Agrobacterium-mediated viral vector-amplified transient gene expression in Nicotiana glutinosa plant tissue culture.

A viral vector based on the bean yellow dwarf virus was investigated for its potential to increase transient gene expression. An intron-containing GUS reporter gene and the cis-acting viral regulatory elements were incorporated in the viral vector and could be complemented by the viral replication-associated proteins provided on a secondary vector. All vectors were delivered to Nicotiana glutinosa plant cell suspension or hairy root cultures via auxotrophic Agrobacterium tumefaciens. Cell culture generated greater yield of reporter gene expression than did root culture, as a result of the limitation imposed on roots to express the protein only in surface tissue containing actively dividing cells. Reporter gene expression increased for cell culture when the reporter gene construct was co-delivered with the construct supplying both viral replication associated proteins (REP and REPA); gene expression decreased when the construct supplying only the viral REP protein was co-delivered. Reporter protein expression increased from 0.091% for the reporter construct alone to 0.22% total soluble protein (% TSP) when the viral Rep-supplying vector was co-delivered with the reporter gene construct. Reporter protein was generated 3 days after the initiation of bacterial co-culture, providing for rapid generation of heterologous protein in cell culture.

Development of auxotrophic agrobacterium tumefaciens for gene transfer in plant tissue culture.

Auxotrophic strains of Agrobacterium tumefaciens were generated for use in liquid co-culture with plant tissue for transient gene expression. Twenty-one auxotrophs were recovered from 1,900 tetracycline-resistant insertional mutants generated with a suicide vector transposon mutagenesis system. Twelve of these auxotrophs were characterized on a nutrient matrix. Isolates were screened for growth in plant cell and root culture, and three auxotrophs were identified that had limited growth: adenine (ade-24), leucine (leu-27), and cysteine (cys-32). Ade-24 displayed poor T-DNA delivery in a transient expression test delivering GUS from a binary vector, while cys-32 displayed the best ability to deliver DNA of these three auxotrophs. The growth yield of cys-32 on cysteine was assessed to provide a quantitative basis for co-culture nutrient supplementation. The utility of cys-32 for delivering T-DNA to plant tissues is demonstrated, where an 85-fold enhancement in GUS expression over wild-type A. tumefaciens was achieved.