Putative Effector Genes Research Articles

A Polyphasic Approach Reveals Novel Genotypes and Updates the Genetic Structure of the Banana Fusarium Wilt Pathogen.

Fusarium oxysporum f. sp. cubense (Foc) is a soil-borne fungus that causes Fusarium wilt, a destructive plant disease that has resulted in devastating economic losses to banana production worldwide. The fungus has a complex evolutionary history and taxonomic repute and consists of three pathogenic races and at least 24 vegetative compatibility groups (VCGs). Surveys conducted in Asia, Africa, the Sultanate of Oman and Mauritius encountered isolates of F. oxysporum pathogenic to banana that were not compatible to any of the known Foc VCGs. Genetic relatedness between the undescribed and known Foc VCGs were determined using a multi-gene phylogeny and diversity array technology (DArT) sequencing. The presence of putative effector genes, the secreted in xylem (SIX) genes, were also determined. Fourteen novel Foc VCGs and 17 single-member VCGs were identified. The multi-gene tree was congruent with the DArT-seq phylogeny and divided the novel VCGs into three clades. Clustering analysis of the DArT-seq data supported the separation of Foc isolates into eight distinct clusters, with the suite of SIX genes mostly conserved within these clusters. Results from this study indicates that Foc is more diverse than hitherto assumed.

Genome-Wide Identification of bZIP Transcription Factor Genes and Functional Analyses of Two Members in Cytospora chrysosperma.

The basic leucine zipper (bZIP) transcription factor (TF) family, one of the largest and the most diverse TF families, is widely distributed across the eukaryotes. It has been described that the bZIP TFs play diverse roles in development, nutrient utilization, and various stress responses in fungi. However, little is known of the bZIP members in Cytospora chrysosperma, a notorious plant pathogenic fungus, which causes canker disease on over 80 woody plant species. In this study, 26 bZIP genes were systematically identified in the genome of C. chrysosperma, and two of them (named CcbZIP05 and CcbZIP23) significantly down-regulated in CcPmk1 deletion mutant (a pathogenicity-related mitogen-activated protein kinase) were selected for further analysis. Deletion of CcbZIP05 or CcbZIP23 displayed a dramatic reduction in fungal growth but showed increased hypha branching and resistance to cell wall inhibitors and abiotic stresses. The CcbZIP05 deletion mutants but not CcbZIP23 deletion mutants were more sensitive to the hydrogen peroxide compared to the wild-type and complemented strains. Additionally, the CcbZIP23 deletion mutants produced few pycnidia but more pigment. Remarkably, both CcbZIP05 and CcbZIP23 deletion mutants were significantly reduced in fungal virulence. Further analysis showed that CcbZIP05 and CcbZIP23 could regulate the expression of putative effector genes and chitin synthesis-related genes. Taken together, our results suggest that CcbZIP05 and CcbZIP23 play important roles in fungal growth, abiotic stresses response, and pathogenicity, which will provide comprehensive information on the CcbZIP genes and lay the foundation for further research on the bZIP members in C. chrysosperma.

Functional analysis of a Phytophthora host-translocated effector using the yeast model system.

Background Phytophthora plant pathogens secrete effector proteins that are translocated into host plant cells during infection and collectively contribute to pathogenicity. A subset of these host-translocated effectors can be identified by the amino acid motif RXLR (arginine, any amino acid, leucine, arginine). Bioinformatics analysis has identified hundreds of putative RXLR effector genes in Phytophthora genomes, but the specific molecular function of most remains unknown.MethodsHere we describe initial studies to investigate the use of Saccharomyces cerevisiae as a eukaryotic model to explore the function of Phytophthora RXLR effector proteins.Results and ConclusionsExpression of individual RXLR effectors in yeast inhibited growth, consistent with perturbation of a highly conserved cellular process. Transcriptome analysis of yeast cells expressing the poorly characterized P. sojae RXLR effector Avh110 identified nearly a dozen yeast genes whose expression levels were altered greater than two-fold compared to control cells. All five of the most down-regulated yeast genes are normally induced under low phosphate conditions via the PHO4 transcription factor, indicating that PsAvh110 perturbs the yeast regulatory network essential for phosphate homeostasis and suggesting likely PsAvh110 targets during P. sojae infection of its soybean host.

