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Plant defense often depends on the synthesis and targeted delivery of antimicrobial metabolites at pathogen contact sites. The pleiotropic drug resistance (PDR) transporter PENETRATION3 (PEN3)/PDR8 in Arabidopsis (Arabidopsis thaliana) has been implicated in resistance to a variety of fungal pathogens. However, the antimicrobial metabolite(s) transported by PEN3 for extracellular defense remains unidentified. Here, we report that PEN3 functions redundantly with another PDR transporter (PDR12) to mediate the secretion of camalexin, the major phytoalexin in Arabidopsis. Consistent with this, the pen3 pdr12 double mutants exhibit dramatically enhanced susceptibility to the necrotrophic fungus Botrytis cinerea as well as severe hypersensitivity to exogenous camalexin. PEN3 and PDR12 are transcriptionally activated upon B. cinerea infection, and their expression is regulated by the mitogen-activated protein kinase 3 (MPK3) and MPK6, and their downstream WRKY33 transcription factor. Further genetic analysis indicated that PEN3 and PDR12 contribute to B. cinerea resistance through exporting not only camalexin but also other unidentified metabolite(s) derived from Trp metabolism, suggesting that PEN3 and PDR12 have multiple functions in Arabidopsis immunity via transport of distinct Trp metabolic products.

• Signaling peptides play crucial roles in plant growth, development and defense. We report here that the Arabidopsis thaliana secreted peptide, ROOT MERISTEM GROWTH FACTOR 7 (RGF7), functions as an endogenous elicitor to trigger plant immunity. • Expression of the RGF7 precursor-encoding gene (preRGF7) is highly induced in Arabidopsis leaves upon infection by the bacterial pathogen Pseudomonas syringae. The pathogen-responsive preRGF7 expression is regulated by the transcription factor WRKY33 and its upstream mitogen-activated protein kinases MPK3/MPK6 and calcium-dependent protein kinases CPK5/CPK6. In the absence of pathogen attack, chemically induced expression of preRGF7 in transgenic Arabidopsis plants was sufficient to trigger immune responses. Pre-induction of preRGF7 expression in transgenic Arabidopsis also led to enhanced immune responses and increased resistance to P. syringae infection. • Biochemical and genetic analyses demonstrated that RGF7 is perceived by the leaf-expressed RGF1 INSENSITIVE (RGI) family receptors RGI4 and RGI5. The SOMATIC EMBRYOGENESIS RECEPTOR KINASES (SERKs) BAK1 and SERK4 are also involved in RGF7 perception via forming RGF7-induced receptor-complexes with RGI4 and RGI5. • These results indicate that the pathogen-induced RGF7 peptide, perceived by the RGI4/RGI5-BAK1/SERK4 receptor complexes, acts as a new damage-associated molecular pattern (DAMP) and plays a significant role in Arabidopsis immunity.

Arabidopsis thaliana MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs or MPKs), play critical roles in plant disease resistance by regulating multiple defense responses. Previously, we characterized the regulation of phytoalexin biosynthesis by Arabidopsis MPK3/MPK6 cascade and its downstream WRKY33 transcription factor. Here, we report another substrate of MPK3/MPK6, ETHYLENE RESPONSE FACTOR6 (ERF6), in regulating Arabidopsis defense gene expression and resistance to the necrotrophic fungal pathogen Botrytis cinerea. Phosphorylation of ERF6 by MPK3/MPK6 in either the gain-of-function transgenic plants or in response to B. cinerea infection increases ERF6 protein stability in vivo. Phosphomimicking ERF6 is able to constitutively activate defense-related genes, especially those related to fungal resistance, including PDF1.1 and PDF1.2, and confers enhanced resistance to B. cinerea. By contrast, expression of ERF6-EAR, in which ERF6 was fused to the ERF-associated amphiphilic repression (EAR) motif, strongly suppresses B. cinerea—induced defense gene expression, leading to hypersusceptibility of the ERF6-EAR transgenic plants to B. cinerea. Different from ERF1, the regulation and function of ERF6 in defensin gene activation is independent of ethylene. Based on these data, we conclude that ERF6, another substrate of MPK3 and MPK6, plays important roles downstream of the MPK3/MPK6 cascade in regulating plant defense against fungal pathogens.

