Biology subjects

Schwessinger, B.

Publications and source records attributed to Schwessinger, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Lineage-specific expansion of a potential novel class of resistance genes in Myrtaceae

Genes encoding intracellular nucleotide-binding site leucine-rich repeat (NBS-LRR) receptors represent the largest class of resistance (R) genes in plants, yet their evolutionary trajectory in trees remains poorly understood. Using high-quality long-read genome assemblies from eight Myrtaceae species, we identified 15,792 NBS-encoding genes with threefold variation in gene content across species. To investigate potential decoy domains for effector proteins of pathogens, we parsed the annotated NBS-encoding genes and determined 181 unique non-canonical domains. Notably, we observed frequent integration of Jacalin domains into TIR-NBS proteins. This novel gene family, named TNJ, is hypothesized to represent a new R gene class. Further exploration of TNJ sequence analysis shows up to seven Jacalin domains per protein and forming a monophyletic clade; however, AF3 modelling confirmed only six domains. Conserved residues in the TIR domain and functional motifs within the NB-ARC domain supports TNJs potential role in immune signalling. Most hypervariable sites and positively selected sites detected to be surface-exposed were clustered in the Jacalin region of TNJ, suggesting that surfaces of Jacalin domain may harbour residues determining pathogen recognition specificity. These findings support TNJ as a potentially new class of R genes in Myrtaceae with Jacalin as a replacement for LRR.

evolutionary biology↗

bioRxiv2026

Inter-haplotype inversions and repeat expansion in the sexually deceptive orchid Chiloglottis trapeziformis

Chiloglottis trapeziformis is a sexually deceptive Australian orchid that provides a valuable system for studying orchid genome evolution, structural variation, and the molecular basis of specialized pollination. However, high-quality nuclear genome resources remain scarce for most orchids, particularly Australias diverse terrestrial lineages. To address this gap, we integrated PacBio HiFi, Oxford Nanopore ultra-long reads, and Hi-C chromatin-contact data to generate the first chromosome-scale, haplotype-resolved nuclear genome assembly for an Australian terrestrial orchid, Chiloglottis trapeziformis. Hi-C guided scaffolding resolved two haplotypes into 20 chromosomes each, consistent with the reported karyotype and genome size (2n=40, haplotype sizes of 1.58 Gb and 1.91 Gb). Genome completeness was high for both haplotypes, recovering 95.1% and 95.5% complete BUSCO genes for haplotype 1 and haplotype 2, respectively. De novo repeat annotation revealed a repeat-rich genome (85.79- 88.25% repetitive sequence), dominated by LTR retrotransposons. Evidence-guided annotation identified 16,287 and 16,548 protein-coding genes in Haplotype 1 and Haplotype 2, respectively. Phylogenetically informed comparisons placed C. trapeziformis as sister to Anoectochilus roxburghii among sampled Orchidoideae and showed broad gene-order conservation. Comparing the two haplotypes for structural variation, we identified large inter-haplotype inversions containing functionally annotated genes with detectable RNA expression, with focal examples further supported by local Hi-C contact patterns and breakpoint-level inspection. Inversion-overlapping genes did not show elevated dS relative to collinear background. This assembly and annotation resource provides a foundation for population and conservation genomics, structural and comparative analyses, and genome-enabled hypothesis testing of molecular traits underlying sexual deception in orchids. Significance statementSexually deceptive orchids use remarkable chemical, visual, and tactile mimicry to attract specific pollinators, but the genome resources needed to understand how these complex traits evolved remain scarce. By generating a chromosome-scale, haplotype-resolved genome for Chiloglottis trapeziformis, we provide the first genomic framework for the large and unique Australasian tribe Diurideae. We show extensive structural variation between haplotypes in an otherwise highly collinear genome, with several large inversions potentially impacting expressed genes. The absence of elevated coding divergence in these regions highlights structural variation as a potentially underappreciated driver of orchid genome evolution. This resource fills a major gap for Australian orchids and provides a foundation for linking genome structure with orchid diversification, conservation, and the evolution of sexual deception.

