Xanthomonas oryzae pv. oryzicola effector Tal10a directly activates rice OsHXK5 expression to facilitate pathogenesis.

Bacterial leaf streak (BLS), caused by Xanthomonas oryzae pv. oryzicola (Xoc), is a major bacterial disease in rice. Transcription activator-like effectors (TALEs) from Xanthomonas can induce host susceptibility (S) genes and facilitate infection. However, knowledge of the function of Xoc TALEs in promoting bacterial virulence is limited. In this study, we demonstrated the importance of Tal10a for the full virulence of Xoc. Through computational prediction and gene expression analysis, we identified the hexokinase gene OsHXK5 as a host target of Tal10a. Tal10a directly binds to the gene promoter region and activates the expression of OsHXK5. CRISPR/Cas9-mediated gene editing in the effector binding element (EBE) of OsHXK5 significantly increases rice resistance to Xoc, while OsHXK5 overexpression enhances the susceptibility of rice plants and impairs rice defense responses. Moreover, simultaneous editing of the promoters of OsSULTR3;6 and OsHXK5 confers robust resistance to Xoc in rice. Taken together, our findings highlight the role of Tal10a in targeting OsHXK5 to promote infection and suggest that OsHXK5 represents a potential target for engineering rice resistance to Xoc.

VmsR, a LuxR-Type Regulator, Contributes to Virulence, Cell Motility, Extracellular Polysaccharide Production and Biofilm Formation in Xanthomonas oryzae pv. oryzicola.

LuxR-type regulators play pivotal roles in regulating numerous bacterial processes, including bacterial motility and virulence, thereby exerting a significant influence on bacterial behavior and pathogenicity. Xanthomonas oryzae pv. oryzicola, a rice pathogen, causes bacterial leaf streak. Our research has identified VmsR, which is a response regulator of the two-component system (TCS) that belongs to the LuxR family. These findings of the experiment reveal that VmsR plays a crucial role in regulating pathogenicity, motility, biofilm formation, and the production of extracellular polysaccharides (EPSs) in Xoc GX01. Notably, our study shows that the vmsR mutant exhibits a reduced swimming motility but an enhanced swarming motility. Furthermore, this mutant displays decreased virulence while significantly increasing EPS production and biofilm formation. We have uncovered that VmsR directly interacts with the promoter regions of fliC and fliS, promoting their expression. In contrast, VmsR specifically binds to the promoter of gumB, resulting in its downregulation. These findings indicate that the knockout of vmsR has profound effects on virulence, motility, biofilm formation, and EPS production in Xoc GX01, providing insights into the intricate regulatory network of Xoc.

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein.

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. At debate is its oligomeric state, let alone its function. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein was previously found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identify only a front-to-front, symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

Dimeric Transmembrane Structure of the SARS-CoV-2 E Protein.

The SARS-CoV-2 E protein is a transmembrane (TM) protein with its N-terminus exposed on the external surface of the virus. Here, the TM structure of the E protein is characterized by oriented sample and magic angle spinning solid-state NMR in lipid bilayers and refined by molecular dynamics simulations. This protein has been found to be a pentamer, with a hydrophobic pore that appears to function as an ion channel. We identified only a symmetric helix-helix interface, leading to a dimeric structure that does not support channel activity. The two helices have a tilt angle of only 6°, resulting in an extended interface dominated by Leu and Val sidechains. While residues Val14-Thr35 are almost all buried in the hydrophobic region of the membrane, Asn15 lines a water-filled pocket that potentially serves as a drug-binding site. The E and other viral proteins may adopt different oligomeric states to help perform multiple functions.

Low-Loss Nanoscopic Spin-Wave Guiding in Continuous Yttrium Iron Garnet Films.

