RESUMEN
Recent studies show that cellular neighborhoods play an important role in evolving biological events such as cancer and diabetes. Therefore, it is critical to accurately and efficiently identify cellular neighborhoods from spatially-resolved single-cell transcriptomic data or single-cell resolution tissue imaging data. In this work, we develop CNTools, a computational toolbox for end-to-end cellular neighborhood analysis on annotated cell images, comprising both the identification and analysis steps. It includes state-of-the-art cellular neighborhood identification methods and post-identification smoothing techniques, with our newly proposed Cellular Neighbor Embedding (CNE) method and Naive Smoothing technique, as well as several established downstream analysis approaches. We applied CNTools on three real-world CODEX datasets and evaluated identification methods with smoothing techniques quantitatively and qualitatively. It shows that CNE with Naive Smoothing overall outperformed other methods and revealed more convincing biological insights. We also provided suggestions on how to choose proper identification methods and smoothing techniques according to input data.
Asunto(s)
Biología Computacional , Procesamiento de Imagen Asistido por Computador , Biología Computacional/métodos , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Análisis de la Célula Individual/métodos , Algoritmos , Programas InformáticosRESUMEN
Human regulatory T cells (Treg) suppress other immune cells. Their dysfunction contributes to the pathophysiology of autoimmune diseases, including type 1 diabetes (T1D). Infusion of Tregs is being clinically evaluated as a novel way to prevent or treat T1D. Genetic modification of Tregs, most notably through the introduction of a chimeric antigen receptor (CAR) targeting Tregs to pancreatic islets, may improve their efficacy. We evaluated CAR targeting of human Tregs to monocytes, a human ß cell line and human islet ß cells in vitro. Targeting of HLA-A2-CAR (A2-CAR) bulk Tregs to HLA-A2+ cells resulted in dichotomous cytotoxic killing of human monocytes and islet ß cells. In exploring subsets and mechanisms that may explain this pattern, we found that CD39 expression segregated CAR Treg cytotoxicity. CAR Tregs from individuals with more CD39low/- Tregs and from individuals with genetic polymorphism associated with lower CD39 expression (rs10748643) had more cytotoxicity. Isolated CD39- CAR Tregs had elevated granzyme B expression and cytotoxicity compared to the CD39+ CAR Treg subset. Genetic overexpression of CD39 in CD39low CAR Tregs reduced their cytotoxicity. Importantly, ß cells upregulated protein surface expression of PD-L1 and PD-L2 in response to A2-CAR Tregs. Blockade of PD-L1/PD-L2 increased ß cell death in A2-CAR Treg co-cultures suggesting that the PD-1/PD-L1 pathway is important in protecting islet ß cells in the setting of CAR immunotherapy. In summary, introduction of CAR can enhance biological differences in subsets of Tregs. CD39+ Tregs represent a safer choice for CAR Treg therapies targeting tissues for tolerance induction.
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Apirasa , Receptores Quiméricos de Antígenos , Linfocitos T Reguladores , Humanos , Apirasa/inmunología , Apirasa/metabolismo , Linfocitos T Reguladores/inmunología , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Citotoxicidad Inmunológica , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Antígeno HLA-A2/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Antígenos CDRESUMEN
Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-BY400*) was inhibited early during initial penetration, with the TaICL-BY400* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.
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Glioxilatos , Isocitratoliasa , Malato Sintasa , Enfermedades de las Plantas , Puccinia , Triticum , Triticum/microbiología , Triticum/genética , Triticum/metabolismo , Triticum/enzimología , Isocitratoliasa/metabolismo , Isocitratoliasa/genética , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/inmunología , Glioxilatos/metabolismo , Malato Sintasa/metabolismo , Malato Sintasa/genética , Puccinia/fisiología , Puccinia/patogenicidad , Regulación de la Expresión Génica de las Plantas , Resistencia a la Enfermedad/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Ten years ago, (black) stem rust - the most damaging of wheat (Triticum aestivum) rusts - re-emerged in western Europe. Disease incidences have since increased in scale and frequency. Here, we investigated the likely underlying causes and used those to propose urgently needed mitigating actions. We report that the first large-scale UK outbreak of the wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt), in 2022 may have been caused by timely arrival of airborne urediniospores from southwest Europe. The drive towards later-maturing wheat varieties in the UK may be exacerbating Pgt incidences, which could have disastrous consequences. Indeed, infection assays showed that two UK Pgt isolates from 2022 could infect over 96% of current UK wheat varieties. We determined that the temperature response data in current disease risk simulation models are outdated. Analysis of germination rates for three current UK Pgt isolates showed substantial variation in temperature response functions, suggesting that the accuracy of disease risk simulations would be substantially enhanced by incorporating data from prevailing Pgt isolates. As Pgt incidences continue to accelerate in western Europe, we advocate for urgent action to curtail Pgt losses and help safeguard future wheat production across the region.
