DNA-functionalized cryogel based colorimetric biosensor for sensitive on-site detection of aflatoxin B1 in food samples.

Hydrogel biosensors present numerous advantages in food safety analysis owing to their remarkable biocompatibility, cargo-loading capabilities and optical properties. However, the current drawbacks (slow target responsiveness and poor mechanical strength) restricted their further utilization at on-site detection of targets. To address these challenges, a DNA-functionalized cryogel with hierarchical pore structures is constructed to improve the reaction rate and the robustness of hydrogel biosensor. During cryogel preparation, ice crystals serve as templates, shaping interconnected hierarchical microporous structures to enhance mass transfer for faster responses. Meanwhile, in the non-freezing zone, concentrated monomers create a dense cross-linked network, strengthening cryogel matrix strength. Accordingly, a colorimetric biosensor based on DNA cryogel has been developed as a proof of concept for rapid detection of aflatoxin B1 (AFB1) in food samples, and an excellent analytical performance was obtained under the optimized conditions with a low detection limit (1 nM), broad detection range (5-100 nM), satisfactory accuracy and precision (recoveries, 81.2-112.6 %; CV, 2.75-5.53 %). Furthermore, by integrating with a smartphone sensing platform, a portable device was created for rapid on-site measurement of target within 45 min, which provided some insight for hydrogel biosensors design.

A Self-Assembled G-Quadruplex/Hemin DNAzyme-Driven DNA Walker Strategy for Sensitive and Rapid Detection of Lead Ions Based on Rolling Circle Amplification.

Herein, a sensitive biosensor is constructed based on a novel rolling circle amplification (RCA) for colorimetric quantification of lead ion (Pb2+). At the detection system, GR5 DNAzymes are modified on the surface of an immunomagnetic bead, and Pb2+ is captured by the aptamer, inducing the disintegration of the GR5 DNAzyme and the release of the DNA walker. After the introduction of the template DNA, T4 DNA ligase, and phi29 DNA polymerase, an RCA is initiated for the sensitivity improvement of this method. Moreover, a G4-hemin DNAzyme is formed as a colorimetric signal, owing to its peroxide-like activity to catalyze the TMB-H2O2 substrate. Under the optimized conditions, the limit of detection (LOD) of this fabricated biosensor could reach 3.3 pM for Pb2+ with a concentration in the range of 0.01-1000 nM. Furthermore, the results of real samples analysis demonstrate its satisfactory accuracy, implying its great potential in the rapid detection of heavy metals in the environment.

BIK1 protein homeostasis is maintained by the interplay of different ubiquitin ligases in immune signaling.

Pathogen-associated molecular patterns (PAMPs) trigger plant innate immunity that acts as the first line of inducible defense against pathogen infection. A receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) functions as a signaling hub immediately downstream of multiple pattern recognition receptors (PRRs). It is known that PLANT U-BOX PROTEIN 25 (PUB25) and PUB26 ubiquitinate BIK1 and mediate BIK1 degradation. However, how BIK1 homeostasis is maintained is not fully understood. Here, we show that two closely related ubiquitin ligases, RING DOMAIN LIGASE 1 (RGLG1) and RGLG2, preferentially associate with the hypo-phosphorylated BIK1 and promote the association of BIK1 with the co-receptor for several PRRs, BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). PUB25 interacts with RGLG2 and mediates its degradation. In turn, RGLG2 represses the ubiquitin ligase activity of PUB25. RGLG1/2 suppress PUB25-mediated BIK1 degradation, promote BIK1 protein accumulation, and positively regulate immune signaling in a ubiquitin ligase activity-dependent manner. Our work reveals how BIK1 homeostasis is maintained by the interplay of different ubiquitin ligases.

Red light-upregulated MPK11 negatively regulates red light-induced stomatal opening in Arabidopsis.

