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WRKY Transcription Factors Associated With NPR1-Mediated Acquired Resistance in Barley Are Potential Resources to Improve Wheat Resistance to Puccinia triticina.

ABSTRACT: Systemic acquired resistance (SAR) in Arabidopsis is established beyond the initial pathogenic infection or is directly induced by treatment with salicylic acid or its functional analogs (SA/INA/BTH). NPR1 protein and WRKY transcription factors are considered the master regulators of SAR. Our previous study showed that NPR1 homologs in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) regulated the expression of genes encoding pathogenesis-related (PR) proteins during acquired resistance (AR) triggered by Pseudomonas syringae pv. tomato DC3000. In the present examination, AR induced by P. syringae DC3000 was also found to effectively improve wheat resistance to Puccinia triticina (Pt). However, with more complex genomes, genes associated with this SAR-like response in wheat and barley are largely unknown and no specific WRKYs has been reported to be involved in this biological process. In our subsequent analysis, barley transgenic line overexpressing wheat wNPR1 (wNPR1-OE) showed enhanced resistance to Magnaporthe oryzae isolate Guy11, whereas AR to Guy11 was suppressed in a barley transgenic line with knocked-down barley HvNPR1 (HvNPR1-Kd). We performed RNA-seq to reveal the genes that were differentially expressed among these transgenic lines and the wild-type barley plants during the AR. Several PR and BTH-induced (BCI) genes were designated as downstream genes of NPR1. The expression of few WRKYs was significantly associated with NPR1 expression during the AR events. The transient expression of three WRKY genes, including HvWRKY6, HvWRKY40, and HvWRKY70, in wheat leaves by Agrobacterium-mediated infiltration enhanced the resistance to Pt. In conclusion, a profile of genes associated with NPR1-mediated AR in barley was drafted and WRKYs discovered in the current study showed a substantial potential for improving wheat resistance to Pt.

SUBMITTER: Gao J

PROVIDER: S-EPMC6199750 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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WRKY Transcription Factors Associated With NPR1-Mediated Acquired Resistance in Barley Are Potential Resources to Improve Wheat Resistance to Puccinia triticina.

Gao Jing J Bi Weishuai W Li Huanpeng H Wu Jiaojiao J Yu Xiumei X Liu Daqun D Wang Xiaodong X

Frontiers in plant science 20181017

Systemic acquired resistance (SAR) in Arabidopsis is established beyond the initial pathogenic infection or is directly induced by treatment with salicylic acid or its functional analogs (SA/INA/BTH). NPR1 protein and WRKY transcription factors are considered the master regulators of SAR. Our previous study showed that NPR1 homologs in wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) regulated the expression of genes encoding pathogenesis-related (PR) prot ...[more]

PMID: 30386355

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Project description:Inoculation of wheat plants with Puccinia triticina (Pt) spores activates a wide range of host responses. Compatible Pt interaction with susceptible Thatcher plants supports all stages of the pathogen life cycle. Incompatible interaction with TcLr9 activates defense responses including oxidative burst and micronecrotic reactions associated with the pathogen's infection structures and leads to complete termination of pathogen development. These two contrasting host-pathogen interactions were a foundation for transcriptome analysis of incompatible wheat-Pt interaction.A suppression subtractive hybridization (SSH) library was constructed using cDNA from pathogen-inoculated susceptible Thatcher and resistant TcLr9 isogenic lines. cDNA represented steps of wheat-brown rust interactions: spore germination, haustorium mother cell (HMC) formation and micronecrotic reactions. All ESTs were clustered and validated by similarity search to wheat genome using BLASTn and sim4db tools. qRT-PCR was used to determine transcript levels of selected ESTs after inoculation in both lines.Out of 793 isolated cDNA clones, 183 were classified into 152 contigs. 89 cDNA clones and encoded proteins were functionally annotated and assigned to 5 Gene Ontology categories: catalytic activity 48 clones (54 %), binding 32 clones (36 %), transporter activity 6 clones (7 %), structural molecule activity 2 clones (2 %) and molecular transducer activity 1 clone (1 %). Detailed expression profiles of 8 selected clones were analyzed using the same plant-pathogen system. The strongest induction after pathogen infection and the biggest differences between resistant and susceptible interactions were detected for clones encoding wall-associated kinase (GenBank accession number JG969003), receptor with leucine-rich repeat domain (JG968955), putative serine/threonine protein kinase (JG968944), calcium-mediated signaling protein (JG968925) and 14-3-3 protein (JG968969).The SSH library represents transcripts regulated by pathogen infection during compatible and incompatible interactions of wheat with P. triticina. Annotation of selected clones confirms their putative roles in successive steps of plant-pathogen interactions. The transcripts can be categorized as defense-related due to their involvement in either basal defense or resistance through an R-gene mediated reaction. The possible involvement of selected clones in pathogen recognition and pathogen-induced signaling as well as resistance mechanisms such as cell wall enforcement, oxidative burst and micronecrotic reactions is discussed.