SARS‐CoV‐2 is responsible for a disruptive worldwide viral pandemic, and renders a severe respiratory disease known as COVID‐19. Spike protein of SARS‐CoV‐2 mediates viral entry into host cells by binding ACE2 through the receptor‐binding domain (RBD). RBD is an important target for development of virus inhibitors, neutralizing antibodies, and vaccines. RBD expressed in mammalian cells suffers from low expression yield and high cost. E. coli is a popular host for protein expression, which has the advantage of easy scalability with low cost. However, RBD expressed by E. coli (RBD‐1) lacks the glycosylation, and its antigenic epitopes may not be sufficiently exposed. In the present study, RBD‐1 was expressed by E. coli and purified by a Ni Sepharose Fast Flow column. RBD‐1 was structurally characterized and compared with RBD expressed by the HEK293 cells (RBD‐2). The secondary structure and tertiary structure of RBD‐1 were largely maintained without glycosylation. In particular, the major β‐sheet content of RBD‐1 was almost unaltered. RBD‐1 could strongly bind ACE2 with a dissociation constant (KD) of 2.98 × 10–8 M. Thus, RBD‐1 was expected to apply in the vaccine development, screening drugs and virus test kit.

Background/Objectives: The wall-associated kinases (WAKs) and WAK-like proteins (WAKLs) comprise a unique receptor-like kinase subfamily in plants, which have been shown to regulate plant development and defense responses by sensing cell wall-derived components, such as pectin or pectin fragments. In this study, we aimed to characterize the function of WAKL10 in flg22-triggered immunity in Arabidopsis. Methods: Through functional analyses of WAKL genes in Arabidopsis, we identified WAKL10 as the most pronouncedly induced WAKL member in response to flg22 treatment. Gain- and loss-of-function genetic analyses were performed to assess its role in flg22-triggered immune responses, including mitogen-activated protein kinase (MAPK) activation, reactive oxygen species (ROS) burst, and defense gene induction. Transgenic Arabidopsis plants expressing a kinase domain-deleted mutant (WAKL10-ΔK) were generated. Co-immunoprecipitation assays were conducted to examine interactions with FLAGELLIN-SENSITIVE 2 (FLS2) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Heterologous overexpression of WAKL10 in tomato was also tested for bacterial disease resistance. Results: WAKL10 positively regulates flg22-triggered immune responses. Interestingly, WAKL10-ΔK retains the capacity to potentiate these responses. Co-immunoprecipitation assays showed that both wild-type WAKL10 and WAKL10-ΔK constitutively associate with FLS2 and BAK1. Overexpression of WAKL10 in tomato confers enhanced bacterial disease resistance. Conclusions: The extracellular domain of WAKL10 promotes FLS2-BAK1 complex formation, thereby contributing to flg22 signaling. This study reveals a new function of WAKLs, distinguished from their proposed role in sensing cell wall components. The functional conservation of WAKL10 suggests its potential application in engineering disease resistance in crop plants.

We have developed an electrochemical sensor for the rapid detection of nitrofurazone (NFZ) in aquaculture seawater, utilizing a glassy carbon electrode (GCE) modified with Au nanoparticles (AuNPs). Compared to the unmodified electrode, the modified electrode exhibited higher electrocatalytic activity than the GCE and could be used efficiently for the detection of NFZ. It was found that the AuNPs modified electrode exhibited a higher specific surface area, as determined by scanning electron microscopy characterization. The effects of various parameters, including the number of electrodeposition cycles for modified electrode preparation and the pH of the electrolyte for NFZ detection were optimized. Under optimized experimental conditions, the peak current and concentration of NFZ exhibited a linear relationship in the range of 3 to 100 μM when analyzed using square wave voltammetry (SWV), with a limit of detection of 0.24 μM. The proposed sensor was successfully utilized for the detection of NFZ in aquaculture seawater. The spiked recoveries ranged from 96.35 to 107% (RSD < 4.13%), indicating that the method demonstrates satisfactory accuracy and performance for detecting NFZ in real seawater.

Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell-cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain-and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs.