Variant-to-gene mapping in human microglial cell models with CRISPR validation implicates RTFDC1 at the Alzheimer's disease 'CASS4' locus.

Alzheimer's disease (AD) research has principally focused on neurons due to their role in neurodegeneration. In contrast, recent studies suggest that genetic mechanisms drive microglia to prolonged inflammation in AD brains, exacerbating neurodegeneration. Indeed, recent genome-wide association studies (GWAS) of AD have identified multiple loci near genes related to microglial function, such as TREM2 and CR1. However, GWAS does not have the sensitivity to identify causal variants or effector genes. We used a combination of ATAC-seq and high-resolution promoter-focused Capture-C in two human microglial cell models to map interactions between GWAS-implicated variants and their putative effector genes. We then validated an observed interaction at the 'CASS4' locus using CRISPR-Cas9 genome editing. Improving on the relatively low resolution of available Hi-C data, we employed high resolution Capture-C to characterize the physical genome-wide interactions of all human promoters. We performed this in the human microglial cell line HMC3 and human iPSC-derived microglia (iMg). We confirmed the enhancer activity of a SNP we elected to pursue and its associated regulatory element using dual-luciferase assays. To confirm the regulatory interaction with the Capture-C implicated effector gene, we used lentiviral CRISPR-Cas9 to delete an approximately 300bp region containing the SNP in HMC3 cells. We then confirmed changes in RNA and protein expression using RNA-seq and Western blotting, respectively. Variant-to-gene mapping in both microglial cell models identified 67 putative effector genes (51 coding) across both cell types, with 14 observed in both models. We identified a novel proxy SNP, rs6024870 (r2 = 0.93 to sentinel SNP rs6014724), at the 'CASS4' locus, which coincided with open chromatin and directly contacted the promoter of RTFDC1, a gene not previously implicated in AD. Deletion of the putative enhancer region harboring rs6024870 by CRISPR-Cas9 in HMC3 reduced the expression of RTFDC1 at both the mRNA and protein level. We also note that CASS4 levels were modestly influenced by this CRISPR-mediated perturbation. We implicate RTFDC1 as a novel effector gene at the AD 'CASS4' GWAS locus. Further efforts will characterize the phenotypic effect of this variant in microglial cell models, including on inflammation and phagocytic activity.

Accelerating Complete Phytoplasma Genome Assembly by Immunoprecipitation-Based Enrichment and MinION-Based DNA Sequencing for Comparative Analyses.

Phytoplasmas are uncultivated plant-pathogenic bacteria with agricultural importance. Those belonging to the 16SrII group, represented by ‘Candidatus P. aurantifolia’, have a wide range of plant hosts and cause significant yield losses in valuable crops, such as pear, sweet potato, peanut, and soybean. In this study, a method that combines immunoprecipitation-based enrichment and MinION long-read DNA sequencing was developed to solve the challenge of phytoplasma genome studies. This approach produced long reads with high mapping rates and high genomic coverage that can be combined with Illumina reads to produce complete genome assemblies with high accuracy. We applied this method to strain NCHU2014 and determined its complete genome sequence, which consists of one circular chromosome with 635,584 bp and one plasmid with 4,224 bp. Although ‘Ca. P. aurantifolia’ NCHU2014 has a small chromosome with only 471 protein-coding genes, it contains 33 transporter genes and 27 putative effector genes, which may contribute to obtaining nutrients from hosts and manipulating host developments for their survival and multiplication. Two effectors, the homologs of SAP11 and SAP54/PHYL1 identified in ‘Ca. P. aurantifolia’ NCHU2014, have the biochemical activities in destabilizing host transcription factors, which can explain the disease symptoms observed in infected plants. Taken together, this study provides the first complete genome available for the 16SrII phytoplasmas and contributes to the understanding of phytoplasma pathogenicity.

Functional Characterization of the Cnidarian Antiviral Immune Response Reveals Ancestral Complexity.