Long-distance transport and control of spin waves through nanochannels is essential for integrated magnonic technology. Current strategies relying on the patterning of single-layer nano-waveguides suffer from a decline of the spin-wave decay length upon downscaling or require large magnetic bias field. Here, we introduce a new waveguiding structure based on low-damping continuous yttrium iron garnet (YIG) films. Rather than patterning the YIG film, we define nanoscopic spin-wave transporting channels within YIG by dipolar coupling to ferromagnetic metal nanostripes. The hybrid material structure offers long-distance transport of spin waves with a decay length of ∼20 µm in 160 nm wide waveguides over a broad frequency range at small bias field. We further evidence that spin waves can be redirected easily by stray-field-induced bends in continuous YIG films. The combination of low-loss spin-wave guiding and straightforward nanofabrication highlights a new approach toward the implementation of magnonic integrated circuits for spin-wave computing.

Tuning of Magnetic Damping in Y3Fe5O12/Metal Bilayers for Spin-Wave Conduit Termination.

In this work, we investigate the structural and dynamic magnetic properties of yttrium iron garnet (YIG) films grown onto gadolinium gallium garnet (GGG) substrates with thin platinum, iridium, and gold spacer layers. Separation of the YIG film from the GGG substrate by a metal film strongly affects the crystalline structure of YIG and its magnetic damping. Despite the presence of structural defects, however, the YIG films exhibit a clear ferromagnetic resonance response. The ability to tune the magnetic damping without substantial changes to magnetization offers attractive prospects for the design of complex spin-wave conduits. We show that the insertion of a 1-nm-thick metal layer between YIG and GGG already increases the effective damping parameter enough to efficiently absorb spin waves. This bilayer structure can therefore be utilized for magnonic waveguide termination. Investigating the dispersionless propagation of spin-wave packets, we demonstrate that a damping unit consisting of the YIG/metal bilayers can dissipate incident spin-wave signals with reflection coefficient R < 0.1 at a distance comparable to the spatial width of the wave packet.

Electric-Field Control of Propagating Spin Waves by Ferroelectric Domain-Wall Motion in a Multiferroic Heterostructure.

Magnetoelectric coupling in multiferroic heterostructures offers a promising platform for electric-field control of magnonic devices based on low-power spin-wave transport. Here, electric-field manipulation of the amplitude and phase of propagating spin waves in a ferromagnetic Fe film on top of a ferroelectric BaTiO3 substrate is demonstrated experimentally. Electric-field effects in this composite material system are mediated by strain coupling between alternating ferroelectric stripe domains with in-plane and perpendicular polarization and fully correlated magnetic anisotropy domains with differing spin-wave transport properties. The propagation of spin waves across the strain-induced magnetic anisotropy domains of the Fe film is directly imaged and it is shown how reversible electric-field-driven motion of ferroelectric domain walls and pinned anisotropy boundaries turns the spin-wave signal on and off. Furthermore, linear electric-field tuning of the spin-wave phase by altering the width of strain-coupled stripe domains is demonstrated. The results provide a new route toward energy-efficient reconfigurable magnonics.

Nanoscale magnonic Fabry-Pérot resonator for low-loss spin-wave manipulation.

Active control of propagating spin waves on the nanoscale is essential for beyond-CMOS magnonic computing. Here, we experimentally demonstrate reconfigurable spin-wave transport in a hybrid YIG-based material structure that operates as a Fabry-Pérot nanoresonator. The magnonic resonator is formed by a local frequency downshift of the spin-wave dispersion relation in a continuous YIG film caused by dynamic dipolar coupling to a ferromagnetic metal nanostripe. Drastic downscaling of the spin-wave wavelength within the bilayer region enables programmable control of propagating spin waves on a length scale that is only a fraction of their wavelength. Depending on the stripe width, the device structure offers full nonreciprocity, tunable spin-wave filtering, and nearly zero transmission loss at allowed frequencies. Our results provide a practical route for the implementation of low-loss YIG-based magnonic devices with controllable transport properties.

Observation of the Imidazole-Imidazolium Hydrogen Bonds Responsible for Selective Proton Conductance in the Influenza A M2 Channel.