genomics↗

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

Sexual recombination under tetrapolar mating can alter host-specialization boundaries between wheat- and barley-adapted stripe rust lineages

O_LIHost specialization is a major driver of genetic structure in fungal plant pathogens, but it remains unclear whether specialization on different cereal hosts prevents sexual recombination when mating-type compatibility is retained. We addressed this question in stripe rust, caused by Puccinia striiformis, by crossing wheat-adapted P. striiformis f. sp. tritici and barley-adapted P. striiformis f. sp. hordei, two divergent host-adapted forms that share common barberry (Berberis vulgaris) as a sexual host. C_LIO_LIControlled reciprocal crosses on barberry produced 18 aeciospore-derived progeny, demonstrating that wheat- and barley-adapted Puccinia striiformis can undergo sexual recombination despite strong host specialization during asexual infection. Chromosome-scale parental assemblies placed the homeodomain (HD) mating-type locus, containing bW-HD1 and bE-HD2, on chromosome 2 and the pheromone receptor (PR) mating-type locus, containing STE3 and mfa genes, on chromosome 6. HD restriction genotyping showed biparental inheritance in all progeny, with each progeny carrying one HD haplotype from each parent. Together with conservation of PR-associated coding sequences and amplification of STE3-associated markers in progeny, these results are consistent with retention of tetrapolar mating across the two host-adapted lineages. C_LIO_LIHost interaction phenotypes were assessed across wheat and barley differentials, near-isogenic lines and wild relatives. The parental isolates retained contrasting wheat- and barley-restricted profiles, whereas progeny did not reproduce either parental virulence profile, but instead showed recombinant infection patterns, including compatibility with both wheat and barley genotypes. C_LIO_LIThese findings indicate that host specialization in Puccinia striiformis does not necessarily prevent sexual compatibility on a shared alternate host. Together with retention of tetrapolar mating, alternate-host sexual reproduction may provide a route for genetic exchange between host-specialized pathogen populations, enabling recombination to generate new combinations of host-interaction traits when divergent pathogen lineages mate on a shared alternate host. C_LI

microbiology↗

bioRxiv2026

Direct effector recognition by a tandem kinase triggers non-canonical immunity in wheat

Tandem kinase proteins (TKPs) are an important class of disease resistance genes in cereal crops alongside nucleotide-binding (NB-ARC) leucine-rich repeat (LRR) (NLR) receptors. Here, we cloned the wheat (Triticum aestivum) resistance gene Lr41, which encodes a TKP with a C-terminal fusion, and its paired effector gene AvrLr41 from the rust pathogen Puccinia triticina. AvrLr41 is unusually large and highly divergent from most fungal effectors and is directly recognized by Lr41 through its pseudokinase domain. Immune activation mediated by Lr41 require auxiliary helper proteins, including either a minimal coiled-coil (CC) helper protein or its full-length NLR ortholog. These findings reveal a non-canonical immune system in which a kinase-type receptor directly recognizes an effector and recruits either CC or NLR helpers, demonstrating that full-length NLRs are not strictly required for NLR-linked immune execution and expanding the functional repertoire of disease resistance proteins in crops.

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 conserved stripe rust effector elicits variable resistance responses in wheat

Plant resistance (R) and pathogen avirulence (Avr) gene interactions are central to pathogen recognition and disease resistance in crops. Functional characterisation of recognised Avr effectors of Puccinia striiformis f. sp. tritici (Pst) lags other key fungal pathogens of wheat. Here, we used a wheat protoplast-based screen to identify Avr/R interactions via the proxy of effector-induced defence responses in a set of diverse wheat cultivars. We identified an Avr candidate, termed AvrPstB48, that triggers defence responses in 16 out of 24 cultivars tested. AvrPstB48 is hemizygous, and the Pst genome carries four divergent paralogs within a gene cluster. Analysis of these paralogs revealed partial redundancy in their ability to activate wheat defences and enabled us to identify a single amino acid in AvrPstB48 that is necessary but not sufficient for defence activation. Notably, the activation of defence signalling by AvrPstB48 in protoplasts did not directly correlate with disease outcomes. Whole-plant infection assays revealed that some cultivars which exhibited strong defence activation in the protoplast assay are susceptible to the Pst isolate Pst104E137A- from which AvrPstB48 is derived. Comparison of infection dynamics of two wheat cultivars that differ in their AvrPstB48 recognition capacity revealed a delay in disease progression in the recognising cultivar Avocet S compared to the non-recognising cultivar Morocco. While correlative only, our observations, combined with other recent reports, support a recognize-then-suppress model of plant-pathogen interaction where disease outcomes are driven not only by simple Avr/R interactions but also by pathogen effectors that suppress defence signalling downstream of effector recognition.

