Time-Course Transcriptome Profiling Reveals Differential Resistance Responses of Tomato to a Phytotoxic Effector of the Pathogenic Oomycete Phytophthora cactorum.

Blight caused by Phytophthora pathogens has a devastating impact on crop production. Phytophthora species secrete an array of effectors, such as Phytophthora cactorum-Fragaria (PcF)/small cysteine-rich (SCR) phytotoxic proteins, to facilitate their infections. Understanding host responses to such proteins is essential to developing next-generation crop resistance. Our previous work identified a small, 8.1 kDa protein, SCR96, as an important virulence factor in Phytophthora cactorum. Host responses to SCR96 remain obscure. Here, we analyzed the effect of SCR96 on the resistance of tomato treated with this recombinant protein purified from yeast cells. A temporal transcriptome analysis of tomato leaves infiltrated with 500 nM SCR96 for 0, 3, 6, and 12 h was performed using RNA-Seq. In total, 36,779 genes, including 2704 novel ones, were detected, of which 32,640 (88.7%) were annotated. As a whole, 5929 non-redundant genes were found to be significantly co-upregulated in SCR96-treated leaves (3, 6, 12 h) compared to the control (0 h). The combination of annotation, enrichment, and clustering analyses showed significant changes in expression beginning at 3 h after treatment in genes associated with defense and metabolism pathways, as well as temporal transcriptional accumulation patterns. Noticeably, the expression levels of resistance-related genes encoding receptor-like kinases/proteins, resistance proteins, mitogen-activated protein kinases (MAPKs), transcription factors, pathogenesis-related proteins, and transport proteins were significantly affected by SCR96. Quantitative reverse transcription PCR (qRT-PCR) validated the transcript changes in the 12 selected genes. Our analysis provides novel information that can help delineate the molecular mechanism and components of plant responses to effectors, which will be useful for the development of resistant crops.

Nlrx1-Regulated Defense and Metabolic Responses to Aspergillus fumigatus Are Morphotype and Cell Type Specific.

The Nlr family member X1 (Nlrx1) is an immuno-metabolic hub involved in mediating effective responses to virus, bacteria, fungi, cancer, and auto-immune diseases. We have previously shown that Nlrx1 is a critical regulator of immune signaling and mortality in several models of pulmonary fungal infection using the clinically relevant fungus Aspergillus fumigatus. In the absence of Nlrx1, hosts produce an enhanced Th2 response primarily by CD103+ dendritic cell populations resulting in enhanced mortality via immunopathogenesis as well as enhanced fungal burden. Here, we present our subsequent efforts showcasing loss of Nlrx1 resulting in a decreased ability of host cells to process A. fumigatus conidia in a cell-type-specific manner by BEAS-2B airway epithelial cells, alveolar macrophages, bone marrow-derived macrophages, but not bone marrow-derived neutrophils. Furthermore, loss of Nlrx1 results in a diminished ability to generate superoxide and/or generic reactive oxygen species during specific responses to fungal PAMPs, conidia, and hyphae. Analysis of glycolysis and mitochondrial function suggests that Nlrx1 is needed to appropriately shut down glycolysis in response to A. fumigatus conidia and increase glycolysis in response to hyphae in BEAS-2B cells. Blocking glycolysis and pentose phosphate pathway (PPP) via 2-DG and NADPH production through glucose-6-phosphate dehydrogenase inhibitor resulted in significantly diminished conidial processing in wild-type BEAS-2B cells to the levels of Nlrx1-deficient BEAS-2B cells. Our findings suggest a need for airway epithelial cells to generate NADPH for reactive oxygen species production in response to conidia via PPP. In context to fungal pulmonary infections, our results show that Nlrx1 plays significant roles in host defense via PPP modulation of several aspects of metabolism, particularly glycolysis, to facilitate conidia processing in addition to its critical role in regulating immune signaling.