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Enfermedades de las Plantas , Tallos de la Planta , Triticum , Triticum/microbiología , Enfermedades de las Plantas/microbiología , Europa (Continente) , Tallos de la Planta/microbiología , Puccinia/patogenicidad , Puccinia/fisiología , Temperatura , Basidiomycota/fisiología , Basidiomycota/patogenicidad , Reino Unido/epidemiologíaRESUMEN
Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs for atlas-scale datasets like Human Pancreas Analysis Program (HPAP), we develop AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX shows the higher performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulates known islet pathobiology and shows differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.
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Algoritmos , Diabetes Mellitus Tipo 1 , Páncreas , Proteómica , Humanos , Proteómica/métodos , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 1/metabolismo , Páncreas/citología , Páncreas/metabolismo , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/citología , Análisis de la Célula Individual/métodos , Redes Neurales de la Computación , Linfocitos T CD8-positivos/metabolismo , Citometría de Imagen/métodosRESUMEN
Type 2 diabetes mellitus (T2D), a major cause of worldwide morbidity and mortality, is characterized by dysfunction of insulin-producing pancreatic islet ß cells1,2. T2D genome-wide association studies (GWAS) have identified hundreds of signals in non-coding and ß cell regulatory genomic regions, but deciphering their biological mechanisms remains challenging3-5. Here, to identify early disease-driving events, we performed traditional and multiplexed pancreatic tissue imaging, sorted-islet cell transcriptomics and islet functional analysis of early-stage T2D and control donors. By integrating diverse modalities, we show that early-stage T2D is characterized by ß cell-intrinsic defects that can be proportioned into gene regulatory modules with enrichment in signals of genetic risk. After identifying the ß cell hub gene and transcription factor RFX6 within one such module, we demonstrated multiple layers of genetic risk that converge on an RFX6-mediated network to reduce insulin secretion by ß cells. RFX6 perturbation in primary human islet cells alters ß cell chromatin architecture at regions enriched for T2D GWAS signals, and population-scale genetic analyses causally link genetically predicted reduced RFX6 expression with increased T2D risk. Understanding the molecular mechanisms of complex, systemic diseases necessitates integration of signals from multiple molecules, cells, organs and individuals, and thus we anticipate that this approach will be a useful template to identify and validate key regulatory networks and master hub genes for other diseases or traits using GWAS data.
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Diabetes Mellitus Tipo 2 , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Predisposición Genética a la Enfermedad , Islotes Pancreáticos , Humanos , Estudios de Casos y Controles , Separación Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Redes Reguladoras de Genes/genética , Estudio de Asociación del Genoma Completo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Reproducibilidad de los ResultadosRESUMEN
The endocrine and exocrine compartments of the pancreas are spatially related but functionally distinct. Multiple diseases affect both compartments, including type 1 diabetes (T1D), pancreatitis, cystic fibrosis, and pancreatic cancer. To better understand how the exocrine pancreas changes with age, obesity, and diabetes, we performed systematic analysis of wellpreserved tissue sections from the pancreatic head, body, and tail of organ donors with T1D (n = 20), type 2 diabetes (T2D, n = 25), and donors with no diabetes (ND, n = 74). Among ND donors, we found that acinar-to-ductal metaplasia (ADM), angiopathy, and pancreatic adiposity increased with age, while ADM and adiposity also increased with BMI. Compared to age- and sex-matched ND organs, T1D pancreata had greater acinar atrophy and angiopathy with fewer intralobular adipocytes. T2D pancreata had greater ADM, angiopathy, and total T lymphocytes, but no difference in adipocyte number, compared to ND organs. While total pancreatic fibrosis was increased in both T1D and T2D, the pattern was different with T1D pancreata having greater periductal and perivascular fibrosis, whereas T2D pancreata had greater lobular and parenchymal fibrosis. Thus, the exocrine pancreas undergoes distinct changes as individuals age or develop T1D or T2D.
