Iron homeostasis and plant immune responses: Recent insights and translational implications.

Iron metabolism and the plant immune system are both critical for plant vigor in natural ecosystems and for reliable agricultural productivity. Mechanistic studies of plant iron home-ostasis and plant immunity have traditionally been carried out in isolation from each other; however, our growing understanding of both processes has uncovered significant connections. For example, iron plays a critical role in the generation of reactive oxygen intermediates during immunity and has been recently implicated as a critical factor for immune-initiated cell death via ferroptosis. Moreover, plant iron stress triggers immune activation, suggesting that sensing of iron depletion is a mechanism by which plants recognize a pathogen threat. The iron deficiency response engages hormone signaling sectors that are also utilized for plant immune signaling, providing a probable explanation for iron-immunity cross-talk. Finally, interference with iron acquisition by pathogens might be a critical component of the immune response. Efforts to address the global burden of iron deficiency-related anemia have focused on classical breeding and transgenic approaches to develop crops biofortified for iron content. However, our improved mechanistic understanding of plant iron metabolism suggests that such alterations could promote or impede plant immunity, depending on the nature of the alteration and the virulence strategy of the pathogen. Effects of iron biofortification on disease resistance should be evaluated while developing plants for iron biofortification.

Draft Assembly of Phytophthora capsici from Long-Read Sequencing Uncovers Complexity.

Resolving complex plant pathogen genomes is important for identifying the genomic shifts associated with rapid adaptation to selective agents such as hosts and fungicides, yet assembling these genomes remains challenging and expensive. Phytophthora capsici is an important, globally distributed plant pathogen that exhibits widespread fungicide resistance and a broad host range. As with other pathogenic oomycetes, P. capsici has a complex life history and a complex genome. Here, we leverage Oxford Nanopore Technologies and existing short-read resources to rapidly generate a low-cost, improved assembly. We generated 10 Gbp from a single MinION flow cell resulting in >1.25 million reads with an N50 of 13 kb. The resulting assembly is 95.2 Mbp in 424 scaffolds with an N50 length of 313 kb. This assembly is approximately 30 Mbp bigger than the current reference genome of 64 Mbp. We confirmed this larger genome size using flow cytometry, with an estimated size of 110 Mbp. BUSCO analysis identified 97.4% complete orthologs (19.2% duplicated). Evolutionary analysis supports a recent whole-genome duplication in this group. Our work provides a blueprint for rapidly integrating benchtop long-read sequencing with existing short-read data, to dramatically improve assembly quality and integrity of complex genomes and offer novel insights into pathogen genome function and evolution.

Oomycetes Used in Arabidopsis Research.

Arabidopsis plants in their natural environment are susceptible to infection by oomycete pathogens, in particular to downy mildew and white rust diseases. These naturally occurring infectious agents have imposed evolutionary pressures on Arabidopsis populations and are therefore highly relevant for the study of host-pathogen co-evolution. In addition, the study of oomycete diseases, including infections caused by several Phytophthora species, has led to many scientific discoveries on Arabidopsis immunity and disease. Herein, we describe the major oomycete species used for experiments on Arabidopsis, and how these pathosystems have been used to provide significant insights into mechanistic and evolutionary aspects of plant-oomycete interactions. We also highlight understudied aspects of plant-oomycete interactions, as well as translational approaches, that can be productively addressed using the reference pathosystems described in this article.

Effector Biology in Focus: A Primer for Computational Prediction and Functional Characterization.

