Biology subjects

Figueroa, M.

Publications and source records attributed to Figueroa, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Zinc coordination by the wheat rust effector AvrSr33 provides a structural scaffold required for immune recognition

Abstract/SummaryInteractions between plants and pathogens drive long-term co-evolution through cycles of effector diversification and immune recognition. Effectors play central roles in this molecular interplay, and zinc-binding folds have been identified in a subset of pathogen effectors, yet the contribution of zinc coordination to effector stability and immune recognition remains unclear. Here, we investigated the structure and recognition of the AvrSr33 effector from the wheat stem rust fungus (Puccinia graminis f. sp. tritici, Pgt). Structural and biochemical analyses show that AvrSr33 adopts a fold containing two zinc-binding sites. Transient expression assays in Nicotiana benthamiana show that AvrSr33 directly interacts with Sr33, and comparison of recognised and non-recognised AvrSr33 variants identifies a polymorphic loop associated with recognition. This loop is positioned adjacent to one of the zinc-binding sites with its orientation constrained by zinc coordination. Reciprocal mutations of key surface residues within this region alter recognition, whereas mutation of the zinc-binding site abolishes recognition. Our data suggest that zinc coordination in AvrSr33 provides a structurally constrained scaffold that supports the surface features associated with Sr33 recognition. These findings provide a mechanistic framework for understanding how zinc coordination contributes to effector recognition and may influence the evolutionary trajectories of pathogen effectors.

Plant Biology↗

bioRxiv2026

Rust fungi secretomes contain structurally diverse effector families including cysteine-rich metal-binding proteins

Rust fungi are significant threats to global food security, causing substantial damage to crops through their ability to adapt and evolve new strains that overcome resistance. These obligate biotrophs infect host plants by secreting effector proteins that manipulate host physiology to promote infection and colonisation. We used AlphaFold2 to investigate structural conservation among effector proteins for the secretomes of Melampsora lini and four Puccinia species. AlphaFold2 yielded high-confidence predictions for 45.7% of the 27,090 secreted proteins, while 19% were poor quality. Comparative analysis revealed extensive structural diversity across the rust secretomes, with all thirteen known rust Avr proteins belonging to different clusters apart from AvrSr13 and AvrSr33. Nevertheless, there were still numerous large clusters of structurally-related proteins, including 59 clusters with over 50 members each, three of which contained known Avr proteins. Of the major structural families defined in other fungi, the rust species studied here only contained FOLD and ToxA-like families. Structural analysis of cysteine-rich proteins revealed over a thousand effector candidates featuring zinc-binding sites, with approximately 75% predicted to be cytoplasmic effectors. In contrast, cysteine-rich apoplastic effector candidates were characterized by a high frequency of disulfide bonds. One family of predicted metal-binding proteins was greatly expanded in P. graminis f. sp. tritici and includes AvrSr13 and AvrSr33. We confirmed that purified AvrSr13 and AvrSr22 proteins bind to zinc in vitro using biochemical assays. Taken together, structural modeling provides new avenues to study sequence-unrelated effectors and highlights the high degree of diversity in the effector repertoires of rust species.

microbiology↗

bioRxiv2026

EffectorGeneP: accurate gene annotation in pathogen genomes from infection transcriptomes

Accurate gene annotation is crucial for inference of biological knowledge from genomes. However, non-canonical genes such as orphan or single-exon genes as well as those residing in rapidly evolving regions are routinely dismissed in annotation pipelines. In filamentous pathogen genomes, this disproportionately affects the annotation of genes encoding disease-promoting effector proteins. We introduce EffectorGeneP, a machine learning tool that self-trains on transcript data, predicts the most likely coding sequence from transcripts and effectively separates bona fide genes from transcriptional noise. EffectorGeneP annotates over 95% of known effectors correctly, while other state-of-the-art methods annotate 15%-78%. We show that EffectorGeneP expands the predicted secretome of pathogens by over 50% and that high-throughput screening of an effector library in plant protoplasts uncovers the previously poorly annotated AvrSr26 gene family in the wheat stem rust fungus. EffectorGeneP decodes genomes at unprecedented resolution and will enable the study of biological processes in important pathogen species.

