Involvement of Arabidopsis Acyl Carrier Protein 1 in PAMP-Triggered Immunity.

Plant fatty acids (FAs) and lipids are essential in storing energy and act as structural components for cell membranes and signaling molecules for plant growth and stress responses. Acyl carrier proteins (ACPs) are small acidic proteins that covalently bind the fatty acyl intermediates during the elongation of FAs. The Arabidopsis thaliana ACP family has eight members. Through reverse genetic, molecular, and biochemical approaches, we have discovered that ACP1 localizes to the chloroplast and limits the magnitude of pattern-triggered immunity (PTI) against the bacterial pathogen Pseudomonas syringae pv. tomato. Mutant acp1 plants have reduced levels of linolenic acid (18:3), which is the primary precursor for biosynthesis of the phytohormone jasmonic acid (JA), and a corresponding decrease in the abundance of JA. Consistent with the known antagonistic relationship between JA and salicylic acid (SA), acp1 mutant plants also accumulate a higher level of SA and display corresponding shifts in JA- and SA-regulated transcriptional outputs. Moreover, methyl JA and linolenic acid treatments cause an apparently enhanced decrease of resistance against P. syringae pv. tomato in acp1 mutants than that in WT plants. The ability of ACP1 to prevent this hormone imbalance likely underlies its negative impact on PTI in plant defense. Thus, ACP1 links FA metabolism to stress hormone homeostasis to be negatively involved in PTI in Arabidopsis plant defense. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Arabidopsis Plasma Membrane ATPase AHA5 Is Negatively Involved in PAMP-Triggered Immunity.

Plants evolve a prompt and robust immune system to defend themselves against pathogen infections. Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) is the first battle layer activated upon the PAMP's perception, which leads to multiple defense responses. The plasma membrane (PM) H+-ATPases are the primary ion pumps to create and maintain the cellular membrane potential that is critical for various essential biological processes, including plant growth, development, and defense. This study discovered that the PM H+-ATPase AHA5 is negatively involved in Arabidopsis PTI against the virulent pathogen Pseudomonas syringae pvr. tomato (Pto) DC3000 infection. The aha5 mutant plants caused the reduced stomata opening upon the Pto infection, which was associated with the salicylic acid (SA) pathway. In addition, the aha5 mutant plants caused the increased levels of callose deposition, defense-related gene expression, and SA accumulation. Our results also indicate that the PM H+-ATPase activity of AHA5 probably mediates the coupling of H2O2 generation and the apoplast alkalization in PTI responses. Moreover, AHA5 was found to interact with a vital defense regulator, RPM1-interacting protein 4 (RIN4), in vitro and in vivo, which might also be critical for its function in PTI. In summary, our studies show that AHA5 functions as a novel and critical component that is negatively involved in PTI by coordinating different defense responses during the Arabidopsis-Pto DC3000 interaction.

A fungal effector and a rice NLR protein have antagonistic effects on a Bowman-Birk trypsin inhibitor.

Bowman-Birk trypsin inhibitors (BBIs) play important roles in animal and plant immunity, but how these protease inhibitors are involved in the immune system remains unclear. Here, we show that the rice (Oryza sativa) BBI protein APIP4 is a common target of a fungal effector and an NLR receptor for innate immunity. APIP4 exhibited trypsin inhibitor activity in vitro and in vivo. Knockout of APIP4 in rice enhanced susceptibility, and overexpression of APIP4 increased resistance to the fungal pathogen Magnaporthe oryzae. The M. oryzae effector AvrPiz-t interacted with APIP4 and suppressed APIP4 trypsin inhibitor activity. By contrast, the rice NLR protein Piz-t interacted with APIP4, enhancing APIP4 transcript and protein levels, and protease inhibitor activity. Our findings reveal a novel host defence mechanism in which a host protease inhibitor targeted by a fungal pathogen is protected by an NLR receptor.

Deciphering the Novel Role of AtMIN7 in Cuticle Formation and Defense against the Bacterial Pathogen Infection.