RESUMEN
Multiplexed antibody-based imaging enables the detailed characterization of molecular and cellular organization in tissues. Advances in the field now allow high-parameter data collection (>60 targets); however, considerable expertise and capital are needed to construct the antibody panels employed by these methods. Organ mapping antibody panels are community-validated resources that save time and money, increase reproducibility, accelerate discovery and support the construction of a Human Reference Atlas.
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Anticuerpos , Recursos Comunitarios , Humanos , Reproducibilidad de los Resultados , Diagnóstico por ImagenRESUMEN
In commemoration of 100 years since the discovery of glucagon, we review current knowledge about the human α cell. Alpha cells make up 30-40% of human islet endocrine cells and play a major role in regulating whole-body glucose homeostasis, largely through the direct actions of their main secretory product - glucagon - on peripheral organs. Additionally, glucagon and other secretory products of α cells, namely acetylcholine, glutamate, and glucagon-like peptide-1, have been shown to play an indirect role in the modulation of glucose homeostasis through autocrine and paracrine interactions within the islet. Studies of glucagon's role as a counterregulatory hormone have revealed additional important functions of the α cell, including the regulation of multiple aspects of energy metabolism outside that of glucose. At the molecular level, human α cells are defined by the expression of conserved islet-enriched transcription factors and various enriched signature genes, many of which have currently unknown cellular functions. Despite these common threads, notable heterogeneity exists amongst human α cell gene expression and function. Even greater differences are noted at the inter-species level, underscoring the importance of further study of α cell physiology in the human context. Finally, studies on α cell morphology and function in type 1 and type 2 diabetes, as well as other forms of metabolic stress, reveal a key contribution of α cell dysfunction to dysregulated glucose homeostasis in disease pathogenesis, making targeting the α cell an important focus for improving treatment.
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Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagón , Células Secretoras de Insulina , Islotes Pancreáticos , Humanos , Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Glucagón/metabolismo , Islotes Pancreáticos/metabolismo , Glucosa/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismoRESUMEN
Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.
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Pandemias , Triticum , Triticum/genética , Fitomejoramiento , Enfermedades de las Plantas/microbiología , Genómica , HongosRESUMEN
Pancreatic islet cells derived from human pluripotent stem cells hold great promise for modeling and treating diabetes. Differences between stem-cell-derived and primary islets remain, but molecular insights to inform improvements are limited. Here, we acquire single-cell transcriptomes and accessible chromatin profiles during in vitro islet differentiation and pancreas from childhood and adult donors for comparison. We delineate major cell types, define their regulomes, and describe spatiotemporal gene regulatory relationships between transcription factors. CDX2 emerged as a regulator of enterochromaffin-like cells, which we show resemble a transient, previously unrecognized, serotonin-producing pre-ß cell population in fetal pancreas, arguing against a proposed non-pancreatic origin. Furthermore, we observe insufficient activation of signal-dependent transcriptional programs during in vitro ß cell maturation and identify sex hormones as drivers of ß cell proliferation in childhood. Altogether, our analysis provides a comprehensive understanding of cell fate acquisition in stem-cell-derived islets and a framework for manipulating cell identities and maturity.
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Células Secretoras de Insulina , Islotes Pancreáticos , Células Madre Pluripotentes , Adulto , Humanos , Páncreas , Diferenciación Celular/genéticaRESUMEN
Wheat production is under constant threat from pests and pathogens, with fungal foliar diseases causing considerable annual yield losses. However, recent improvements in genomic tools and resources provide an unprecedented opportunity to enhance wheat's resilience in the face of these biotic constraints. Here, we discuss the impact of these advances on three key areas of managing fungal diseases of wheat: (i) enhancing the abundance of resistance traits available for plant breeding, (ii) accelerating the identification of novel fungicide targets and (iii) developing better tools for disease diagnostics and surveillance. Embracing these new genomics-led technological innovations in crop protection could revolutionise our wheat production system to improve resilience and prevent yield losses.
