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Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici.

ABSTRACT: Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is an airborne disease that endangers wheat during its entire growth period. In this study, the Pst134EA_003354 uncharacterized protein (GenBank: XM_047941824.1) of Pst was used as the target sequence, and the primers PS-RPA-F and PS-RPA-R, as well as the probe PS-LF-probe, were designed for recombinase polymerase amplification (RPA) technology. Flow chromatography was combined with the process to establish an RPA detection method for Pst. This method successfully established visual detection within 10 min under a constant temperature of 39 °C, and the detection results were consistent with those of ordinary PCR analysis. However, it only had high specificity for Pst, and the detection limit was 10 fg/μL. In addition, this rapid method successfully detected Pst from wheat leaves during the field incubation period, indicating substantial benefits for applied use. In summary, the RPA detection method established in this study has the favourable characteristics of high efficiency, simple functionality, and rapid and universal practicability, providing a theoretical basis for the early detection and prevention of Pst.

SUBMITTER: Liu Y

PROVIDER: S-EPMC10522615 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici.

Liu Yaoxia Y Hao Jianyun J Guo Qingyun Q Yan Jiahui J Yao Qiang Q

Scientific reports 20230926 1

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is an airborne disease that endangers wheat during its entire growth period. In this study, the Pst134EA_003354 uncharacterized protein (GenBank: XM_047941824.1) of Pst was used as the target sequence, and the primers PS-RPA-F and PS-RPA-R, as well as the probe PS-LF-probe, were designed for recombinase polymerase amplification (RPA) technology. Flow chromatography was combined with the process to establish an RPA detection me ...[more]

PMID: 37752159

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Project description:Stripe rust [caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst)] is a destructive disease of wheat (Triticum aestivum L.) worldwide. Genetic resistance is the preferred method for control and the Yr5 gene, originally identified in Triticum spelta var. album, represents a major resistance (R) gene that confers all-stage resistance to all currently known races of Pst in the United States. To identify transcripts associated with the Yr5-mediated incompatible interaction and the yr5-compatible interaction, the Wheat GeneChip was used to profile the changes occurring in wheat isolines that differed for the presence of the Yr5 gene after inoculation with Pst. This time-course study (6, 12, 24 and 48 h post-inoculation) identified 115 transcripts that were induced during the R-gene-mediated incompatible interaction, and 73 induced during the compatible interaction. Fifty-four transcripts were induced in both interactions and were considered as basal defence transcripts, whilst 61 transcripts were specific to the incompatible interaction [hypersensitive response (HR)-specific transcripts] and 19 were specific to the compatible interaction (biotrophic interaction-specific transcripts). The temporal pattern of transcript accumulation showed a peak at 24 h after infection that may reflect haustorial penetration by Pst at ~16 h. An additional 12 constitutive transcript differences were attributed to the presence of Yr5 after eliminating those considered as incomplete isogenicity. Annotation of the induced transcripts revealed that the presence of Yr5 resulted in a rapid and amplified resistance response involving signalling pathways and defence-related transcripts known to occur during R-gene-mediated responses, including protein kinase signalling and the production of reactive oxygen species leading to a hypersensitive response. Basal defence also involved substantial induction of many defence-related transcripts but the lack of R-gene signalling resulted in weaker response.

Project description:BackgroundPuccinia 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.ResultsA 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.ConclusionGenes 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.

Project description:Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungus that causes extensive damage in wheat. The pathogen is now known to be a heteroecious fungus with an intricate life cycle containing sexual and asexual stages. Orthologues of the STE12 transcription factor that regulate mating and filamentation in Saccharomyces cerevisiae, as well as virulence in other fungi, have been extensively described. Because reliable transformation and gene disruption methods are lacking for Pst, knowledge about the function of its STE12 orthologue is limited. In this study, we identified a putative orthologue of STE12 from Pst in haustoria-enriched transcripts and designated it as PstSTE12. The gene encodes a protein of 879 amino acids containing three helices in the homeodomain, conserved phenylalanine and tryptophan sites, and two C2 /H2 -Zn2+ finger domains. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that the expression of PstSTE12 was highly induced during the early infection stages and peaked during haustorium formation and the pycniospore stage in the aecial host barberry. Subcellular localization assays indicated that PstSTE12 is localized in the nucleus and functions as a transcriptional activator. Yeast one-hybrid assays revealed that PstSTE12 exhibits transcriptional activity, and that its C-terminus is necessary for the activation of transcription. PstSTE12 complemented the mating defect in an α ste12 mutant of S. cerevisiae. In addition, it partially complemented the defects of the Magnaporthe oryzae mst12 mutant in plant infection. Knocking down PstSTE12 via host-induced gene silencing (HIGS) mediated by Barley stripe mosaic virus (BSMV) resulted in a substantial reduction in the growth and spread of hyphae in Pst and weakened the virulence of Pst on wheat. Our results suggest that PstSTE12 probably acts at an intersection participating in the invasion and mating processes of Pst, and provide new insights into the comprehension of the variation of virulence in cereal rust fungi.

Dataset Information

Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici.

Publications

Establishment of a recombinase polymerase amplification detection method for Puccinia striiformis f. sp. tritici.

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