Stomatal movements allow the uptake of CO2 for photosynthesis and water loss through transpiration, therefore play a crucial role in determining water use efficiency. Both red and blue lights induce stomatal opening, and the stomatal apertures under light are finetuned by both positive and negative regulators in guard cells. However, the molecular mechanisms for precisely adjusting stomatal apertures under light have not been completely understood. Here, we provided evidence supporting that Arabidopsis thaliana mitogen-activated protein kinase 11 (MPK11) plays a negative role in red light-induced stomatal opening. First, MPK11 was found to be highly expressed in guard cells, and MPK11-GFP signals were detected in both nuclear and cytoplasm of guard cells. The transcript levels of MPK11 in guard cells were upregulated by white light, and the stomata of mpk11 opened wider than that of wild type under white light. Consistent with the larger stomatal aperture, mpk11 mutant exhibited higher stomatal conductance and CO2 assimilation rate under white light. The transcript levels of the genes responsible for osmolytes increases were higher in guard cells of mpk11 than that of wild type, which may contribute to the larger stomatal aperture of mpk11 under white light. Furthermore, MPK11 transcript levels in guard cells were upregulated by red light, and mpk11 mutant showed a larger stomatal aperture under red light. Taken together, these results demonstrate that red light-upregulated MPK11 plays a negative role in stomatal opening, which finetuning the stomatal opening apertures and preventing excessive water loss by transpiration under light.

High-capacity recovery of Cs+ ions by facilely synthesized layered vanadyl oxalatophosphates with the clear insight into remediation mechanism.

Radiocesium remediation is of great significance for the sustainable development of nuclear energy and ecological protection. It is very challenging for the effective recovery of 137Cs from aqueous solutions due to its strong radioactivity, solubility and mobility. Herein, the efficient recovery of Cs+ ions has been achieved by three layered vanadyl oxalatophosphates, namely (NH4)2[(VO)2(HPO4)2C2O4]·5 H2O (NVPC), Na2[(VO)2(HPO4)2C2O4]·2 H2O (SVPC), and K2.5[(VO)2(HPO4)1.5(PO4)0.5(C2O4)]·4.5 H2O (KVPC). NVPC exhibits the ultra-fast kinetics (within 5 min) and high adsorption capacity for Cs+ (qmCs = 471.58 mg/g). It also holds broad pH durability and excellent radiation stability. Impressively, the entry of Cs+ can be directly visualized by the single-crystal structural analysis, and thus the underlying mechanism of Cs+ capture by NVPC from aqueous solutions has been illuminated at the molecular level. This is a pioneering work in the removal of radioactive ions by metal oxalatophosphate materials which highlights the great potential of metal oxalatophosphates for radionuclide remediation.

The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signaling in Arabidopsis.

Immune responses are triggered when pattern recognition receptors recognize microbial molecular patterns. The Arabidopsis (Arabidopsis thaliana) receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) acts as a signaling hub of plant immunity. BIK1 homeostasis is maintained by a regulatory module in which CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) regulates BIK1 turnover via the activities of two E3 ligases. Immune-induced alternative splicing of CPK28 attenuates CPK28 function. However, it remained unknown whether CPK28 is under proteasomal control. Here, we demonstrate that CPK28 undergoes ubiquitination and 26S proteasome-mediated degradation, which is enhanced by flagellin treatment. Two closely related ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, specifically interact with CPK28 at the plasma membrane; this association is enhanced by flagellin elicitation. ATL31/6 directly ubiquitinate CPK28, resulting in its proteasomal degradation. Furthermore, ATL31/6 promotes the stability of BIK1 by mediating CPK28 degradation. Consequently, ATL31/6 positively regulate BIK1-mediated immunity. Our findings reveal another mechanism for attenuating CPK28 function to maintain BIK1 homeostasis and enhance immune responses.

Indirect competitive-structured electrochemical immunosensor for tetrabromobisphenol A sensing using CTAB-MnO2 nanosheet hybrid as a label for signal amplification.

Tetrabromobisphenol A (TBBPA) is a kind of brominated flame retardant that is usually added to products to reduce their flame retardancy. However, its extensive use has resulted in their residues being found in the environment, which is very harmful. Herein, an indirect competitive immunosensor has been established for TBBPA detection based on the signal amplification system. Pd nanospheres in situ reduced on the surface of MnO2 nanosheet hybrid (MnO2/Pd) was used as the label for the secondary antibody through the Pd-N bond, and gold-toluidine blue composite was loaded onto MWCNTs (MWCNTs/Au-TB), which functioned as the platform for the immunosensor. The spherical structure of Pd had abundant catalytic active sites, which enhanced the catalytic activity of MnO2/Pd as the label, hence amplifying the signal response. Besides, MWCNTs/Au-TB improved electron transfer and produced a strong signaling pathway for immobilizing antigens through the Au-NH2 bond, which can specifically recognize primary antibodies to improve sensitivity. The immunosensor had a linear concentration range of 0-81 ng/mL, a low detection limit of 0.17 ng/mL (S/N = 3), with good stability, selectivity, and reproducibility based on the above advantages. Additionally, the acceptable accuracy and recoveries (recoveries, 92-124%; CV, 3.3-8.8%) in the real water sample analysis indicated that this strategy is promising for emerging pollutant analysis.