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Grano Comestible , Triticum , Triticum/genética , Genómica , FenotipoRESUMEN
Since emerging in Brazil in 1985, wheat blast has spread throughout South America and recently appeared in Bangladesh and Zambia. Here we show that two wheat resistance genes, Rwt3 and Rwt4, acting as host-specificity barriers against non-Triticum blast pathotypes encode a nucleotide-binding leucine-rich repeat immune receptor and a tandem kinase, respectively. Molecular isolation of these genes will enable study of the molecular interaction between pathogen effector and host resistance genes.
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Magnaporthe , Triticum , Triticum/genética , Triticum/microbiología , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Brasil , BangladeshRESUMEN
Plant pathogens deliver effector proteins to reprogramme a host plants circuitry, supporting their own growth and development, whilst thwarting defence responses. A subset of these effectors are termed avirulence factors (Avr) and can be recognised by corresponding host resistance (R) proteins, creating a strong evolutionary pressure on pathogen Avr effectors that favours their modification/deletion to evade the immune response. Hence, identifying Avr effectors and tracking their allele frequencies in a population is critical for understanding the loss of host recognition. However, the current systems available to confirm Avr effector function, particularly for obligate biotrophic fungi, remain limited and challenging. Here, we explored the utility of the genetically tractable wheat blast pathogen Magnaporthe oryzae pathotype Triticum (MoT) as a suitable heterologous expression system in wheat. Using the recently confirmed wheat stem rust pathogen (Puccina graminis f. sp. tritici) avirulence effector AvrSr50 as a proof-of-concept, we found that delivery of AvrSr50 via MoT could elicit a visible Sr50-dependant cell death phenotype. However, activation of Sr50-mediated cell death correlated with a high transgene copy number and transcript abundance in MoT transformants. This illustrates that MoT can act as an effective heterologous delivery system for fungal effectors from distantly related fungal species, but only when enough transgene copies and/or transcript abundance is achieved.
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Ascomicetos , Basidiomycota , Magnaporthe , Triticum/genética , Triticum/metabolismo , Ascomicetos/metabolismo , Basidiomycota/metabolismo , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismoRESUMEN
Cellular composition and anatomical organization influence normal and aberrant organ functions. Emerging spatial single-cell proteomic assays such as Image Mass Cytometry (IMC) and Co-Detection by Indexing (CODEX) have facilitated the study of cellular composition and organization by enabling high-throughput measurement of cells and their localization directly in intact tissues. However, annotation of cell types and quantification of their relative localization in tissues remain challenging. To address these unmet needs, we developed AnnoSpat (Annotator and Spatial Pattern Finder) that uses neural network and point process algorithms to automatically identify cell types and quantify cell-cell proximity relationships. Our study of data from IMC and CODEX show the superior performance of AnnoSpat in rapid and accurate annotation of cell types compared to alternative approaches. Moreover, the application of AnnoSpat to type 1 diabetic, non-diabetic autoantibody-positive, and non-diabetic organ donor cohorts recapitulated known islet pathobiology and showed differential dynamics of pancreatic polypeptide (PP) cell abundance and CD8+ T cells infiltration in islets during type 1 diabetes progression.