LYSMD3: A mammalian pattern recognition receptor for chitin.

Chitin, a major component of fungal cell walls, has been associated with allergic disorders such as asthma. However, it is unclear how mammals recognize chitin and the principal receptor(s) on epithelial cells that sense chitin remain to be determined. In this study, we show that LYSMD3 is expressed on the surface of human airway epithelial cells and demonstrate that LYSMD3 is able to bind chitin, as well as ß-glucan, on the cell walls of fungi. Knockdown or knockout of LYSMD3 also sharply blunts the production of inflammatory cytokines by epithelial cells in response to chitin and fungal spores. Competitive inhibition of the LYSMD3 ectodomain by soluble LYSMD3 protein, multiple ligands, or antibody against LYSMD3 also blocks chitin signaling. Our study reveals LYSMD3 as a mammalian pattern recognition receptor (PRR) for chitin and establishes its role in epithelial cell inflammatory responses to chitin and fungi.

Accurate and efficient gene function prediction using a multi-bacterial network.

MOTIVATION: Nearly 40% of the genes in sequenced genomes have no experimentally or computationally derived functional annotations. To fill this gap, we seek to develop methods for network-based gene function prediction that can integrate heterogeneous data for multiple species with experimentally based functional annotations and systematically transfer them to newly sequenced organisms on a genome-wide scale. However, the large sizes of such networks pose a challenge for the scalability of current methods. RESULTS: We develop a label propagation algorithm called FastSinkSource. By formally bounding its rate of progress, we decrease the running time by a factor of 100 without sacrificing accuracy. We systematically evaluate many approaches to construct multi-species bacterial networks and apply FastSinkSource and other state-of-the-art methods to these networks. We find that the most accurate and efficient approach is to pre-compute annotation scores for species with experimental annotations, and then to transfer them to other organisms. In this manner, FastSinkSource runs in under 3 min for 200 bacterial species. AVAILABILITY AND IMPLEMENTATION: An implementation of our framework and all data used in this research are available at https://github.com/Murali-group/multi-species-GOA-prediction. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

"Small" Intestinal Immunopathology Plays a "Big" Role in Lethal Cytokine Release Syndrome, and Its Modulation by Interferon-γ, IL-17A, and a Janus Kinase Inhibitor.

Chimeric antigen receptor T cell (CART) therapy, administration of certain T cell-agonistic antibodies, immune check point inhibitors, coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) and Toxic shock syndrome (TSS) caused by streptococcal as well as staphylococcal superantigens share one common complication, that is T cell-driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). It is not understood whether the failure of a particular organ contributes more significantly to the severity of CRS. Also not known is whether a specific cytokine or signaling pathway plays a more pathogenic role in precipitating MOD compared to others. As a result, there is no specific treatment available to date for CRS, and it is managed only symptomatically to support the deteriorating organ functions and maintain the blood pressure. Therefore, we used the superantigen-induced CRS model in HLA-DR3 transgenic mice, that closely mimics human CRS, to delineate the immunopathogenesis of CRS as well as to validate a novel treatment for CRS. Using this model, we demonstrate that (i) CRS is characterized by a rapid rise in systemic levels of several Th1/Th2/Th17/Th22 type cytokines within a few hours, followed by a quick decline. (ii) Even though multiple organs are affected, small intestinal immunopathology is the major contributor to mortality in CRS. (iii) IFN-γ deficiency significantly protected from lethal CRS by attenuating small bowel pathology, whereas IL-17A deficiency significantly increased mortality by augmenting small bowel pathology. (iv) RNA sequencing of small intestinal tissues indicated that IFN-γ-STAT1-driven inflammatory pathways combined with enhanced expression of pro-apoptotic molecules as well as extracellular matrix degradation contributed to small bowel pathology in CRS. These pathways were further enhanced by IL-17A deficiency and significantly down-regulated in mice lacking IFN-γ. (v) Ruxolitinib, a selective JAK-1/2 inhibitor, attenuated SAg-induced T cell activation, cytokine production, and small bowel pathology, thereby completely protecting from lethal CRS in both WT and IL-17A deficient HLA-DR3 mice. Overall, IFN-γ-JAK-STAT-driven pathways contribute to lethal small intestinal immunopathology in T cell-driven CRS.

Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells.

Aspergillus fumigatus is a ubiquitous mold that produces small airborne conidia capable of traversing deep into the respiratory system. Recognition, processing, and clearance of A. fumigatus conidia by bronchial airway epithelial cells are thought to be relevant to host defense and immune signaling. Using z-stack confocal microscopy, we observed that only 10 to 20% of adherent conidia from the AF293 clinical isolate are internalized by BEAS-2B cells 6 h postchallenge and not prior. Similar percentages of internalization were observed for the CEA10 clinical isolate. A large subset of both AF293 and CEA10 conidia are rendered metabolically inactive without internalization at 3 h postchallenge by BEAS-2B cells. A significantly larger percentage of CEA10 conidia are metabolically active at 9 and 12 h postchallenge in comparison to the AF293 isolate, demonstrating heterogeneity among clinical isolates. We identified 7 host markers (caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1) that consistently localized around internalized conidia 9 h postchallenge. Transient gene silencing of RAB5C, PIK3C3, and flotillin-2 resulted in a larger population of metabolically active conidia. Our findings emphasize the abundance of both host phosphatidylinositol 3-phosphate (PI3P) and PI4P around internalized conidia, as well as the importance of class III PI3P kinase for conidial processing. Therapeutic development focused on RAB5C-, PIK3C3-, and flotillin-2-mediated pathways may provide novel opportunities to modulate conidial processing and internalization. Determination of how contacted, external conidia are processed by airway epithelial cells may also provide a novel avenue to generate host-targeted therapeutics.IMPORTANCE Conidia from the fungus Aspergillus fumigatus are notorious for their ability to stay airborne. This characteristic is believed to allow conidia to penetrate into the cleanest environments. Several hundred conidia are thought to be inhaled each day by a given individual and then expelled by mucociliary clearance. Given that airway epithelial cells make up a significant portion of the pulmonary-air interface, we set out to determine the percentage of conidia that are actually internalized after initial contact with airway epithelial cells. We determined this through an in vitro assay using an immortalized bronchial airway epithelial cell line known as BEAS-2B. Our results suggest a small fraction of conidia are internalized by BEAS-2B cells, while the majority stay adherent to the surface of cells or are washed away during sample processing. Internalization of conidia was observed at 6 h postchallenge and not prior. Our data also indicate conidia are rendered metabolically inactive within 3 h of challenge, suggesting BEAS-2B cells process a large number of conidia without internalization in this early time frame. We have also identified several host endocytosis markers that localize around internalized conidia as well as contribute to the processing of conidia. Understanding how these host endocytosis markers affect the processing of internal and/or external conidia may provide a novel avenue for therapeutic development.

Large-scale protein function prediction using heterogeneous ensembles.

Heterogeneous ensembles are an effective approach in scenarios where the ideal data type and/or individual predictor are unclear for a given problem. These ensembles have shown promise for protein function prediction (PFP), but their ability to improve PFP at a large scale is unclear. The overall goal of this study is to critically assess this ability of a variety of heterogeneous ensemble methods across a multitude of functional terms, proteins and organisms. Our results show that these methods, especially Stacking using Logistic Regression, indeed produce more accurate predictions for a variety of Gene Ontology terms differing in size and specificity. To enable the application of these methods to other related problems, we have publicly shared the HPC-enabled code underlying this work as LargeGOPred ( https://github.com/GauravPandeyLab/LargeGOPred).

Comparative genome analyses reveal sequence features reflecting distinct modes of host-adaptation between dicot and monocot powdery mildew.