microbiology↗

bioRxiv2026

Haplotype-phased genomes of the barley leaf rust pathogen reveal evidence of repeat element expansion and somatic hybridization

Barley leaf rust disease, caused by Puccinia hordei, leads to substantial yield losses and diminished malting quality of barley across temperate growing regions worldwide. To address the paucity of high-resolution genomic resources for this pathogen, we generated haplotype-phased, chromosome-scale assemblies for ten globally distributed isolates using PacBio HiFi and Hi-C sequencing. Phylogenomic analysis revealed seven distinct lineages of P. hordei, including evidence of nuclear exchange, with a shared nuclear haplotype detected between two US lineages. Nuclear genome sizes ranged from [~]140-147Mbp, with the exception of isolate 90ISR03 from Israel ([~]163Mbp), which also harbored a 6.2Mbp supernumerary scaffold in one nucleus exhibiting chromosomal characteristics. Consistent with its larger genome, P. hordei had a higher repeat content ([~]70%) than related cereal rust fungi, driven primarily by the proliferation of LTR retroelements and DNA transposons. Across the global pan-genome of 13 unique nuclear haplotypes, approximately one-third of all protein orthogroups were conserved across all isolates. Only 18% of predicted effector orthogroups were shared between all haplotypes, reflecting the highly dynamic and variable nature of the effector repertoire. The long-term propagation of clonal P. hordei lineages is apparent both within the US and globally, and nuclear exchange is important for generating novel diversity and virulence profiles. The high degree of genome plasticity is evident in extensive structural variation, including large-scale translocations and inversions as well as a putative accessory chromosome. These chromosome-level, haplotype-resolved genomes provide a foundational resource for exploring the evolution, diversity, and avirulence gene repertoire of P. hordei.

genomics↗

bioRxiv2026

Isolation and metabolomic analysis of the culturable human gut mycobiota during dysbiosis

The human gut mycobiome, though less diverse than the bacterial microbiome, plays a significant role in health and disease. This study investigates the culturable fungal communities in fecal samples from hospitalized patients with diarrhea in Mexico City. We isolated and characterized 26 fungal strains using culture-dependent methods, including 20 yeasts and six filamentous fungi. The most prevalent organisms were Candida albicans, Rhodotorula mucilaginosa, Penicillium spp., and Paecilomyces spp. Fungal isolates were tested for their ability to withstand gut-like conditions, including temperature, pH, oxidative stress, and bile salts. Notably, Paecilomyces variotii demonstrated thermotolerance, surviving at 42{degrees}C, and exhibited competitive growth against other fungi. Co-occurrence analysis revealed associations between fungal isolates and bacterial pathogens such as Salmonella and Clostridioides difficile, suggesting potential interkingdom interactions. Cytotoxicity assays on Caco-2 cells showed that cell-free supernatants from Candida inospicua and filamentous fungi reduced cell viability by up to 40%. Finally, dereplication and untargeted metabolomic analyses of P. variotii, Penicillium crustosum, and Penicillium chrysogenum revealed the presence of several bioactive metabolites, including mycotoxins and antimicrobial compounds, highlighting their potential roles in gut ecology and disease. Overall, this study underscores the importance of the gut mycobiome in dysbiosis and its interactions with bacterial pathogens. The findings suggest that fungi, particularly thermotolerant species such as P. variotii, may contribute to gut dysbiosis and disease progression, particularly in immunocompromised patients. Further research is needed to elucidate the functional roles of these fungi and their metabolites in gut health and disease.

