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The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks.

Leaf rust (Puccinia triticina) is a common disease that causes significant yield losses in wheat. The most frequently used methods to control leaf rust are the application of fungicides and the cultivation of resistant genotypes. However, high genetic diversity and associated adaptability of pathogen populations hamper achieving durable resistance in wheat. Emerging alternatives, such as microbial priming, may represent an effective measure to stimulate plant defense mechanisms and could serve as a means of controlling a broad range of pathogens. In this study, 175 wheat genotypes were inoculated with two bacterial strains: Ensifer meliloti strain expR+ch (producing N-acyl homoserine lactone (AHL)) or transformed E. meliloti carrying the lactonase gene attM (control). In total, 21 genotypes indicated higher resistance upon bacterial AHL priming. Subsequently, the phenotypic data of 175 genotypes combined with 9917 single-nucleotide polymorphisms (SNPs) in a genome-wide association study to identify quantitative trait loci (QTLs) and associated markers for relative infection under attM and expR+ch conditions and priming efficiency using the Genome Association and Prediction Integrated Tool (GAPIT). In total, 15 QTLs for relative infection under both conditions and priming efficiency were identified on chromosomes 1A, 1B, 2A, 3A, 3B, 3D, 6A, and 6B, which may represent targets for wheat breeding for priming and leaf-rust resistance.

Abstract Background The enteric pathogen Salmonella is the causative agent of the majority of food-borne bacterial poisonings. Resent research revealed that colonization of plants by Salmonella is an active infection process. Salmonella changes the metabolism and adjust the plant host by suppressing the defense mechanisms. In this report we developed an automatic algorithm to quantify the symptoms caused by Salmonella infection on Arabidopsis. Results The algorithm is designed to attribute image pixels into one of the two classes: healthy and unhealthy. The task is solved in three steps. First, we perform segmentation to divide the image into foreground and background. In the second step, a support vector machine (SVM) is applied to predict the class of each pixel belonging to the foreground. And finally, we do refinement by a neighborhood-check in order to omit all falsely classified pixels from the second step. The developed algorithm was tested on infection with the non-pathogenic E. coli and the plant pathogen Pseudomonas syringae and used to study the interaction between plants and Salmonella wild type and T3SS mutants. We proved that T3SS mutants of Salmonella are unable to suppress the plant defenses. Results obtained through the automatic analyses were further verified on biochemical and transcriptome levels. Conclusion This report presents an automatic pixel-based classification method for detecting “unhealthy” regions in leaf images. The proposed method was compared to existing method and showed a higher accuracy. We used this algorithm to study the impact of the human pathogenic bacterium Salmonella Typhimurium on plants immune system. The comparison between wild type bacteria and T3SS mutants showed similarity in the infection process in animals and in plants. Plant epidemiology is only one possible application of the proposed algorithm, it can be easily extended to other detection tasks, which also rely on color information, or even extended to other features.

Abstract Background In nature, beneficial bacteria triggering induced systemic resistance (ISR) may protect plants from potential diseases, reducing yield losses caused by diverse pathogens. However, little is known about how the host plant initially responds to different beneficial bacteria. To reveal the impact of different bacteria on barley (Hordeum vulgare), bacterial colonization patterns, gene expression, and composition of seed endophytes were explored. Results This study used the soil-borne Ensifer meliloti, as well as Pantoea sp. and Pseudomonas sp. isolated from barley seeds, individually. The results demonstrated that those bacteria persisted in the rhizosphere but with different colonization patterns. Although root-leaf translocation was not observed, all three bacteria induced systemic resistance (ISR) against foliar fungal pathogens. Transcriptome analysis revealed that ion- and stress-related genes were regulated in plants that first encountered bacteria. Iron homeostasis and heat stress responses were involved in the response to E. meliloti and Pantoea sp., even if the iron content was not altered. Heat shock protein-encoding genes responded to inoculation with Pantoea sp. and Pseudomonas sp. Furthermore, bacterial inoculation affected the composition of seed endophytes. Investigation of the following generation indicated that the enhanced resistance was not heritable. Conclusions Here, using barley as a model, we highlighted different responses to three different beneficial bacteria as well as the influence of soil-borne Ensifer meliloti on the seed microbiome. In total, these results can help to understand the interaction between ISR-triggering bacteria and a crop plant, which is essential for the application of biological agents in sustainable agriculture.