BACKGROUND: Powdery mildew (PM) is one of the most important and widespread plant diseases caused by biotrophic fungi. Notably, while monocot (grass) PM fungi exhibit high-level of host-specialization, many dicot PM fungi display a broad host range. To understand such distinct modes of host-adaptation, we sequenced the genomes of four dicot PM biotypes belonging to Golovinomyces cichoracearum or Oidium neolycopersici. RESULTS: We compared genomes of the four dicot PM together with those of Blumeria graminis f.sp. hordei (both DH14 and RACE1 isolates), B. graminis f.sp. tritici, and Erysiphe necator infectious on barley, wheat and grapevine, respectively. We found that despite having a similar gene number (6620-6961), the PM genomes vary from 120 to 222 Mb in size. This high-level of genome size variation is indicative of highly differential transposon activities in the PM genomes. While the total number of genes in any given PM genome is only about half of that in the genomes of closely related ascomycete fungi, most (~ 93%) of the ascomycete core genes (ACGs) can be found in the PM genomes. Yet, 186 ACGs were found absent in at least two of the eight PM genomes, of which 35 are missing in some dicot PM biotypes, but present in the three monocot PM genomes, indicating remarkable, independent and perhaps ongoing gene loss in different PM lineages. Consistent with this, we found that only 4192 (3819 singleton) genes are shared by all the eight PM genomes, the remaining genes are lineage- or biotype-specific. Strikingly, whereas the three monocot PM genomes possess up to 661 genes encoding candidate secreted effector proteins (CSEPs) with families containing up to 38 members, all the five dicot PM fungi have only 116-175 genes encoding CSEPs with limited gene amplification. CONCLUSIONS: Compared to monocot (grass) PM fungi, dicot PM fungi have a much smaller effectorome. This is consistent with their contrasting modes of host-adaption: while the monocot PM fungi show a high-level of host specialization, which may reflect an advanced host-pathogen arms race, the dicot PM fungi tend to practice polyphagy, which might have lessened selective pressure for escalating an with a particular host.

Modulation of Immune Signaling and Metabolism Highlights Host and Fungal Transcriptional Responses in Mouse Models of Invasive Pulmonary Aspergillosis.

Incidences of invasive pulmonary aspergillosis, an infection caused predominantly by Aspergillus fumigatus, have increased due to the growing number of immunocompromised individuals. While A. fumigatus is reliant upon deficiencies in the host to facilitate invasive disease, the distinct mechanisms that govern the host-pathogen interaction remain enigmatic, particularly in the context of distinct immune modulating therapies. To gain insights into these mechanisms, RNA-Seq technology was utilized to sequence RNA derived from lungs of 2 clinically relevant, but immunologically distinct murine models of IPA on days 2 and 3 post inoculation when infection is established and active disease present. Our findings identify notable differences in host gene expression between the chemotherapeutic and steroid models at the interface of immunity and metabolism. RT-qPCR verified model specific and nonspecific expression of 23 immune-associated genes. Deep sequencing facilitated identification of highly expressed fungal genes. We utilized sequence similarity and gene expression to categorize the A. fumigatus putative in vivo secretome. RT-qPCR suggests model specific gene expression for nine putative fungal secreted proteins. Our analysis identifies contrasting responses by the host and fungus from day 2 to 3 between the two models. These differences may help tailor the identification, development, and deployment of host- and/or fungal-targeted therapeutics.

GraphSpace: stimulating interdisciplinary collaborations in network biology.

SUMMARY: Networks have become ubiquitous in systems biology. Visualization is a crucial component in their analysis. However, collaborations within research teams in network biology are hampered by software systems that are either specific to a computational algorithm, create visualizations that are not biologically meaningful, or have limited features for sharing networks and visualizations. We present GraphSpace, a web-based platform that fosters team science by allowing collaborating research groups to easily store, interact with, layout and share networks. AVAILABILITY AND IMPLEMENTATION: Anyone can upload and share networks at http://graphspace.org. In addition, the GraphSpace code is available at http://github.com/Murali-group/graphspace if a user wants to run his or her own server. CONTACT: murali@cs.vt.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Nucleolar Dominance and Repression of 45S Ribosomal RNA Genes in Hybrids between Xenopus borealis and X. muelleri (2n = 36).