plant biology↗

bioRxiv2025

Benchmarking fungal species classification using Oxford Nanopore Technologies long-read ITS metabarcodes

The gold standard for fungal classification has long been specimen or culture-based, however, many Fungi display simple morphological characteristics, or are unculturable by current methods. Using DNA metabarcodes, many taxa can be identified from a single sample simultaneously, but annotating these sequences with species-level taxonomy often requires more information than short-read sequences afford. Oxford Nanopore Technologies (ONT) long-read sequencing has achieved species-level resolution in metabarcoding of bacteria and invertebrates, but complex fungal taxonomy and biology has been a hurdle to the application of this technology for Fungi. In this work, we use a mock community of real ONT long-read metabarcodes from 54 fungi from the Dikarya subkingdom to extensively benchmark classification approaches to assign species-level taxonomy. We compare eight classification approaches spanning alignment and k-mer based algorithms, to emerging machine learning methods, and assess the sensitivity, precision, and diversity estimation of each classifier at the species level. Our results indicate that classifiers which determine informed thresholds of sequence similarity based on a provided reference database are not only more accurate at the species level, but more consistent to correct species abundance distributions, and better able to place sequences from unknown species taxonomically closer to their true origin. We demonstrate the power of machine learning classifiers to leverage long-read metabarcodes, and their promise as emerging methods in DNA sequence classification. We present our results in the context of real-world use cases, to demonstrate that species-level taxonomic inference is achievable, precise, and reliable with ONT long-read fungal metabarcodes.

molecular biology↗

bioRxiv2025

Host adaptation and genome evolution of the broad host range fungal rust pathogen, Austropuccinia psidii

Rust diseases on plants are caused by fungi in the order Pucciniales. Typically, rust fungi have narrow host specificity however the pandemic biotype of Austropuccinia psidii has an unusually broad host range causing disease on over 480 myrtaceous species globally. We assembled and analysed a fully phased chromosome-level genome for the pandemic A. psidii and addressed key outstanding questions of its infection biology. Our research revealed a conserved rust fungal karyotype of 18 haploid chromosomes, in line with fungi for distantly related cereal rusts. We observed chromosomal re-assortment between the two nuclei, with one nucleus carrying 19 and the other 17 chromosomes. The synteny of universal single-copy orthologs is mostly maintained with the distantly related rust fungus Puccinia graminis f. sp. tritici. In contrast, nucleotide composition and methylation profiles of A. psidii are distinct compared to rust fungi with smaller genome sizes that have not undergone massive transposable element expansions. Our analysis of mating type loci supports a tetrapolar mating system for A. psidii with a novel finding of expanded numbers of pheromone peptide precursors. We show that infection dynamics of A. psidii are consistent on four different susceptible host species separated by 65 mya of evolution and that transcriptional regulation during infection reveals two distinct waves of gene expression in early and late infection, including allele-specific expression of candidate effectors. Together, these findings enhance the understanding of the genome biology and pathology of A. psidii, while also providing a valuable resource for future research on this serious rust pathogen.

microbiology↗

bioRxiv2025

Long-term surveillance suggests multiple hybridization events by nuclear reassortment and accelerated intercontinental spread of wheat yellow rust

