A conserved oomycete effector RxLR23 triggers plant defense responses by targeting ERD15La to release NbNAC68.

Oomycete pathogens deliver many effectors to enhance virulence or suppress plant immunity. Plant immune networks are interconnected, in which a few effectors can trigger a strong defense response when recognized by immunity-related proteins. How effectors activate plant defense response remains poorly understood. Here we report Phytophthora capsici effector RxLR23KM can induce plant cell death and plant immunity. RxLR23KM specifically binds to ERD15La, a regulator of abscisic acid and salicylic acid pathway, and the binding intensity depends on the amino acid residues (K93 and M320). NbNAC68, a downstream protein of ERD15La, can stimulate plant immunity that is compromised after binding with ERD15La. Silencing of NbNAC68 substantially prevents the activation of plant defense response. RxLR23KM binds to ERD15La, releasing NbNAC68 to activate plant immunity. These findings highlight a strategy of plant defense response that ERD15La as a central regulator coordinates RxLR23KM to regulate NbNAC68-triggered plant immunity.

Three-dimensional polymer phenylethnylcopper/nitrogen doped graphene aerogel electrode coupled with Fe3O4 NPs nanozyme: Toward sensitive and robust photoelectrochemical detection of glyphosate in agricultural matrix.

BACKGROUND: Presently, glyphosate (Gly) is the most extensively used herbicide globally, Nevertheless, its excessive usage has increased its accumulation in off-target locations, and aroused concerns for food and environmental safety. Commonly used detection methods, such as high-performance liquid chromatography and gas chromatography, have limitations due to expensive instruments, complex pre-processing steps, and inadequate sensitivity. Therefore, a facile, sensitive, and reliable Gly detection method should be developed. RESULTS: A photoelectrochemical (PEC) sensor consisting of a three-dimensional polymer phenylethnylcopper/nitrogen-doped graphene aerogel (PPhECu/3DNGA) electrode coupled with Fe3O4 NPs nanozyme was constructed for sensitive detection of Gly. The microscopic 3D network of electrodes offered fast transfer routes for photo-generated electrons and a large surface area for nanozyme loading, allowing high signal output and analytical sensitivity. Furthermore, the use of peroxidase-mimicking Fe3O4 NPs instead of natural enzyme improved the stability of the sensor against ambient temperature changes. Based on the inhibitory effect of Gly on the catalytic activity Fe3O4 NPs, the protocol achieved Gly detection in the range of 5 × 10-10 to 1 × 10-4 mol L-1. Additionally, feasibility of the detection was confirmed in real agricultural matrix including tea, maize seedlings, maize seeds and soil. SIGNIFICANCE: This work achieved facile, sensitive and reliable analysis towards Gly, and it was expected to inspire the design and utilization of 3D architectures in monitoring agricultural chemicals in food and environmental matrix.

Measurement Methods for Droplet Adhesion Characteristics and Micrometer-Scale Quantification of Contact Angle on Superhydrophobic Surfaces: Challenges and Opportunities.

Inspired by nature, superhydrophobic surfaces have been widely studied. Usually the wettability of a superhydrophobic surface is quantified by the macroscopic contact angle. However, this method has various limitations, especially for precision micro devices with superhydrophobic surfaces, such as biomimetic artificial compound eyes and biomimetic water strider robots. These precision micro devices with superhydrophobic surfaces proposed a higher demand for the quantification of contact angles, requiring contact angle quantification technology to have micrometer-scale measurement capabilities. In this review, it is proposed to achieve micrometer-scale quantification of superhydrophobic surface contact angles through droplet adhesion characteristics (adhesion force and contact radius). Existing contact angle quantification techniques and droplet characteristics' measurement methods were described in detail. The advancement of micrometer-scale quantification technology for the contact angle of superhydrophobic surfaces will enhance our understanding of superhydrophobic surfaces.

A disturbance suppression micro-Newton force sensor based on shadow method.

