Fine dissection of limber pine resistance to Cronartium ribicola using targeted sequencing of the NLR family

Background Proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) domains (NLR) make up one of most important resistance (R) families for plants to resist attacks from various pathogens and pests. The available transcriptomes of limber pine (Pinus flexilis) allow us to characterize NLR genes and related resistance gene analogs (RGAs) in host resistance against Cronartium ribicola, the causal fungal pathogen of white pine blister rust (WPBR) on five-needle pines throughout the world. We previously mapped a limber pine major gene locus (Cr4) that confers complete resistance to C. ribicola on the Pinus consensus linkage group 8 (LG-8). However, genetic distribution of NLR genes as well as their divergence between resistant and susceptible alleles are still unknown. Results To identify NLR genes at the Cr4 locus, the present study re-sequenced a total of 480 RGAs using targeted sequencing in a Cr4-segregated seed family. Following a call of single nucleotide polymorphisms (SNPs) and genetic mapping, a total of 541 SNPs from 155 genes were mapped across 12 LGs. Three putative NLR genes were newly mapped in the Cr4 region, including one that co-segregated with Cr4. The tight linkage of NLRs with Cr4-controlled phenotypes was further confirmed by bulked segregation analysis (BSA) using extreme-phenotype genome-wide association study (XP-GWAS) for significance test. Local tandem duplication in the Cr4 region was further supported by syntenic analysis using the sugar pine genome sequence. Significant gene divergences have been observed in the NLR family, revealing that diversifying selection pressures are relatively higher in local duplicated genes. Most genes showed similar expression patterns at low levels, but some were affected by genetic background related to disease resistance. Evidence from fine genetic dissection, evolutionary analysis, and expression profiling suggests that two NLR genes are the most promising candidates for Cr4 against WPBR. Conclusion This study provides fundamental insights into genetic architecture of the Cr4 locus as well as a set of NLR variants for marker-assisted selection in limber pine breeding. Novel NLR genes were identified at the Cr4 locus and the Cr4 candidates will aid deployment of this R gene in combination with other major/minor genes in the limber pine breeding program.

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

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.

Characterization of Cronartium ribicola dsRNAs reveals novel members of the family Totiviridae and viral association with fungal virulence

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.

Whitebark Pine in Crater Lake and Lassen Volcanic National Parks: Assessment of Stand Structure and Condition in a Management and Conservation Perspective

Whitebark pine (Pinus albicaulis. Engelm.) is vulnerable to a number of threats including an introduced pathogen (Cronartium ribicola J.C. Fisch.), epidemic levels of native mountain pine beetle (Dendroctonus ponderosae Hopkins), fire suppression, and climate change. To describe the structure of whitebark pine populations in two national parks in the southern Cascades (Crater Lake, Oregon, USA (CRLA) and Lassen Volcanic, California, USA (LAVO) National Parks), we surveyed trees in 30 × 50 × 50 m plots in both parks. We used these plots to describe the extent of white pine blister rust (the disease caused by Cronartium ribicola), mountain pine beetle occurrence, and to elucidate factors influencing the presence of pests and pathogens, cone production, and canopy kill. In each plot, we recorded data related to tree health, including symptoms of blister rust and mountain pine beetle, and reproductive vigor (cone production). In both parks, encroachment from other species, particularly mountain hemlock (Tsuga mertensiana (Bong.) Carrière), was negatively associated with cone production. In CRLA, water stress was a good predictor of blister rust infection and cone production. For CRLA and LAVO, the presence of mountain pine beetle and blister rust was associated with higher canopy kill for whitebark pine. Lastly, we found evidence for a pest-pathogen interaction, mountain pine beetle attack was greater for trees that showed symptoms of blister rust infection in CRLA. Our results indicate that whitebark pine populations in the southern Cascade Range are experiencing moderate levels of blister rust infection compared with other sites across the species range, and that competition from shade-tolerant species may result in an additional threat to whitebark pine in both parks. We present our findings in the context of park management and situate them in range-wide and regional conservation strategies aimed at the protection and restoration of a declining species.

Identification and Functional Characterization of an Effector Secreted by Cronartium ribicola

Cri-9402 was identified as a protein effector from Cronartium ribicola, based on the effect of its expression on growth of Pseudomonas syringae Psm ES4326 introduced into transiently transformed tobacco leaves and stably transformed Arabidopsis seedlings. In tobacco leaves transiently expressing its coding sequence, growth of P. syringae Psm ES4326 was inhibited. Expression of pathogenesis-related (PR) protein 2 (PR2), PR4a, endochitinase B, hypersensitive-related 201 (HSR201), HSR203J, and proteinase inhibitor 1 was upregulated but expression of PR1, coronatine insensitive 1, and abscisic acid 1 was significantly suppressed. In transformed Arabidopsis seedlings, the effector stimulated growth of P. syringae Psm ES4326; significantly suppressed expression of PR1, PR2, nonexpresser of pathogenesis-related genes 1 (NPR1), NPR3, NPR4, phytoalexin deficient 4, and salicylic acid induction deficient 2; and enhanced expression of plant defensin 1.2 (PDF1.2). The above results showed that the majority of responses to this effector in tobacco leaves were converse to those in transformed Arabidopsis. We could conclude that Cri-9402 promoted disease resistance in tobacco leaves and disease susceptibility in Arabidopsis seedlings. Its transcript was mainly expressed in aeciospores of C. ribicola and was probably involved in production or germination of aeciospores, and it was an effector potentially functioning in white pine–blister rust interactions.