The cuticle is the outermost layer of plant aerial tissue that interacts with the environment and protects plants against water loss and various biotic and abiotic stresses. ADP ribosylation factor guanine nucleotide exchange factor proteins (ARF-GEFs) are key components of the vesicle trafficking system. Our study discovers that AtMIN7, an Arabidopsis ARF-GEF, is critical for cuticle formation and related leaf surface defense against the bacterial pathogen Pseudomonas syringae pathovar tomato (Pto). Our transmission electron microscopy and scanning electron microscopy studies indicate that the atmin7 mutant leaves have a thinner cuticular layer, defective stomata structure, and impaired cuticle ledge of stomata compared to the leaves of wild type plants. GC-MS analysis further revealed that the amount of cutin monomers was significantly reduced in atmin7 mutant plants. Furthermore, the exogenous application of either of three plant hormones-salicylic acid, jasmonic acid, or abscisic acid-enhanced the cuticle formation in atmin7 mutant leaves and the related defense responses to the bacterial Pto infection. Thus, transport of cutin-related components by AtMIN7 may contribute to its impact on cuticle formation and related defense function.

The Kinase OsCPK4 Regulates a Buffering Mechanism That Fine-Tunes Innate Immunity.

The calcium-dependent protein kinase OsCPK4 has been demonstrated to play important roles in salt and drought tolerance, plant growth, and development in rice (Oryza sativa). However, little is known about molecular mechanisms underlying OsCPK4 function in rice immunity. In this study, we demonstrated that the generation of oxidative burst and pathogenesis-related gene expression triggered by microbe-associated molecular patterns were significantly enhanced in the oscpk4 mutants. These mutant lines are more resistant to bacterial blight and fungal blast diseases than the wild-type plants, indicating that OsCPK4 negatively regulates innate immunity in rice. OsCPK4 was further identified to interact with a receptor-like cytoplasmic kinase OsRLCK176. OsRLCK176 accumulation is negatively regulated by OsCPK4. Interestingly, the kinase-dead OsCPK4 promotes OsRLCK176 degradation more strongly than the wild-type protein. OsCPK4 and OsRLCK176 mutually phosphorylate each other and form a feedback loop. Moreover, the kinase activity and phosphorylation of OsCPK4 and OsRLCK176 contribute to the stability of OsRLCK176. These findings indicate that the kinase-inactive OsCPK4 promotes OsRLCK176 degradation and restricts plant defenses, whereas the activation of OsCPK4-OsRLCK176 phosphorylation circuit invalidates the OsRLCK176 degradation machinery, thus enhancing plant immunity. Collectively, the study proposes a novel defense buffering mechanism mediated by OsCPK4, which fine-tunes microbe-associated molecular pattern-triggered immunity in rice.

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling.

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

Electrostatic potentials of the S-locus F-box proteins contribute to the pollen S specificity in self-incompatibility in Petunia hybrida.

Self-incompatibility (SI) is a self/non-self discrimination system found widely in angiosperms and, in many species, is controlled by a single polymorphic S-locus. In the Solanaceae, Rosaceae and Plantaginaceae, the S-locus encodes a single S-RNase and a cluster of S-locus F-box (SLF) proteins to control the pistil and pollen expression of SI, respectively. Previous studies have shown that their cytosolic interactions determine their recognition specificity, but the physical force between their interactions remains unclear. In this study, we show that the electrostatic potentials of SLF contribute to the pollen S specificity through a physical mechanism of 'like charges repel and unlike charges attract' between SLFs and S-RNases in Petunia hybrida. Strikingly, the alteration of a single C-terminal amino acid of SLF reversed its surface electrostatic potentials and subsequently the pollen S specificity. Collectively, our results reveal that the electrostatic potentials act as a major physical force between cytosolic SLFs and S-RNases, providing a mechanistic insight into the self/non-self discrimination between cytosolic proteins in angiosperms.

Combining Sprague-Dawley rat uterus cell membrane chromatography with HPLC/MS to screen active components from Leonurus artemisia.