Abstract Background N-3-oxo-tetradecanoyl-L-homoserine lactone (oxo-C14-HSL) is one of the N-acyl homoserine lactones (AHL) that mediate quorum sensing in Gram-negative bacteria. In addition to bacterial communication, AHL are involved in interactions with eukaryotes. Short-chain AHL are easily taken up by plants and transported over long distances. They promote root elongation and growth. Plants typically do not uptake hydrophobic long sidechain AHL such as oxo-C14-HSL, although they prime plants for enhanced resistance to biotic and abiotic stress. Many studies have focused on priming effects of oxo-C14-HSL for enhanced plant resistance to stress. However, specific plant factors mediating oxo-C14-HSL responses in plants remain unexplored. Here, we identify the Arabidopsis protein ALI1 as a mediator of oxo-C14-HSL-induced priming in plants. Results We compared oxo-C14-HSL-induced priming between wild-type Arabidopsis Col-0 and an oxo-C14-HSL insensitive mutant ali1. The function of the candidate protein ALI1 was assessed through biochemical, genetic, and physiological approaches to investigate if the loss of the ALI1 gene resulted in subsequent loss of AHL priming. Through different assays, including MAP kinase activity assay, gene expression and transcriptome analysis, and pathogenicity assays, we revealed a loss of AHL priming in ali1. This phenomenon was reverted by the reintroduction of ALI1 into ali1. We also investigated the interaction between ALI1 protein and oxo-C14-HSL using biochemical and biophysical assays. Although biophysical assays did not reveal an interaction between oxo-C14-HSL and ALI1, a pull-down assay and an indirect method employing biosensor E. coli LuxCDABE support such interaction. We expressed fluorescently tagged ALI1 in tobacco leaves to assess the localization of ALI1 and demonstrate that ALI1 colocalizes with the plasma membrane, tonoplast, and endoplasmic reticulum. Conclusions These results suggest that the candidate protein ALI1 is indispensable for oxo-C14-HSL-dependent priming for enhanced resistance in Arabidopsis and that the ALI1 protein may interact with oxo-C14-HSL. Furthermore, ALI1 protein is localized in the cell periphery. Our findings advance the understanding of interactions between plants and bacteria and provide an avenue to explore desired outcomes such as enhanced stress resistance, which is useful for sustainable crop protection.

Salmonella enterica, a foodborne and human pathogen, is a constant threat to human health. Agricultural environments, for example, soil and plants, can be ecological niches and vectors for Salmonella transmission. Salmonella persistence in such environments increases the risk for consumers. Therefore, it is necessary to investigate the mechanisms used by Salmonella to adapt to agricultural environments. We assessed the adaptation strategy of S. enterica serovar Typhimurium strain 14028s to agricultural-relevant situations by analyzing the abundance of intermediates in glycolysis and the tricarboxylic acid pathway in tested environments (diluvial sand soil suspension and leaf-based media from tomato and lettuce), as well as in bacterial cells grown in such conditions. By reanalyzing the transcriptome data of Salmonella grown in those environments and using an independent RT-qPCR approach for verification, several genes were identified as important for persistence in root or leaf tissues, including the pyruvate dehydrogenase subunit E1 encoding gene aceE. In vivo persistence assay in tomato leaves confirmed the crucial role of aceE. A mutant in another tomato leaf persistence-related gene, aceB, encoding malate synthase A, displayed opposite persistence features. By comparing the metabolites and gene expression of the wild-type strain and its aceB mutant, fumarate accumulation was discovered as a potential way to replenish the effects of the aceB mutation. Our research interprets the mechanism of S. enterica adaptation to agriculture by adapting its carbon metabolism to the carbon sources available in the environment. These insights may assist in the development of strategies aimed at diminishing Salmonella persistence in food production systems.

Abstract Structural variants (SVs) underlie genomic variation but are often overlooked due to difficult detection from short reads. Most algorithms have been tested on humans, and it remains unclear how applicable they are in other organisms. To solve this, we develop perSVade (personalized structural variation detection), a sample-tailored pipeline that provides optimally called SVs and their inferred accuracy, as well as small and copy number variants. PerSVade increases SV calling accuracy on a benchmark of six eukaryotes. We find no universal set of optimal parameters, underscoring the need for sample-specific parameter optimization. PerSVade will facilitate SV detection and study across diverse organisms.

