Profiling of Gene Expression During Early Stages of A Compatible Plant-Oomycete Interaction via cDNA-AFLP^® Technology

C.B. Lawrence¹, W.T. Bass², J. Kovar³, and J. Qui⁴
1. Plant-Fungal Genomics Laboratory, Dept. of Bioagricultural Sciences and Pest Management,
Colorado State University, Fort Collins, CO 80523-1177
2. THRI, University of Kentucky, Lexington, KY 40546
3. LI-COR Biosciences, Lincoln, NE 68504-1325

Abstract

With the advent of molecular biology, genetic engineering, and now, genomics-based approaches, it may be possible to initiate studies which address questions regarding more complex relationships of plant-microbe interactions such as obligate parasitism. The model system we have been investigating is the Peronospora tabacina-Nicotiana spp. interaction. The obligate Oomycete, P. tabacina causes downy mildew (blue mold) of Nicotiana spp. and is representative of a class of disease-causing organisms of high economic importance to multitudes of agronomic and horticultural crops. We have hypothesized that, due to the very specialized nature of the interaction existing in this plant-pathosystem, differential host gene expression and subsequent metabolic changes may be necessary for the establishment of compatibility. Gene expression profiling was accomplished using an AFLP^® (amplified fragment length polymorphism)-based cDNA technique and allowed for the amplification of gene sequences both up- and down-regulated during the interaction. Axenically grown tobacco plants (c.v. Ky14), highly susceptible to blue mold, were inoculated with P. tabacina sporangiospores.

Total RNA was isolated from leaf tissue both prior to and 6 hours following inoculation and used as templates for cDNA-AFLP^® analysis. AFLP^® fragments from healthy and infected leaves were separated in parallel by gel electrophoresis and detected using NEN Global Edition IR2 DNA Analyzer. Individual target bands were excised from the gel using an optical infrared imaging system. Fragments were re-amplified using PCR, ligated into a T/A cloning vector, and sequenced using LI-COR sequencing technology. Putative identification of sequence was determined using database homology searches (Blastn and Blastp). The possible role of the differential expression of these genes in relation to disease susceptibility are presented.