Differential gene expression in incompatible interaction between wheat and stripe rust fungus revealed by cDNA-AFLP and comparison to compatible interaction

Abstract

Background: Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. Due to special features of hexaploid wheat with large and complex genome and difficulties for transformation, and of Pst without sexual reproduction and hard to culture on media, the use of most genetic and molecular techniques in studying genes involved in the wheat-Pst interactions has been largely limited. The objective of this study was to identify transcriptionally regulated genes during an incompatible interaction between wheat and Pst using cDNA-AFLP technique

Results: A total of 52,992 transcript derived fragments (TDFs) were generated with 64 primer pairs and 2,437 (4.6%) of them displayed altered expression patterns after inoculation with 1,787 up-regulated and 650 down-regulated. We obtained reliable sequences (>100 bp) for 255 selected TDFs, of which 113 (44.3%) had putative functions identified. A large group (17.6%) of these genes shared high homology with genes involved in metabolism and photosynthesis; 13.8% to genes with functions related to disease defense and signal transduction; and those in the remaining groups (12.9%) to genes involved in transcription, transport processes, protein metabolism, and cell structure, respectively. Through comparing TDFs identified in the present study for incompatible interaction and those identified in the previous study for compatible interactions, 161 TDFs were shared by both interactions, 94 were expressed specifically in the incompatible interaction, of which the specificity of 43 selected transcripts were determined using quantitative real-time polymerase chain reaction (qRT-PCR). Based on the analyses of homology to genes known to play a role in defense, signal transduction and protein metabolism, 20 TDFs were chosen and their expression patterns revealed by the cDNA-AFLP technique were confirmed using the qRT-PCR analysis.

Conclusion: We uncovered a number of new candidate genes possibly involved in the interactions of wheat and Pst, of which 11 TDFs expressed specifically in the incompatible interaction. Resistance to stripe rust in wheat cv. Suwon11 is executed after penetration has occurred. Moreover, we also found that plant responses in compatible and incompatible interactions are qualitatively similar but quantitatively different soon after stripe rust fungus infection.

Figure 1

Histology in wheat cv. 'Suwon 11' after inoculation with P. striiformis f. sp. tritici pathotype CY23 (incompatible interaction). (A) hypersensitive cell death (HR) was only observed in the mesophyll cells in contact with the haustorial mother cell at few infection sites, 18 hpi. (B) HR could be observed in most of infection sites, 24 hpi. (C) Second hyphae were formed and mesophll cells which around intercellular hyphae showed cell death, 72 hpi. (D) Host cell death and fungal spread inhibit at infection sites, 120 hpi. Bars = 50 μm, SV, substomatal vesicle; IH, infection hypha; HMC, haustorial mother cell; SH, secondary hyphae; NC, necrotic cell.

Figure 2

Expression of wheat (cv. Suwon 11) genes in leaves inoculated with Puccinia striiformis f. sp. tritici (pathotype CY23) transcripts displayed by cDNA-AFLP. An example showing selective amplification with primers MTT+TAC. Lanes 1~11 are: 6, 12, 18, 24, 36, 48, 72, 96, 120, 144 and 168 hpi, respectively; lane 12: 0 h (control plants of mock inoculation with sterile water near 0 hpi; M = molecular weight marker.

Figure 3

Classification of differentially accumulated transcript derived fragments (TDFs) after inoculation with Puccinia striiformis f. sp. tritici. A total of 255 TDFs were classified based on the BLASTX homology search.

Figure 4

Quantitative real-time PCR (qRT-PCR) analyses of 12 selected genes. Leaf tissues were sampled for both inoculated and mock-inoculated plants at 12, 18, 24, 48, 72 and 120 hpi, as well as mock-inoculated near 0 hpi. Three independent biological replications were performed. Relative gene quantification was calculated by comparative ΔΔCT method. All data were normalized to the 18S rRNA expression level. The mean expression value was calculated for every transcript derived fragment (TDF) with three replications.

Figure 5

Quantitative real-time PCR (qRT-PCR) analyses of 8 selected genes. Leaf tissues were sampled for both inoculated and mock-inoculated plants at 12, 18, 24, 48, 72 and 120 hpi, as well as mock-inoculated near 0 hpi. Three independent biological replications were performed. Relative gene quantification was calculated by comparative ΔΔCT method. All data were normalized to the 18S rRNA expression level. The mean expression value was calculated for every transcript derived fragment (TDF) with three replications.