Transcriptional profiling of sorghum midge defense responses in sorghum using CDNA-AFLP

Abstract

Sorghum midge (Stenodiplosis sorghicola (Coquillett), is the most ubiquitous damaging pest of grain sorghum [Sorghum bicolor (L.) Moench] worldwide. In spite of its economic importance, little is known about the molecular basis of compatible interaction between the pest and sorghum host. Monitoring the transcriptome changes in sorghum in response to developing embryofeeding sorghum midge will provide a solid foundation to understand the genetic mechanisms of the plant defense against a sorghum midge attack. To identify genes that are differentially expressed in sorghum upon sorghum midge attack, a transcriptome analysis using cDNA-AFLP was profiled in the sorghum midge resistant and susceptible genotypes at time intervals 0, 3, 6 and 12 hours. In total, 12 different primer combinations were used, generating cDNA fragments ranging from 50 bp to 500 bp in size. Of approximately 6,720 cDNA fragments generated, 118 were novel, 17 up-regulated and 23 were down regulated genes. These identified cDNAs were cloned, sequenced, and aligned against GenBank databases using National Centre for Biotechnology Information BlastX and BlastN tools. Database analysis predicted that the proteins encoded by these cDNAs are involved in signal transduction, secondary metabolism, oxidative response, photosynthesis, cell maintenance, and other cellular activities. There are also a large number of identified genes with unknown function. Of the 53 reliable cDNA-AFLP transcripts, 4 homologous to genes known to play a role in defense genes were further selected for validation of cDNA-AFLP expression patterns using qRT-PCR analyses. Overall, the profiles of sorghum genes responsive to sorghum midge feeding indicated that defense genes in sorghum were co-ordinately regulated. Collectively, the results suggest that the transcripts differentially expressed during sorghum midge feeding represent candidate genes of sorghum defense response to sorghum midge stress. Maize protease inhibitor, subtilisin/chymotrypsin inhibitor CI-IB, and cinnamoyl-CoA reductase 2 proteins were hypothesized to be vital for enabling the plant to overcome the stresses inflicted by sorghum midge feeding. The results show that cDNA-AFLP is a reliable technique for studying expression patterns of genes involved in the sorghum-sorghum midge interactions. This study provides the first global catalogue of genes expressed during sorghum and sorghum midge infestation, together with their functional annotations. The results will help to elucidate the molecular basis of the attack process and identify genes and chemicals that could help to inhibit the growth of the pest.