Efficient Removal of Cs+ and Sr2+ Ions by Granulous (Me2NH2)4/3(Me3NH)2/3Sn3S7·1.25H2O/Polyacrylonitrile Composite.

The need to effectively and selectively remove radioactive 137Cs and 90Sr from nuclear waste solutions persists to mitigate their environmental mobility and high radiotoxicity. Because it is difficult to effectively remove them from acidic environments that degrade most sorbents, new sorbent materials are highly desirable. Here, efficient removal of Cs+ and Sr2+ is achieved by the composite of layered tin sulfide (Me2NH2)4/3(Me3NH)2/3Sn3S7·1.25H2O (FJSM-SnS) and polyacrylonitrile (PAN) (FJSM-SnS/PAN). The granulous composite possesses regular particle morphology and good mechanical strength as an engineered form. It shows excellent acid-base and γ-irradiation resistance, high maximum adsorption capacities (qm) of 296.12 and 62.88 mg/g for Cs+ and Sr2+ ions, respectively, and high selectivity even in the presence of excess Na+ ions or using lake water. Impressively, qmCs of FJSM-SnS/PAN reaches 89.29 mg/g under even acidic conditions (pH = 2.5). The column loaded with FJSM-SnS/PAN granules exhibits high removal rates (R) toward low-concentration Cs+ and Sr2+ ions under both neutral and acidic conditions. Moreover, the composite can be recycled and reused with high RCs and RSr. This work highlights the great potential of metal sulfide ion-exchangers in engineered form for the efficient removal of Cs+ or Sr2+ ions, especially under acidic conditions, for radionuclide remediation.

MiR172b-TOE1/2 module regulates plant innate immunity in an age-dependent manner.

Plant innate immunity varies with age and plant developmental stages. Recently, we reported that Arabidopsis thaliana microRNA miR172b regulates FLS2 transcription through two transcription factors: TARGET OF EAT1 (TOE1) and TOE2. Although the flg22-triggered immune responses were investigated in 2-d-old or even younger toe1/toe2 mutant and miR172b over expression (OE) transgenic plants, the FLS2-mediated immune responses in older plants remain uncharacterized yet. In this work, we analyzed the flg22-triggered immune response in 6-d-old toe1/toe2 and miR172b OE plants. We found that unlike 2-d-old plants, 6-d-old Col-0, toe1/toe2 and miR172b OE plants exhibit comparable flg22-triggered immune responses. Strikingly, miR172b precursor in 6-d-old Col-0 plants upon flg22 treatment reached to a very high level, consequently, the TOE1/2 protein level under this condition was very low or almost undetectable, which explains why 6-d-old WT seedlings are very similar to toe1/toe2 seedlings or miR172b OE plants with respect to the flg22-triggered immune responses. Taken together, our study reveals that miR172b-TOE1/2 module regulates plant innate immunity in an age-dependent manner.

Ratiometric fluorescence immunoassay based on FAM-DNA-functionalized CdSe/ZnS QDs for the sensitive detection of tetrabromobisphenol A in foodstuff and the environment.

A simple indirectly competitive ratiometric fluorescent immunoassay was designed based on fluorescein amidite (FAM)-DNA-functionalized CdSe/ZnS quantum dots (QDs) for the sensitive determination of tetrabromobisphenol A (TBBPA). At the detection system, catalase (CAT) was labeled on the secondary antibody (Ab2), which served as a controller of the H2O2 concentration. After the competitive binding step, the emitted red fluorescence (excitation at 490 nm) from FAM-DNA-functionalized CdSe/ZnS QDs could be effectively quenched by the H2O2 added. Under the optimized conditions, the limit of detection (LOD) reached 0.118 µg/L with a linear range of 0.34-45.34 µg/L, which was approximately 1 order of magnitude lower than that by HRP-based traditional ELISA. Furthermore, the combination of the dual-output ratiometric fluorescence assays with ELISA improved the inherent built-in rectification to the environment, which brought about satisfactory accuracy and precision (recoveries, 83.16-112.4%; CV, 2.42-7.28%), indicating great potential for the determination of trace TBBPA from food and environmental samples. Graphical abstract.

