AUTHOR=Liu Jun-Jun , Sniezko Richard A. , Zamany Arezoo , Williams Holly , Omendja Kangakola , Kegley Angelia , Savin Douglas P. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.602812

DOI=10.3389/fmicb.2021.602812

ISSN=1664-302X

ABSTRACT=<p>Breeding programs of five-needle pines have documented both major gene resistance (MGR) and quantitative disease resistance (QDR) to <italic>Cronartium ribicola</italic> (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 (<italic>R</italic>) genes, including <italic>vcr2</italic> that overcomes MGR conferred by the western white pine (WWP, <italic>Pinus monticola</italic>) R gene (<italic>Cr2</italic>). In the absence of a reference genome, the present study generated a <italic>vcr2</italic> 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 <italic>vcr2</italic> 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 <italic>vcr2</italic> pathotype for its overcoming <italic>Cr2</italic>. 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 <italic>vcr2</italic> and avirulent (<italic>Avcr2</italic>) pathotypes. An examination of the distribution of minor allele frequency (MAF) uncovered a high level of genomic divergence between <italic>vcr2</italic> and <italic>Avcr2</italic> pathotypes. By integration of extreme-phenotypic genome-wide association (XP-GWAS) analysis and allele frequency directional difference (AFDD) mapping, we identified a set of <italic>vcr2</italic>-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 <italic>vcr2</italic>-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.</p>