The precision of micro-force measurement depends on the force sensor sensitivity and the environmental disturbance magnitude. However, micro-force sensors generally have the poor anti-disturbance ability. Inspired by the shadow formed by water striders walking on water surface under sunlight, a viscoelastic-polymer micro-force (VPMF) sensor based on the shadow method was proposed, which could suppress disturbances effectively due to the high damping ratio of 0.22. The shadow diameter change and the applied force were proportional. The experimental results indicated that the sensitivity could reach 2.15 µN/pixel with a good linear performance. Furthermore, compared with the cantilever, it was capable of the reduction of the disturbance influence by approximately 96.35%. Therefore, the VPMF sensor can be applied to reliable micro-force measurement in complex environments such as industrial sites.

Facile and sensitive photoelectrochemical detection of kojic acid in food products: a general strategy for immobilization-free analysis of enzyme inhibitors.

As one of the representative secondary metabolites in fermentation processes and common additives in modern industry, kojic acid (KA) has been mired in controversy in recent years due to its potential toxicity and carcinogenicity. Hence, it is of high importance to develop novel analysis strategies for KA to surveil its rational utilization and ensure public health. Based on enzyme modulated sensitization of TiO2 NPs and the inhibition effect of KA towards tyrosinase (Tyr), we report a facile and sensitive photoelectrochemical (PEC) sensor for KA in food samples. On an operational level, this protocol excluded the tedious immobilization process of traditional PEC enzyme sensors, allowing the crucial catalysis and sensitization process to take place in a small centrifuge tube, making the experimental process concise and fast. Under optimized conditions, a linear detection range of 10-7 M to 10-3 M was achieved, with a detection limit of 3.2 × 10-8 M. Furthermore, the feasibility of the strategy in food samples was validated in vinegar and wheat flour. This protocol would hold great promise for real application in diverse food products, and offer a general prototype for future immobilization-free and quick analysis of other enzyme inhibitors.

Structural Insights into the NAD(P)H:Quinone Oxidoreductase from Phytophthora capsici.

Soluble quinone oxidoreductases catalyze transfer of electrons from NADPH to quinones. Transfer of electrons is essential for detoxification of synthetic compounds. Here, we present the crystal structure of a NADPH-dependent QOR from Phytophthora capsici (Pc) complexed with NADPH at 2.4 Å resolution. The enzyme exhibits a bi-modular architecture, containing a NADPH-binding groove and a substrate-binding pocket in each subunit. In the crystal, each asymmetric unit of PcQOR contains two molecules stabilized by intermolecular interactions. Gel filtration and ultracentrifugation analyses reveal that it functions as a tetramer in solution. Alignment of homologous structures exhibits a conserved topology. However, the active sites vary among the homologues, indicating differences in substrate specificities. Enzymatic assays indicate that PcQOR tends to catalyze the large substrates, like 9,10-phenanthrenequinone. Computational simulation associated with site-directed mutagenesis and enzymatic activity analysis declares a potential quinone-binding channel. The ability to reduce quinones probably helps P. capsici to detoxify some harmful chemicals encountered during invasion.

Efficient preparation of c-di-AMP at gram-scale using an immobilized Vibrio cholerae dinucleotide cyclase DncV.

Cyclic di-AMP is a bacterial nucleotide second messenger and evaluated as a potential vaccine adjuvant candidate. Here, we report a practical and economical enzymatic method for gram-scale preparation of c-di-AMP using an immobilized Vibrio cholerae dinucleotide cyclase DncV. The method mainly includes four steps: preparation of DncV-immobilized resin, enzymatic synthesis of c-di-AMP, purification using macroporous absorption resin SP207, and desiccation using rotary evaporation and lyophilization. Enzymatic synthesis is the most critical step, and almost all substrate ATP was converted to c-di-AMP under an optimum condition in which 300 mL of 300 mM NH4Ac/NH3 pH 9.5 buffer supplemented with 20 mM MnCl2, 10 mM ATP and 4 mL of DncV-immobilized resin containing ∼19 mg DncV were incubated at 30 °C overnight. After purification, up to 1 g of the diammonium salt of c-di-AMP with weight purity of ≥98% was obtained as white powder, which corresponds to an overall yield of ∼80% based on the ATP input into the reaction. The method is easily performed in laboratory to prepare c-di-AMP on a gram scale and could be used in industry on a large scale.

Crystal structure and expression patterns of prolyl 4-hydroxylases from Phytophthora capsici.

