessary for viral replication. The 3a protein encoded by RNA3 is involved in virus movement, and a subgenomic RNA, RNA4, which is generated from the 3' half of RNA3 serves as a messenger RNA for the viral coat protein (CP).

There are now a number of studies that describe the establishment of CMV resistance in transgenic plants. For example, transgenic plants that express the CMV coat protein gene are often virus resistant, and plants highly resistant to CMV infection can be generated by expressing non-structural genes such as gene 2a. Recent experiments using other viruses suggest that transgenes expressing untranslatable sense RNAs can confer effective resistance; this is called 'RNA-mediated' or 'homology-dependent' resistance [1]. Recently, we reported that the transgenic tobacco expressing both RNA1 and RNA2 of CMV exhibit CMV-resistance [2]. In the present study, the mechanism of this resistance is discussed based on the results of additional experiments.

Materials and methods
Tobacco plants (cv. BY-4) were transformed with either cDNA of RNA1 or RNA2 of CMV Y strain. The Agrobacterium-mediated gene transfer was used with plasmid pBIYRI or pBIYR2 containing full-length cDNA of RNA1 or RNA2, respectively, under the CaMV 358 promoter. Plants transformed with either cDNA of RNA1 or RNA2 were designated V1 and V2, respectively. Plants homozygous for each of the RNAs were generated and crossed to produce V1V2 lines that expressed both RNA1 and RNA2. Then the haploid plants of V1V2 were generated by culture of anthers from V1V2 plants, and the diploid plants were obtained by root tissue culture from them. Finally, the diploid plants and the plants expressing CMV CP (V4) were crossed to produce V1V2V4 plants. All the transgenic plants were assayed for viral resistance by mechanical inoculation with CMV virions or with CMV RNA.

Results and conclusions
An RNase protection assay indicated that RNA1 and RNA2 multiplied in V1V2 (V2V1) plants. V1V2 (V2V1) plants, unlike their parent lines, showed a remarkably high level of resistance to CMV; this resistance was more effective against RNA inoculation than against virion inoculation. Experiments using protoplasts showed that the resistance was expressed at the single cell level. The mechanism for this resistance does not fit the criteria for 'RNA-mediated' resistance. The expression of transgenes and resistance of V1V2V4 plants is under investigation. Possible mechanisms of the observed resistance will be discussed.

References
1. Smith HA, Swaney SL, Parks TD, Wernsman EA, Dougherty WG, 1994. Plant Cell 6, 1441-1453.
2. Suzuki M, Masuta C, Takamani Y, Kuwata S, 1996. FEBS Letters 379, 26-30.