A novel mannose-based selection system for plant transformation using celery mannose-6-phosphate reductase gene.

To investigate its potential application as a selectable marker for plant transformation, the mannitol producing, celery mannose-6-phosphate reductase gene (M6PR) was transformed into Arabidopsis and tobacco using Agrobacterium tumefaciens-mediated transformation. Mannose-tolerance assays in transgenic materials revealed that the M6PR can act as a selectable marker gene in either a positive or a negative selection mode depending on the plant species. For mannose sensitive species, such as Arabidopsis, expression of M6PR enhanced mannose tolerance and provided a positive selection for transgenic seeds. On medium containing 2 g/L mannose, transgenic seeds germinated, whereas wild type (WT) seeds did not. For mannose-tolerant species, expression of M6PR increased mannose sensitivity in tobacco and enabled a negative selection for transgenic leaves and seeds. Mannose at 30 g/L blanched leaf explants from all 29 transgenic tobacco events with M6PR. In contrast, 30 g/L mannose did not inhibit shoot regeneration from leaf explants of WT or transgenic plants with either an antisense M6PR or a plasmid control. Similarly, mannose at 30 g/L inhibited seed germination of transgenic tobacco seeds with M6PR but not that of WT or transgenic tobacco with either the antisense M6PR or the plasmid control. Northern blot confirmed transcripts of the M6PR in transgenic tobacco, and accumulation of mannitol verified activity of the M6PR in tobacco leaves. Either positive or negative selection using the celery M6PR is versatile for plant transformation. Additionally, the celery M6PR is a potential target gene for improving salt-tolerance in plants due to mannitol accumulation.

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes.

BACKGROUND: Agrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of alpha-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here. RESULTS: TFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio. CONCLUSIONS: TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.