Protein kinase CK2α is overexpressed in classical hodgkin lymphoma, regulates key signaling pathways, PD-L1 and may represent a new target for therapy.

Introduction: In classical Hodgkin lymphoma (cHL), the survival of neoplastic cells is mediated by the activation of NF-κB, JAK/STAT and PI3K/Akt signaling pathways. CK2 is a highly conserved serine/threonine kinase, consisting of two catalytic (α) and two regulatory (ß) subunits, which is involved in several cellular processes and both subunits were found overexpressed in solid tumors and hematologic malignancies. Methods and results: Biochemical analyses and in vitro assays showed an impaired expression of CK2 subunits in cHL, with CK2α being overexpressed and a decreased expression of CK2ß compared to normal B lymphocytes. Mechanistically, CK2ß was found to be ubiquitinated in all HL cell lines and consequently degraded by the proteasome pathway. Furthermore, at basal condition STAT3, NF-kB and AKT are phosphorylated in CK2-related targets, resulting in constitutive pathways activation. The inhibition of CK2 with CX-4945/silmitasertib triggered the de-phosphorylation of NF-κB-S529, STAT3-S727, AKT-S129 and -S473, leading to cHL cell lines apoptosis. Moreover, CX-4945/silmitasertib was able to decrease the expression of the immuno-checkpoint CD274/PD-L1 but not of CD30, and to synergize with monomethyl auristatin E (MMAE), the microtubule inhibitor of brentuximab vedotin. Conclusions: Our data point out a pivotal role of CK2 in the survival and the activation of key signaling pathways in cHL. The skewed expression between CK2α and CK2ß has never been reported in other lymphomas and might be specific for cHL. The effects of CK2 inhibition on PD-L1 expression and the synergistic combination of CX-4945/silmitasertib with MMAE pinpoints CK2 as a high-impact target for the development of new therapies for cHL.

Sucrose supplements modulate the Pseudomonas chlororaphis-Arabidopsis thaliana interaction via decreasing the production of phenazines and enhancing the root auxin response.

Management of the plant microbiome may help support food needs for the human population. Bacteria influence plants through enhancing nutrient uptake, metabolism, photosynthesis, biomass production and/or reinforcing immunity. However, information into how these microbes behave under different growth conditions is missing. In this work, we tested how carbon supplements modulate the interaction of Pseudomonas chlororaphis with Arabidopsis thaliana. P. chlororaphis streaks strongly repressed primary root growth, lateral root formation and ultimately, biomass production. Noteworthy, increasing sucrose availability into the media from 0 to 2.4% restored plant growth and promoted lateral root formation in bacterized seedlings. This effect could not be observed by supplementing sucrose to leaves only, indicating that the interaction was strongly modulated by bacterial access to sugar. Total phenazine content decreased in the bacteria grown in high (2.4%) sucrose medium, and conversely, the expression of phzH and pslA genes were diminished by sugar supply. Pyocyanin antagonized the promoting effects of sucrose in lateral root formation and biomass production in inoculated seedlings, indicating that this virulence factor accounts for growth repression during the plant-bacterial interaction. Defence reporter transgenes PR-1::GUS and LOX2::GUS were induced in leaves, while the expression of the auxin-inducible, synthetic reporter gene DR5::GUS was enhanced in the roots of bacterized seedlings at low and high sucrose treatments, which suggests that growth/defence trade-offs in plants are critically modulated by P. chlororaphis. Collectively, our data suggest that bacterial carbon nutrition controls the outcome of the relation with plants.

Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation.

The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered the biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.

Dihydrophenazine: a multifunctional new weapon that kills multidrug-resistant Acinetobacter baumannii and restores carbapenem and oxidative stress susceptibilities.