O_LIEvolutionary forces affecting crop pathogens, including hybridization and long-distance dispersal (LDD), may have strong implications for food security and sustainable plant disease control at global scales. However, consolidated evidence is often lacking due to absence of consistent pathogen surveys beyond national capacities. C_LIO_LIOur study documents world-wide connectivity between populations of Puccinia striiformis, causing yellow rust on cereals and grasses, when analysing 3240 pathogen samples collected in 41 countries on six continents from 2009-2023. Our analyses revealed twelve cases of inter-continental spread of Puccinia striiformis, including seven cases with major impact on disease epidemics in recipient areas. C_LIO_LISomatic hybridization by nuclear reassortment between co-existing multi-locus genotypes (MLGs) on a common host was the most plausible mechanism for the emergence of three novel clonal groups of global relevance, first detected in Europe. Subsequently, onwards spread to South America and Australia was observed. Several high-impact incursions from South Asia into East Africa were also observed, including a genotype with a dramatic impact on wheat breeding programs of global relevance. C_LIO_LIOur study stresses an urgent need for coordinated crop pathogens monitoring across borders. Only global efforts will enable prevention and control of pathogens that represent major challenges for food security at regional and global scales. C_LI

evolutionary biology↗

bioRxiv2024

Long-read genomics reveal extensive nuclear-specific evolution and allele-specific expression in a dikaryotic fungus

Phased telomere to telomere (T2T) genome assemblies are revolutionising our understanding of long hidden genome biology "dark matter" such as centromeres, rDNA repeats, inter-haplotype variation, and allele specific expression (ASE). Yet insights into dikaryotic fungi that separate their haploid genomes into distinct nuclei is limited. Here we explore the impact of dikaryotism on the genome biology of a long-term asexual clone of the wheat pathogenic fungus Puccinia striiformis f. sp. tritici. We use Oxford Nanopore (ONT) duplex sequencing combined with Hi-C to generate a T2T nuclear-phased assembly with >99.999% consensus accuracy. We show that this fungus has large regional centromeres enriched in LTR retrotransposons, with a single centromeric dip in methylation that suggests one kinetochore attachment site per chromosomes. The centromeres of chromosomes pairs are most often highly diverse in sequence and kinetochore attachment sites are not always positionally conserved. Each nucleus carries a unique array of rDNAs with >200 copies that harbour nucleus-specific sequence variations. The inter-haplotype diversity between the two nuclear genomes is caused by large-scale structural variations linked to transposable elements. Nanopore long-read cDNA analysis across distinct infection conditions revealed pervasive allele specific expression for nearly 20% of all heterozygous gene pairs. Genes involved in plant infection were significantly enriched in ASE genes which appears to be mediated by elevated CpG gene body methylation of the lower expressed pair. This suggests that epigenetically regulated ASE is likely a previously overlooked mechanism facilitating plant infection. Overall, our study reveals how dikaryotism uniquely shapes key eukaryotic genome features.

genomics↗

bioRxiv2024

Intragenomic variants of a putative effector drive early-stage infection in a broad host-range rust fungus

Rust fungi are pathogens that impact plants of environmental, agricultural, cultural, and economic importance. Their mechanisms of pathogenicity are not well-understood but are likely governed by effectors, secreted proteins that manipulate host cellular processes to facilitate infection and suppress immune responses. We sought to understand how three effector candidates (EFC1, EFC2, and EFC3) expressed in the first stages of Austropuccinia psidii (myrtle rust) infection influence pathogenicity. We experimentally tested gene function through application of double-stranded RNA (dsRNA) and characterised the genomic landscape of putative effectors expressed during infection to assess whether putative effectors are needed for infection, and whether they are under selection pressure. One of the three screened candidates, EFC1, met our criteria of an effector in that it was predicted to be secreted, and was needed to cause but not maintain infection. We identified that this effector belongs to a gene family of intragenomic variants in tandem repeats flanked by transposable elements. Single nucleotide polymorphisms among these variants have signatures of non-neutral selection. This effector has predicted structural homology to a glycosaminoglycan-binding domain and may have a role in pectin or chitin-binding. We hypothesise that intragenomic variability in this family of effector genes facilitates host-range versatility in the A. psidii-Myrtaceae pathosystem.

microbiology↗

bioRxiv2024

High-throughput optimisation of protein secretion in yeast via an engineered biosensor.