With the growth in distance education offerings, instructors who now teach quantitative material via the web have been faced with many challenges. Foremost has been the need to develop appropriate methods for teaching such material to students who are not physically in the classroom. Methodologies that have traditionally been taught in a highly interactive mode in the classroom must now be presented effectively in a far more asynchronous environment. Tutorials and detailed handouts are one way to accomplish this. We present a written tutorial for creating quality control charts using Excel. The tutorial guides students through the process of creating X-bar and R charts in such a way as to reinforce the theoretical basis of quality control already taught. Students apply their knowledge in hands-on activity, learn how to improve Excel's default charts to create visually effective control charts, and learn to reuse/recycle their work to easily create additional charts for different sets of problem data.

Plants may harbor the human pathogen Salmonella enterica. Interactions between S. enterica and different plant species have been studied in individual reports. However, disparities arising from the distinct experimental conditions may render a meaningful comparison very difficult. This study explored interaction patterns between different S. enterica strains including serovars Typhimurium 14028s and LT2 and serovar Senftenberg, and different plants (Arabidopsis, lettuce, and tomato) in one approach. Better persistence of S. enterica serovar Typhimurium strains was observed in all tested plants, whereas the resulting symptoms varied depending on plant species. Genes encoding pathogenesis-related proteins were upregulated in plants inoculated with Salmonella. Furthermore, transcriptome of tomato indicated dynamic responses to Salmonella, with strong and specific responses already 24 h after inoculation. By comparing with publicly accessible Arabidopsis and lettuce transcriptome results generated in a similar manner, constants and variables were displayed. Plants responded to Salmonella with metabolic and physiological adjustments, albeit with variability in reprogrammed orthologues. At the same time, Salmonella adapted to plant leaf-mimicking media with changes in biosynthesis of cellular components and adjusted metabolism. This study provides insights into the Salmonella-plant interaction, allowing for a direct comparison of responses and adaptations in both organisms.

Leaf rust caused by Puccinia hordei is one of the major diseases of barley (Hordeum vulgare L.) leading to yield losses up to 60%. Even though, resistance genes Rph1 to Rph28 are known, most of these are already overcome. In this context, priming may promote enhanced resistance to P. hordei. Several bacterial communities such as the soil bacterium Ensifer (syn. Sinorhizobium) meliloti are reported to induce resistance by priming. During quorum sensing in populations of gram negative bacteria, they produce N-acyl homoserine-lactones (AHL), which induce resistance in plants in a species- and genotype-specific manner. Therefore, the present study aims to detect genotypic differences in the response of barley to AHL, followed by the identification of genomic regions involved in priming efficiency of barley. A diverse set of 198 spring barley accessions was treated with a repaired E. meliloti natural mutant strain expR+ch producing a substantial amount of AHL and a transformed E. meliloti strain carrying the lactonase gene attM from Agrobacterium tumefaciens. For P. hordei resistance the diseased leaf area and the infection type were scored 12 dpi (days post-inoculation), and the corresponding relative infection and priming efficiency were calculated. Results revealed significant effects (p

Neuromyelitis optica spectrum disorder (NMOSD) is associated with pathological aquaporin-4 immunoglobulin G (AQP4-IgG), which cause brain damage. However, the impact of AQP4-IgG on retinal tissue remains unclear. Additionally, dysregulated complement anaphylatoxins C3a and C5a, known to modulate the endothelial barrier, are implicated in NMOSD. This study evaluates the susceptibility of human brain microvascular endothelial cells (HBMEC) and human retinal endothelial cells (HREC) to C3a- and C5a-mediated stress using real-time cell barrier analysis, immunocytochemical staining, qPCR and IgG transmigration assays. The findings reveal that C3a induced a concentration-dependent paracellular barrier breakdown and increased transcellular permeability in HBMEC, while HREC maintained barrier integrity under the same conditions. C5a attenuated C3a-induced disruption in HBMEC, indicating a protective role. Anaphylatoxin treatment elevated transcript levels of complement component C3 and increased C5 gene and protein expression in HREC, with no changes observed in HBMEC. In HBMEC, C5a treatment led to a transient upregulation of C3a receptor (C3AR) mRNA and an early decrease in C5a receptor 1 (C5AR1) protein detection. Conversely, HREC exhibited a late increase in C5aR1 protein levels. These results indicate that the retinal endothelial barrier is more stable under anaphylatoxin-induced stress compared to the brain, potentially offering better protection against paracellular AQP4-IgG transport. [ABSTRACT FROM AUTHOR]

