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.
Highlights
White pine blister rust (WPBR) caused by Cronartium ribicola (Cri) J.C
A total of 102 and 161 million 100-bp RNA-seq PE reads were generated from RNA prepared from vcr2 aeciospores and cankered western white pine (WWP) stems with Cr2/− genotypes, respectively, (Supplementary Table S1)
Because infected host tissues contained a mixture of mRNAs expressed by both C. ribicola mycelia and WWP cells, transcriptomes were de-novo assembled for the spore samples and cankered WWP stem samples separately
Summary
White pine blister rust (WPBR) caused by Cronartium ribicola (Cri) J.C. Fisch is the most severe and damaging disease of five-needle pine species (subgenus Strobus) world-wide. Fisch is the most severe and damaging disease of five-needle pine species (subgenus Strobus) world-wide This fungal pathogen depends on pine trees as its primary hosts and Ribes as the principal alternate hosts to complete its life-cycle (Geils et al, 2010). Cronartium ribicola has a complex life cycle with five spore stages (aeciospores, urediniospores, teliospores, basidiospores, and spermatia), requiring an alternate hosts (mainly species of Ribes in North America, and Pedicularis in some areas of Asia) to complete its life cycle over at least 2 years. Don) and other native five-needle pines of all ages across North America and predisposes older trees to secondary attack by insects and other fungi
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