Biology subjects

Rathjen, J.

Publications and source records attributed to Rathjen, J..

11 recordsLinked to original sources

bioRxiv2026

The variable wheat stripe rust effector AvrYr7 evades Yr7 recognition through sequence and expression polymorphisms

Introductory paragraphWheat provides about 20% of total dietary calories worldside1. Wheat diseases, including wheat stripe (yellow) rust, cause billions of dollars in losses each year2. Wheat stripe (yellow) rust is caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst) which is best controlled by fungicide application and disease resistant wheat cultivars3. To-date, there are over 80 catalogued and >10 cloned yellow rust resistance genes (Yr genes)4. Yet our knowledge of corresponding avirulence (Avr) genes lags far behind5-8. The absence of cloned Avrs reflects Psts complex genome and the lack of robust transformation and genetic systems3. Recent advances in generating high-quality genome assemblies and the development of wheat defense assays have addressed these challenges9-11. Here we clone AvrYr7 which is recognized by Yr712. We further identify six additional alleles of AvrYr7 that escape recognition due to non-synonymous genetic variations, transposable element activity, missense mutation, and expression polymorphism. These findings provide critical insights into virulence evolution in one of the worlds most important wheat pathogens.

plant biology↗

bioRxiv2026

A chromosome-scale, haplotype-resolved genome assembly of the barley stripe rust pathogen Puccinia striiformis f. sp. hordei

Puccinia striiformis f. sp. hordei (Psh) causes barley stripe rust, an economically important disease affecting barley production across multiple temperate regions. Unlike its well-studied wheat-infecting relative P. striiformis f. sp. tritici, genomic resources for Psh remain limited, with no fully documented chromosome-scale, haplotype-resolved reference genomes publicly available. Here we present a high-quality, haplotype-resolved genome assembly of the dikaryotic Psh isolate NP85002, generated using Oxford Nanopore long-read sequencing combined with Hi-C chromatin conformation capture. The assembly comprises 18 chromosomes per haplotype (151.3 Mb total) including 33 telomere-to-telomere chromosomes, telomeric repeats at 69/72 chromosome ends, and six internal gaps. The assembly shows high consensus accuracy (QV >72) and strong haplotype separation (97.54% within-haplotype Hi-C contacts). Genome completeness reached 90.1% complete BUSCOs, and a combined lift-over plus ab initio annotation achieved 94.4% complete BUSCOs. Repeat annotation indicates 45% repetitive content. We additionally provide a 102,058 bp mitochondrial genome assembly with 40 annotated genes. This genome resource provides a chromosome-scale framework for comparative and population genomic analyses of barley stripe rust and related cereal rust pathogens.

microbiology↗

bioRxiv2025

A chromosome-scale genome assembly of the flax rust fungus reveals the two unusually large effector proteins, AvrM3 and AvrN

Rust fungi comprise thousands of species many of which cause disease on important crop plants. The flax rust fungus Melampsora lini has been a model species for the genetic dissection of plant immunity since the 1940s, however the highly fragmented and incomplete reference genome has so far hindered progress in effector gene discovery. Here, we generate a fully-phased, chromosome-scale assembly of the two nuclear genomes of M. lini strain CH5, resolving an additional 320 Mbp of sequence compared to the previous short-read assembly. The 482 Mbp dikaryotic genome is at least 79% repetitive with a large proportion ([~]40%) of the genome comprised of young, highly similar transposable elements. The assembly resolves the known effector gene loci some of which carry complex duplications that were collapsed in the previous assembly. Using a genetic map followed by manual correction of gene models, we identify the AvrM3 and AvrN genes which encode unusually large fungal effector proteins and trigger defense responses when co-expressed with the corresponding resistance genes in Nicotiana tabacum. We locate the genes linked to the tetrapolar mating system on chromosomes 4 and 9, but in contrast to the cereal rusts which have one pheromone receptor gene per haplotype, in flax rust three pheromone receptor genes are found with two of them closely linked on one haplotype. Taken together, we show that a high-quality assembly is crucial for resolving complex gene loci and given the increasing number of fungal effectors of large size, the commonly applied criterion for effector candidates as being small proteins as needs to be reconsidered.

microbiology↗

bioRxiv2023

Multiplexed effector screening for recognition by endogenous resistance genes using positive defense reporters in wheat protoplasts