microbiology↗

bioRxiv2025

Novel clonal lineages of wheat stem rust in the Southern Cone of South America

Puccinia graminis f. sp. tritici (Pgt) causes wheat stem rust, a devastating disease of cereals. Recent approaches to examine populations at a genomic level have provided valuable information on the genotypic diversity of Pgt populations in Africa, Europe and North America and evolutionary mechanisms underlying the emergence of new races. However, an in-depth characterisation of Pgt populations in South America has been lacking. To bridge this knowledge gap, 91 Pgt isolates were collected from Argentina and Uruguay in 2020 and 2021 and used to generate transcriptome and whole genome sequence data and pathotype information. Phylogenetic analyses revealed that this South American Pgt population includes three clonal lineages, two of which have not been detected elsewhere. The third lineage is globally dispersed, including isolates from Africa and the Middle East. The predominant lineage, unique to South America, encompassed 90% of the samples and showed related pathotypes differing by virulence on single resistance genes consistent with evolution by stepwise mutation within the clonal lineage. There was no evidence of sexual recombination giving rise to new genetic diversity in this population. These observations urge to routinely incorporate and compare genotypic data from South American Pgt isolates to surveillance data from other geographic regions.

evolutionary biology↗

bioRxiv2025

Detection of a novel Puccinia coronata f. sp. avenae genotypic lineage in Australia during the 2024 oat growing season

Crown rust, caused by Puccinia coronata f. sp. avenae (Pca), remains a persistent threat to oat production in Australia. To monitor recent shifts in virulence and population structure, 30 Pca isolates collected during the 2024 growing season across major Australian oat-producing regions were analysed. Virulence analysis of 30 isolates using 52 oat differential lines identified 25 unique races that were not detected in previous years. Whole-genome sequencing of 28 of these isolates were analysed in the context of a broader historical Australian and international genomic datasets including isolates from Taiwan, South Africa, USA. Results confirmed the uniqueness of the Australian Pca population and revealed well-established genotypic lineages persisting over multiple years, with L18 and L16 being dominant. Notably, L16 was again present in Western Australia after being undetected in 2023, while L18 maintained its prevalence for a third consecutive season. Beyond these dominant groups, phylogenetic analysis and a k-mer containment analysis also identified a novel and genetically distinct lineage, designated as L19, represented by one isolate collected in WA. To add to the characterisation of lineage L19, we recorded virulence phenotypes on a small collection of current commercial cultivars. These findings enhance understanding of Pca diversity and emphasise the importance of surveillance approaches that integrate phenotypic and genomic surveillance.

pathology↗

bioRxiv2025

The MLLT3 YEATS domain is a dual reader of histone marks (H3K9/18/27ac/cr) and ncRNA (7SK), linking epigenetic and RNA signaling to regulate hematopoiesis

The protein MLLT3 (AF9) is a critical regulator of hematopoiesis. The N-terminal YEATS domain of MLLT3 is an epigenetic reader that binds to acetylated as well as crotonylated lysine. Using PAR-CLIP, biochemical assays, and NMR based mapping of binding, we demonstrate that the YEATS domain of MLLT3 binds to a specific stem-loop region of the noncoding RNA 7SK. 7SK is a noncoding RNA with a well-documented function in transcriptional elongation. We developed point mutations in the YEATS domain that disrupt RNA binding while having no effect on binding of acetylated histone peptides to probe the specific role of RNA binding in MLLT3 function. Our results show loss of RNA binding by MLLT3 skews hematopoietic differentiation away from the myeloid lineage and toward the lymphoid lineage and has substantial effects on gene expression, confirming the essential nature of MLLT3-RNA binding for function.

biochemistry↗

bioRxiv2025

Haplotype-resolved genomes of diverse oat crown rust isolates reveal both global dispersion of long-lived clonal haplotypes and limited recombination between haplotypes.