High-throughput chemiluminescence immunoassay based on Co2+/hemin synergistic catalysis for sensitive detection tetrabromobisphenol A bis(2-hydroxyethyl) ether in the environments.

Here, a novel chemiluminescence (CL) immunoassay was fabricated for sensitive determination of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE), one of typical tetrabromobisphenol A derivatives. At the indirectly competitive method, the synthesized PS@hemin@Co2+ was labelled by secondary antibody (Ab2) instead of common natural enzymes, which showed excellent catalysis towards the decomposition of luminol-H2O2 for producing CL signal. Furthermore, the CL signal was greatly amplified owing to the synergistic catalysis of hemin and Co2+ in the detection system. Under the optimized conditions, the established method offered (i) low detection limit (LOD, 0.9 µg/L), which was almost 5 times lower than that using a conventional ELISA with the same antibody; (ii) a good linearity (1.6-14.3 µg/L); (iii) satisfactory accuracy and precision (recoveries, 89.67-125.33%; CV, 2.75-8.37%). The proposed CL immunoassay was applied for analysis of environmental samples from various sources collected from Jiangsu and Zhejiang province, China. And the detected concentrations were ranged in 2.4-3.7 µg/L in environmental waters and 1.8-2.4 ng/g (dry weight, dw) in soil samples, indicating great potential for trace TBBPA-DHEE detection from environmental samples.

A novel signal amplification strategy based on the competitive reaction between 2D Cu-TCPP(Fe) and polyethyleneimine (PEI) in the application of an enzyme-free and ultrasensitive electrochemical immunosensor for sulfonamide detection.

Nanozymes with peroxidase-like activity have been widely used as signal labels in electrochemical immunosensors. However, these sensors always suffer from some shortcomings during the processes underlying nanozyme labeling, including complex reactions, nanozyme inactivation after being decorated on the antibodies. To solve these problems, a novel electrochemical immunosensor was designed for ultrasensitive detection of sulfonamides (SAs), in which the synthesized 2D Cu-TCPP(Fe) with peroxidase-like property was used as a nanozyme that was directly modified on the electrode surface. Meanwhile, the structure of 2D Cu-TCPP(Fe) could be destroyed by the polyethyleneimine (PEI) from PEI-GO@Ab2 due to the stronger affinity between PEI and Cu2+, leading to an activity change of the prepared nanozyme. When H2O2 was introduced to the system, the electrochemical current was significantly declined owing to the peroxidase activity of 2D Cu-TCPP(Fe) decreased, which led to signal amplifications. Under the optimized conditions, this strategy had a wide detection range (1.186-28.051 ng/mL), satisfactory accuracy and precision (recoveries, 64-118%; CV, 2.16-7.27%) with a low detection limit of 0.395 ng/mL. The findings of this study indicate that the electrochemical immunosensor we developed has great potential and can be used for enzyme-free detection of SAs in environmental samples.

Identification of critical cysteine sites in brassinosteroid-insensitive 1 and novel signaling regulators using a transient expression system.

The plant hormones brassinosteroids (BRs) modulate plant growth and development. Cysteine (Cys) residues located in the extracellular domain of a protein are of importance for protein structure by forming disulfide bonds. To date, the systematic study of the functional significance of Cys residues in BR-insensitive 1 (BRI1) is still lacking. We used brassinolide-induced exogenous bri1-EMS-Suppressor 1 (BES1) dephosphorylation in Arabidopsis thaliana protoplasts as a readout, took advantage of the dramatic decrease of BRI1 protein levels during protoplast isolation, and of the strong phosphorylation of BES1 by BR-insensitive 2 (BIN2) in protoplasts, and developed a protoplast transient system to identify critical Cys sites in BRI1. Using this system, we identified a set of critical Cys sites in BRI1, as substitution of these Cys residues with alanine residues greatly compromised the function of BRI1. Moreover, we identified two negative regulators of BR signaling, pattern-triggered immunity compromised RLCK1 (PCRK1) and PCRK2, that were previously known to positively regulate innate immunity signaling. This work not only provides insight into the functional importance of critical Cys residues in stabilizing the superhelical conformation of BRI1-leucine-rich-repeat, but also reveals that PCRK1/2 can inversely modulate BR and plant immune signaling pathways.