AIMS: The current work aims to fully characterize a new antimicrobial agent against Acinetobacter baumannii, which continues to represent a growing threat to healthcare settings worldwide. With minimal treatment options due to the extensive spread of resistance to almost all the available antimicrobials, the hunt for new antimicrobial agents is a high priority. METHODS AND RESULTS: An Egyptian soil-derived bacterium strain NHM-077B proved to be a promising source for a new antimicrobial agent. Bio-guided fractionation of the culture supernatants of NHM-077B followed by chemical structure elucidation identified the active antimicrobial agent as 1-hydroxy phenazine. Chemical synthesis yielded more derivatives, including dihydrophenazine (DHP), which proved to be the most potent against A. baumannii, yet it exhibited a marginally safe cytotoxicity profile against human skin fibroblasts. Proteomics analysis of the cells treated with DHP revealed multiple proteins with altered expression that could be correlated to the observed phenotypes and potential mechanism of the antimicrobial action of DHP. DHP is a multipronged agent that affects membrane integrity, increases susceptibility to oxidative stress, interferes with amino acids/protein synthesis, and modulates virulence-related proteins. Interestingly, DHP in subinhibitory concentrations re-sensitizes the highly virulent carbapenem-resistant A. baumannii strain AB5075 to carbapenems providing great hope in regaining some of the benefits of this important class of antibiotics. CONCLUSIONS: This work underscores the potential of DHP as a promising new agent with multifunctional roles as both a classical and nonconventional antimicrobial agent that is urgently needed.

Study on the Design, Synthesis, Bioactivity and Translocation of the Conjugates of Phenazine-1-carboxylic Acid and N-Phenyl Alanine Ester.

The natural pesticide phenazine-1-carboxylic acid (PCA) is known to lack phloem mobility, whereas Metalaxyl is a representative phloem systemic fungicide. In order to endow PCA with phloem mobility and also enhance its antifungal activity, thirty-two phenazine-1-carboxylic acid-N-phenylalanine esters conjugates were designed and synthesized by conjugating PCA with the active structure N-acylalanine methyl ester of Metalaxyl. All target compounds were characterized by 1H NMR, 13C NMR and HRMS. The antifungal evaluation results revealed that several target compounds exhibited moderate to potent antifungal activities against Sclerotinia sclerotiorum, Bipolaris sorokiniana, Phytophthora parasitica, Phytophthora citrophthora. In particular, compound F7 displayed excellent antifungal activity against S. sclerotiorum with an EC50 value of 6.57 µg/mL, which was superior to that of Metalaxyl. Phloem mobility study in castor bean system indicated good phloem mobility for the target compounds F1-F16. Particularly, compound F2 exhibited excellent phloem mobility; the content of compound F2 in the phloem sap of castor bean was 19.12 µmol/L, which was six times higher than Metalaxyl (3.56 µmol/L). The phloem mobility tests under different pH culture solutions verified the phloem translocation of compounds related to the "ion trap" effect. The distribution of the compound F2 in tobacco plants further suggested its ambimobility in the phloem, exhibiting directional accumulation towards the apical growth point and the root. These results provide valuable insights for developing phloem mobility fungicides mediated by exogenous compounds.

Phenazine Cations as Anticancer Theranostics†.

The biological properties of two water-soluble organic cations based on polypyridyl structures commonly used as ligands for photoactive transition metal complexes designed to interact with biomolecules are investigated. A cytotoxicity screen employing a small panel of cell lines reveals that both cations show cytotoxicity toward cancer cells but show reduced cytotoxicity to noncancerous HEK293 cells with the more extended system being notably more active. Although it is not a singlet oxygen sensitizer, the more active cation also displayed enhanced potency on irradiation with visible light, making it active at nanomolar concentrations. Using the intrinsic luminescence of the cations, their cellular uptake was investigated in more detail, revealing that the active compound is more readily internalized than its less lipophilic analogue. Colocalization studies with established cell probes reveal that the active cation predominantly localizes within lysosomes and that irradiation leads to the disruption of mitochondrial structure and function. Stimulated emission depletion (STED) nanoscopy and transmission electron microscopy (TEM) imaging reveal that treatment results in distinct lysosomal swelling and extensive cellular vacuolization. Further imaging-based studies confirm that treatment with the active cation induces lysosomal membrane permeabilization, which triggers lysosome-dependent cell-death due to both necrosis and caspase-dependent apoptosis. A preliminary toxicity screen in the Galleria melonella animal model was carried out on both cations and revealed no detectable toxicity up to concentrations of 80 mg/kg. Taken together, these studies indicate that this class of synthetically easy-to-access photoactive compounds offers potential as novel therapeutic leads.

CX-4945 (Silmitasertib) Induces Cell Death by Impairing Lysosomal Utilization in KRAS Mutant Cholangiocarcinoma Cell Lines.