Project description:The Canada Western Red Spring wheat (Triticum aestivum L.) cultivars AAC Concord, AAC Prevail, CDC Hughes, Lillian, Glenlea, and elite line BW961 express a spectrum of resistance to leaf rust caused by Puccinia triticina Eriks. This study aimed to identify and map the leaf rust resistance of the cultivars using three doubled haploid populations, AAC Prevail/BW961 (PB), CDC Hughes/AAC Concord (HC), and Lillian/Glenlea (LG). The populations were evaluated for seedling resistance in the greenhouse and adult plant disease response in the field at Morden, MB for 3 years and genotyped with the 90K wheat Infinium iSelect SNP array. Genetic maps were constructed to perform QTL analysis on the seedling and field leaf rust data. A total of three field leaf rust resistance QTL segregated in the PB population, five in the HC, and six in the LG population. In the PB population, BW961 contributed two QTL on chromosomes 2DS and 7DS, and AAC Prevail contributed a QTL on 4AL consistent across trials. Of the five QTL in HC, AAC Concord contributed two QTL on 4AL and 7AL consistent across trials and a QTL on 3DL.1 that provided seedling resistance only. CDC Hughes contributed two QTL on 1DS and 3DL.2. Lillian contributed four QTL significant in at least two of the three trials on 2BS, 4AL, 5AL, and 7AL, and Glenlea two QTL on 4BL and 7BL. The 1DS QTL from CDC Hughes, the 2DS from BW961, the 4AL from the AAC Prevail, AAC Concord, and Lillian, and the 7AL from AAC Concord and Lillian conferred seedling leaf rust resistance. The QTL on 4AL corresponded with Lr30 and was the same across cultivars AAC Prevail, AAC Concord, and Lillian, whereas the 7AL corresponding with LrCen was coincident between AAC Concord and Lillian. The 7DS and 2DS QTL in BW961 corresponded with Lr34 and Lr2a, respectively, and the 1DS QTL in CDC Hughes with Lr21. The QTL identified on 5AL could represent a novel gene. The results of this study will widen our knowledge of leaf rust resistance genes in Canadian wheat and their utilization in resistance breeding.

Project description:The resistance to leaf rust (Lr) caused by Puccinia triticina in wheat (Triticum aestivum L.) has been well studied over the past decades with over 70 Lr genes being mapped on different chromosomes and numerous QTLs (quantitative trait loci) being detected or mapped using DNA markers. Such resistance is often divided into race-specific and race-nonspecific resistance. The race-nonspecific resistance can be further divided into resistance to most or all races of the same pathogen and resistance to multiple pathogens. At the molecular level, these three types of resistance may cover across the whole spectrum of pathogen specificities that are controlled by genes encoding different protein families in wheat. The objective of this study is to predict and analyze genes in three such families: NBS-LRR (nucleotide-binding sites and leucine-rich repeats or NLR), START (Steroidogenic Acute Regulatory protein [STaR] related lipid-transfer) and ABC (ATP-Binding Cassette) transporter. The focus of the analysis is on the patterns of relationships between these protein-coding genes within the gene families and QTLs detected for leaf rust resistance.We predicted 526 ABC, 1117 NLR and 144 START genes in the hexaploid wheat genome through a domain analysis of wheat proteome. Of the 1809 SNPs from leaf rust resistance QTLs in seedling and adult stages of wheat, 126 SNPs were found within coding regions of these genes or their neighborhood (5 Kb upstream from transcription start site [TSS] or downstream from transcription termination site [TTS] of the genes). Forty-three of these SNPs for adult resistance and 18 SNPs for seedling resistance reside within coding or neighboring regions of the ABC genes whereas 14 SNPs for adult resistance and 29 SNPs for seedling resistance reside within coding or neighboring regions of the NLR gene. Moreover, we found 17 nonsynonymous SNPs for adult resistance and five SNPs for seedling resistance in the ABC genes, and five nonsynonymous SNPs for adult resistance and six SNPs for seedling resistance in the NLR genes. Most of these coding SNPs were predicted to alter encoded amino acids and such information may serve as a starting point towards more thorough molecular and functional characterization of the designated Lr genes. Using the primer sequences of 99 known non-SNP markers from leaf rust resistance QTLs, we found candidate genes closely linked to these markers, including Lr34 with distances to its two gene-specific markers being 1212 bases (to cssfr1) and 2189 bases (to cssfr2).This study represents a comprehensive analysis of ABC, NLR and START genes in the hexaploid wheat genome and their physical relationships with QTLs for leaf rust resistance at seedling and adult stages. Our analysis suggests that the ABC (and START) genes are more likely to be co-located with QTLs for race-nonspecific, adult resistance whereas the NLR genes are more likely to be co-located with QTLs for race-specific resistance that would be often expressed at the seedling stage. Though our analysis was hampered by inaccurate or unknown physical positions of numerous QTLs due to the incomplete assembly of the complex hexaploid wheat genome that is currently available, the observed associations between (i) QTLs for race-specific resistance and NLR genes and (ii) QTLs for nonspecific resistance and ABC genes will help discover SNP variants for leaf rust resistance at seedling and adult stages. The genes containing nonsynonymous SNPs are promising candidates that can be investigated in future studies as potential new sources of leaf rust resistance in wheat breeding.

