Title: Applications of Suppression Subtractive Hybridization (SSH) in Identifying differentially expressed transcripts in Ascochyta rabiei Authors
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 28, 2009
Publication Date: June 21, 2009
Citation: White, D., Vandemark, G.J., Chen, W. 2009. Applications of Suppression Subtractive Hybridization (SSH) in Identifying differentially expressed transcripts in Ascochyta rabiei. Meeting Abstract. Technical Abstract: Introduction – Ascochyta rabiei, casual agents of chickpea ascochyta blight, is divided into two pathotypes based on virulence levels. Genetic mechanisms of this phenotypic differentiation are poorly understood. This research is directed toward understanding molecular differences between the two pathotypes during early stages of conidial germination by employing SSH. Materials and Methods – cDNA pools were synthesized from mRNAs from germinating conidia of strains AR19 (pathotype I) and AR628 (pathotype II). Two distinct cDNA subtractions were performed to generate pools of pathotype I and pathotype II-enriched transcripts. Pools of enriched transcript from each pathotype were cloned into the pCR2.1 TOPO vector to create libraries of enriched transcript. In addition, differential display (virtual Northerns) was performed on each pathotype-specific library using probes generated from total or subtracted RNA from each pathotype. Clones from each library that exhibited differential expression were sequenced. Southern hybridizations were performed to determine if each pathotype carried the genetic material for each enriched transcript. Temporal expression of three pathotype II transcripts, a ubiquitin, an ADP-ribosylation factor, and a translation initiation factor, were monitored using RT-PCR over 36 h of early infection on chickpea material. Expression of each transcript was compared between four pathotype I and four pathotype II isolates. Results and Discussion – Three up-regulated transcripts in pathotype I were identified, which are highly homologous to a hypothetical protein, a transaldolase, and an alcohol oxidase of other fungi. Five up-regulated transcripts in pathotype II were identified with homology to fungal gene products for ubiquitin, an ADP-ribosylation factor, a translation initiation factor, and a gene involved in nitrate assimilation. The genomic DNA pathotype I isolates carries the genetic component of each up-regulated pathotype II transcript. Expression over the first 36 h of germination on plant material using RT-PCR showed that pathotype I isolates exhibited high expression of the ubiquitin, ADP-ribosylation factor, and translation initiation factor transcripts during the first 12 hours followed by a rapid decrease between 12 and 24 h. Conversely, pathotype II isolates exhibited low initial expression of the 3 transcripts during the first 12 h and increased dramatically between 12 and 24 h. Results show that the differences between the two pathotypes are not only in levels of expression of certain transcripts during conidial germination, but also in temporal expression during the first 36 hours of early stages of germination. Exploring the roles of the transcripts will help us understand the different pathogenic mechanisms of A. rabiei pathotypes.