DOES EXPRESSION OF A DEFENSE TRANSGENE ALTER NONTARGET RHIZOSPHERE MICROFLORA?

R.M. Goodman^a, L. Jacobson^a, B. Quirino^a, J. Handelsman^a, and D.A. Joseph^b

^aDepartment of Plant Pathology, and ^bDepartments of Computer Sciences and Mathematics, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706.

SUMMARY

Sensitive methods for assessment and description of microbial communities may contribute to environmental monitoring and risk assessment in various ways. Our interest is in the potential of microbial indicators to assess whether and how transgeic plants expressing a defense gene influence rhizosphere microflora. We are using computer-based sequence analysis to develop an array of oligonucleotides based on phylogenetically meaningful sequences from 16S ribosomal RNA genes (rRNA). The array will be hybridized with labeled rRNA gene sequences extracted and amplified from total rhizosphere microbial communities. The goal is development of an array that will reproducibly detect changes in microbial populations associated with roots of plants expressing defense transgenes. We will determine baseline variability and then through an iterative process refine the hybridization array to assess changes caused by transgene expression.

The transgene we are using is one of the systemic acquired resistance (SAR) related genes, designated SAR 8.2. When SAR 8.2 is expressed as a transgene in tobacco, it confers tolerance to several oomycete diseases, including those caused by Peronospora tabacina, Phytophthora parasitica, and Pythium spp. Our study is focused on SAR 8.2 transgenic potatoes in which the potential for control of the late blight pathogen, P. infestans, is also being evaluated.

SAR is correlated with the expression of a suite of genes, some of which like SAR 8.2 confer resistance to specific pathogens when individually and constitutively expressed as transgenes. Moreover, certain chemicals induce effective expression levels of SAR. Both approaches are under development as crop protection alternatives to pesticides. SAR is thus a scientifically and practically appropriate target for assessment of nontarget effects of defense transgene expression on rhizosphere microflora.

We will report on our progress in developing methods to isolate PCR-amplifiable microbial DNA from rhizospheres, the use of an RNase protection assay to select SAR 8.2-expressing and non-expressing transgenic potatoes for field testing of 16S rRNA-based assessment methods, and the design of our first year field trial.