Stage-specific gene expression during urediniospore germination in Puccinia striiformis f. sp tritici

Abstract

Background: Puccinia striiformis f. sp. tritici is an obligate biotrophic pathogen that causes leaf stripe rust on wheat. Although it is critical to understand molecular mechanisms of pathogenesis in the wheat stripe rust fungus for developing novel disease management strategies, little is known about its genome and gene functions due to difficulties in molecular studies with this important pathogen. To identify genes expressed during early infection stages, in this study we constructed a cDNA library with RNA isolated from urediniospores of P. striiformis f. sp. tritici germinated for 10 h.

Results: A total of 4798 ESTs were sequenced from the germinated urediniospore library and assembled into 315 contigs and 803 singletons. About 23.9% and 13.3% of the resulting 1118 unisequences were homologous to functionally characterized proteins and hypothetical proteins, respectively. The rest 62.8% unisequences had no significant homologs in GenBank. Several of these ESTs shared significant homology with known fungal pathogenicity or virulence factors, such as HESP767 of the flax rust and PMK1, GAS1, and GAS2 of the rice blast fungus. We selected six ESTs (Ps28, Ps85, Ps87, Ps259, Ps261, and Ps159) for assaying their expression patterns during urediniospore germination and wheat infection by quantitative real-time PCR. All of them had the highest transcript level in germinated urediniospores and a much less transcript level in un-germinated urediniospores and infected wheat tissues (1-7 dpi). The transcript level of Ps159 increased at later infection stages (6-7 dpi). Our data indicated that these genes were highly expressed in germinated urediniospores and may play important roles in fungal-plant interactions during early infection stages in the wheat stripe rust fungus.

Conclusion: Genes expressed in germinated urediniospores of P. striiformis f. sp. tritici were identified by EST analysis. Six of them were confirmed by quantitative real-time PCR assays to be highly expressed in germinated urediniospores.

Figure 1

Urediniospores and germ tubes of P. striiformis f. sp tritici (race CY32) after incubating for 10 h on plastic surface. Solid arrowheads marked germinated and un-germinated urediniospores. Dashed arrowhead indicated the swelling of germ tubes at the tip. Bar = 100 μm.

Figure 2

Classification of the P. striiformis f. sp. tritici uniseqs homologous to proteins of known functions. A total of 267 uniseqs (23.9% of 1118 uniseqs) with significant homologs in GenBank were categorized according to the functions of their homologs. Over 60% of them were involved in primary metabolism (42.59%) and protein synthesis (21.58%).

Figure 3

Assays for the transcript levels of ESTs Ps28, Ps85, Ps159, Ps259, and Ps261 in P. striiformis f. sp. tritici. RNA samples were isolated from urediniospores, germinated urediniospores, or leaves of wheat cultivar Huixianhong inoculated with CY32 and collected at the indicated time points. The expression level of these genes was calculated by the comparative Ct method with the actin gene of P. striiformis f. sp. tritici as the endogenous reference for normalization. Relative quantification was computed with their expression levels in different stages in comparison to that in urediniospores. The average and standard error were calculated from three biological replicates. S, urediniospores; GS, germinated urediniospores; dpi, days post inoculation.

Figure 4

Assays for the transcript level of Ps87 during incompatible interactions. RNA samples were isolated from urediniospores, germinated urediniospores, or leaves of wheat cultivar Shuiyuan 11 inoculated with CY23 and collected at the indicated time points. The expression level of Ps87 was calculated by the comparative Ct method with the actin gene of P. striiformis f. sp. tritici as the endogenous reference for normalization. Relative quantification was computed with its expression levels in different stages in comparison to that in urediniospores. The average and standard error were calculated from three biological replicates. S, urediniospores; GS, germinated urediniospores; dpi, days post inoculation.