Nucleolar dominance is a dramatic disruption in the formation of nucleoli and the expression of ribosomal RNA (rRNA) genes, characteristic of some plant and animal hybrids. Here, we report that F1 hybrids produced from reciprocal crosses between 2 sister species of Xenopus clawed frogs, X. muelleri and X. borealis, undergo nucleolar dominance somewhat distinct from a pattern previously reported in hybrids between phylogenetically more distant Xenopus species. Patterns of nucleolar development, 45S rRNA expression, and gene copy inheritance were investigated using a combination of immunostaining, pyrosequencing, droplet digital PCR, flow cytometry, and epigenetic inhibition. In X. muelleri × X. borealis hybrids, typically only 1 nucleolus is formed, and 45S rRNA genes are predominantly expressed from 1 progenitor's alleles, X. muelleri, regardless of the cross-direction. These changes are accompanied by an extensive (∼80%) loss of rRNA gene copies in the hybrids relative to their parents, with the transcriptionally underdominant variant (X. borealis) being preferentially lost. Chemical treatment of hybrid larvae with a histone deacetylase inhibitor resulted in a partial derepression of the underdominant variant. Together, these observations shed light on the genetic and epigenetic basis of nucleolar dominance as an underappreciated manifestation of genetic conflicts within a hybrid genome.

Selective advantage of trisomic human cells cultured in non-standard conditions.

An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes.

Pathways on demand: automated reconstruction of human signaling networks.

Signaling pathways are a cornerstone of systems biology. Several databases store high-quality representations of these pathways that are amenable for automated analyses. Despite painstaking and manual curation, these databases remain incomplete. We present PATHLINKER, a new computational method to reconstruct the interactions in a signaling pathway of interest. PATHLINKER efficiently computes multiple short paths from the receptors to transcriptional regulators (TRs) in a pathway within a background protein interaction network. We use PATHLINKER to accurately reconstruct a comprehensive set of signaling pathways from the NetPath and KEGG databases. We show that PATHLINKER has higher precision and recall than several state-of-the-art algorithms, while also ensuring that the resulting network connects receptor proteins to TRs. PATHLINKER's reconstruction of the Wnt pathway identified CFTR, an ABC class chloride ion channel transporter, as a novel intermediary that facilitates the signaling of Ryk to Dab2, which are known components of Wnt/ß-catenin signaling. In HEK293 cells, we show that the Ryk-CFTR-Dab2 path is a novel amplifier of ß-catenin signaling specifically in response to Wnt 1, 2, 3, and 3a of the 11 Wnts tested. PATHLINKER captures the structure of signaling pathways as represented in pathway databases better than existing methods. PATHLINKER's success in reconstructing pathways from NetPath and KEGG databases point to its applicability for complementing manual curation of these databases. PATHLINKER may serve as a promising approach for prioritizing proteins and interactions for experimental study, as illustrated by its discovery of a novel pathway in Wnt/ß-catenin signaling. Our supplementary website at http://bioinformatics.cs.vt.edu/~murali/supplements/2016-sys-bio-applications-pathlinker/ provides links to the PATHLINKER software, input datasets, PATHLINKER reconstructions of NetPath pathways, and links to interactive visualizations of these reconstructions on GraphSpace.

Corrigendum: Pathway on demand: automated reconstruction of human signaling networks.

[This corrects the article DOI: 10.1038/npjsba.2016.2.].

Crystal structure of the effector AvrLm4-7 of Leptosphaeria maculans reveals insights into its translocation into plant cells and recognition by resistance proteins.

The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.