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Comparative Transcriptomics and RNA-Seq-Based Bulked Segregant Analysis Reveals Genomic Basis Underlying Cronartium ribicola vcr2 Virulence.

ABSTRACT: Breeding programs of five-needle pines have documented both major gene resistance (MGR) and quantitative disease resistance (QDR) to Cronartium ribicola (Cri), a non-native, invasive fungal pathogen causing white pine blister rust (WPBR). WPBR is one of the most deadly forest diseases in North America. However, Cri virulent pathotypes have evolved and can successfully infect and kill trees carrying resistance (R) genes, including vcr2 that overcomes MGR conferred by the western white pine (WWP, Pinus monticola) R gene (Cr2). In the absence of a reference genome, the present study generated a vcr2 reference transcriptome, consisting of about 20,000 transcripts with 1,014 being predicted to encode secreted proteins (SPs). Comparative profiling of transcriptomes and secretomes revealed vcr2 was significantly enriched for several gene ontology (GO) terms relating to oxidation-reduction processes and detoxification, suggesting that multiple molecular mechanisms contribute to pathogenicity of the vcr2 pathotype for its overcoming Cr2. RNA-seq-based bulked segregant analysis (BSR-Seq) revealed genome-wide DNA variations, including about 65,617 single nucleotide polymorphism (SNP) loci in 7,749 polymorphic genes shared by vcr2 and avirulent (Avcr2) pathotypes. An examination of the distribution of minor allele frequency (MAF) uncovered a high level of genomic divergence between vcr2 and Avcr2 pathotypes. By integration of extreme-phenotypic genome-wide association (XP-GWAS) analysis and allele frequency directional difference (AFDD) mapping, we identified a set of vcr2-associated SNPs within functional genes, involved in fungal virulence and other molecular functions. These included six SPs that were top candidate effectors with putative activities of reticuline oxidase, proteins with common in several fungal extracellular membrane (CFEM) domain or ferritin-like domain, polysaccharide lyase, rds1p-like stress responsive protein, and two Cri-specific proteins without annotation. Candidate effectors and vcr2-associated genes provide valuable resources for further deciphering molecular mechanisms of virulence and pathogenicity by functional analysis and the subsequent development of diagnostic tools for monitoring the virulence landscape in the WPBR pathosystems.

SUBMITTER: Liu JJ

PROVIDER: S-EPMC7990074 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Mycoviruses were recently discovered in the white pine blister rust (WPBR) fungus Cronartium ribicola (J.C. Fisch.). Detection and characterization of their double stranded RNA (dsRNA) would facilitate understanding of pathogen virulence and disease pathogenesis in WPBR systems. METHODS:Full-length cDNAs were cloned from the dsRNAs purified from viral-infected C. ribicola, and their cDNA sequences were determined by DNA sequencing. Evolutionary relationships of the dsRNAs with related mycoviruses were determined by phylogenetic analysis. Dynamic distributions of the viral RNAs within samples of their fungal host C. ribicola were investigated by measurement of viral genome prevalence and viral gene expression. RESULTS:In this study we identified and characterized five novel dsRNAs from C. ribicola, designated as Cronartium ribicola totivirus 1-5 (CrTV1 to CrTV5). These dsRNA sequences encode capsid protein and RNA-dependent RNA polymerase with significant homologies to dsRNA viruses of the family Totiviridae. Phylogenetic analysis showed that the CrTVs were grouped into two distinct clades. CrTV2 through CrTV5 clustered within the genus Totivirus. CrTV1 along with a few un-assigned dsRNAs constituted a distinct phyletic clade that is genetically distant from presently known genera in the Totiviridae family, indicating that CrTV1 represents a novel genus in the Totiviridae family. The CrTVs were prevalent in fungal samples obtained from infected western white pine, whitebark pine, and limber pines. Viral RNAs were generally expressed at higher levels during in planta mycelium growth than in aeciospores and urediniospores. CrTV4 was significantly associated with C. ribicola virulent pathotype and specific C. ribicola host tree species, suggesting dsRNAs as potential tools for dissection of pathogenic mechanisms of C. ribicola and diagnosis of C. ribicola pathotypes. CONCLUSION:Phylogenetic and expression analyses of viruses in the WPBR pathogen, C. ribicola, have enchanced our understanding of virus diversity in the family Totiviridae, and provided a potential strategy to utilize pathotype-associated mycoviruses to control fungal forest diseases.