The integral membrane M2 protein is a 97-residue membrane protein that assembles as a tetramer to conduct protons at a slow rate (102-103/s) when activated by low pH. The proton conductance mechanism has been extensively debated in the literature, but it is accepted that the proton conductance is facilitated by hydrogen bonds involving the His37 residues. However, the hydrogen bonding partnership remains unresolved. Here, we report on the measurement of 15N-15N J-couplings of 15N His37-labeled full length M2 (M2FL) protein from Influenza A virus embedded in synthetic liquid crystalline lipid bilayers using two-dimensional J-resolved NMR spectroscopy. We experimentally observed the hydrogen-bond mediated J-couplings between Nδ1 and Nε2 of adjacent His37 imidazole rings, providing direct evidence for the existence of various imidazolium-imidazole hydrogen-bonding geometries in the histidine tetrad at low pH, thus validating the proton conduction mechanism in the M2FL protein by which the proton is transferred through the breaking and reforming of the hydrogen bonds between pairs of His37 residues.

Low-loss YIG-based magnonic crystals with large tunable bandgaps.

Control of spin waves in magnonic crystals is essential for magnon-based computing. Crystals made of ferromagnetic metals offer versatility in band structure design, but strong magnetic damping restricts their transmission efficiency. Yttrium iron garnet (YIG) with ultralow damping is the palpable alternative, yet its small saturation magnetization limits dipolar coupling between discrete units. Here, we experimentally demonstrate low-loss spin-wave manipulation in magnonic crystals of physically separated nanometer-thick YIG stripes. We enhance the transmission of spin waves in allowed minibands by filling the gaps between YIG stripes with CoFeB. Thus-formed magnonic crystals exhibit tunable bandgaps of 50-200 MHz with nearly complete suppression of the spin-wave signal. We also show that Bragg scattering on only two units produces clear frequency gaps in spin-wave transmission spectra. The integration of strong ferromagnets in nanometer-thick YIG-based magnonic crystals provides effective spin-wave manipulation and low-loss propagation, a vital parameter combination for magnonic technologies.

Control of spin-wave transmission by a programmable domain wall.

Active manipulation of spin waves is essential for the development of magnon-based technologies. Here, we demonstrate programmable spin-wave filtering by resetting the spin structure of pinned 90° Néel domain walls in a continuous CoFeB film with abrupt rotations of uniaxial magnetic anisotropy. Using micro-focused Brillouin light scattering and micromagnetic simulations, we show that broad 90° head-to-head or tail-to-tail magnetic domain walls are transparent to spin waves over a broad frequency range. In contrast, magnetic switching to a 90° head-to-tail configuration produces much narrower and strongly reflecting domain walls at the same pinning locations. Based on these results, we propose a magnetic spin-wave valve with two parallel domain walls. Switching the spin-wave valve from an open to a closed state changes the transmission of spin waves from nearly 100 to 0%. Active control over spin-wave transport through programmable domain walls could be utilized in magnonic logic devices or non-volatile memory elements.

Exchange-torque-induced excitation of perpendicular standing spin waves in nanometer-thick YIG films.

Spin waves in ferrimagnetic yttrium iron garnet (YIG) films with ultralow magnetic damping are relevant for magnon-based spintronics and low-power wave-like computing. The excitation frequency of spin waves in YIG is rather low in weak external magnetic fields because of its small saturation magnetization, which limits the potential of YIG films for high-frequency applications. Here, we demonstrate how exchange-coupling to a CoFeB film enables efficient excitation of high-frequency perpendicular standing spin waves (PSSWs) in nanometer-thick (80 nm and 295 nm) YIG films using uniform microwave magnetic fields. In the 295-nm-thick YIG film, we measure intense PSSW modes up to 10th order. Strong hybridization between the PSSW modes and the ferromagnetic resonance mode of CoFeB leads to characteristic anti-crossing behavior in broadband spin-wave spectra. We explain the excitation of PSSWs by exchange coupling between forced magnetization precessions in the YIG and CoFeB layers. If the amplitudes of these precessions are different, a dynamic exchange torque is generated, causing the emission of spin waves from the interface. PSSWs form when the wave vector of the spin waves matches a perpendicular confinement condition. PSSWs are not excited if exchange coupling between YIG and CoFeB is eliminated by a 10 nm Ta spacer layer. Micromagnetic simulations confirm the exchange-torque-driven mechanism.

