ARS | Publication request: Application of subtractive suppression hybridization in studying differentially expressed genes between pathotypes of Ascochyta rabiei.

Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: May 1, 2008
Publication Date: June 1, 2008
Citation: White, D., Chen, W. 2008. Application of subtractive suppression hybridization in studying differentially expressed genes between pathotypes of Ascochyta rabiei.. Phytopathology.97:S170

Technical Abstract: Ascochyta rabiei causes ascochyta blight of chickpea. Two pathotypes have been identified. The two pathotypes are inter-mating, and no neutral molecular markers were found specific for either pathotype. Here we report using subtractive suppressive hybridization in an attempt to identify pathotype-specific transcripts. Conidia of each pathotype are allowed to germinate on an artificial medium amended with chickpea plant extract. Total RNAs were isolated from the germinating conidia. Representative cDNA pools were generated from the mRNA isolated from each pathotype. A subtractive hybridization procedure was used to eliminate cDNAs that are common to both pathgotypes, to produce enriched populations of cDNA that contain rare and unique transcripts for each pathotype. Differential screening showed that many of the enriched libraries contained transcripts that were common to both pathotypes. However, two unique transcripts were isolated from the pathotype I cDNA library. Two additional transcripts were highly expressed in pathotype I relative to pathotype II. Conversely, four transcripts showed significantly higher levels of expression in pathotype II relative to pathotype I. Sequence analysis of these eight differentially expressed transcripts and homology searches matched gene products ranging from general metabolism to previously identified virulence factors in other fungal species. These sequences provide the information necessary to quantitatively study their differential expression between the two pathotypes.