O_LIPlant resistance (R) and pathogen avirulence (Avr) gene interactions play a vital role in pathogen resistance. Efficient molecular screening tools for crops lack far behind their model organism counterparts, yet they are essential to rapidly identify agriculturally important molecular interactions that trigger host resistance. C_LIO_LIHere, we have developed a novel wheat protoplast assay that enables efficient screening of Avr/R interactions at scale. Our assay allows access to the extensive gene pool of phenotypically described R genes because it does not require the overexpression of cloned R genes. It is suitable for multiplexed Avr screening, with interactions tested in pools of up to fifty Avr candidates. C_LIO_LIWe identified Avr/R-induced defense genes to create promoter-luciferase reporter. Then, we combined this with a dual-color ratiometric reporter system that normalizes read-outs accounting for experimental variability and Avr/R-induced cell-death. Moreover, we introduced a self-replicative plasmid reducing the amount of plasmid used in the assay. C_LIO_LIOur assay increases the throughput of Avr candidate screening, accelerating the study of cellular defense signaling and resistance gene identification in wheat. We anticipate that our assay will significantly accelerate Avr identification for many wheat pathogens, leading to improved genome-guided pathogen surveillance and breeding of disease-resistant crops. C_LI

plant biology↗

bioRxiv2021

Inferring species compositions of complex fungal communities from long- and short-read sequence data

BackgroundThe kingdom fungi is crucial for life on earth and is highly diverse. Yet fungi are challenging to characterize. They can be difficult to culture and may be morphologically indistinct in culture. They can have complex genomes of over 1 Gb in size and are still underrepresented in whole genome sequence databases. Overall their description and analysis lags far behind other microbes such as bacteria. At the same time, classification of species via high throughput sequencing without prior purification is increasingly becoming the norm for pathogen detection, microbiome studies, and environmental monitoring. However, standardized procedures for characterizing unknown fungi from complex sequencing data have not yet been established. ResultsWe compared different metagenomics sequencing and analysis strategies for the identification of fungal species. Using two fungal mock communities of 44 phylogenetically diverse species, we compared species classification and community composition analysis pipelines using shotgun metagenomics and amplicon sequencing data generated from both short and long read sequencing technologies. We show that regardless of the sequencing methodology used, the highest accuracy of species identification was achieved by sequence alignment against a fungi-specific database. During the assessment of classification algorithms, we found that applying cut-offs to the query coverage of each read or contig significantly improved the classification accuracy and community composition analysis without significant data loss. ConclusionOverall, our study expands the toolkit for identifying fungi by improving sequence-based fungal classification, and provides a practical guide for the design of metagenomics analyses.

bioinformatics↗

bioRxiv2021

Identification and correction of phase switches with Hi-C data in the Nanopore and HiFi chromosome-scale assemblies of the dikaryotic leaf rust fungus Puccinia triticina

BackgroundMost animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false positive signals. ResultsWe generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome (~0.7% heterozygosity) and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. ConclusionsThis first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

genomics↗

bioRxiv2019

Distinct life histories impact dikaryotic genome evolution in the rust fungus Puccinia striiformis causing stripe rust in wheat

Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici, is a major threat to wheat production world-wide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole genome comparative genomics of two P. striiformis f. sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural and allele levels. At the whole genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates while 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f. sp. tritici are the first steps in understanding the evolution of dikaryotic rust fungi at a whole genome level.

evolutionary biology↗

bioRxiv2018

Pathogen Detection and Microbiome Analysis of Infected Wheat Using a Portable DNA Sequencer

Fungal diseases of plants are responsible for major losses in agriculture, highlighting the need for rapid and accurate identification of plant pathogens. Disease outcomes are often defined not only by the main pathogen but are influenced by diverse microbial communities known as the microbiome at sites of infection. Here we present the first use of whole genome sequencing from a portable DNA sequencing device as a method for detection of fungal pathogens from wheat (Triticum aestivum). The data revealed that our method is robust and applicable to the diagnosis of fungal diseases including wheat stripe rust (caused by Puccinia striiformis f. sp. tritici), septoria tritici blotch (caused by Zymoseptoria tritici) and yellow leaf spot (caused by Pyrenophora tritici repentis). We also identified a bacterial genus Pseudomonas co-present with Puccinia and Zymoseptoria infections but not Pyrenophora infections. One limitation of the method is the over-representation of redundant wheat genome sequences in the sample. This could be addressed by amplicon-based sequencing approaches in future studies. Our work outlines a new approach for the detection of a broad range of plant pathogens and associated microbes using a portable sequencer, providing the basis for future development of a system for on-site disease monitoring.