Beyond Structural Biology to Functional Biology: Solid-State NMR Experiments and Strategies for Understanding the M2 Proton Channel Conductance.

In terms of structural biology, solid-state NMR experiments and strategies have been well established for resonance assignments, leading to the determination of three-dimensional structures of insoluble membrane proteins in their native-like environment. It is also known that NMR has the unique capabilities to characterize structure-function relationships of membrane-bound biological systems beyond structural biology. Here, we report on solid-state NMR experiments and strategies for extracting functional activities on a sub-millisecond time scale. Specifically, we use the His37-labeled full length M2 (M2FL) protein of the Influenza A virus embedded in synthetic lipid bilayers as an example to characterize the proton conduction mechanism and kinetics. The integral membrane M2 protein assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH and is essential for the viral lifecycle. Our results present convincing evidence for the formation of imidazolium-imidazole hydrogen bonds in the His37 tetrad at low pH and that these hydrogen bonds have a low barrier that facilitates the proton conduction mechanism in the M2FL protein. Moreover, it has been possible to measure hydronium ion exchange between water and the protons in the His37 NH bonds based on chemical exchange spectroscopy with minimized spin diffusion. The results identify an exchange rate constant of ∼4000 s-1 for pH 5.8 at -10 °C.

Probing Hydronium Ion Histidine NH Exchange Rate Constants in the M2 Channel via Indirect Observation of Dipolar-Dephased 15N Signals in Magic-Angle-Spinning NMR.

Water-protein chemical exchange in membrane-bound proteins is an important parameter for understanding how proteins interact with their aqueous environment, but has been difficult to observe in membrane-bound biological systems. Here, we demonstrate the feasibility of probing specific water-protein chemical exchange in membrane-bound proteins in solid-state MAS NMR. By spin-locking the 1H magnetization along the magic angle, the 1H spin diffusion is suppressed such that a water-protein chemical exchange process can be monitored indirectly by dipolar-dephased 15N signals through polarization transfer from 1H. In the example of the Influenza A full length M2 protein, the buildup of dipolar-dephased 15N signals from the tetrad of His37 side chains have been observed as a function of spin-lock time. This confirms that hydronium ions are in exchange with protons in the His37 NH bonds at the heart of the M2 proton conduction mechanism, with an exchange rate constant of ∼1750 s-1 for pH 6.2 at -10 °C.

[Influence of different slope position and profile in Disporopsis pernyi forest land on soil microbial biomass and enzyme activity in southwest Karst mountain of China ].

Soil microbial biomass and enzyme activity are important parameters to evaluate the quality of the soil environment. The goal of this study was to determine the influence of different slope position and section in Disporopsis pernyi forest land on the soil microbial biomass and enzyme activity in southwest Karst Mountain. In this study, we chose the Dip forest land at Yunfo village Chengdong town Liangping country Chongqing Province as the study object, to analyze the influence of three different slope positions [Up Slope(US), Middle Slope(MS), Below Slope(BS)] and two different sections-upper layer(0-15 cm) and bottom layer(15-30 cm) on the soil microbial biomass carbon (SMBC), soil microbial biomass nitrogen (SMBN), microbial carbon entropy (qMBC), microbial nitrogen entropy (qMBN) , catalase(CAT), alkaline phosphatase (ALK), urease(URE), and invertase(INV). The results showed that the same trend (BS > MS > US) was found for SMBC, SMBN, qMBC, qMBN, CAT and INV of upper soil layer, while a different trend (BS > US > MS) was observed for ALK. In addition, another trend (MS > US > BS) was observed for URE. The same trend (BS > MS >US) was observed for SMBN, qMBN, CAT, ALK, URE and INV in bottom layer, but a different trend (MS > BS > US) was observed for SMBC and qMBC. The SMBC, SMBN, CAT, ALK, URE and INV manifested as upper > bottom with reduction of the section, while qMBC and qMBN showed the opposite trend. Correlation analysis indicated that there were significant (P <0.05) or highly significant (P < 0.01) positive correlations among SMBC in different slope position and section, soil enzyme activity and moisture. According to the two equations of regression analysis, SMBC tended to increase with the increasing CAT and ALK, while decreased with the increasing pH. Then SMBN tended to increase with the increasing URE and INV.

