Analysis of enriched transcripts induced during velvetleaf - Colletotrichum coccodes interaction

Abstract

We used suppression subtractive hybridization (SSH) and differential screening (DS) to generate cDNA libraries enriched for up-regulated host and pathogen genes following inoculation of velvetleaf leaves with the mycoherbicide Colletotrichum coccodes. A total of 116 expressed sequence tags (EST), representing preferentially expressed genes, were analyzed, of which 89 sequences were assigned putative functions including transcription and translation, energy, cell growth and maintenance, and oxidative stress and defense. The EST sequences were assembled into 20 contigs and 29 singletons/singlets. Except for contig 26 that had the highest hit to fungal Histone 4, all other sequences had top hits to plant sequences. Genes related to oxidative stress and defense formed the largest category corresponding to nine contigs and three singlets (or 48% of the ESTs). The metallothioneins (MT) type 3 proteins were represented by six contigs, three singletons, and two singlets. We also identified genes for the ethylene response element binding (EREB) protein, WRKY, and bZIP proteins, which are likely to play roles in transcription, as well as genes for proteins such as ascorbate peroxidase and reticuline oxidase. The up-regulation of seven selected genes were validated by quantitative real-time PCR. Relative to uninfected velvetleaf leaves, these genes were significantly induced by the presence of C. coccodes on velvetleaf leaves.

Keywords:

• high-throughput cDNA screening
• SSH
• plant-microbe interaction
• gene expression
• gene up-regulation