BACKGROUND/AIM: Macropinocytosis is a non-selective form of endocytosis that facilitates the uptake of extracellular substances, such as nutrients and macromolecules, into the cells. In KRAS-driven cancers, including pancreatic ductal adenocarcinoma, macropinocytosis and subsequent lysosomal utilization are known to be enhanced to overcome metabolic stress. In this study, we investigated the role of Casein Kinase 2 (CK2) inhibition in macropinocytosis and subsequent metabolic processes in KRAS mutant cholangiocarcinoma (CCA) cell lines. MATERIALS AND METHODS: The bovine serum albumin (BSA) uptake indicating macropinocytosis was performed by flow cytometry using the HuCCT1 KRAS mutant CCA cell line. To validate macropinosome, the Rab7 and LAMP2 were labeled and analyzed via immunocytochemistry and western blot. The CX-4945 (Silmitasertib), CK2 inhibitor, was used to investigate the role of CK2 in macropinocytosis and subsequent lysosomal metabolism. RESULTS: The TFK-1, a KRAS wild-type CCA cell line, showed only apoptotic morphological changes. However, the HuCCT1 cell line showed macropinocytosis. Although CX-4945 induced morphological changes accompanied by the accumulation of intracellular vacuoles and cell death, the level of macropinocytosis did not change. These intracellular vacuoles were identified as late macropinosomes, representing Rab7+ vesicles before fusion with lysosomes. In addition, CX-4945 suppressed LAMP2 expression following the inhibition of the Akt-mTOR signaling pathway, which interrupts mature macropinosome and lysosomal metabolic utilization. CONCLUSION: Macropinocytosis is used as an energy source in the KRAS mutant CCA cell line HuCCT1. The inhibition of CK2 by CX-4945 leads to cell death in HuCCT1 cells through alteration of the lysosome-dependent metabolism.

Quorum-sensing regulation of phenazine production heightens Pseudomonas aeruginosa resistance to ciprofloxacin.

Quorum sensing is a type of cell-cell communication that modulates various biological activities of bacteria. Previous studies indicate that quorum sensing contributes to the evolution of bacterial resistance to antibiotics, but the underlying mechanisms are not fully understood. In this study, we grew Pseudomonas aeruginosa in the presence of sub-lethal concentrations of ciprofloxacin, resulting in a large increase in ciprofloxacin minimal inhibitory concentration. We discovered that quorum sensing-mediated phenazine biosynthesis was significantly enhanced in the resistant isolates, where the quinolone circuit was the predominant contributor to this phenomenon. We found that production of pyocyanin changed carbon flux and showed that the effect can be partially inhibited by the addition of pyruvate to cultures. This study illustrates the role of quorum sensing-mediated phenotypic resistance and suggests a strategy for its prevention.

Performance evaluation of machine-assisted interpretation of Gram stains from positive blood cultures.

Manual microscopy of Gram stains from positive blood cultures (PBCs) is crucial for diagnosing bloodstream infections but remains labor intensive, time consuming, and subjective. This study aimed to evaluate a scan and analysis system that combines fully automated digital microscopy with deep convolutional neural networks (CNNs) to assist the interpretation of Gram stains from PBCs for routine laboratory use. The CNN was trained to classify images of Gram stains based on staining and morphology into seven different classes: background/false-positive, Gram-positive cocci in clusters (GPCCL), Gram-positive cocci in pairs (GPCP), Gram-positive cocci in chains (GPCC), rod-shaped bacilli (RSB), yeasts, and polymicrobial specimens. A total of 1,555 Gram-stained slides of PBCs were scanned, pre-classified, and reviewed by medical professionals. The results of assisted Gram stain interpretation were compared to those of manual microscopy and cultural species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The comparison of assisted Gram stain interpretation and manual microscopy yielded positive/negative percent agreement values of 95.8%/98.0% (GPCCL), 87.6%/99.3% (GPCP/GPCC), 97.4%/97.8% (RSB), 83.3%/99.3% (yeasts), and 87.0%/98.5% (negative/false positive). The assisted Gram stain interpretation, when compared to MALDI-TOF MS species identification, also yielded similar results. During the analytical performance study, assisted interpretation showed excellent reproducibility and repeatability. Any microorganism in PBCs should be detectable at the determined limit of detection of 105 CFU/mL. Although the CNN-based interpretation of Gram stains from PBCs is not yet ready for clinical implementation, it has potential for future integration and advancement.