Plant-pathogen interactions are controlled by a multilayered immune system, which is activated by pathogen recognition in the host. Pathogens secrete effector molecules to interfere with the immune recognition or signaling network and reprogram cell structure or metabolism. Understanding the effector repertoires of diverse pathogens will contribute to unraveling the molecular mechanism of virulence and developing sustainable disease-control strategies for crops and natural ecosystems. Effector functionality has been investigated extensively in only a small number of pathogen species. However, many more pathogen genomes are becoming available, and much can be learned from a broader view of effector biology in diverse pathosystems. The purpose of this review is to summarize methodology for computational prediction of protein effectors, functional characterization of effector proteins and their targets, and the use of effectors as probes to screen for new sources of host resistance. Although these techniques were generally developed in model pathosystems, many of the approaches are directly applicable for exploration and exploitation of effector biology in pathosystems that are less well studied. We hope to facilitate such exploration, which will broaden understanding of the mechanisms that underpin the biological diversity of plant-pathogen interactions, and maximize the impact of new approaches that leverage effector biology for disease control.

Synthesis of Water-Soluble Imidazolium Polyesters as Potential Nonviral Gene Delivery Vehicles.

The inherent hydrolytic reactivity of polyesters renders them excellent candidates for a variety of biomedical applications. Incorporating ionic groups further expands their potential impact, encompassing charge-dependent function such as deoxyribonucleic acid (DNA) binding, antibacterial properties, and pH-responsiveness. Catalyst-free and solvent-free polycondensation of a bromomethyl imidazolium-containing (BrMeIm) diol with neopentylglycol (NPG) and adipic acid (AA) afforded novel charged copolyesters with pendant imidazolium sites. Varying ionic content influenced thermal properties and offered a wide-range, -41 to 40 °C, of composition-dependent glass transition temperatures (Tgs). In addition to desirable melt and thermal stability, polyesters with ionic concentrations ≥15 mol % readily dispersed in water, suggesting potential as nonviral gene delivery vectors. An electrophoretic gel shift assay confirmed the novel cationic copolyesters successfully bound DNA at an N/P ratio of 4 for 50 mol % and 75 mol % charged copolyesters (P(NA50-co-ImA50) and P(NA25-co-ImA75)), and an N/P ratio of 5 for 100 mol % Im (PImA). Polyplexes exhibited insignificant cytotoxicity even at high concentrations (200 µg/mL), and a Luciferase transfection assay revealed the ionic (co)polyesters transfected DNA significantly better than the untreated controls. The successful transfection of these novel (co)polyesters inspires future imidazolium-containing polyester design.

A distal enhancer controls cytokine-dependent human cPLA2α gene expression.

Specific control of group IVA cytosolic phospholipase A2 (cPLA2α or PLA2G4A) expression modulates arachidonic acid production, thus tightly regulating the downstream effects of pro- and anti-inflammatory eicosanoids. The significance of this pathway in human disease is apparent in a range of pathologies from inflammation to tumorigenesis. While much of the regulation of cPLA2α has focused on posttranslational phosphorylation of the protein, studies on transcriptional regulation of this gene have focused only on proximal promoter regions. We have identified a DNase I hypersensitive site encompassing a 5' distal enhancer element containing a highly conserved consensus AP-1 site involved in transcriptional activation of cPLA2α by interleukin (IL)-1ß. Chromatin immunoprecipitation (ChIP), knockdown, knockout, and overexpression analyses have shown that c-Jun acts both in a negative and positive regulatory role. Transcriptional activation of cPLA2α occurs through the phosphorylation of c-Jun in conjunction with increased association of C/EBPß with the distal novel enhancer. The association of C/EBPß with the transcriptional activation complex does not require an obvious DNA binding site. These data provide new and important contributions to the understanding of cPLA2α regulation at the transcriptional level, with implications for eicosanoid metabolism, cellular signaling, and disease pathogenesis.

Effect of allergy and inflammation on eicosanoid gene expression in CFTR deficiency.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2-15% of patients. We hypothesized that sensitization/challenge of CFTR(-/-) mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF. METHODS: FABP-hCFTR(+/-)-CFTR(-/-) mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines. RESULTS: Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR(-/-) mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells. CONCLUSIONS: The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.

Regulation of human microsomal prostaglandin E synthase-1 by IL-1ß requires a distal enhancer element with a unique role for C/EBPß.