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The autonomic nervous system regulates pancreatic function. Islet capillaries are essential for the extension of axonal projections into islets, and both of these structures are important for appropriate islet hormone secretion. Because beta cells provide important paracrine cues for islet glucagon secretion and neurovascular development, we postulated that beta cell loss in type 1 diabetes (T1D) would lead to a decline in intraislet capillaries and reduction of islet innervation, possibly contributing to abnormal glucagon secretion. To define morphological characteristics of capillaries and nerve fibers in islets and acinar tissue compartments, we analyzed neurovascular assembly across the largest cohort of T1D and normal individuals studied thus far. Because innervation has been studied extensively in rodent models of T1D, we also compared the neurovascular architecture between mouse and human pancreas and assembled transcriptomic profiles of molecules guiding islet angiogenesis and neuronal development. We found striking interspecies differences in islet neurovascular assembly but relatively modest differences at transcriptome level, suggesting that posttranscriptional regulation may be involved in this process. To determine whether islet neurovascular arrangement is altered after beta cell loss in T1D, we compared pancreatic tissues from non-diabetic, recent-onset T1D (<10-yr duration), and longstanding T1D (>10-yr duration) donors. Recent-onset T1D showed greater islet and acinar capillary density compared to non-diabetic and longstanding T1D donors. Both recent-onset and longstanding T1D had greater islet nerve fiber density compared to non-diabetic donors. We did not detect changes in sympathetic axons in either T1D cohort. Additionally, nerve fibers overlapped with extracellular matrix (ECM), supporting its role in the formation and function of axonal processes. These results indicate that pancreatic capillaries and nerve fibers persist in T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components.NEW & NOTEWORTHY Defining the neurovascular architecture in the pancreas of individuals with type 1 diabetes (T1D) is crucial to understanding the mechanisms of dysregulated glucagon secretion. In the largest T1D cohort of biobanked tissues analyzed to date, we found that pancreatic capillaries and nerve fibers persist in human T1D despite beta cell loss, suggesting that alpha cell secretory changes may be decoupled from neurovascular components. Because innervation has been studied extensively in rodent T1D models, our studies also provide the first rigorous direct comparisons of neurovascular assembly in mouse and human, indicating dramatic interspecies differences.
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Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Células Secretoras de Glucagón , Islotes Pancreáticos , Humanos , Ratones , Animales , Diabetes Mellitus Tipo 1/metabolismo , Islotes Pancreáticos/metabolismo , Glucagón/metabolismo , Capilares/metabolismo , Células Secretoras de Glucagón/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fibras Nerviosas/metabolismoRESUMEN
Stripe rust disease of wheat, caused by Puccinia striiformis f. sp. tritici, (Pst) is one of the most serious diseases of wheat worldwide. In India, virulent stripe rust races have been constantly evolving in the North-Western Plains Zone leading to the failure of some of the most widely grown resistant varieties in the region. With the goal of studying the recent evolution of virulent races in this region, we conducted whole-genome re-sequencing of three prevalent Indian Pst pathotypes Pst46S119, Pst78S84 and Pst110S119. We assembled 58.62, 58.33 and 55.78 Mb of Pst110S119, Pst46S119 and Pst78S84 genome, respectively and found that pathotypes were highly heterozygous. Comparative phylogenetic analysis indicated the recent evolution of pathotypes Pst110S119 and Pst78S84 from Pst46S119. Pathogenicity-related genes classes (CAZyme, proteases, effectors, and secretome proteins) were identified and found to be under positive selection. Higher rate of gene families expansion were also observed in the three pathotypes. A strong association between the effector genes and transposable elements may be the source of the rapid evolution of these strains. Phylogenetic analysis differentiated the Indian races in this study from other known United States, European, African, and Asian races. Diagnostic markers developed for the identification of three Pst pathotypes will help tracking of yellow rust at farmers field and strategizing resistance gene deployment.
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Basidiomycota , Enfermedades de las Plantas , Estados Unidos , Filogenia , Enfermedades de las Plantas/genética , Basidiomycota/genética , PucciniaRESUMEN
Functional characterization of effector proteins of fungal obligate biotrophic pathogens, especially confirmation of avirulence (Avr) properties, has been notoriously difficult, due to the experimental intractability of many of these organisms. Previous studies in wheat have shown promising data suggesting the type III secretion system (T3SS) of bacteria may be a suitable surrogate for delivery and detection of Avr properties of fungal effectors. However, these delivery systems were tested in the absence of confirmed Avr effectors. Here, we tested two previously described T3SS-mediated delivery systems for their suitability when delivering two confirmed Avr effectors from two fungal pathogens of wheat, Puccinia graminis f. sp. tritici and Magnaporthe oryzae pathotype tritici. We showed that both effectors (AvrSr50 and AvrRmg8) were unable to elicit a hypersensitive response on wheat seedlings with the corresponding resistance gene when expressed by the Pseudomonas fluorescens "Effector to Host Analyser" (EtHAn) system. Furthermore, we found the utility of Burkholderia glumae for screening Avr phenotypes is severely limited, as the wild-type strain elicits nonhost cell death in multiple wheat accessions. These results provide valuable insight into the suitability of these systems for screening fungal effectors for Avr properties that may help guide further development of surrogate bacterial delivery systems in wheat. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.