Leaf rust (Puccinia triticina) is a common disease that causes significant yield losses in wheat. The most frequently used methods to control leaf rust are the application of fungicides and the cultivation of resistant genotypes. However, high genetic diversity and associated adaptability of pathogen populations hamper achieving durable resistance in wheat. Emerging alternatives, such as microbial priming, may represent an effective measure to stimulate plant defense mechanisms and could serve as a means of controlling a broad range of pathogens. In this study, 175 wheat genotypes were inoculated with two bacterial strains: Ensifer meliloti strain expR+ch (producing N-acyl homoserine lactone (AHL)) or transformed E. meliloti carrying the lactonase gene attM (control). In total, 21 genotypes indicated higher resistance upon bacterial AHL priming. Subsequently, the phenotypic data of 175 genotypes combined with 9917 single-nucleotide polymorphisms (SNPs) in a genome-wide association study to identify quantitative trait loci (QTLs) and associated markers for relative infection under attM and expR+ch conditions and priming efficiency using the Genome Association and Prediction Integrated Tool (GAPIT). In total, 15 QTLs for relative infection under both conditions and priming efficiency were identified on chromosomes 1A, 1B, 2A, 3A, 3B, 3D, 6A, and 6B, which may represent targets for wheat breeding for priming and leaf-rust resistance. [ABSTRACT FROM AUTHOR]

Beneficial bacteria in the rhizosphere are known to trigger faster and stronger plant immune responses to biotic and abiotic stressors. In the present study, we aimed to test the hypothesis that a rhizosphere microbiome transplant (RMT) may improve the immune response and reduce the disease rates of barley (Hordeum vulgare). This hypothesis was tested in a greenhouse system with the powdery mildew-causing fungus Blumeria graminis f. sp. hordei (Bgh). Detached rhizosphere microbiome from barley grown in a field soil was transplanted to barley seedlings grown in potting soil with reduced microbial diversity. Saline-treated plants served as control. At the three-leaf stage, barley was infected with Bgh. Decreased susceptibility to Bgh was observed for barley treated with the RMT as displayed by lower Bgh pustule counts in a detached leaf assay. A trend toward enhanced relative transcript abundances of the defense-related genes PR1b and PR17b was observed in leaves, 24 h after the Bgh challenge, when compared to the control. Moreover, 10 days after the Bgh challenge, the barley rhizosphere microbiome was harvested and analyzed by sequencing of 16S rRNA gene amplicons. The microbial community composition was significantly influenced by the RMT and displayed higher microbial diversity compared to the control. Furthermore, microbial beta-diversity and predicted functional profiles revealed a treatment-dependent clustering. Bacterial isolates from the RMT showed in vitro plant beneficial traits related to induced resistance. Our results showed that transplantation of a rhizosphere microbiome could be a sustainable strategy to improve the health of plants grown in potting soil with low microbial diversity under greenhouse conditions.

The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks. [ABSTRACT FROM AUTHOR]

Oxidative phosphorylation is among the most conserved mitochondrial pathways. However, one of the cornerstones of this pathway, the multi-protein complex NADH : ubiquinone oxidoreductase (complex I) has been lost multiple independent times in diverse eukaryotic lineages. The causes and consequences of these convergent losses remain poorly understood. Here, we used a comparative genomics approach to reconstruct evolutionary paths leading to complex I loss and infer possible evolutionary scenarios. By mining available mitochondrial and nuclear genomes, we identified eight independent events of mitochondrial complex I loss across eukaryotes, of which six occurred in fungal lineages. We focused on three recent loss events that affect closely related fungal species, and inferred genomic changes convergently associated with complex I loss. Based on these results, we predict novel complex I functional partners and relate the loss of complex I with the presence of increased mitochondrial antioxidants, higher fermentative capabilities, duplications of alternative dehydrogenases, loss of alternative oxidases and adaptation to antifungal compounds. To explain these findings, we hypothesize that a combination of previously acquired compensatory mechanisms and exposure to environmental triggers of oxidative stress (such as hypoxia and/or toxic chemicals) induced complex I loss in fungi.