Secretion of high value proteins and enzymes is fundamental to the synthetic biology economy; allowing continuous fermentation during production and protein purification without cell lysis. Most eukaryotic protein secretion is encoded by an N-terminal signal peptide; however, the strong impact of signal peptide sequence variation on the secretion efficiency of a given protein is not well defined. Despite high natural signal peptide sequence diversity, most recombinant protein secretion systems employ only a few well characterised signal peptides. Additionally, the selection of promoters and terminators can significantly affect secretion efficiency, yet screening numerous genetic constructs for optimal sequences remains inefficient. Here, we have adapted a yeast G-protein coupled receptor biosensor, to measure the concentration of a peptide tag that is co-secreted with any protein of interest. Protein secretion efficiency can thus be quantified via the induction of a fluorescent reporter that is upregulated downstream of receptor activation. This enables high-throughput screening of over 6000 combinations of promoters, signal peptides and terminators, assembled using one-pot Combinatorial Golden Gate cloning. We demonstrate this biosensor can quickly identify best combinations for secretion and quantify secretion levels. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/594099v2_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@87d612org.highwire.dtl.DTLVardef@1f73d24org.highwire.dtl.DTLVardef@cf35a7org.highwire.dtl.DTLVardef@1cb8a3b_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

Mating-compatibility genes employed as diagnostic markers to identify novel incursions of the myrtle rust pathogen Austropuccinia psidii

Austropuccinia psidii is the causal agent of myrtle rust in over 480 species within the family Myrtaceae. Lineages of A. psidii are structured by host in its native range, and some have success on new-encounter hosts. For example, the pandemic biotype has spread beyond South America, and proliferation of other lineages is an additional risk to biodiversity and industries. Efforts to manage A. psidii incursions, including lineage differentiation, relies on variable microsatellite markers. Testing these markers is time-consuming and complex, particularly on a large scale. We designed a novel diagnostic approach targeting the fungal mating-type HD (homeodomain) transcription factor locus to address these limitations. The HD locus (bW1/2-HD1 and bE1/2-HD2) is highly polymorphic, facilitating clear biological predictions about its inheritance from founding populations. To be considered the same lineage, all four HD alleles must be identical. Our lineage diagnostics relies on PCR amplification of the HD locus in different genotypes of A. psidii followed by amplicon sequencing using Oxford Nanopore Technologies (ONT) and comparative analysis. The lineage-specific assay was validated on four isolates with existing genomes, uncharacterized isolates, and directly from infected leaf material. We reconstructed HD alleles from amplicons and confirmed their sequence identity relative to their reference. Genealogies using HD alleles confirmed the variations at the HD loci among lineages/isolates. Our study establishes a robust diagnostic tool, for differentiating known lineages of A. psidii based biological predictions. This tool holds promise for detecting new pathogen incursions and can be refined for broader applications, including air-sample detection and mixed-isolate infections.

plant biology↗

bioRxiv2023

Exploring polymorphic interspecies structural variants in Eucalyptus: Unravelling Their Role in Reproductive Isolation and Adaptive Divergence.

Structural variants (SVs) play a significant role in speciation and adaptation in many species, yet few studies have explored the prevalence and impact of different categories of SVs. We conducted a comparative analysis of long-read assembled reference genomes of closely related Eucalyptus species to identify candidate SVs potentially influencing speciation and adaptation. Interspecies SVs can be either fixed differences, or polymorphic in one or both species. To describe SV patterns, we employed short-read whole-genome sequencing on over 600 individuals of E. melliodora and E. sideroxylon, along with recent high quality genome assemblies. We aligned reads and genotyped interspecies SVs predicted between species reference genomes. Our results revealed that 49,756 of 58,025 and 39,536 of 47,064 interspecies SVs could be typed with short reads, in E. melliodora and E. sideroxylon respectively. Focusing on inversions and translocations, symmetric SVs which are readily genotyped within both populations, 24 were found to be structural divergences, 2,623 structural polymorphisms, and 928 shared structural polymorphisms. We assessed the functional significance of fixed interspecies SVs by examining differences in estimated recombination rates and genetic differentiation between species, revealing a complex history of natural selection. Shared structural polymorphisms displayed enrichment of potentially adaptive genes. Understanding how different classes of genetic mutations contribute to genetic diversity and reproductive barriers is essential for understanding how organisms enhance fitness, adapt to changing environments, and diversify. Our findings reveal the prevalence of interspecies SVs and elucidate their role in genetic differentiation, adaptive evolution, and species divergence within and between populations.