Animals evolved a broad repertoire of innate immune sensors and downstream effector cascades for defense against RNA viruses. Yet, this system varies greatly among different bilaterian animals, masking its ancestral state. In this study, we aimed to characterize the antiviral immune response of the cnidarian Nematostella vectensis and decipher the function of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) known to detect viral double-stranded RNA (dsRNA) in bilaterians but activate different antiviral pathways in vertebrates and nematodes. We show that polyinosinic:polycytidylic acid (poly(I:C)), a mimic of long viral dsRNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), triggers a complex antiviral immune response bearing features distinctive for both vertebrate and invertebrate systems. Importantly, a well-characterized agonist of the vertebrate RIG-I receptor does not induce a significant transcriptomic response that bears signature of the antiviral immune response, which experimentally supports the results of a phylogenetic analysis indicating clustering of the two N. vectensis RLR paralogs (NveRLRa and NveRLRb) with MDA5. Furthermore, the results of affinity assays reveal that NveRLRb binds poly(I:C) and long dsRNA and its knockdown impairs the expression of putative downstream effector genes including RNA interference components. Our study provides for the first time the functional evidence for the conserved role of RLRs in initiating immune response to dsRNA that originated before the cnidarian–bilaterian split and lay a strong foundation for future research on the evolution of the immune responses to RNA viruses.

Historical genomics reveals the evolutionary mechanisms behind multiple outbreaks of the host-specific coffee wilt pathogen Fusarium xylarioides

BackgroundNearly 50% of crop yields are lost to pests and disease, with plants and pathogens locked in an amplified co-evolutionary process of disease outbreaks. Coffee wilt disease, caused by Fusarium xylarioides, decimated coffee production in west and central Africa following its initial outbreak in the 1920s. After successful management, it later re-emerged and by the 2000s comprised two separate epidemics on arabica coffee in Ethiopia and robusta coffee in east and central Africa.ResultsHere, we use genome sequencing of six historical culture collection strains spanning 52 years to identify the evolutionary processes behind these repeated outbreaks. Phylogenomic reconstruction using 13,782 single copy orthologs shows that the robusta population arose from the initial outbreak, whilst the arabica population is a divergent sister clade to the other strains. A screen for putative effector genes involved in pathogenesis shows that the populations have diverged in gene content and sequence mainly by vertical processes within lineages. However, 15 putative effector genes show evidence of horizontal acquisition, with close homology to genes from F. oxysporum. Most occupy small regions of homology within wider scaffolds, whereas a cluster of four genes occupy a 20Kb scaffold with strong homology to a region on a mobile pathogenicity chromosome in F. oxysporum that houses known effector genes. Lacking a match to the whole mobile chromosome, we nonetheless found close associations with DNA transposons, especially the miniature impala type previously proposed to facilitate horizontal transfer of pathogenicity genes in F. oxysporum. These findings support a working hypothesis that the arabica and robusta populations partly acquired distinct effector genes via transposition-mediated horizontal transfer from F. oxysporum, which shares coffee as a host and lives on other plants intercropped with coffee.ConclusionOur results show how historical genomics can help reveal mechanisms that allow fungal pathogens to keep pace with our efforts to resist them. Our list of putative effector genes identifies possible future targets for fungal control. In turn, knowledge of horizontal transfer mechanisms and putative donor taxa might help to design future intercropping strategies that minimize the risk of transfer of effector genes between closely-related Fusarium taxa.

247-OR: Variant-to-Gene-Mapping Analyses Reveal a Role for Pancreatic Islet Cells in Conferring Genetic Susceptibility to Sleep-Related Traits

