Comparative gene expression analysis of susceptible and resistant near-isogenic lines in common wheat infected by Puccinia triticina

Abstract

Gene expression after leaf rust infection was compared in near-isogenic wheat lines differing in the Lr10 leaf rust resistance gene. RNA from susceptible and resistant plants was used for cDNA library construction. In total, 55 008 ESTs were sequenced from the two libraries, then combined and assembled into 14 268 unigenes for further analysis. Of these ESTs, 89% encoded proteins similar to (E value of < or =10(-5)) characterized or annotated proteins from the NCBI non-redundant database representing diverse molecular functions, cellular localization and biological processes based on gene ontology classification. Further, the unigenes were classified into susceptible and resistant classes based on the EST members assembled from the respective libraries. Several genes from the resistant sample (14-3-3 protein, wali5 protein, actin-depolymerization factor and ADP-ribosylation factor) and the susceptible sample (brown plant hopper resistance protein, caffeic acid O-methyltransferase, pathogenesis-related protein and senescence-associated protein) were selected and their differential expression in the resistant and susceptible samples collected at different time points after leaf rust infection was confirmed by RT-PCR analysis. The molecular pathogenicity of leaf rust in wheat was studied and the EST data generated made a foundation for future studies.

Figure 1

EST distribution in each class of contigs.

Figure 2

Venn diagram showing the number of susceptible, resistant and common genes expressed during leaf rust infection in susceptible and resistant samples.

Figure 3

Functional annotation of contig sequences based on GO categorization. Sequences were evaluated for their predicted involvement in a biological process (a), molecular function (b), and cellular localization (c).

Figure 4

Gene expression studies of candidate genes. Representative contigs were selected and analyzed by RT–PCR. Susceptible (Thatcher) and resistant (ThatcherLr10) samples were collected 0, 8, 16, 20 and 48 h after leaf rust infection. Resistant and susceptible samples are shown for their respective genes. Actin was used as the internal control.

Figure 5

Flow chart for EST assembly and analysis.

Figure 6

A simple pathway for leaf rust interaction in wheat. The genes identified in the present study were indicated in bold and italic letter. NBS-LRR, nucleolar binding site-leucine-rich repeats; GST, glutathione S-transferase; PI, proteinase inhibitors; PAL, phenylalanine ammonia-lyase; OASL, O-acetylserine(thiol)lyase; OMT, O-methyltransferase.