Project description:BackgroundFive-needle pines are important forest species that have been devastated by white pine blister rust (WPBR, caused by Cronartium ribicola) across North America. Currently little transcriptomic and genomic data are available to understand molecular interactions in the WPBR pathosystem.ResultsWe report here RNA-seq analysis results using Illumina deep sequencing of primary needles of western white pine (Pinus monticola) infected with WPBR. De novo gene assembly was used to generate the first P. monticola consensus transcriptome, which contained 39,439 unique transcripts with an average length of 1,303 bp and a total length of 51.4 Mb. About 23,000 P. monticola unigenes produced orthologous hits in the Pinus gene index (PGI) database (BLASTn with E values < e-100) and 6,300 genes were expressed actively (at RPKM ≥ 10) in the healthy tissues. Comparison of transcriptomes from WPBR-susceptible and -resistant genotypes revealed a total of 979 differentially expressed genes (DEGs) with a significant fold change > 1.5 during P. monticola- C. ribicola interactions. Three hundred and ten DEGs were regulated similarly in both susceptible and resistant seedlings and 275 DEGs showed regulatory differences between susceptible and resistant seedlings post infection by C. ribicola. The DEGs up-regulated in resistant seedlings included a set of putative signal receptor genes encoding disease resistance protein homologs, calcineurin B-like (CBL)-interacting protein kinases (CIPK), F-box family proteins (FBP), and abscisic acid (ABA) receptor; transcriptional factor (TF) genes of multiple families; genes homologous to apoptosis-inducing factor (AIF), flowering locus T-like protein (FT), and subtilisin-like protease. DEGs up-regulated in resistant seedlings also included a wide diversity of down-stream genes (encoding enzymes involved in different metabolic pathways, pathogenesis-related -PR proteins of multiple families, and anti-microbial proteins). A large proportion of the down-regulated DEGs were related to photosystems, the metabolic pathways of carbon fixation and flavonoid biosynthesis.ConclusionsThe novel P. monticola transcriptome data provide a basis for future studies of genetic resistance in a non-model, coniferous species. Our global gene expression profiling presents a comprehensive view of transcriptomic regulation in the WPBR pathosystem and yields novel insights on molecular and biochemical mechanisms of disease resistance in conifers.

Project description:Wheat powdery mildew, caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt), is a destructive disease leading to huge yield losses in production. Host resistance can greatly contribute to the control of the disease. To explore potential genes related to the powdery mildew (Pm) resistance, in this study, we used a resistant genotype YD588 to investigate the potential resistance components and profiled its expression in response to powdery mildew infection. Genetic analysis showed that a single dominant gene, tentatively designated PmYD588, conferred resistance to powdery mildew in YD588. Using bulked segregant RNA-Seq (BSR-Seq) and single nucleotide polymorphism (SNP) association analysis, two high-confidence candidate regions were detected in the chromosome arm 2B, spanning 453,752,054-506,356,791 and 584,117,809-664,221,850 bp, respectively. To confirm the candidate region, molecular markers were developed using the BSR-Seq data and mapped PmYD588 to an interval of 4.2 cM by using the markers YTU588-004 and YTU588-008. The physical position was subsequently locked into the interval of 647.1-656.0 Mb, which was different from those of Pm6, Pm33, Pm51, Pm52, Pm63, Pm64, PmQ, PmKN0816, MlZec1, and MlAB10 on the same chromosome arm in its position, suggesting that it is most likely a new Pm gene. To explore the potential regulatory genes of the R gene, 2,973 differentially expressed genes (DEGs) between the parents and bulks were analyzed using gene ontology (GO), clusters of orthologous group (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Based on the data, we selected 23 potential regulated genes in the enriched pathway of plant-pathogen interaction and detected their temporal expression patterns using an additional set of wheat samples and time-course analysis postinoculation with Bgt. As a result, six disease-related genes showed distinctive expression profiles after Bgt invasion and can serve as key candidates for the dissection of resistance mechanisms and improvement of durable resistance to wheat powdery mildew.

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Comparative Transcriptomics and RNA-Seq-Based Bulked Segregant Analysis Reveals Genomic Basis Underlying Cronartium ribicola vcr2 Virulence.

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