Preclinical Targeting of the PGRMC1-CK2 Axis with Silmitasertib: A Potential Strategy for Lung Adenocarcinoma Therapy.

Progesterone receptor membrane component 1 (PGRMC1) is a pleiotropic protein over-expressed in lung adenocarcinoma (LUAD). The precise molecular mechanisms underlying the signature motif of Casein kinase (CK2) presence in PGRMC1 and their role in LUAD remain unclear. X-ray crystallographic structure for CK2 and PGRMC1 from the PubChem database was obtained and subjected to protein-protein interaction (PPI) analysis to identify their interactions. In addition, the CK2 inhibitor - Silmitasertib was also utilised to understand the interaction between PGRMC1-CK2. The PPI complex (PGRMC1-CK2) and the PPI-ligand interaction analysis and their Molecular Dynamics (MD) studies revealed the stability of their interactions and critical amino acid contacts within the 5Ǻ vicinity of the CK2 signature motif "T/S-x-x-E/D". Moreover, in-vitro colony formation assay, migration assay, and gene expression analysis using quantitative Real-time PCR revealed that Silmitasertib (IC50-2.5 µM) was highly influential in suppressing the PGRMC1-CK2 expression axis. In conclusion, our study infers that PGRMC1-CK-2 axis inhibition could be a potential therapeutic option to limit the promotion and progression of lung cancer.

Role of UDP-Glycosyltransferase (ugt) Genes in Detoxification and Glycosylation of 1-Hydroxyphenazine (1-HP) in Caenorhabditis elegans.

Caenorhabditis elegans is a useful model organism to study the xenobiotic detoxification pathways of various natural and synthetic toxins, but the mechanisms of phase II detoxification are understudied. 1-Hydroxyphenazine (1-HP), a toxin produced by the bacterium Pseudomonas aeruginosa, kills C. elegans. We previously showed that C. elegans detoxifies 1-HP by adding one, two, or three glucose molecules in N2 worms. Our current study evaluates the roles that some UDP-glycosyltransferase (ugt) genes play in 1-HP detoxification. We show that ugt-23 and ugt-49 knockout mutants are more sensitive to 1-HP than reference strains N2 or PD1074. Our data also show that ugt-23 knockout mutants produce reduced amounts of the trisaccharide sugars, while the ugt-49 knockout mutants produce reduced amounts of all 1-HP derivatives except for the glucopyranosyl product compared to the reference strains. We characterized the structure of the trisaccharide sugar phenazines made by C. elegans and showed that one of the sugar modifications contains an N-acetylglucosamine (GlcNAc) in place of glucose. This implies broad specificity regarding UGT function and the role of genes other than ogt-1 in adding GlcNAc, at least in small-molecule detoxification.

Tepuazines A-E: Phenazine Glycosides from a Venezuelan Quartz-Rich (Tepui) Cave Soil-Derived Streptomyces virginiae CMB-CA091.

Investigation of the secondary metabolites of Streptomyces virginiae CMB-CA091 isolated from the quartz-rich (tepui) soil of a cave in Venezuela yielded two new dimeric phenazine glycosides, tepuazines A and B (1 and 2); three new monomeric phenazine glycosides, tepuazines C-E (3-5); and a series of known analogues, baraphenazine G (6), phenazinolin D (7), izumiphenazine C (8), 4-methylaminobenzoyl-l-rhamnopyranoside (9), and 2-acetamidophenol (10). Structures were assigned to 1-10 on the basis of detailed spectroscopic analysis and biosynthetic considerations, with 1 and 2 featuring a rare 2-oxabicyclo[3.3.1]nonane-like ring C/D bridge shared with only a handful of known Streptomyces natural products. We propose a plausible convergent biosynthetic relationship linking all known members of this structure class that provides a rationale for the observed ring C/D configuration.

Efficient nitrate and Cr(VI) removal by denitrifier: The mechanism of S. oneidensis MR-1 promoting electron production, transportation and consumption.