The studies of PGE2 (prostaglandin E2) biosynthesis have focused primarily on the role of cyclo-oxygenases. Efforts have shifted towards the specific PGE2 terminal synthases, particularly mPGES-1 (microsomal PGE synthase 1), which has emerged as the crucial inducible synthase with roles in pain, cancer and inflammation. mPGES-1 is induced by pro-inflammatory cytokines with studies focusing on the proximal promoter, mediated specifically through Egr-1 (early growth-response factor 1). Numerous studies demonstrate that the mPGES-1 promoter (PTGES) alone cannot account for the level of IL-1ß (interleukin 1ß) induction. We identified two DNase I-hypersensitive sites within the proximal promoter near the Egr-1 element and a novel distal site near -8.6 kb. Functional analysis of the distal site revealed two elements that co-operate with basal promoter expression and a stimulus-dependent enhancer. A specific binding site for C/EBPß (CCAAT/enhancer-binding protein ß) in the enhancer was directly responsible for inducible enhancer activity. ChIP (chromatin immunoprecipitation) analysis demonstrated constitutive Egr-1 binding to the promoter and induced RNA polymerase II and C/EBPß binding to the promoter and enhancer respectively. Knockout/knockdown studies established a functional role for C/EBPß in mPGES-1 gene regulation and the documented interaction between Egr-1 and C/EBPß highlights the proximal promoter co-operation with a novel distal enhancer element in regulating inducible mPGES-1 expression.

Induction of group IVC phospholipase A2 in allergic asthma: transcriptional regulation by TNFα in bronchoepithelial cells.

Airway inflammation in allergen-induced asthma is associated with eicosanoid release. These bioactive lipids exhibit anti- and pro-inflammatory activities with relevance to pulmonary pathophysiology. We hypothesized that sensitization/challenge using an extract from the ubiquitous fungus Aspergillus fumigatus in a mouse model of allergic asthma would result in altered phospholipase gene expression, thus modulating the downstream eicosanoid pathway. We observed the most significant induction in the group IVC PLA2 (phospholipase A2) [also known as cPLA2γ (cytosolic PLA2γ) or PLA2G4C]. Our results infer that A. fumigatus extract can induce cPLA2γ levels directly in eosinophils, whereas induction in lung epithelial cells is most likely to be a consequence of TNFα (tumour necrosis factor α) secretion by A. fumigatus-activated macrophages. The mechanism of TNFα-dependent induction of cPLA2γ gene expression was elucidated through a combination of promoter deletions, ChIP (chromatin immunoprecipitation) and overexpression studies in human bronchoepithelial cells, leading to the identification of functionally relevant CRE (cAMP-response element), NF-κB (nuclear factor κB) and E-box promoter elements. ChIP analysis demonstrated that RNA polymerase II, ATF-2 (activating transcription factor 2)-c-Jun, p65-p65 and USF (upstream stimulating factor) 1-USF2 complexes are recruited to the cPLA2γ enhancer/promoter in response to TNFα, with overexpression and dominant-negative studies implying a strong level of co-operation and interplay between these factors. Overall, our results link cytokine-mediated alterations in cPLA2γ gene expression with allergic asthma and outline a complex regulatory mechanism.

cPLA(2)α gene activation by IL-1ß is dependent on an upstream kinase pathway, enzymatic activation and downstream 15-lipoxygenase activity: a positive feedback loop.

Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA(2)α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca(2+) levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1ß stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA(2)α is phosphorylated within 10min of IL-1ß treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA(2)α and a downstream lipoxygenase (15-LOX2) are required for IL-1ß-dependent induction of cPLA(2)α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA(2)α→15-LOX2-dependent, positive feedback loop where a protein's enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.

Gene delivery to intestinal epithelial cells in vitro and in vivo with recombinant adeno-associated virus types 1, 2 and 5.

