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Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to Lr20 in Phenotype-Paired Puccinia triticina Isolates from Australia.

ABSTRACT: Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, Puccinia triticina (Pt). In the present study, 20 Pt isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for Lr20, were analyzed using whole genome sequencing. Compared to the reference genome of the American Pt isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) per isolate was found and the proportion of heterozygous SNPs was above 87% in the majority of the isolates, demonstrating a high level of polymorphism and a high rate of heterozygosity. From the genome-wide SNPs, a phylogenetic tree was inferred, which consisted of a large clade of 15 isolates representing diverse presumed clonal lineages including 14 closely related isolates and the more diverged isolate 670028, and a small clade of five isolates characterized by lower heterozygosity level. Principle component analysis detected three distinct clusters, corresponding exactly to the two major subsets of the small clade and the large clade comprising all 15 isolates without further separation of isolate 670028. While genome-wide association analysis identified 302 genes harboring at least one SNP associated with Lr20 virulence (p < 0.05), a Wilcoxon rank sum test revealed that 36 and 68 genes had significant (p < 0.05) and marginally significant (p < 0.1) differences in the counts of non-synonymous mutations between Lr20 avirulent and virulent groups, respectively. Twenty of these genes were predicted to have a signal peptide without a transmembrane segment, and hence identified as candidate effector genes corresponding to Lr20. SNP analysis also implicated the potential involvement of epigenetics and small RNA in Pt pathogenicity. Future studies are thus warranted to investigate the biological functions of the candidate effectors as well as the gene regulation mechanisms at epigenetic and post-transcription levels. Our study is the first to integrate phenotype-genotype association with effector prediction in Pt genomes, an approach that may circumvent some of the technical difficulties in working with obligate rust fungi and accelerate avirulence gene identification.

SUBMITTER: Wu JQ

PROVIDER: S-EPMC5298990 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to Lr20 in Phenotype-Paired Puccinia triticina Isolates from Australia.

Wu Jing Qin JQ Sakthikumar Sharadha S Dong Chongmei C Zhang Peng P Cuomo Christina A CA Park Robert F RF

Frontiers in plant science 20170209

Leaf rust is one of the most common and damaging diseases of wheat, and is caused by an obligate biotrophic basidiomycete, Puccinia triticina (Pt). In the present study, 20 Pt isolates from Australia, comprising 10 phenotype-matched pairs with contrasting pathogenicity for Lr20, were analyzed using whole genome sequencing. Compared to the reference genome of the American Pt isolate 1-1 BBBD Race 1, an average of 404,690 single nucleotide polymorphisms (SNPs) pe ...[more]

PMID: 28232843

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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.

Project description:Wheat leaf rust caused by Puccinia triticina is one of the most common and serious diseases in wheat production. The constantly changing pathogens overcome the plant resistance to P. triticina. Plant pathogens secrete effector proteins that alter the structure of the host cell, interfere plant defenses, or modify the physiology of plant cells. Therefore, the identification of effector proteins is critical to reveal the pathogenic mechanism. We used SignalP v4.1, TargetP v1.1, TMHMM v2.0, and EffectorP v2.0 to screen the candidate effector proteins in P. triticina isolates - KHTT, JHKT, and THSN. As a result, a total of 635 candidate effector proteins were obtained. Structural analysis showed that effector proteins were small in size (50AA to 422AA) and of diverse sequences, and the conserved sequential elements or clear common elements were not involved, regardless of their secretion from the pathogen to the host. There were 427 candidate effector proteins that contain more than or equal to 4 cysteine residues, and 339 candidate effector proteins contained the known motifs. Sixteen families, 9 domains, and 53 other known functional types were found in 186 candidate effector proteins using the Pfam search. Three novel motifs were found by MEME. Heterogeneous expression system was performed to verify the functions of 30 candidate effectors by inhibiting the programmed cell death (PCD) induced by BAX (the mouse-apoptotic gene elicitor) on Nicotiana benthamiana. Hypersensitive response (HR) can be induced by the six effectors in the wheat leaf rust resistance near isogenic lines, and this would be shown by the method of transient expression through Agrobacterium tumefaciens infiltration. The quantitative reverse transcription PCR (qRT-PCR) analysis of 14 candidate effector proteins secreted after P. triticina inoculation showed that the tested effectors displayed different expression patterns in different stages, suggesting that they may be involved in the wheat-P. triticina interaction. The results showed that the prediction of P. triticina effector proteins based on transcriptomic analysis and multiple bioinformatics software is effective and more accurate, laying the foundation of revealing the pathogenic mechanism of Pt and controlling disease.

Dataset Information

Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to Lr20 in Phenotype-Paired Puccinia triticina Isolates from Australia.

Publications

Comparative Genomics Integrated with Association Analysis Identifies Candidate Effector Genes Corresponding to <i>Lr20</i> in Phenotype-Paired <i>Puccinia triticina</i> Isolates from Australia.

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