Altered glucose metabolism observed in sleep dysregulation has been reported to contribute to obesity and type 2 diabetes (T2D) risk. We elected to investigate if known sleep loci exert any of their effect in the pancreatic alpha and beta-cell setting. To establish genetic commonalities between sleep and metabolic disorders, we conducted linkage disequilibrium (LD) genetic correlation analyses with publicly available GWAS summary statistics for 4 sleep-associated traits (insomnia, chronotype, short and long sleep), T2D and obesity. Insomnia was significantly correlated with both T2D (genetic correlation (rg)=0.2; P=2.4x10-11) and obesity (rg=0.13; P=0.0021), as was short sleep (T2D, rg=0.2; P=1.8x10-10; obesity, rg=0.22; and P=5.1x10-7) and long sleep (T2D, rg=0.23; P=3.1x10-10; obesity rg=0.09; P=0.019). Chronotype was significantly correlated with T2D only (rg=0.05; P=0.038). Using partitioned LD score regression we then intersected summary statistics with our ATAC-seq and high-resolution promoter-focused Capture C data to assess enrichment of sleep trait SNPs within promoter-interacting open chromatin regions in both the human beta-cell line, EndoC-BH1, and sorted human alpha-cells. EndoC-BH1 was highly significantly enriched for chronotype loci (P=2.2x10-7). Furthermore there was significant enrichment for insomnia (P=0.01) and short sleep (P=0.017). The alpha-cells were also significantly enriched for chronotype (P=0.0016), insomnia (P=0.0076), short sleep (P=0.022), along with long sleep (P=0.034). Motivated by these observations, subsequent variant-to-gene mapping implicated 32, 37 and 6 putative effector genes for insomnia, chronotype and short sleep, respectively, in EndoC-BH1. For alpha-cells, 55, 54, 10 and 5 genes were implicated for insomnia, chronotype, short and long sleep, respectively. Our data suggest that a subset of sleep-related loci confer their effects via cells in pancreatic islets. Disclosure C. Lasconi: None. M. C. Pahl: None. J. A. Pippin: None. C. Su: None. M. Johnson: None. A. Chesi: None. A. D. Wells: None. K. H. Kaestner: None. S. F. A. Grant: None. Funding National Institutes of Health (UM1DK126194)

Detection of Plasmopara halstedii in sunflower seeds: A case study using molecular testing

Plasmopara halstedii (Farl.) Berl. & De Toni causing downy mildew of sunflower is responsible for considerable economic losses worldwide. Because P. halstedii can be seed-transmitted, monitoring of seeds for pathogen contamination is important for the sunflower seed trade. The relevance of asymptomatic or latent infections as factors of disease spread have not been studied by molecular techniques. A molecular marker based on a putative effector gene of P. halstedii was used to examine the pathogen’s presence in asymptomatic sunflowers growing near patches of mildewed plants in naturally infected fields. The method based on conventional PCR was highly sensitive for detection of P. halstedii in DNA from whole seeds. By the application of this protocol, we found that all seed samples obtained from symptomatic plants amplified the expected fragment, whereas the diagnostic marker identified the presence of pathogen in one out of 21 asymptomatic plants. Possible uses of this marker to detect downy mildew in seed from asymptomatic plants or for race identification are discussed.

Gut Microbiota Accelerates Bone Growth and Marrow Adiposity of the Adolescent Gnotobiotic Rat

Skeletal growth and bone homeostasis are significant holobiome components strongly tied to the gut microbiota. We aimed to explore the requirement of gut microbiome for skeletal maturation in an adolescent organism. For our approach, we co-housed young (7-week-old) germ-free (GF) rats with conventional rats of the same age, sex (male), and strain (Sprague Dawley) for 10 days to naturally conventionalize GF (GFC) rats with the gut microbiota by coprophagy. Bone morphometry by micro-CT scanning and histological staining revealed accelerated longitudinal and radial bone growth in GFC rats when compared to GF rats. Changes in bone mass and structure for GFC rats encompassed an increase of bone accrual in cortical and trabecular compartments, greater tissue mineral density, improved proliferation and hypertrophy of growth plate chondrocytes, bone lengthening, and preferential deposition of periosteal bone in the tibia diaphysis. Analysis of bone marrow at the tibia midshaft depicted more adipocyte number, greater adipocyte density, and altered cell size distribution to favor small adipocytes, but reduced megakaryocytes, in GFC rats. Interestingly, putative effector genes responsible for bone mass accumulation were found to be comparable between GF and GFC rats, which shifted our focus onto systemic host and microbial regulatory factors. Assessment of serum markers indicated that the active bone formation could be due to elevated levels of 25-hydroxy vitamin D and alkaline phosphatase. Importantly, 16S ribosomal RNA gene sequencing demonstrated that the prominent bacterial colonizers were Clostridiaceae and Clostridium members, which are potent short chain fatty acid (SCFA) producers. 1H-NMR metabolomics in the cecal contents showed a dramatic increase of SCFA, specifically butyrate, following bacterial colonization. In relation with the literature, our observation of increased hepatic insulin-like growth factor-1 (IGF-1) expression and systemic IGF-1 levels suggest that butyrate promotes bone growth through the somatotropic axis. Collectively, our study demonstrates that gut microbiota can deliver signals to the skeleton for accelerating bone expansion and accumulating marrow fat.

