Efficient transformation of pinaceous gymnosperm cells by Agrobacterium

Abstract

Agrobacterium tumefaciens and Agrobacteriwn rhizogenes, the causal agents of crown gall disease and hairy root disease, are known for their ability to transfer part of their DNA (the T-DNA) from the bacterial tumor-inducing (Ti) plasmid to the genome of the host plant. Interest has focused on these bacteria because they provide one of the more efficient means of introducing foreign DNA into susceptible plants. Although long recognized as a pathogen of dicotyledonous angiosperms, Agrobacteriwn strains have not generally been regarded as strongly tumorigenic on gymnosperms. Studies were undertaken which identified Agrobacteriwn strains which were both strongly tumongenic and weakly tumorigenic on four pinaceous gymnosperms; A bies procera, Pinusponderosa, Pseudotsuga menziesii and Tsuga heterophylla. Identification of opmes in the tumor tissue confirmed host transformation. Strongly tumongenic bacterial strains were also isolated from native galls found on P. menziesü. Certain native bacterial isolates harbored Ti-plasmid sized plasmids and incited rapid tumor development. Successful plant transformation by Agrobacterium requires the induction and expression of a cascade of bacterial virulence genes. This cascade is initiated when periplasmic or transmembrane regions of the bacterial virA protein interact with certain phenolic compounds originating from the host plant. Because it was not known whether the range in bacterial tumorigenicity observed on conifers was due to differences in strain sensitivity to host phenolic compounds, or was due to other causes, studies were undertaken to identify compounds in P. menziesii extracts capable of inducing expression of the Agrobacterium virulence genes. Coniferin was identified as the major inducing compound in P. menziesii extracts. As a glucoside, coniferin has a novel structure in comparison to other Agrobacterium virulence gene inducers which are aglycones. In a representative set ofAgrobacterium strains, interesting parallels exist between levels of bacterial beta-glucosidase, virulence gene induction by coniferin and tumorigenicity levels on conifer hosts. Further, betaglucosidase activity is enhanced when strongly tumorigenic bacteria are cultured in the presence of coniferin, but not in the presence of coniferyl alcohol, the aglycone.