Membrane protein structural validation by oriented sample solid-state NMR: diacylglycerol kinase.

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.

A systematic assessment of mature MBP in membrane protein production: overexpression, membrane targeting and purification.

Obtaining enough membrane protein in native or native-like status is still a challenge in membrane protein structure biology. Maltose binding protein (MBP) has been widely used as a fusion partner in improving membrane protein production. In the present work, a systematic assessment on the application of mature MBP (mMBP) for membrane protein overexpression and purification was performed on 42 membrane proteins, most of which showed no or poor expression level in membrane fraction fused with an N-terminal Histag. It was found that most of the small membrane proteins were overexpressed in the native membrane of Escherichia coli when using mMBP. In addition, the proteolysis of the fusions were performed on the membrane without solubilization with detergents, leading to the development of an efficient protocol to directly purify the target membrane proteins from the membrane fraction through a one-step affinity chromatography. Our results indicated that mMBP is an excellent fusion partner for overexpression, membrane targeting and purification of small membrane proteins. The present expression and purification method may be a good solution for the large scale preparation of small membrane proteins in structural and functional studies.

Functional reconstitution of influenza A M2(22-62).

Amantadine-sensitive proton uptake by liposomes is currently the preferred method of demonstrating M2 functionality after reconstitution, to validate structural determination with techniques such as solid-state NMR. With strong driving forces (two decades each of both [K(+)] gradient-induced membrane potential and [H(+)] gradient), M2(22-62) showed a transport rate of 78 H(+)/tetramer-s (pH(o) 6.0, pH(i) 8.0, nominal V(m)=-114 mV), higher than previously measured for similar, shorter, and full-length constructs. Amantadine sensitivity of the conductance domain at pH 6.8 was also comparable to other published reports. Proton flux rate was optimal at protein densities of 0.05-1.0% (peptide wt.% in lipid). Rundown of total proton uptake after addition of valinomycin and CCCP, as detected by delayed addition of valinomycin, indicated M2-induced K(+) flux of 0.1K(+)/tetramer-s, and also demonstrated that the K(+) permeability, relative to H(+), was 2.8 × 10(-6). Transport rate, amantadine and cyclooctylamine sensitivity, acid activation, and H(+) selectivity were all consistent with full functionality of the reconstituted conductance domain. Decreased external pH increased proton uptake with an apparent pK(a) of 6.

Insight into the mechanism of the influenza A proton channel from a structure in a lipid bilayer.

The M2 protein from the influenza A virus, an acid-activated proton-selective channel, has been the subject of numerous conductance, structural, and computational studies. However, little is known at the atomic level about the heart of the functional mechanism for this tetrameric protein, a His(37)-Trp(41) cluster. We report the structure of the M2 conductance domain (residues 22 to 62) in a lipid bilayer, which displays the defining features of the native protein that have not been attainable from structures solubilized by detergents. We propose that the tetrameric His(37)-Trp(41) cluster guides protons through the channel by forming and breaking hydrogen bonds between adjacent pairs of histidines and through specific interactions of the histidines with the tryptophan gate. This mechanism explains the main observations on M2 proton conductance.