Genetic diversity of pathogen populations plays an important role in host adaptation. While single high quality genome references are a valuable resource, a compilation of genome references from individuals increases the breadth of genetic variation within a species that is captured. Puccinia coronata f. sp. avenae (Pca), a fungal pathogen causing crown rust of oat, demonstrates rapid virulence evolution and adaptation to newly released cultivars. To broaden the geographic and temporal distribution of available Pca genomes, we generated nuclear haplotype-resolved genome references for ten isolates from Europe, Africa and the Middle East and compared these with existing references for USA and Australian isolates. Of the full collection of 52 haplotypes, 40 were unique. Importantly, the presence of a nearly identical haplotype in a UK isolate collected in 1984 and in USA isolates from 1990 and 2017 supports the existence of long-lived clonal haplotypes in the global population that have been exchanged between lineages. Taken together with the identification of infrequent recombination between haplotypes from geographically dispersed isolates, this evidence reflects a globally mobile population of Pca that is mostly comprised of persistent clonal lineages with some influence from rare recombination events. Analysis of the core and non-core proteome suggests that while the core proteome is enriched for predicted secreted and effector proteins, sequence and expression variation are most prevalent in non-core orthogroups. We anticipate that this expanded collection of haplotypes will facilitate the development of new surveillance technologies and identification of virulence loci.

genomics↗

bioRxiv2025

QTL mapping of oat crown rust resistance in Australian fields and identification of a seedling resistance locus in oat line GS7

The development of oat cultivars with resistance to crown rust caused by Puccinia coronata f. sp. Avenae (Pca) is key for sustainable disease control. This study examined two recombinant inbred line populations, Provena x GS7 and Boyer x GS7, to identify adult plant resistance QTL in Australian fields. Seven distinct QTL associated with rust resistance were identified, with KASP markers developed for single nucleotide polymorphisms (SNPs) tightly linked to the four most significant QTL on chromosomes 4A and 7A. A major QTL named QPc_GS7_4A.2 with a resistance allele derived from line GS7 was mapped to chromosome 4A, overlapping with genomic regions previously associated with both resistance gene Pc61 and adult plant resistance. Genetic mapping for rust resistance at seedling stage using a subset of Provena x GS7 lines with contrasting alleles at QPc_GS7_4A.2 confirmed the role of this locus on seedling resistance, likely by Pc61. Furthermore, we found similar resistance profiles between GS7 and the Pc61 differential line against 20 Pca isolates at the seedling stage. Haplotype analysis of QPc_GS7_4A.2 in the oat crown rust differential set and an oat collection revealed the resistance haplotype in lines previously postulated to carry resistance gene Pc61. These results suggest that the QTL QPc_GS7_4A.2 is closely linked to the Pc61 locus on chromosome 4A. The KASP markers associated with Pc61 and QTL identified in this study will be valuable tools, allowing breeders to efficiently integrate the resistance allele for gene combinations in new cultivars, particularly in regions where Pc61 remains effective.

plant biology↗

bioRxiv2025

Virulence on Pm4 kinase-based resistance is determined by two divergent wheat powdery mildew effectors

The wheat resistance gene Pm4 encodes a kinase fusion protein and has gained particular attention as it confers race-specific resistance against two major wheat pathogens: powdery mildew and blast. Here, we describe the identification of AvrPm4, the mildew avirulence effector recognised by Pm4, using UV- mutagenesis, and its functional validation in wheat protoplasts. We show that AvrPm4 directly interacts with and is phosphorylated by Pm4. Using genetic association and QTL mapping, we furthermore demonstrate that evasion of Pm4 resistance by virulent mildew isolates relies on a second fungal component, SvrPm4, which suppresses AvrPm4-induced cell death. Surprisingly, SvrPm4 was previously described as AvrPm1a. We show that SvrPm4, but not its inactive variant svrPm4, is recognised by the NLR immune receptor Pm1a. These multiple roles of a single effector provide a new perspective on fungal (a)virulence proteins and their combinatorial interactions with different types of immune receptors.

genetics↗

bioRxiv2024

Identification of diagnostic KASP markers for selection of crown rust resistance in oats