When Cr(VI) and nitrate coexist, the efficiency of both bio-denitrification and Cr(VI) bio-reduction is poor because chromate hinders bacterial normal functions (i.e., electron production, transportation and consumption). Moreover, under anaerobic condition, the method about efficient nitrate and Cr(VI) removal remained unclear. In this paper, the addition of Shewanella oneidensis MR-1 to promote the electron production, transportation and consumption of denitrifier and cause an increase in the removal of nitrate and Cr(VI). The efficiency of nitrate and Cr(VI) removal accomplished by P. denitrificans as a used model denitrifier increased respectively from 51.3% to 96.1% and 34.3% to 99.8% after S. oneidensis MR-1 addition. The mechanism investigations revealed that P. denitrificans provided S. oneidensis MR-1 with lactate, which was utilized to secreted riboflavin and phenazine by S. oneidensis MR-1. The riboflavin served as coenzymes of cellular reductants (i.e., thioredoxin and glutathione) in P. denitrificans, which created favorable intracellular microenvironment conditions for electron generation. Meanwhile, phenazine promoted biofilm formation, which increased the adsorption of Cr(VI) on the cell surface and accelerated the Cr(VI) reduction by membrane bound chromate reductases thereby reducing damage to other enzymes respectively. Overall, this strategy reduced the negative effect of chromate, thus improved the generation, transportation, and consumption of electrons. SYNOPSIS: The presence of S. oneidensis MR-1 facilitated nitrate and Cr(VI) removal by P. denitrificans through decreasing the negative effect of chromate due to the metabolites' secretion.

Highly Sensitive Measurement of Horseradish Peroxidase Using Surface-Enhanced Raman Scattering of 2,3-Diaminophenazine.

The development of various enzyme-linked immunosorbent assays (ELISAs) coupled with surface-enhanced Raman scattering (SERS) detection is a growing area in analytical chemistry due to their potentially high sensitivity. A SERS-based ELISA with horseradish peroxidase (HRP) as an enzymatic label, an o-phenylenediamine (oPD) substrate, and a 2,3-diaminophenazine (DAP) enzymatic product was one of the first examples of such a system. However, the full capabilities of this long-known approach have yet to be revealed. The current study addresses a previously unrecognized problem of SERS detection stage performance. Using silver nanoparticles and model mixtures of oPD and DAP, the effects of the pH, the concentration of the aggregating agent, and the particle surface chloride stabilizer were extensively evaluated. At the optimal mildly acidic pH of 3, a 0.93 to 1 M citrate buffer, and AgNPs stabilized with 20 mM chloride, a two orders of magnitude advantage in the limits of detection (LODs) for SERS compared to colorimetry was demonstrated for both DAP and HRP. The resulting LOD for HRP of 0.067 pmol/L (1.3 amol per assay) underscores that the developed approach is a highly sensitive technique. We suppose that this improved detection system could become a useful tool for the development of SERS-based ELISA protocols.

Pseudomonas aeruginosa heme metabolites biliverdin IXß and IXδ are integral to lifestyle adaptations associated with chronic infection.

Pseudomonas aeruginosa is a versatile opportunistic pathogen requiring iron for its survival and virulence within the host. The ability to switch to heme as an iron source and away from siderophore uptake provides an advantage in chronic infection. We have recently shown the extracellular heme metabolites biliverdin IXß (BVIXß) and BVIXδ positively regulate the heme-dependent cell surface signaling cascade. We further investigated the role of BVIXß and BVIXδ in cell signaling utilizing allelic strains lacking a functional heme oxygenase (hemOin) or one reengineered to produce BVIXα (hemOα). Compared to PAO1, both strains show a heme-dependent growth defect, decreased swarming and twitching, and less robust biofilm formation. Interestingly, the motility and biofilm defects were partially rescued on addition of exogenous BVIXß and BVIXδ. Utilizing liquid chromatography-tandem mass spectrometry, we performed a comparative proteomics and metabolomics analysis of PAO1 versus the allelic strains in shaking and static conditions. In shaking conditions, the hemO allelic strains showed a significant increase in proteins involved in quorum sensing, phenazine production, and chemotaxis. Metabolite profiling further revealed increased levels of Pseudomonas quinolone signal and phenazine metabolites. In static conditions, we observed a significant repression of chemosensory pathways and type IV pili biogenesis proteins as well as several phosphodiesterases associated with biofilm dispersal. We propose BVIX metabolites function as signaling and chemotactic molecules integrating heme utilization as an iron source into the adaptation of P. aeruginosa from a planktonic to sessile lifestyle. IMPORTANCE: The opportunistic pathogen Pseudomonas aeruginosa causes long-term chronic infection in the airways of cystic fibrosis patients. The ability to scavenge iron and to establish chronic infection within this environment coincides with a switch to utilize heme as the primary iron source. Herein, we show the heme metabolites biliverdin beta and delta are themselves important signaling molecules integrating the switch in iron acquisition systems with cooperative behaviors such as motility and biofilm formation that are essential for long-term chronic infection. These significant findings will enhance the development of viable multi-targeted therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.

