Using cDNA-AFLP and microarray analysis for rapid identification of Diuraphis noxia induced genes from near-isogenic Triticum aestivum lines

Abstract

This is a study of transcriptional gene regulation in wheat (Triticum aestivum, L.) in response to Russian wheat aphid (RWA) (Diuraphis noxia, Kurdjumov) infestation. The Russian wheat aphid feeds on the phloem sap in the leaves of wheat plants, and causes the leaves of susceptible wheat plants to curl. This forms a protective barrier for the RWA from insecticides and natural enemies. Chlorosis also results from the RWA feeding. In cases of high infestation, death of susceptible plants can also occur. Eleven wheat genes that confer resistance to the Russian wheat aphid have been identified, but their mechanism at molecular level is still not clearly understood. Wheat near-isogenic lines (NILs) were used in a genome-wide, transcriptome analysis using cDNA-AFLP technology. RWA-resistant cultivar ‘Tugela DN’ and RWA-susceptible cultivar ‘Tugela’ were infested with the RWA and leaves were collected from the infested plants at different (0-, 1-, 2-, 6-, 12-, 24-, 48- and 120-) hours post infestation. cDNA samples derived from these leaves was then analyzed by cDNA-AFLP which revealed 18 clusters of differential gene regulation between the two NILs. The results of this experiment show that differential regulation of transcripts occur even within the first hour of infestation. All types of regulation were observed within the clusters. Differentially expressed transcript derived fragments (TDFs) that were randomly isolated from PAGE gels and sequenced (41 TDFs) included sequences in the functional groups similar to those observed in the microarray analysis. The functional categories are cell structure and maintenance [protein synthesis (14%), chaperone (2%), protein degradation (2%), transcription factor (5%)]; photosynthesis [sugar metabolism (5%), carbohydrate metabolism (2%), energy related (7%)]; defenserelated [signaling (7%), defense-related (10%)] while the rest did not have any significant homology to any known or characterized proteins. Previous suppressive subtractive hybridization experiments identified transcripts that are differentially expressed in wheat in response to RWA feeding. More transcripts were identified by PCR from cDNA pools derived from RWA-infested plants as having conserved motifs common in pathenogenesis related proteins. The isolated transcripts were used to generate a defense response-biased microarray chip that was used to investigate the regulation of these transcripts during infestation of RWA resistant wheat plants (‘Tugela DN’) in a time trial. Dual hybridization of CyDye labeled probes derived from the induced ‘Tugela DN’ plants to the microarray chips revealed differential regulation of the immobilized transcripts in wheat, at different time points post infestation with the RWA. Statistical analysis of the CyDye intensities on the 380 spots mounted on the cDNA microarray slides showed 29 transcripts to be significantly regulated (P≤0.05) during the time of the experiment. These included ESTs that were grouped into four functional categories, namely cell structure and maintenance (9 ESTs); photosynthesis (8 ESTs); defense-related (4 ESTs) and those with no significant homology found or proteins with unknown function (8 ESTs). Patterns of regulation of these transcripts in all of the functional categories included all types of regulation e.g. mainly down-regulation, mainly up-regulation, and a combination of up-/up-/down-regulation in response to RWA feeding. In conclusion, data obtained utilizing cDNA microarray and cDNA-AFLP analyses in infested wheat suggest that the ability to maintain structures involved in photosynthesis by regulating the relevant transcripts through-out infestation is an important determinant in plant survival during RWA feeding. The timing of regulation is also important as some of the transcripts are also regulated in RWA susceptible ‘Tugela’ plants but not in a timely manner which leads to loss of energy and subsequent death of susceptible plants.