Oat crown rust, caused by Puccinia coronata f. sp. avenae, poses a significant threat to global oat production (Avena sativa L.). Molecular markers are essential to assist in the integration of multiple resistance genes into a single oat cultivar to achieve genetic resistance durability. Here, we validated previously reported markers for the race-specific resistance genes Pc39, Pc45/PcKM, Pc54, and Pc68, and developed new KASP markers closely linked to these loci. These markers were subsequently screened across a collection of 150 oat cultivars. Analysis of molecular marker data, pedigree information, and disease resistance profile indicated several oat cultivars likely carrying Pc39 and Pc68. Newly identified carriers of Pc68 include the cultivars Galileo and Graza80, while Warrego, Kowari, Possum, Forester, Drummond, Hokonui, Glider, and Culgoa were identified as potential carriers of Pc39. Discrepancies between the previous postulation of Pc gene carriers and our phenotyping and genotyping analysis were also found. For example, the oat line Glider was previously postulated to carry Pc58 or Pc59, but it was positive for the Pc39-associated marker and had a similar resistance profile to Pc39 carriers. These findings underscore the importance of the utilisation of molecular markers in tracking the resistance genes in breeding germplasm. Key messageThis study has developed and validated KASP molecular markers associated with crown rust resistance genes Pc39, Pc45/PcKM, Pc54, and Pc68 that could be used for marker-assisted selection in oats.

genetics↗

bioRxiv2024

Characterisation of virulence of Puccinia coronata f. sp. avenae in Australia in the 2023 growing season

Crown rust caused by the basidiomycete fungus Puccinia coronata f. sp. avenae (Pca) results in significant crop losses worldwide. Genetic solutions to protect against this disease require disease resistance gene discovery and introduction of resistance genes into elite germplasm by breeders. To inform disease resistance breeding activities, it is paramount to monitor changes of virulence in the Pca population and link those to genotypes of the pathogen. In 2023, a collection of 37 Pca isolates from diverse regions in Australia were gathered and their infection types across a commonly used set of oat differential lines determined to assign virulence pathotypes. We compared those virulence phenotypes to data collected in previous collections. While some of the virulence phenotypes had been reported in 2022, our analysis detected new virulence or an increase in frequency for virulence on some resistance genes. Notably, some of the frequency increases in virulence were recorded in Western Australia (WA), a region of interest due to its role in oat production for milling.

plant biology↗

bioRxiv2024

A wheat tandem kinase sensor activates an NLR helper to trigger immunity

Most plant resistance genes encode membrane-anchored receptor-like proteins or intracellular nucleotide-binding and leucine-rich repeat (NLR) receptors. In wheat and barley, tandem kinases (TKs) have emerged as a new class of resistance determinants. To understand the modus operandi of the wheat stem rust resistance protein Sr62TK, we identified two genetic interactors-- a host gene required for Sr62TK function and the corresponding fungal AvrSr62 effector. We discovered that the SR62 locus consists of a digenic module encoding Sr62TK and an NLR (Sr62NLR). AvrSr62 binds to the N-terminal kinase of Sr62TK. This triggers displacement of the C-terminal kinase allowing it to recruit Sr62NLR for activation of immune responses. Understanding the mechanism of this two-component resistance complex will help engineering and breeding for durable resistance.

plant biology↗

bioRxiv2024

A high-resolution haplotype pangenome uncovers somatic hybridization, recombination and intercontinental migration in oat crown rust