Project description:Leaf rust, caused by Puccinia triticina (Ptr), is a significant disease of spring wheat spread in Kazakhstan. The development of resistant cultivars importantly requires the effective use of leaf rust resistance genes. This study aims to: (i) determine variation in Ptr population using races from the East Kazakhstan, Akmola, and Almaty regions of Kazakhstan; (ii) examine resistance during seedling and adult plant stages; and (iii) identify the sources of Lr resistance genes among the spring wheat collection using molecular markers. Analysis of a mixed population of Ptr identified 25 distinct pathotypes. Analysis of these pathotypes using 16 Thatcher lines that are near-isogenic for leaf rust resistance genes (Lr) showed different virulence patterns, ranging from least virulent "CJF/B" and "JCL/G" to highly virulent "TKT/Q". Most of the pathotypes were avirulent to Lr9, Lr19, Lr24, and Lr25 and virulent to Lr1, Lr2a, Lr3ka, Lr11, and Lr30. The Ptr population in Kazakhstan is diverse, as indicated by the range of virulence observed in five different races analyzed in this study. The number of genotypes showed high levels of seedling resistance to each of the five Ptr races, thus confirming genotypic diversity. Two genotypes, Stepnaya 62 and Omskaya 37, were highly resistant to almost all five tested Ptr pathotypes. Stepnaya 62, Omskaya 37, Avangard, Kazakhstanskaya rannespelaya, and Kazakhstanskaya 25 were identified as the most stable genotypes for seedling resistance. However, most of the varieties from Kazakhstan were susceptible in the seedling stage. Molecular screening of these genotypes showed contrasting differences in the genes frequencies. Among the 30 entries, 22 carried leaf rust resistance gene Lr1, and two had Lr9 and Lr68. Lr10 and Lr28 were found in three and four cultivars, respectively. Lr19 was detected in Omskaya 37. Two single cultivars separately carried Lr26 and Lr34, while Lr37 was not detected in any genotypes within this study. Field evaluation demonstrated that the most frequent Lr1 gene is ineffective. Kazakhstanskaya 19 and Omskaya 37 had the highest number of resistance genes: three and four Lr genes, respectively. Two gene combinations (Lr1, Lr68) were detected in Erythrospermum 35 and Astana. The result obtained may assist breeders in incorporating effective Lr genes into new cultivars and developing cultivars resistant to leaf rust.

Project description:Effectors are considered to be virulence factors secreted by pathogens, which play an important role during host-pathogen interactions. In this study, the candidate effector Pt9226 was cloned from genomic DNA of Puccinia triticina (Pt) pathotype THTT, and there were six exons and five introns in the 877 bp sequence, with the corresponding open reading frame of 447 bp in length, encoding a protein of 148 amino acids. There was only one polymorphic locus of I142V among the six Pt pathotypes analyzed. Bioinformatics analysis showed that Pt9226 had 96.46% homology with the hypothetical putative protein PTTG_26361 (OAV96349.1) in the Pt pathotype BBBD. RT-qPCR analyses showed that the expression of Pt9226 was induced after Pt inoculation, with a peak at 36 hpi, which was 20 times higher than the initial expression at 0 hpi, and another high expression was observed at 96 hpi. No secretory function was detected for the Pt9226-predicted signal peptide. The subcellular localization of Pt9226Δsp-GFP was found to be multiple, localized in the tobacco leaves. Pt9226 could inhibit programmed cell death (PCD) induced by BAX/INF1 in tobacco as well as DC3000-induced PCD in wheat. The transient expression of Pt9226 in 26 wheat near-isogenic lines (NILs) by a bacterial type III secretion system of Pseudomonas fluorescens EtHAn suppressed callose accumulation triggered by Ethan in wheat near-isogenic lines TcLr15, TcLr25, and TcLr30, and it also suppressed the ROS accumulation in TcLr15. RT-qPCR analysis showed that the expression of genes coded for pathogenesis-related protein TaPR1, TaPR2, and thaumatin-like protein TaTLP1, were suppressed, while the expression of PtEF-1α was induced, with 1.6 times at 72 h post inoculation, and TaSOD was induced only at 24 and 48 h compared with the control, when the Pt pathotype THTT was inoculated on a transient expression of Pt9226 in wheat TcLr15. Combining all above, Pt9226 acts as a virulence effector in the interaction between the Pt pathotype THTT and wheat.

Dataset Information

WRKY Transcription Factors Associated With NPR1-Mediated Acquired Resistance in Barley Are Potential Resources to Improve Wheat Resistance to Puccinia triticina.

Publications

<i>WRKY</i> Transcription Factors Associated With <i>NPR1</i>-Mediated Acquired Resistance in Barley Are Potential Resources to Improve Wheat Resistance to <i>Puccinia triticina</i>.

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