genomics↗

bioRxiv2023

Transcriptional acclimation to warming temperatures of the Australian alpine herb Wahlenbergia ceracea

Understanding the molecular mechanisms of heat tolerance could help to predict the impacts of climate change on our native flora. However, much of our current understanding is derived from Arabidopsis thaliana and select crops exposed to short and intense periods of heat stress. Here, we characterise the transcriptomic response of Wahlenbergia ceracea, a perennial herb found in Australias subalpine regions, to sustained moderate warming. We contrasted responses between relatively heat-tolerant and heat-sensitive lines grown under cool (24/15 {o}C day/night) or warm (30/20 {o}C) temperatures. We observed that sustained warming up-regulated genes related to RNA regulation, including modification and splicing, and down-regulated genes associated with photosynthesis and plastid organization. Interestingly, heat-tolerant lines demonstrated a more pronounced repression of photosynthesis-associated genes, compared to heat-sensitive lines, suggesting that the regulation of the light-harvesting machinery may contribute to photosystem thermal tolerance. Co-expression analyses revealed only weak module-level correlations with direct measures of thermal tolerance. However, stronger associations were evident with chlorophyll content and photosynthetic efficiency, which might indirectly influence thermal tolerance. Prediction of transcription factors targeting warming-responsive genes implicated hormone signaling networks, especially ethylene, in contributing to differences in thermal tolerance. In conclusion, we present genomic resources for transcriptome analyses in W. ceracea and highlight the contrasting gene regulatory patterns between relatively heat-tolerant and heat-sensitive W. ceracea lines experiencing sustained moderate warming.

plant biology↗

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↗

bioRxiv2023

A high-quality pseudo-phased genome for Melaleuca quinquenervia shows allelic diversity of NLR-type resistance genes

BackgroundThe coastal wetland tree species Melaleuca quinquenervia (Cav.) S.T.Blake (Myrtaceae), commonly named the broad-leaved paperbark, is a foundation species in eastern Australia, Indonesia, Papua New Guinea, and New Caledonia. The species has been widely grown as an ornamental, becoming invasive in areas such as Florida in the United States. Long-lived trees must respond to a wide range pests and pathogens throughout their lifespan, and immune receptors encoded by the nucleotide- binding domain and leucine-rich repeat containing (NLR) gene family play a key role in plant stress responses. Expansion of this gene family is driven largely by tandem duplication, resulting in a clustering arrangement on chromosomes. Due to this clustering and their highly repetitive domain structure, comprehensive annotation of NLR encoding genes within genomes has been difficult. Additionally, as many genomes are still presented in their haploid, collapsed state, the full allelic diversity of the NLR gene family has not been widely published for outcrossing tree species. ResultsWe assembled a chromosome-level pseudo-phased genome for M. quinquenervia and describe the full allelic diversity of plant NLRs using the novel FindPlantNLRs pipeline. Analysis reveals variation in the number of NLR genes on each haplotype, differences in clusters and in the types and numbers of novel integrated domains. ConclusionsWe anticipate that the high quality of the genome for M. quinquenervia will provide a new framework for functional and evolutionary studies into this important tree species. Our results indicate a likely role for maintenance of NLR allelic diversity to enable response to environmental stress, and we suggest that this allelic diversity may be even more important for long-lived plants.

genomics↗