CcPmk1 is a regulator of pathogenicity in Cytospora chrysosperma and can be used as a potential target for disease control.

Fus3/Kss1, also known as Pmk1 in several pathogenic fungi, is a component of the mitogen‐activated protein kinase (MAPK) signalling pathway that functions as a regulator in fungal development, stress response, mating, and pathogenicity. Cytospora chrysosperma, a notorious woody plant‐pathogenic fungus, causes canker disease in many species, and its Pmk1 homolog, CcPmk1, is required for fungal development and pathogenicity. However, the global regulation network of CcPmk1 is still unclear. In this study, we compared transcriptional analysis between a CcPmk1 deletion mutant and the wild type during the simulated infection process. A subset of transcription factor genes and putative effector genes were significantly down‐regulated in the CcPmk1 deletion mutant, which might be important for fungal pathogenicity. Additionally, many tandem genes were found to be regulated by CcPmk1. Eleven out of 68 core secondary metabolism biosynthesis genes and several gene clusters were significantly down‐regulated in the CcPmk1 deletion mutant. GO annotation of down‐regulated genes showed that the ribosome biosynthesis‐related processes were over‐represented in the CcPmk1 deletion mutant. Comparison of the CcPmk1‐regulated genes with the Pmk1‐regulated genes from Magnaporthe oryzae revealed only a few overlapping regulated genes in both CcPmk1 and Pmk1, while the enrichment GO terms in the ribosome biosynthesis‐related processes were also found. Subsequently, we calculated that in vitro feeding artificial small interference RNAs of CcPmk1 could silence the target gene, resulting in inhibited fungal growth. Furthermore, silencing of BcPmk1 in Botrytis cinerea with conserved CcPmk1 and BcPmk1 fragments could significantly compromise fungal virulence using the virus‐induced gene silencing system in Nicotiana benthamiana. These results suggest that CcPmk1 functions as a regulator of pathogenicity and can potentially be designed as a target for broad‐spectrum disease control, but unintended effects on nonpathogenic fungi need to be avoided.

A Sge1 homolog in Cytospora chrysosperma governs conidiation, virulence and the expression of putative effectors.

SIX Gene Expression 1 (Sge1) is an important and well-recognized fungal-specific transcription regulator from the Gti1/Pac2 family that exhibits a conserved function in the vegetative growth, regulating the expression of effector genes and pathogenicity in plant pathogenic fungi. However, its functions in Cytospora chrysosperma, a notorious phytopathogenic fungus in forestry, remain poorly understood. Here, we characterized a Sge1 orthologue, CcSge1, in C. chrysosperma and deleted its Gti1/Pac2 domain for functional analysis. The CcSge1 deletion mutants showed obvious defects in hyphal growth, conidial production and response to hydrogen peroxide. Correspondingly, significantly lower expression of conidiation related genes were found in deletion mutants compared to that of the wild type. Importantly, the CcSge1 deletion mutants totally lost their pathogenicity to the host. Further analysis demonstrated that CcSge1 was responsible for the expression of putative effector genes and the transcription of CcSge1 was under tight control by pathogenicity-related MAP Kinase 1 (CcPmk1). What's more, one of the putative effector gene CCG_07874 was positively regulated by both CcSge1 and CcPmk1. Taken together, these data indicate that CcSge1is indispensable for hyphal radial growth, conidiation, the expression of effector genes and fungal virulence.

3D promoter architecture re-organization during iPSC-derived neuronal cell differentiation implicates target genes for neurodevelopmental disorders.

Neurodevelopmental disorders are thought to arise from interrupted development of the brain at an early age. Genome-wide association studies (GWAS) have identified hundreds of loci associated with susceptibility to neurodevelopmental disorders; however, which noncoding variants regulate which genes at these loci is often unclear. To implicate neuronal GWAS effector genes, we performed an integrated analysis of transcriptomics, epigenomics and chromatin conformation changes during the development from Induced pluripotent stem cell-derived neuronal progenitor cells (NPCs) into neurons using a combination of high-resolution promoter-focused Capture-C, ATAC-seq and RNA-seq. We observed that gene expression changes during the NPC-to-neuron transition were highly dependent on both promoter accessibility changes and long-range interactions which connect distal cis-regulatory elements (enhancer or silencers) to developmental-stage-specific genes. These genome-scale promoter-cis-regulatory-element atlases implicated 454 neurodevelopmental disorder-associated, putative causal variants mapping to 600 distal targets. These putative effector genes were significantly enriched for pathways involved in the regulation of neuronal development and chromatin organization, with 27 % expressed in a stage-specific manner. The intersection of open chromatin and chromatin conformation revealed development-stage-specific gene regulatory architectures during neuronal differentiation, providing a rich resource to aid characterization of the genetic and developmental basis of neurodevelopmental disorders.