Basidiomycetes like rust fungi have complex reproductive cycles and dikaryotic life stages which influence their population structure and evolution. Puccinia coronata f. sp. avenae (Pca), the causal agent of oat crown rust, is a pathogen of global economic importance. To investigate the genetic diversity of the species, as well as the role of mating type system and nuclear exchange (somatic hybridization) in host adaptation of Pca we acquired whole genome sequencing data of Taiwanese and Australian isolates, adding to existing data for USA and South African populations. An atlas of 30 chromosome-level, fully-phased nuclear haplotypes from six USA isolates and nine Australian isolates was generated to capture the genomic composition of key oat crown rust lineages. This study provides evidence of nuclear exchange and recombination of haplotypes in both the USA and Australian Pca populations as mechanisms for the introduction of genetic diversity. The limitations of assuming clonal evolutionary history from virulence phenotyping is demonstrated by the detection of either sexual or cryptic genetic recombination in the Pca Australian population. Overall, the characterization of intercontinental migration of Pca at the haplotype level provides further impetus for molecular monitoring of rust pathogen populations on a global scale.

genomics↗

bioRxiv2024

Detection of oat crown rust disease in Taiwan (2019-2021)

Oat is a minor forage crop grown in Taiwan. Only a few historical records of oat rust disease have been reported in the country, therefore the pathogen population remains poorly characterized. A rust-like disease outbreak was detected at the Experimental Farm of National Taiwan University in 2019, which caused significant damage to the field experiments. To determine the identity of the pathogen responsible for this disease outbreak, we collected infected foliar material. Disease signs suggested infection by the oat crown rust fungus. Hence, common procedures in rust pathology were applied to confirm the identity of the pathogen with phenotypic and molecular diagnostic techniques. A total of 50 field samples from infected oat cultivars were collected in 2019 and five rust isolates were purified in 2020 and 2021. Phylogenetic analysis based on ITS sequences indicated that the pathogen was likely Puccinia coronata f. sp. avenae (Pca), which was further supported by the placement of Taiwanese isolate NTU-01 with other Pca representatives in a phylogenetic tree of Basidiomycete fungi. Phenotyping assays across 36 oat differential lines demonstrated that Taiwanese isolates are phenotypically similar with relatively limited virulence. This study presents the first molecular confirmation of Pca in Taiwan and reports the virulence profiles of Taiwanese Pca population.

microbiology↗

bioRxiv2023

AvrSr27 is a zinc-bound effector with a modular structure important for immune recognition

Stem rust, caused by the fungal pathogen Puccinia graminis f. sp.tritici (Pgt) is a major threat for wheat production and global food security. Central to the success of Pgt is the secretion of proteinaceous effectors that promote infection and colonisation, while immunity in wheat is driven by receptor-mediated recognition of these effectors resulting in pathogen avirulence. Here, we report the crystal structure of the cysteine-rich effector AvrSr27, the third experimentally derived structure of a Pgt effector. The AvrSr27 structure reveals a novel {beta}-strand rich modular fold consisting of two structurally similar domains and confirms the poor prediction we obtained from the AlphaFold2-derived model. The highly prevalent cysteine residues within the protein facilitate the co-ordination of 4 zinc molecules. Utilising the structure, we show that the N-terminal domain of AvrSr27 is sufficient for immune recognition and interaction by Sr27. The 7-cys motif sequence in each AvrSr27 domain, which facilitates zinc binding, was also found in two haustorially-expressed, structurally homologous Pgt proteins. Remarkably, despite relatively low sequence identity, we show that these proteins can associate with Sr27 and trigger cell death in heterologous systems and wheat protoplasts, albeit weaker than AvrSr27. Collectively, our findings have important implications for the field embarking on bespoke engineering of immunity receptors as solutions to plant disease.

plant biology↗

bioRxiv2023

Genotypic and resistance profile analysis of two oat crown rust differential sets urge coordination and standardisation

Puccinia coronata f. sp. avenae (Pca) is the causal agent of the disease known as crown rust which represents a bottleneck in oat production worldwide. Characterisation of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared virulence profiles of 25 Pca isolates from Australia using two host differential sets, one from Australia and one from the USA. These differential sets were also genotyped using DArT sequencing technology. Phenotypic and genotypic discrepancies were detected on eight out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterise materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies.

plant biology↗