A Near-Complete Haplotype-Phased Genome of the Dikaryotic Wheat Stripe Rust FungusPuccinia striiformisf. sp.triticiReveals High Interhaplotype Diversity

Abstract

A long-standing biological question is how evolution has shaped the genomic architecture of dikaryotic fungi. To answer this, high-quality genomic resources that enable haplotype comparisons are essential. Short-read genome assemblies for dikaryotic fungi are highly fragmented and lack haplotype-specific information due to the high heterozygosity and repeat content of these genomes. Here, we present a diploid-aware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long reads using the FALCON-Unzip assembler. Transcriptome sequencing data sets were used to infer high-quality gene models and identify virulence genes involved in plant infection referred to as effectors. This represents the most complete Puccinia striiformis f. sp. tritici genome assembly to date (83 Mb, 156 contigs, N50 of 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high interhaplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When we investigated genome features that potentially promote the rapid evolution of virulence, we found that candidate effector genes are spatially associated with conserved genes commonly found in basidiomycetes. Yet, candidate effectors that lack an allelic counterpart are more distant from conserved genes than allelic candidate effectors and are less likely to be evolutionarily conserved within the P. striiformis species complex and Pucciniales. In summary, this haplotype-phased assembly enabled us to discover novel genome features of a dikaryotic plant-pathogenic fungus previously hidden in collapsed and fragmented genome assemblies.

Dissecting the first phased dikaryotic genomes of the wheat rust pathogenPuccinia triticinareveals the mechanisms of somatic exchange in nature

“…For each isolate, we predicted about 2,000 effectors on the primary contigs (Supplementary file 4) and haplotigs ( Table 2; Supplementary file 4), respectively, comprising about 8% of the total proteins in each genome, in line with previous reports for Pt Race1, Pst, and Pca (11,18,19). A majority (57-70%) of the effectors belonged to either synteny or chromosome allele pairs, and only 6-9% of the effectors were singletons ( Table 2).…”

“…In summary, our assemblies are clearly significantly better than previously published genome assemblies, for example the Pt Race1 genome (14,818 scaffolds with N50 of 544 kb) (11) and the recently reported genomes of Pst and Pca based on PacBio long-read sequencing (N50 of 1.5 Mb and 268 kb, respectively) (18,19). To date, the three Pt assemblies reported here exhibit the highest level of contiguity and completeness of any rust fungus genome available, providing a fundamental platform for in-depth investigation of the genome function of this destructive pathogen and, more generally, of dikaryotic fungi evolution in the absence of sexual cycle in nature.…”

“…While these resequencing studies have identified a small list of effector candidates for further validation, a more recent comparative study between an isolate of Pgt (Pgt279) and a derivative (Pgt632) isolate with virulence for Sr50 successfully identified AvrSr50 (17). Recently, third generation long-read sequencing has been used to assemble the first haplotype-phased reference genomes for the dikaryotic rust fungi Pst and Pca, which produced reference genomes with much higher contiguity than those based on short-read sequencing and uncovered novel genome features in a dikaryotic fashion (18,19).…”

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567‐A

“…For example, Ustilago maydis , a hemibiotrophic fungal pathogen, secretes the effector Cmu1, affecting salicylic acid (SA) biosynthesis in plants to inhibit SA‐mediated immunity (Djamei et al , ). In contrast with the relatively small set of effectors produced by individual species of bacterial pathogens, whole‐genome sequencing of biotrophic and hemibiotrophic fungi has identified hundreds of potential effectors (Dean et al , ; Duplessis et al , ; Kamper et al , ; Miller et al , ; Nemri et al , ; Pedersen et al , ; Plissonneau et al , ; Schwessinger et al , ; Spanu et al , ; Yoshida et al , ). Functional characterization of fungal effectors is challenging, mainly because fungal effector mutants often display no associated phenotype, either as a result of functional redundancy or of difficulty in accurately measuring small changes in phenotype (Selin et al , ).…”

Chromium Sequencing: The Doors Open for Genomics of Obligate Plant Pathogens

“…For example, PacBio sequencing was recently tested to assemble and phase dikaryotic genomes of two rust fungi [17,18]. This research showed that long-read sequencing could resolve challenges for genome assembly of dikaryotic taxa.…”