Putative Novel Effector Genes Revealed by the Genomic Analysis of the Phytopathogenic Fungus Fusarium oxysporum f. sp. physali (Foph) That Infects Cape Gooseberry Plants.

The vascular wilt disease caused by the fungus Fusarium oxysporum f. sp. physali (Foph) is one of the most limiting factors for the production and export of cape gooseberry (Physalis peruviana) in Colombia. A transcriptomic analysis of a highly virulent strain of F. oxysporum in cape gooseberry plants, revealed the presence of secreted in the xylem (SIX) effector genes, known to be involved in the pathogenicity of other formae speciales (ff. spp.) of F. oxysporum. This pathogenic strain was classified as a new f. sp. named Foph, due to its specificity for cape gooseberry hosts. Here, we sequenced and assembled the genome of five strains of F. oxysporum from a fungal collection associated to the cape gooseberry crop (including Foph), focusing on the validation of the presence of SIX homologous and on the identification of putative effectors unique to Foph. By comparative and phylogenomic analyses based on single-copy orthologous, we found that Foph is closely related to F. oxysporum ff. spp., associated with solanaceous hosts. We confirmed the presence of highly identical homologous genomic regions between Foph and Fol that contain effector genes and identified six new putative effector genes, specific to Foph pathogenic strains. We also conducted a molecular characterization using this set of putative novel effectors in a panel of 36 additional stains of F. oxysporum including two of the four sequenced strains, from the fungal collection mentioned above. These results suggest the polyphyletic origin of Foph and the putative independent acquisition of new candidate effectors in different clades of related strains. The novel effector candidates identified in this genomic analysis, represent new sources involved in the interaction between Foph and cape gooseberry, that could be implemented to develop appropriate management strategies of the wilt disease caused by Foph in the cape gooseberry crop.

Genome-wide association study implicates novel loci and reveals candidate effector genes for longitudinal pediatric bone accrual

BackgroundBone accrual impacts lifelong skeletal health, but genetic discovery has been primarily limited to cross-sectional study designs and hampered by uncertainty about target effector genes. Here, we capture this dynamic phenotype by modeling longitudinal bone accrual across 11,000 bone scans in a cohort of healthy children and adolescents, followed by genome-wide association studies (GWAS) and variant-to-gene mapping with functional follow-up.ResultsWe identify 40 loci, 35 not previously reported, with various degrees of supportive evidence, half residing in topological associated domains harboring known bone genes. Of several loci potentially associated with later-life fracture risk, a candidate SNP lookup provides the most compelling evidence for rs11195210 (SMC3). Variant-to-gene mapping combining ATAC-seq to assay open chromatin with high-resolution promoter-focused Capture C identifies contacts between GWAS loci and nearby gene promoters. siRNA knockdown of gene expression supports the putative effector gene at three specific loci in two osteoblast cell models. Finally, using CRISPR-Cas9 genome editing, we confirm that the immediate genomic region harboring the putative causal SNP influences PRPF38A expression, a location which is predicted to coincide with a set of binding sites for relevant transcription factors.ConclusionsUsing a new longitudinal approach, we expand the number of genetic loci putatively associated with pediatric bone gain. Functional follow-up in appropriate cell models finds novel candidate genes impacting bone accrual. Our data also raise the possibility that the cell fate decision between osteogenic and adipogenic lineages is important in normal bone accrual.

Putative Effector Genes Distinguish Two Pathogenicity Groups of Fusarium oxysporum f. sp. spinaciae.