Intestinal disorders such as inflammatory bowel disease (IBD) result in chronic illness requiring lifelong therapy. Our aim was to evaluate the efficacy of recombinant adeno-associated virus (AAV) vector-mediated gene delivery to intestinal epithelial cells in vitro and in vivo. Human colon epithelial cell lines and colon biopsies were transduced using AAV pseudotypes 2/1, 2/2, and 2/5 encoding green fluorescence protein (GFP). Mice were administered the same vectors through oral, enema, intraperitoneal (IP) injection and superior mesenteric artery (SMA) injection routes. Tropism and efficiency were determined by microscopy, flow cytometry, immunohistochemistry and PCR. Caco2 cells were more permissive to AAV transduction. Human colon epithelial cells in organ culture were more effectively transduced by AAV2/2. SMA injection provided the most effective means of vector gene transfer to small intestine and colonic epithelial cells in vivo. Transgene detection 80 days post AAV treatment suggests transduction of crypt progenitor cells. This study shows the feasibility of AAV-mediated intestinal gene delivery, applicable for the investigation of IBD pathogenesis and novel therapeutic options, but also revealed the need for further studies to identify more efficient pseudotypes.

Enhanced IgE allergic response to Aspergillus fumigatus in CFTR-/- mice.

To gain insight into aberrant cytokine regulation in cystic fibrosis (CF), we compared the phenotypic manifestations of allergen challenge in gut-corrected CFTR-deficient mice with background-matched C57Bl6 (B6) mice. Aspergillus fumigatus (Af) antigen was used to mimic allergic bronchopulmonary aspergillosis, a peculiar hyper-IgE syndrome with a high prevalence in CF patients. CFTR-/-, C57BL/6 and FVB/NJ mice were sensitized with Af antigen by serial intraperitoneal injections. Control mice were mock sensitized with PBS. Challenges were performed by inhalation of Af antigen aerosol. After Af antigen challenge, histologic analysis showed goblet cell hyperplasia and lymphocytic infiltration in both strains. However, total serum IgE levels were markedly elevated in CF mice. Sensitized CF mice showed a five-fold greater IgE response to sensitization as compared with B6- and FVB-sensitized controls. Additional littermate controls to fully normalize for B6-FVB admixture in the strain background confirmed the role of CFTR mutation in the hyper-IgE syndrome. Cytokine mRNA levels of IL-5 and GM-CSF in the bronchoalveolar lavage (BAL) fluid, and BAL cell differentials indicated that CFTR mutation caused a shift from an IL-5-predominant to an IL-4-predominant cytokine profile. This system models a very specific type of airway inflammation in CF and could provide insights into pathogenesis and treatment of the disease.

Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell Line.

Mutations in CFTR lead to a complex phenotype that includes increased susceptibility to Pseudomonas infections, a functional deficiency of IL-10, and an exaggerated proinflammatory cytokine response. We examined the effects of CFTR gene correction on the gene expression profile of a CF bronchial epithelial cell line (IB3-1) and determined which CF-related gene expression changes could be reversed by IL-10 expression. We performed microarray experiments to monitor the gene expression profile of three cell lines over a time course of exposure to Pseudomonas. At baseline, we identified 843 genes with statistically different levels of expression in CFTR-corrected (S9) cells compared to the IB3-1 line or the IL-10-expressing line. K-means clustering and functional group analysis revealed a primary up-regulation of ubiquitination enzymes and TNF pathway components and a primary down-regulation of protease inhibitors and protein glycosylation enzymes in CF. Key gene expression changes were confirmed by real-time RT-PCR. Massive reprogramming of gene expression occurred 3 h after Pseudomonas exposure. Changes specific to CF included exaggerated activation of cytokines, blunted activation of anti-proteases, and repression of protein glycosylation enzymes. In conclusion, the CFTR genotype changes the expression of multiple genes at baseline and in response to bacterial challenge, and only a subset of these changes is secondary to IL-10 deficiency.