Although the project was terminated earlier than originally planned, substantial technical and scientific progress was made towards all three objectives. A range of molecular and genomic approaches were employed to explore how the multinuclear genome architecture of Puccinia striiformis f. sp. tritici (Pst) shapes its evolution and pathogenicity, with several key datasets generated that will continue to support future discoveries.
Objective 1: Define epigenetic modifications of DNA and histones in both haplotypes of a Pst founder isolate of an important global epidemic lineage.
This objective was partially achieved. Using Oxford Nanopore long-read sequencing, I successfully mapped DNA modifications, specifically 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), to the genomes of fifteen historical PstS0 lineage isolates collected between 1950 and 2010 that differed in virulence profiles in addition to a reference PstS0 isolate. The high-quality long reads, coupled with the natural heterozygosity of this lineage, allowed for haplotype-resolved methylation profiling across all isolates. These analyses revealed striking differences in DNA methylation not only between isolates, but also between haplotypes within the same isolate, even in the absence of underlying genetic variation, pointing to a potential role for epigenetic divergence in mediating virulence.
Objective 2: Uncover mechanisms of how modifications change haplotype-specific transcription, RNA modifications, and translation during wheat infection.
This objective was also partially achieved. In collaboration with Bioplatforms Australia, I generated a comprehensive long-read cDNA dataset representing four Pst lineages found in Europe and Australia. Samples covered the full wheat infection time course (4 to 12 days post-inoculation) plus dormant spores, with four biological replicates per timepoint. The long-read transcriptome data provide a unique resource for haplotype-specific expression analysis during host colonization. While not all planned functional analyses were completed, expanding the study to multiple lineages increased the scope and potential impact of the work, enabling comparative investigations of expression dynamics and regulatory differences across diverse genetic backgrounds.
Objective 3: Understand how epigenetic DNA and histone modifications influence mutational profiles and transcript levels by studying a unique collection of asexually evolved Pst isolates spanning 50 years of evolution in the field.
Significant progress was made toward this objective despite the planned activities being scheduled for the project’s return phase. Prior to the formal start of the fellowship, I visited Aarhus University to extract DNA from fifteen carefully selected PstS0 isolates based on sampling date, geographic origin, and virulence profile. Oxford Nanopore sequencing and subsequent genome assembly enabled comparative mutation analyses, revealing a relatively low number of SNPs (~1,000–12,000 per isolate) mostly located in transposable elements and non-coding regions. Notably, some isolates exhibited a haplotype-specific mutation bias, with one nucleus accumulating more mutations than the other. Isolates and haplotypes within the same isolate differed in methylation profiles (see objective 1). Based on these findings, seven isolates were selected for RNA sequencing to correlate genetic and epigenetic variation with transcriptional activity; these RNA samples have been submitted for sequencing and will contribute to future analysis.
In summary, while the early termination of the project limited completion of all planned experimental work, major datasets were generated that advance our understanding of haplotype-specific regulation and evolution in this important plant pathogen. The foundational work completed will support downstream analyses and future publications, with significant potential for improving our broader understanding of genome dynamics and adaptive evolution in multinuclear fungi.