Transcriptional Regulation of the Immune Receptor FLS2 Controls the Ontogeny of Plant Innate Immunity.

Innate immunity plays a vital role in protecting plants and animals from pathogen infections. Immunity varies with age in both animals and plants. However, little is known about the ontogeny of plant innate immunity during seedling development. We report here that the Arabidopsis (Arabidopsis thaliana) microRNA miR172b regulates the transcription of the immune receptor gene FLAGELLIN-SENSING2 (FLS2) through TARGET OF EAT1 (TOE1) and TOE2, which directly bind to the FLS2 promoter and inhibit its activity. The level of miR172b is very low in the early stage of seedling development but increases over time, which results in decreased TOE1/2 protein accumulation and, consequently, increased FLS2 transcription and the ontogeny of FLS2-mediated immunity during seedling development. Our study reveals a role for the miR172b-TOE1/2 module in regulating plant innate immunity and elucidates a regulatory mechanism underlying the ontogeny of plant innate immunity.plantcell;30/11/2779/FX1F1fx1.

A novel and sensitive chemiluminescence immunoassay based on AuNCs@pepsin@luminol for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether and tetrabromobisphenol A mono(hydroxyethyl) ether.

A novel chemiluminescence immunoassay based on luminol-modified gold nanoclusters (AuNCs@Peps@luminol) was developed for simultaneous detection of tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA-DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA-MHEE), an important derivative and byproduct of tetrabromobisphenol A (TBBPA), respectively. In the system, alkaline phosphatase (ALP) was labeled on the second antibody (Ab2) for signal amplification. When ALP-Ab2 was captured by antigen-primary antibody (Ab1) complex, disodium phenyl phosphate (PPNa) generated massive phenol under the catalysis of ALP, markedly inhibiting the chemiluminescence intensity of AuNCs@Peps@luminol. Under the optimized conditions, the calculated detection of limit (LOD, 90% inhibition) was 0.078 µg/L for TBBPA-DHEE with a linear range of 0.23-9.32 µg/L, which was lower 9 times than that of conventional ELISA with the same antibody. In addition, our method showed satisfactory accuracy and precision (recoveries, 88.00-113.4%; CV, 2.75-8.14%), it can be applied to systematically investigate the concentration of the trace TBBPA-DHEE and TBBPA-MHEE in environmental and food samples.

A sensitive, colorimetric immunosensor based on Cu-MOFs and HRP for detection of dibutyl phthalate in environmental and food samples.

A sensitive and artful colorimetric immunosensor based on horseradish peroxidase (HRP) was designed by labelling metal-organic frameworks (Cu-MOFs) on the second antibody (Cu-MOFs@Ab2) as signal amplification for the detection of trace dibutyl phthalate (DBP). In this system, when Cu-MOFs@Ab2 was captured by antigen- primary antibody (Ab1) complex, tremendous Cu(II) will be released from Cu-MOFs in the presence of nitric acid (HNO3), and Cu(II) will be further reduced to Cu(I) after the addition of sodium ascorbate (SA), consequently, inhibiting the HRP to catalyse the colorless 3,3',5,5'-tetramethylbenzidine (TMB) into blue oxidized TMB (ox TMB). Under the optimized conditions, the limit of detection (LOD) was 1 µg L-1, which was almost 60 times lower than that using a conventional ELISA with the same antibody. In addition, our method showed good accuracy and reproducibility (recoveries of 87.73-103.4%; CV values of 1.46-5.95%) through a spike-recovery analysis. The proposed immunosensor indicated great potential for trace DBP determination from environmental and food samples.

A novel, enzyme-linked immunosorbent assay based on the catalysis of AuNCs@BSA-induced signal amplification for the detection of dibutyl phthalate.

