5.3.1
DEVELOPMENT OF CMV COAT PROTEIN, MOVEMENT PROTEIN AND REPLICASE RESISTANCE CONSTRUCTS FOR THE AUSTRALIAN GRAIN INDUSTRY

R YANG1, GI DWYER1, SJ WYLIE1,2 and MGK JONES1

1SABC, Murdoch University, Perth 6150, Western Australia; 2CLIMA, University of Western Australia, Nedlands 6009, Western Australia

Background and objectives
Cucumber mosaic virus (CMV) is one of the most serious virus diseases of narrow-leafed lupin, Lupinus angustifolius, in Australia. There is no known natural resistance to CMV in narrow-leafed lupin germplasm. Integration of virus-encoded gene sequences into the host genome has been shown to provide resistance to pathogenic viruses in a broad range of plants [1, 2]. The exact mechanism underlying resistance is not known; however, it is thought to be active mostly at the level of the transcript, although some evidence has been presented for a role of the expressed protein. We have developed a series of molecular constructs containing various gene sequences from the CMV subgroup 2 strain LY. The constructs are based on the movement protein (MP), the replicase (Rep) and the coat protein (CP) coding sequences and have been designed to express translatable and untranslatable gene sequences.

Materials and methods
CMV Rep, MP and CP sequences were amplified from genomic RNA by RT-PCR using primers designed from aligned CMV sequences. The PCR products were ligated into the TA cloning vectors pGEM-T and pGEM-T Easy (Promega) and transformed into E. coli JM109. Each of the inserts was sequenced in both directions to ensure that no mutations were incorporated during PCR, that they remained in-frame with the start codon, and that the additional sequences added were present. For the Rep gene, the conserved GDD motif was removed from the cloned insert using an inverse PCR mutagenesis protocol. The finished constructs were transformed into Agrobacterium tumefaciens (strain AGL0) and are currently being used to produce transgenic lupins and tobacco.

Results and conclsions
A total of eight different constructs were developed: (i) the MP gene plus the RNA 3a 5'-untranslated region (UNMP); (ii) the MP gene alone (MP); (iii) the MP gene rendered untranslatable by insertion of a downstream stop codon (MPS); (iv) the N-terminal half of the MP gene plus the RNA 3a 5' untranslated region (TRUNMP); (v) the Rep gene with the conserved GDD motif removed (MUTREP); (vi) the CP gene containing part of the RNA 3b 3' untranslatable region (CP); (vii) the CP gene rendered untranslatable by insertion of a downstream stop codon (CPS); and (viii) the N-terminal 600 bp of the CP gene (TRUNCP).

All of the above genes have been cloned into the binary vector pYR2, a derivative of pPZB101, recently developed in the laboratory. The T-DNA region of this vector comprises two plant expression cassettes. The first cassette contains a chimaeric CaMV 35S-bar-ocs-3' resistance gene for selection. The second cassette contains the transgene under the control of the subterranean clover stunt virus, particle 4 promoter (SC4) and the particle 5 terminator (SC4-insert-SC5). These contructs are now being used to produce transgenic lupins for the Australian grains industry. Six resistance genes, MUTREP, MP, MPS, CP, CPS and TRUNCP, were cloned into a different binary vector, pART27. The T-DNA region of this vector is comprised of two plant expression cassettes. The first cassette contains a chimaeric pnos-nptII-nos resistance gene for selection. The second cassette contains the transgene under the control of the CaMV 35S promoter and the ocs3' terminator (35S-insert-ocs 3'). These contructs are now being used to produce transgenic tobacco plants. The progeny from trangenic tobacco experiments will be used to analyse the basis of resistance.

We thank Murdoch University for providing Mr Rongchang Yang with an Overseas Postgraduate Research Scholarship. We also appreciate the support of the Centre for Legumes in Mediterranean Agriculture (CLIMA) and the Grains Research and Development Corporation (GRDC).

References
1. Carr JP, Zaitlin M, 1993. Seminars in Virology 4, 339-347.
2. Prins M, Goldbach R, 1996. Archives of Virology 141, 2259-2276.