Identification of expressed genes during compatible interaction between stripe rust (Puccinia striiformis) and wheat using a cDNA library

Abstract

Background: Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. To establish compatibility with the host, Pst forms special infection structures to invade the plant with minimal damage to host cells. Although compatible interaction between wheat and Pst has been studied using various approaches, research on molecular mechanisms of the interaction is limited. The aim of this study was to develop an EST database of wheat infected by Pst in order to determine transcription profiles of genes involved in compatible wheat-Pst interaction.

Results: Total RNA, extracted from susceptible infected wheat leaves harvested at 3, 5 and 8 days post inoculation (dpi), was used to create a cDNA library, from which 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,160 singletons to give a set of 2,743 unisequences (GenBank accessions: GR302385 to GR305127). The BLASTx program was used to search for homologous genes of the unisequences in the GenBank non-redundant protein database. Of the 2,743 unisequences, 52.8% (the largest category) were highly homologous to plant genes; 16.3% to fungal genes and 30% of no-hit. The functional classification of all ESTs was established based on the database entry giving the best E-value using the Bevan's classification categories. About 50% of the ESTs were significantly homologous to genes encoding proteins with known functions; 20% were similar to genes encoding proteins with unknown functions and 30% did not have significant homology to any sequence in the database. The quantitative real-time PCR (qRT-PCR) analysis determined the transcription profiles and their involvement in the wheat-Pst interaction for seven of the gene.

Conclusion: The cDNA library is useful for identifying the functional genes involved in the wheat-Pst compatible interaction, and established a new database for studying Pst pathogenesis genes and wheat defense genes. The transcription patterns of seven genes were confirmed by the qRT-PCR assay to be differentially expressed in wheat-Pst compatible and incompatible interaction.

Figure 1

A fully susceptible symptom of Puccinia striiformis f. sp. tritici-wheat compatible interaction. The 15 dpi wheat leaves from wheat plants Suwon 11 inoculated with P. striiforms f. sp. tritici race CYR31, which generates a compatible interaction with heavy sporulation from rust uredia.

Figure 2

Frequencies of EST clones derived from a Puccinia striiformis f. sp. tritici-wheat compatible interaction. The number of ESTs is shown above the histograph for each number of occurrences. After sequencing 6,002 clones, 5,793 ESTs with high quality were obtained and clustered into 583 contigs and 2,163 singletons, resulting in 2,746 unique sequences (unisequences).

Figure 3

The functional classification of ESTs from Puccinia striiformis f. sp. tritici-wheat compatible interaction with significant similarities to plant genes in the GenBank database. The homologies of unigenes to plant genes were classified into 13 functional categories based on their cellular roles. About 26% of the ESTs showed significant homology to enzymes involved in energy and metabolism, representing the largest category. 18% ESTs showed homologies to genes of unknown function and hypothetic proteins in the Genbank database.

Figure 4

Quantitative RT-PCR assay for the transcription levels of four cell death suppressor-related proteins in the compatible and incompatible interactions of wheat- P. striiformis f. sp. tritici. RNA derived from wheat-P. striiformis f. sp. tritici. compatible (wheat cultivar Suwon 11 inoculated with CYR31) and incompatible (wheat cultivar Suwon 11 inoculated with CYR23) interactions was extracted from infected wheat leaves harvested at 0, 24, 48, 72 and 120 hpi. The Y ax shows relative expressions as folds of the mean expression level of the mock treatment. The mock values for compatible and incompatible interactions are mean values for all time-points as there was no significant difference among the different time points.

Figure 5

Quantitative RT-PCR assay for the transcription levels of three ESTs with significant homologies to fungal genes in the compatible interactions of wheat- Puccinia striiformis f. sp. tritici and urediniospores. RNA derived from compatible (wheat cultivar Suwon 11 inoculated with CYR31) interaction was extracted from infected wheat leaves harvested at 0, 6, 24, 48, 72, 120, 168, 192 hpi and urediniospores. The Y ax of the figure shows relative expressions of three fungal homologues genes compared with the mean expression level of the mock treatment. The mock values for compatible interactions are mean values for all time-points as there is no significant difference among the different time points. S, urediniospores.