A novel, competitive, enzyme-linked immunosorbent assay (ELISA) was presented in this paper based on the inhibition of catalysis of AuNCs@BSA triggered by dissolved Ag+ for the detection of dibutyl phthalate (DBP). In the immunoassay system, numerous Ag+ was released from AgNPs (labelled on the second antibody, AgNPs@Ab2) in the presence of H2O2 after the competition step, preventing AuNCs@BSA from inducing a color change of 3,3',5,5'-tetramethylbenzidine (TMB) to blue. Due to the signal amplification by the principle, the sensitivity of the modified ELISA was improved with the low limit of detection (LOD) of 4.017µg/L for DBP, which was decreased 16 times relative to that using conventional ELISA with the same antibody. In addition, the established method showed satisfactory accuracy and reliability (recoveries, 85.75-117.73%; CV, 1.33-6.79%) in spike-recovery analysis. To the best of our knowledge, this is the first time that AuNCs@BSA has been used in ELISA as a peroxidase-like catalyst. Our method shows great potential for trace DBP detection from environmental and food samples.

Plasmonic ELISA Based on Nanospherical Brush-Induced Signal Amplification for the Ultrasensitive Naked-Eye Simultaneous Detection of the Typical Tetrabromobisphenol A Derivative and Byproduct.

On the basis of H2O2-mediated growth of gold nanoparticle (AuNPs), a novel plasmonic enzyme-linked immunosorbent assay (pELISA) was developed with a polyclonal antibody for the ultrasensitive simultaneous naked-eye detection of tetrabromobisphenol A bis(2-hydroxyetyl) ether (TBBPA DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA MHEE), one of the major derivatives and byproducts of tetrabromobisphenol A (TBBPA), respectively. In this modified indirect competitive pELISA, glucose oxidase (GOx) played an important role leading to the growth of AuNPs through a reaction between GOx and glucose to produce hydrogen peroxide (H2O2). In addition, further signal amplification was achieved via a large number of GOx molecules, which were immobilized on silica nanoparticles carrying poly brushes (SiO2@PAA) to increase the enzyme load, and the whole complex was conjugated on the second antibody. Under the optimized conditions, 10-3 µg/L TBBPA DHEE can be distinguished via the observation of a colored solution, and the limit of detection (LOD) of the method using a microplate reader reaches 3.3 × 10-4 µg/L. In contrast, the sensitivity of the method was 3 orders of magnitude higher than that using conventional colorimetric ELISA with the same antibody. Furthermore, the proposed approach showed good repeatability and reliability after a recovery test fortified with a variety of targets was performed (recoveries, 78.00-102.79%; coefficient of variation (CV), 4.38-9.87%). To our knowledge, this is the first case in which pELISA was applied for the detection of small molecules via the production of H2O2 from GOx and glucose. The method will be widely used for the investigation of TBBPA DHEE and TBBPA MHEE in real environments.

Comparative study of Arabidopsis PBS1 and a wheat PBS1 homolog helps understand the mechanism of PBS1 functioning in innate immunity.

Arabidopsis AVRPPHB SUSCEPTIBLE1 (PBS1) serves as a "decoy" in activating RESISTANCE TO PSEUDOMONAS SYRINGAE5 (RPS5) upon cleavage by Pseudomonas phaseolicola B (AvrPphB), a Pseudomonas syringae effector. The SEMPH motif in PBS1 was thought to allow it to be distinguished by RPS5 from the closely related Arabidopsis kinases. However, the underlying mechanism is not fully understood. Here, we isolated and characterized a wheat PBS1 homolog, TaPBS1. Although this plasma membrane-localized kinase could be cleaved by AvrPphB and could associate with RPS5, it failed to trigger RPS5-mediated hypersensitive response (HR) in a transient assay. TaPBS1 harbors a STRPH motif. The association of RPS5 with TaPBS1 was weaker than with PBS1. Change of the STRPH motif to the SEMPH motif allowed TaPBS1 to trigger HR. However, the SEMPH motif is not required for association of PBS1 with RPS5. The difference between "SEMPH" and "STRPH" points to the importance of "EM" in PBS1. Furthermore we found that a negatively charged amino acid at the position of "E" in the SEMPH motif was required for recognition of PBS1 by RPS5. Additionally, both PBS1 and TaPBS1 undergo the flagellin-induced phosphorylation. Therefore, our work will help understand the mechanism of PBS1 functioning in plant innate immunity.