Development of chitosan biopolymer by chemically modified orange peel for safranin O dye removal: A sustainable adsorbent and adsorption modeling using RSM-BBD.

This study aimed to develop a biocomposite (hereinafter, CHI/OP-H2SO4) via the functionalization of chitosan (CHI) biopolymer by chemically modified orange peel (OP-H2SO4). The physicochemical characteristics of CHI/OP-H2SO4 were studied using methods such as pHpzc, XRD, FTIR, BET, and FESEM-EDX. The efficacy of the CHI/OP-H2SO4 biocomposite in removing cationic dye (safranin O, SAF-O) from aqueous solutions was assessed. The Box-Behnken Design (BBD) based on response surface methodology (RSM) was employed to optimize the adsorption performance of CHI/OP-H2SO4, considering factors such as A: CHI/OP-H2SO4 dose (0.02-0.08 g), B: pH (4-10), and C: time (10-60 min). The pseudo-first-order and Freundlich isotherm models align well with the experimental data of SAF-O adsorption by CHI/OP-H2SO4. The excellent adsorption capacity for CHI/OP-H2SO4 was recorded (321.2 mg/g). The notable adsorption of SAF-O onto CHI/OP-H2SO4 is attributed primarily to electrostatic forces between the acidic groups of CHI/OP-H2SO4 and the SAF-O cation, along with H-bonding, and n-π interactions. By transforming waste materials into valuable resources, this approach not only mitigates environmental impact but also produces a promising and sustainable adsorbent for the removal of cationic dyes, exemplified here by the effective removal of SAF-O dye.

Formation of phenazines, phenoxazines, and benzoxazoles in the browning reactions of o-quinones.

Quinone-induced browning is widely produced in foods and is mostly considered a consequence of quinone/nucleophile reactions. However, even in the absence of amino acids or proteins, o-quinones develop browning. In an attempt to better understand the reaction pathways involved in this browning development, this study describes the reactions of 4-methyl-1,2-benzoquinone with alcohols, ammonia, and short chain aldehydes. These reaction mixtures developed browning at 37 °C and the main produced compounds were isolated by semipreparative HPLC and characterized by NMR and MS as phenazines, phenoxazines, and benzoxazoles. A reaction pathway that explains the formation of all these compounds is proposed. The formation of phenazines is responsible, at least partially, for the produced browning, and the formation of benzoxazoles inhibits such browning. Browning development seems to be a consequence of a competition among the reactions of formation of phenazines, phenoxazines, and benzoxazoles, which appear to be produced from a single intermediate.

Phenazine biosynthesis protein MoPhzF regulates appressorium formation and host infection through canonical metabolic and noncanonical signaling function in Magnaporthe oryzae.

Microbe-produced secondary metabolite phenazine-1-carboxylic acid (PCA) facilitates pathogen virulence and defense mechanisms against competitors. Magnaporthe oryzae, a causal agent of the devastating rice blast disease, needs to compete with other phyllosphere microbes and overcome host immunity for successful colonization and infection. However, whether M. oryzae produces PCA or it has any other functions remains unknown. Here, we found that the MoPHZF gene encodes the phenazine biosynthesis protein MoPhzF, synthesizes PCA in M. oryzae, and regulates appressorium formation and host virulence. MoPhzF is likely acquired through an ancient horizontal gene transfer event and has a canonical function in PCA synthesis. In addition, we found that PCA has a role in suppressing the accumulation of host-derived reactive oxygen species (ROS) during infection. Further examination indicated that MoPhzF recruits both the endoplasmic reticulum membrane protein MoEmc2 and the regulator of G-protein signaling MoRgs1 to the plasma membrane (PM) for MoRgs1 phosphorylation, which is a critical regulatory mechanism in appressorium formation and pathogenicity. Collectively, our studies unveiled a canonical function of MoPhzF in PCA synthesis and a noncanonical signaling function in promoting appressorium formation and host infection.