Fusarium wilt of spinach, caused by Fusarium oxysporum f. sp. spinaciae, is an important disease during warm conditions in production regions with acid soils, yet little is known about what confers pathogenicity to spinach in F. oxysporum f. sp. spinaciae genetically. To identify candidate fungal genes that contribute to spinach Fusarium wilt, each of 69 geographically diverse F. oxysporum isolates was tested for pathogenicity on each of three spinach inbreds. Thirty-nine isolates identified as F. oxysporum f. sp. spinaciae caused quantitative differences in disease severity among the inbreds that revealed two distinct pathogenicity groups of F. oxysporum f. sp. spinaciae. Putative effector gene profiles, predicted from whole-genome sequences generated for nine F. oxysporum f. sp. spinaciae isolates and five nonpathogenic, spinach-associated F. oxysporum (NPS) isolates, distinguished the F. oxysporum f. sp. spinaciae isolates from the NPS isolates, and separated the F. oxysporum f. sp. spinaciae isolates into two groups. Five of the putative effector genes appeared to be unique to F. oxysporum f. sp. spinaciae, as they were not found in 222 other publicly available genome assemblies of F. oxysporum, implicating potential involvement of these genes in pathogenicity to spinach. In addition, two combinations of the 14 known Secreted in Xylem (SIX) genes that have been affiliated with host pathogenicity in other formae speciales of F. oxysporum were identified in genome assemblies of the nine F. oxysporum f. sp. spinaciae isolates, either SIX8 and SIX9 or SIX4, SIX8, and SIX14. Characterization of these putative effector genes should aid in understanding mechanisms of pathogenicity in F. oxysporum f. sp. spinaciae, developing molecular tools for rapid detection and quantification of F. oxysporum f. sp. spinaciae, and breeding for resistance to Fusarium wilt in spinach.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

High‐resolution, genome‐wide, promoter‐focused Capture C in astrocytes implicates causal genes for Alzheimer’s disease

AbstractBackgroundPrevious studies of Alzheimer’s Disease (AD) have primarily implicated neurons and microglia as disease‐conferring cell types due to obvious links with degeneration and inflammation, respectively. However, astrocytes may also play a role in AD pathogenesis, but have been less studied in this context. Indeed, genome‐wide association studies (GWAS) have identified multiple genomic variants that reside near genes with astrocyte‐related functions, such as lipid processing and synaptic function. However, GWAS only reports genomic variants associated with a given trait and not necessarily the precise localization of culprit genes. High resolution chromatin conformation capture‐based techniques detect 3D genomic interactions between GWAS‐implicated signals and their effector genes, and allow for the characterization of non‐coding variants in the context of their regulatory activity.MethodTo improve on the low resolution of available Hi‐C data, we employed a high resolution DpnII‐based Capture‐C method to simultaneously characterize the physical genome‐wide interactions of 36,691 baited regions covering human promoters genome‐wide (including non‐coding transcripts) ‐ which we applied to a human primary astrocyte line (NHA, Lonza). Following sequencing, we investigated significant interactions at two different resolutions (1‐fragment and 4‐fragment resolutions; median fragment size = 265bp and 1,441bp, respectively). In parallel, we generated ATAC‐seq open chromatin maps to filter for informative (r2>0.7) proxy single nucleotide polymorphisms (SNPs) for each of the 45 AD independent signals reported to date.ResultATAC‐seq fine‐mapping yielded 100 candidate SNPs in open chromatin for 30 of these loci. By further constraining on our promoter Capture C data in astrocytes, at both one and four DpnII fragment resolution (median distance between interacting regions 9kb and 71kb, respectively), we observed contacts to “open” promoters for 6 putative target genes, corresponding to 6 of the original GWAS loci. These included PICALM, FERMT2, CASS4, and CLU. ConclusionWe observed informative contacts between proxy SNPs and putative effector genes in the human astrocyte context for ∼13% of AD GWAS loci. Further efforts in other relevant cell types, where many of the other loci may in fact be principally operating, should shed light on additional signals. Follow‐up functional studies are warranted to validate these findings.

A single gene in Fusarium oxysporum limits host range.