Prolyl 4-hydroxylases (P4Hs) are members of the Fe2+ and 2-oxoglutarate- dependent oxygenases family, which play central roles in the collagen stabilization, hypoxia sensing, and translational regulation in eukaryotes. Thus far, nothing is known about the role of P4Hs in development and pathogenesis in oomycetes. Here we show that the Phytophthora capsici genome contains five putative prolyl 4-hydroxylases. In mycelia, all P4Hs were downregulated in response to hypoxia, but the expression of PcP4H1 was most affected. Strikingly, Pc4H1 was upregulated more than 110 fold at the onset of infection, and Pc4H5 was upregulated seven fold, while the expression of other P4H's were unchanged. Similar to well-characterized P4H proteins, the crystallographic structure of PcP4H1 contains a highly conserved double-stranded ß-helix core fold and catalytic residues. However, the binding affinity of 2-oxoglutarate to PcP4H1 is very low. The extended C-terminal α-helix bundle and longer ß2-ß3 disordered substrate binding loop may help in confirming the peptide target of this enzyme.

Crystal structure of the RxLR effector PcRxLR12 from Phytophthora capsici.

RxLR genes are a prominent class of effectors in oomycetes, and almost half of these proteins contain a conserved sequence motif termed the WY domain, that may exist singly, or as divergent tandem repeats in different effectors. Here we describe the crystal structure of PcRxLR12 (63-488) from Phytophthora capsici at 3.0 Šresolution. The structure consists of five tandemly arrayed WY-domains linked to each other by short connecting helices. Superposition of the WY-2 domain on the other four domains of PcRxLR12, show that the first α-helix termed the K motif, and Loop 3 which connects α3 and α4 are the key regions of structural divergence between the WY domains. A similar pattern was observed when WY-2 was superposed on the 11 WY domains from other oomycete effectors. We also note that an added connecting helix between WY domains in some RXLR effectors, ensures that the WY domains are oriented in the same direction.

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology, mycelial growth and plant infection.

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4',6-diamidino-2-phenylindole (DAPI) staining and PcSDA1-green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non-filamentous yeasts and human cells.

Characterization of Cell-Death-Inducing Members of the Pectate Lyase Gene Family in Phytophthora capsici and Their Contributions to Infection of Pepper.

Pectate lyases (PL) play a critical role in pectin degradation. PL have been extensively studied in major bacterial and fungal pathogens of a wide range of plant species. However, the contribution of PL to infection by oomycete pathogens remains largely unknown. Here, we cloned 22 full-length pectate lyase (PcPL) genes from a highly aggressive strain of Phytophthora capsici SD33. Of these, PVX agroinfiltration revealed that 12 PcPL genes were found to be highly induced during infection of pepper by SD33 but the induction level was twofold less in a mildly aggressive strain, YN07. The four genes with the highest transcript levels as measured by by quantitative reverse-transcription polymerase chain reaction (PcPL1, PcPL15, PcPL16, and PcPL20) also produced a severe cell death response following transient expression in pepper leaves but the other eight PcPL genes did not. Overexpression of these four genes increased the virulence of SD33 on pepper slightly, and increased it more substantially during infection of tobacco. Overexpression of the genes in YN07 restored its aggressiveness to near that of SD33. Gene silencing experiments with the 12 PcPL genes produced diverse patterns of silencing of PcPL genes, from which it could be inferred from regression analysis that PcPL1, PcPL16, and PcPL20 could account for nearly all of the contributions of the PcPL genes to virulence.

Structural insights into the T6SS effector protein Tse3 and the Tse3-Tsi3 complex from Pseudomonas aeruginosa reveal a calcium-dependent membrane-binding mechanism.

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex.

[Scrotal calculi: advances in clinical research].

Scrotal calculi are freely mobile calcified bodies or stones located between the layers of the tunica vaginalis of the testis. The literature on this relatively rare benign lesion consists mostly of case reports. In most cases, scrotal calculi are found incidentally during ultrasound examination. Now with the application of high-frequency ultrasonography, the detection rate of scrotal calculi is gradually increasing. This article summarizes the etiology, pathogenesis, clinical manifestations, diagnosis, and treatment of scrotal calculi.

Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function.

Tse1 (Tse is type VI secretion exported), an effector protein produced by Pseudomonas aeruginosa, is an amidase that hydrolyses the γ-D-glutamyl-DAP (γ-D-glutamyl-L-meso-diaminopimelic acid) linkage of the peptide bridge of peptidoglycan. P. aeruginosa injects Tse1 into the periplasm of recipient cells, degrading their peptidoglycan, thereby helping itself to compete with other bacteria. Meanwhile, to protect itself from injury by Tse1, P. aeruginosa expresses the cognate immunity protein Tsi1 (Tsi is type VI secretion immunity) in its own periplasm to inactivate Tse1. In the present paper, we report the crystal structures of Tse1 and the Tse1-(6-148)-Tsi1-(20-end) complex at 1.4 Å and 1.6 Å (1 Å=0.1 nm) resolutions respectively. The Tse1 structure adopts a classical papain-like α+ß fold. A cysteine-histidine catalytic diad is identified in the reaction centre of Tse1 by structural comparison and mutagenesis studies. Tsi1 binds Tse1 tightly. The HI loop (middle finger tip) from Tsi1 inserts into the large pocket of the Y-shaped groove on the surface of Tse1, and CD, EF, JK and LM loops (thumb, index finger, ring finger and little finger tips) interact with Tse1, thus blocking the binding of enzyme to peptidoglycan. The catalytic and inhibition mechanisms provide new insights into how P. aeruginosa competes with others and protects itself.

Crystal structures of STING protein reveal basis for recognition of cyclic di-GMP.

STING functions as both an adaptor protein signaling cytoplasmic double-stranded DNA and a direct immunosensor of cyclic diguanylate monophosphate (c-di-GMP). The crystal structures of the C-terminal domain of human STING (STING(CTD)) and its complex with c-di-GMP reveal how STING recognizes c-di-GMP. In response to c-di-GMP binding, two surface loops, which serve as a gate and latch of the cleft formed by the dimeric STING(CTD), undergo rearrangements to interact with the ligand.

Crystal structure of periplasmic catecholate-siderophore binding protein VctP from Vibrio cholerae at 1.7 Å resolution.

VctP, one of the two essential siderophore-binding PBPs from the pathogen Vibrio cholerae, plays an important role in the transport of enterobactin and vibriobactin, which have quite different configurations of iron coordination, from the periplasm to the inner membrane. The current study reports the crystal structure of VctP from V. cholerae N16961 at 1.7Å resolution. A structural comparison of VctP with its homologues and the results of molecular docking indicate that enterobactin and vibriobactin share the same binding pocket. Significantly, a basic triad consisting of Arg137, Arg226 and Arg270 is used to balance the three negative charges of ferric-enterobactin, while a basic dyad consisting of Arg137 and Arg270 is used to balance the two negative charges of ferric-vibriobactin.

Synthesis and carbohydrate binding studies of fluorescent alpha-amidoboronic acids and the corresponding bisboronic acids.

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent alpha-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-alpha-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make alpha-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.

Identification of the first fluorescent alpha-amidoboronic acids that change fluorescent properties upon sugar binding.

The first amidoboronic acids were identified that show significant fluorescent property changes upon binding with various carbohydrates.

Computer-based de novo design, synthesis, and evaluation of boronic acid-based artificial receptors for selective recognition of dopamine.

Dopamine is an important neurotransmitter that plays important roles in various physiological and pathological processes, such as Parkinson's disease. Chemosensors for dopamine have a number of potential applications. On the basis both of the strong and reversible complexation between the boronic acid moiety and a diol functional group and computational chemistry studies, we have designed a series of four compounds for selective three-point recognition of dopamine, which include boronic acid-diol complexation, aromatic-hydrophobic interactions, and ionic interactions between a carboxylate and a protonated amino group. These compounds were synthesized in seven or eight linear steps and showed dopamine selectivity of up to tenfold over epinephrine. NMR spectroscopy experiments were conducted to probe the structures of the receptor-dopamine complexes. These receptors are the first to show such significant selectivity for dopamine over epinephrine in aqueous solution under near physiological conditions.