Two-pore domain potassium channels (K2P) are a large family of “background” channels that allow outward “leak” of potassium ions. The NALCN/UNC80/UNC79 complex is a non-selective channel that allows inward flow of sodium and other cations. It is unclear how K2Ps and NALCN differentially modulate animal behavior. Here, we found that loss of function (lf) in the K2P gene twk-40 suppressed the reduced body curvatures of C . elegans NALCN(lf) mutants. twk-40(lf) caused a deep body curvature and extended backward locomotion, and these phenotypes appeared to be associated with neuron-specific expression of twk-40 and distinct twk-40 transcript isoforms. To survey the functions of other less studied K2P channels, we examined loss-of-function mutants of 13 additional twk genes expressed in the motor circuit and detected defective body curvature and/or locomotion in mutants of twk-2 , twk-17 , twk-30 , twk-48 , unc-58 , and the previously reported twk-7 . We generated presumptive gain-of-function (gf) mutations in twk-40 , twk-2 , twk-7 , and unc-58 and found that they caused paralysis. Further analyses detected variable genetic interactions between twk-40 and other twk genes, an interdependence between twk-40 and twk-2 , and opposite behavioral effects between NALCN and twk-2 , twk-7 , or unc-58 . Finally, we found that the hydrophobicity/hydrophilicity property of TWK-40 residue 159 could affect the channel activity. Together, our study identified twk-40 as a novel modulator of the motor behavior, uncovered potential behavioral effects of five other K2P genes and suggests that NALCN and some K2Ps can oppositely affect C . elegans behavior.

Data interpolation methods are important statistical analysis tools that can fill in data gaps and missing areas by predicting and estimating unknown data points, thereby improving the accuracy and credibility of data analysis and research. Different interpolation methods are widely used in related fields, but the error between different interpolation methods and their interpolation fusion optimization have a significant impact on the interpolation accuracy, which still deserves further exploration. This study is based on two different types of point data: PM2.5 (PM2.5 refers to particulate matter in the atmosphere with a diameter of 2.5 μm or less, also known as inhalable particles or fine particulate matter) in Xinyang City, Henan Province, and the elevation of typical gullies in Yuanmou County, Yunnan Province. Using relative difference coefficients and hotspot analysis methods, the differences in error characteristics among four interpolation methods, ordinary kriging (OK), universal kriging (UK), inverse distance weighted (IDW), and radial basis functions (RBFs), were compared, and the influence of interpolation fusion methods on the accuracy of interpolation results was explored. The results show that after interpolation of PM2.5 concentration and gully elevation, the error difference between OK and UK is the smallest in both datasets. For PM2.5 concentration data, IDW and UK interpolation errors have the largest difference; for elevation data, the differences between RBF and UK interpolation are the largest. The weighted fusion results show that the interpolation error accuracy of PM2.5 concentration data with an interpolation point density of 0.009 points per square kilometer is improved, and the root mean square error (RMSE) after fusion is reduced from 0.374 μg/m3 to 0.004 μg/m3. However, the error accuracy of the elevation data of the gully with an interpolation point density of 0.76 points/m2 did not improve significantly. This indicates that characteristics such as the density of the original data are important factors that affect the accuracy of interpolation. In the case of sparse interpolation points, it is possible to consider fusing the interpolation results with different error patterns to improve their accuracy. This study provides a new idea for improving the accuracy of interpolation errors.

Confronted with the expansion of local governments’ implicit debt and the associated risks induced by the practice of “land-based financing”, substantial alterations occurred in China’s land reserve financing policy during 2016–2017. These modifications led to an entire cessation of land reserve loans and the initiation of specialized bonds designated solely for land reserves. Empirical evidence, gathered through the approximate application of the difference-in-differences method, reveals that the reform of the land reserve financing policy can markedly reduce local governments’ implicit debt level. Based upon this foundation, the results of the triple-difference regression demonstrate that the diminution effect of the land reserve financing policy reform on local governments’ implicit debt is more pronounced in regions characterized by lower levels of marketization and more substantial legal financing constraints. This research enriches the comprehensive understanding of the impact of land reserve financing policy reform, possessing considerable referential value for the prevention and resolution of local governments’ implicit debt.