Fusarium oxysoporum f. sp. radicis‐cucumerinum (Forc) is able to cause disease in cucumber, melon, and watermelon, while F. oxysporum f. sp. melonis (Fom) can only infect melon plants. Earlier research showed that mobile chromosomes in Forc and Fom determine the difference in host range between Forc and Fom. By closely comparing these pathogenicity chromosomes combined with RNA‐sequencing data, we selected 11 candidate genes that we tested for involvement in the difference in host range between Forc and Fom. One of these candidates is a putative effector gene on the Fom pathogenicity chromosome that has nonidentical homologs on the Forc pathogenicity chromosome. Four independent Forc transformants with this gene from Fom showed strongly reduced or no pathogenicity towards cucumber, while retaining pathogenicity towards melon and watermelon. This suggests that the protein encoded by this gene is recognized by an immune receptor in cucumber plants. This is the first time that a single gene has been demonstrated to determine a difference in host specificity between formae speciales of F. oxysporum.

Variant-to-Gene-Mapping Analyses Reveal a Role for the Hypothalamus in Genetic Susceptibility to Inflammatory Bowel Disease.

Background & AimsInflammatory bowel disease (IBD) is a polygenic disorder characterized principally by dysregulated inflammation impacting the gastrointestinal tract. However, there also is increasing evidence for a clinical association with stress and depression. Given the role of the hypothalamus in stress responses and in the pathogenesis of depression, useful insights could be gleaned from understanding its genetic role in IBD.MethodsWe conducted genetic correlation analyses on publicly available genome-wide association study summary statistics for depression and IBD traits to identify genetic commonalities. We used partitioned linkage disequilibrium score regression, leveraging our ATAC sequencing and promoter-focused Capture C data, to measure enrichment of IBD single-nucleotide polymorphisms within promoter-interacting open chromatin regions of human embryonic stem cell-derived hypothalamic-like neurons (HNs). Using the same data sets, we performed variant-to-gene mapping to implicate putative IBD effector genes in HNs. To contrast these results, we similarly analyzed 3-dimensional genomic data generated in epithelium-derived colonoids from rectal biopsy specimens from donors without pathologic disease noted at the time of colonoscopy. Finally, we conducted enrichment pathway analyses on the implicated genes to identify putative IBD dysfunctional pathways.ResultsWe found significant genetic correlations (rg) of 0.122 with an adjusted P (Padj) = 1.4 × 10-4 for IBD: rg = 0.122; Padj = 2.5 × 10-3 for ulcerative colitis and genetic correlation (rg) = 0.094; Padj = 2.5 × 10-3 for Crohn’s disease, and significant approximately 4-fold (P = .005) and approximately 7-fold (P = .03) enrichment of IBD single-nucleotide polymorphisms in HNs and colonoids, respectively. We implicated 25 associated genes in HNs, among which CREM, CNTF, and RHOA encode key regulators of stress. Seven genes also additionally were implicated in the colonoids. We observed an overall enrichment for immune and hormonal signaling pathways, and a colonoid-specific enrichment for microbiota-relevant terms.ConclusionsOur results suggest that the hypothalamus warrants further study in the context of IBD pathogenesis.

The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia

Abstract. Herlina L, Istiaji B. 2020. The use of effector gene based-markers to facilitate identification of Fusarium sp. infected shallot in Java, Indonesia. Biodiversitas 21: 4677-4685. One of the most important diseases and become challenge in breeding resistant variety of shallots in Indonesia is Fusarium disease, caused by Fusarium oxysporum f.sp. cepae (FOC). To discriminate the F. oxysporum into forma speciales is uneasy, often managed through laborious and time-intensive disease assays., therefore molecular approaches become the most relevant choice. FOCs from Indonesia were examined by molecular criteria based on putative and effector genes-based markers, i.e.: SIX-genes, C5-gene, and CRX1-2-genes. The alignment analysis discovered some regions which sequences highly contained conserved bases, while amplification result bands vary in size, between 400 - 500 bp. The combined of 7 SIX-genes primers and 3 effector primers (C5 and CRX1-2) used in the clustering analysis of 15 FOC isolates in this study showed that they succeeded in separating 15 FOC isolates into 4 groups through NTSys. Clustering analysis showed that those markers succeeded in grouping 15 FOC isolates into different clades (by coefficient of similarity: 0.69). Phylogenetic analysis based on CRX genes sequence as putative effector genes confirmed that CRX1 and CRX2 genes were able to classify the FOC into their forma speciales. Those effectors genes are potential to serve as marker-templates to facilitate identification of FOC which infested shallots in Indonesia.