Steve Wylie

Bean yellow mosaic virus (BYMV) is a serious pathogen of cultivated Lupinus species in Western Australia and elsewhere, causing significant crop losses. Natural resistance genes to BYMV have not been found in lupin germplasm. The main aim of this study was to design and construct binary plasmids that could be used to develop pathogen-derived resistance to BYMV in host plants, including lupins. A cDNA fragment corresponding to part of the 6kDa protein gene, the NIa gene, the Nib gene, the coat protein gene, and the 3' untranslated region of a Western Australian isolate, BYMV-MI, was isolated by high fidelity polymerase chain reaction (PCR) and cloned to yield plasmid pDAMl. A fragment of cDNA corresponding to part of the 6kDa protein gene, the entire NIa gene, and the 5' end of the NIb gene of BYMV, was sequenced and determined to be 1446 nucleotides in length. The nucleotide sequence of the coat protein gene was also determined and found to be 819 nucleotides in length. A comparison of the deduced amino acid sequence of the NIa gene of BYMV-MI with that of other BYMV isolates from Australia, Japan and Denmark showed 93.1 % to 97 .9% homology. A novel binary vector, pPZBexp, was developed to facilitate cloning of potyviral gene sequences for expression in plants. Plasmid pPZBexp contains a 35S-bar-ocs3' cassette for Basta herbicide selection of transformants, and a 35S-CMV5'-CaMV3' expression cassette with a Ncol cloning site between the CMV5' and CaMV3' untranslated regions for insertion of virus resistance sequences. NIa gene sequences were modified to have an in-frame initiation codon for translation. Fourteen binary vectors, containing different configurations of the NIa gene, were produced. These include the full-length NIa gene, each of the two domains of the NIa gene (VPg and protease), untranslatable forms of the full-length gene and the domains, and antisense orientations of these constructs. A further two binary vectors, one containing a point mutation in an active site of the protease domain, and another containing a frame-shift mutation at the 5' end of the NIa gene, are in the process of construction. Twelve of these plasmids are based on pPZBexp, and two are based on the binary vector, pTAB 10. These vectors were used to transform Agrobacterium tumefaciens strains AGL0, AGLl and LBA4404 by electroporation. Trifolium subterraneum hypocotyls, and stem and leaf pieces of Nicotiana benthamiana, were cocultivated with Agrobacterium tumefaciens containing one of six of these binary vectors. Following selection on medium containing Basta, fifty nine T. subterraneum explants_ and sixteen N. benthamiana explants containing full-length NIa gene constructs in sense and antisense orientations, or control sequences lacking virus resistance genes, were transferred to a genetic manipulation glasshouse. Fourteen T. subterraneum plants and seven N. benthamiana lines containing a full-length, translatable NIa gene sequence were analysed for the presence of transgenes. Four T. subterraneum plant lines, Ts20.3, Ts20.10, T320.13 and Ts20.14, were shown to contain the NIa and bar genes by PCR and Southern analysis. Cuttings taken from two of these lines, Ts20.3 and Ts20.10, were challenged with BYMV. Symptoms were apparent on control plants 10-14 days postinoculation but three of the transgenic plants, Ts20.3C, Ts20.3E and Ts20.1 0C, remained free of BYMV disease symptoms after three virus inoculations. BYMV could not be detected by ELISA after 31 days in plants Ts20.3C and Ts20.3E, and only in mature leaves of Ts20.10C. However, leaves of transgenic T. subterraneum plants were not resistant to an application of l00mg/1 phosphinothricin (PPT) (the active ingredient of Basta), and they did not express the PAT product of the bar gene despite containing the gene. All of seven N. benthamiana lines tested were shown to be transgenic by PCR, and a PAT assay showed they also expressed the bar gene product. All transgenic N. benthamiana lines tested were tolerant of leaf applications of 100mg/1 PPT, and some plants were tolerant of concentrations of l000mg/1 PPT. Virus resistance testing of these plants is in progress. The results of virus testing of T. subterraneum plants indicate that the binary vectors developed here can confer resistance to BYMV. As a result, several of these binary vectors are now being utilised in lupin transformation programs in the State Agricultural Biotechnology Centre, Murdoch University, and the Centre for Legumes in Mediterranean Agriculture (CLIMA), University of Western Australia.