plant biology↗

bioRxiv2018

A comprehensive toolkit to enable MinION long-read sequencing in any laboratory

Long-read sequencing technologies are transforming our ability to assemble highly complex genomes. Realising their full potential relies crucially on extracting high quality, high molecular weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which potentiates all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time consuming and costly. Here we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimising DNA extractions from a challenging plant tissue as a case study. Following the workflow illustrated we were able to reliably and repeatedly obtain > 8.5 Gb of long read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open-source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls, will be useful to others who plan to establish long-read sequencing in their own laboratories.

genomics↗

bioRxiv2017

A near complete haplotype-phased genome of the dikaryotic wheat stripe rust fungus Puccinia striiformis f. sp. tritici reveals high inter-haplome diversity

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 diploidaware assembly of the wheat stripe rust fungus Puccinia striiformis f. sp. tritici based on long-reads using the FALCON-Unzip assembler. RNA-seq datasets 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 1.5 Mb) and provides phased haplotype information for over 92% of the genome. Comparisons of the phase blocks revealed high inter-haplotype diversity of over 6%. More than 25% of all genes lack a clear allelic counterpart. When investigating 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.\n\nImportanceCurrent representations of eukaryotic microbial genomes are haploid, hiding the genomic diversity intrinsic to diploid and polyploid life forms. This hidden diversity contributes to the organisms evolutionary potential and ability to adapt to stress conditions. Yet it is challenging to provide haplotype-specific information at a whole-genome level. Here, we take advantage of long-read DNA sequencing technology and a tailored-assembly algorithm to disentangle the two haploid genomes of a dikaryotic pathogenic wheat rust fungus. The two genomes display high levels of nucleotide and structural variations, which leads to allelic variation and the presence of genes lacking allelic counterparts. Non-allelic candidate effector genes, which likely encode important pathogenicity factors, display distinct genome localization patterns and are less likely to be evolutionary conserved than those which are present as allelic pairs. This genomic diversity may promote rapid host adaptation and/or be related to the age of the sequenced isolate since last meiosis.

microbiology↗

bioRxiv2017

De novo assembly and phasing of dikaryotic genomes from two isolates of Puccinia coronata f. sp. avenae, the causal agent of oat crown rust

Oat crown rust, caused by the fungus Puccinia coronata f. sp. avenae (Pca), is a devastating disease that impacts worldwide oat production. For much of its life cycle, Pca is dikaryotic, with two separate haploid nuclei that may vary in virulence genotype, highlighting the importance of understanding haplotype diversity in this species. We generated highly contiguous de novo genome assemblies of two Pca isolates, 12SD80 and 12NC29, from long-read sequences. In total, we assembled 603 primary contigs for a total assembly length of 99.16 Mbp for 12SD80 and 777 primary contigs with a total length of 105.25 Mbp for 12NC29, and approximately 52% of each genome was assembled into alternate haplotypes. This revealed structural variation between haplotypes in each isolate equivalent to more than 2% of the genome size, in addition to about 260,000 and 380,000 heterozygous single-nucleotide polymorphisms in 12SD80 and 12NC29, respectively. Transcript-based annotation identified 26,796 and 28,801 coding sequences for isolates 12SD80 and 12NC29, respectively, including about 7,000 allele pairs in haplotype-phased regions. Furthermore, expression profiling revealed clusters of co-expressed secreted effector candidates, and the majority of orthologous effectors between isolates showed conservation of expression patterns. However, a small subset of orthologs showed divergence in expression, which may contribute to differences in virulence between 12SD80 and 12NC29. This study provides the first haplotype-phased reference genome for a dikaryotic rust fungus as a foundation for future studies into virulence mechanisms in Pca.\n\nImportanceDisease management strategies for oat crown rust are challenged by the rapid evolution of Puccinia coronata f. sp. avenae (Pca), which renders resistance genes in oat varieties ineffective. Despite the economic importance of understanding Pca, resources to study the molecular mechanisms underpinning pathogenicity and emergence of new virulence traits are lacking. Such limitations are partly due to the obligate biotrophic lifestyle of Pca as well as the dikaryotic nature of the genome, features that are also shared with other important rust pathogens. This study reports the first release of a haplotype-phased genome assembly for a dikaryotic fungal species and demonstrates the amenability of using emerging technologies to investigate genetic diversity in populations of Pca.

genomics↗