CONTEXT: Leonurus artemisia (Lour.) S.Y.Hu (Lamiaceae) (YiMuCao in Chinese) is a traditional Chinese medicine. Leonurus artemisia has been shown to have many pharmacological effects such as increasing uterine contraction amplitude, and tension, but the active components are still unknown. OBJECTIVE: The objective of this study is to determine active components of L. Artemisia that are responsible for the biological activity using HPLC and cell membrane-based system. MATERIALS AND METHODS: The whole L. artemisia ethanol extract and its eight fractions were screened using Sprague-Dawley rat uterus cell membrane chromatography (CMC) combined with the HPLC/MS system. Oxytocin was used to investigate the activity of CMC column. The effect of active components screened from L. artemisia was studied by tension measurement of isolated rat uterine strips in vitro at a dose of 10(-7)-10(-4 )mol/L with oxytocin as a control. RESULTS: The acetone extract showed obvious activity when compared with the eight extracts of L. artemisia. From the acetone extract, in the negative ionization mode, the active compound was identified as genkwanin, with a molecular weight of 283. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions at a dose from 10(-7) to 10(-4 )mol/L. The EC50 value was 4.86 ± 4.21 µmol/L for genkwanin and 4.30 ± 3.65 µmol/L for oxytocin on the contractile amplitude of uterine strips isolated from rats. DISCUSSION AND CONCLUSION: Genkwanin was identified as the active compound in L. artemisia by this method. In vitro pharmacological experiments proved that genkwanin promoted uterine contractions. Genkwanin may be used to uterine inertia and may have an effect on postpartum hemorrhage.

Identification and characterization of suppressor mutants of spl11- mediated cell death in rice.

Lesion mimic mutants have been used to dissect programmed cell death (PCD) and defense-related pathways in plants. The rice lesion-mimic mutant spl11 exhibits race nonspecific resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. Spl11 encodes an E3 ubiquitin ligase and is a negative regulator of PCD in rice. To study the regulation of Spl11-mediated PCD, we performed a genetic screen and identified three spl11 cell-death suppressor (sds) mutants. These suppressors were characterized for their resistance to X. oryzae pv. oryzae and M. oryzae and for their expression of defense-related genes. The suppression of the cell-death phenotypes was generally correlated with reduced expression of defense-related genes. When rice was challenged with avirulent isolates of M. oryzae, the disease phenotype was unaffected in the sds mutants, indicating that the suppression might be Spl11-mediated pathway specific and may only be involved in basal defense. In addition, we mapped one of the suppressor mutations to a 140-kb interval on the long arm of rice chromosome 1. Identification and characterization of these sds mutants should facilitate our efforts to elucidate the Spl11-mediated PCD pathway.

Online LC-ESI-MS/MS comparative analysis of N/O-glycopatterns in human colostrum from different ethnic groups in Northwest China.

Human milk oligosaccharides, including free oligosaccharides and glycoconjugates, exert a key role in neonatal health and development. Changes in free oligosaccharides of milk from different ethnic groups have been documented. In this study, human milk was collected from Han, Hui, and Tibetan populations in northwest China, and differences in N/O-glycome among these three ethnic groups were systematically compared using online high-performance liquid chromatography-tandem mass spectrometry. Among the 63 detected N-glycans, 35 showed significant differences between the three ethnic groups (p < 0.05). Among the 70 detected O-glycans, four neutral O-glycans and six acidic O-glycans exhibited significant differences among the three ethnic groups (p < 0.05), with six acidic O-glycans reported for the first time. Overall, the extent of milk N/O-glycosylation was higher in the Han population than in the Hui or Tibetan groups. This trend was particularly pronounced for the main sialylated N/O-glycans. Except for sulfated O-glycans, which were higher in the milk from Tibetan mothers, the other types of N/O-glycans were present in similar proportions across all ethnic groups. Understanding the composition of N/O-glycans in human milk can help research on the structure-function relationship of glycans.

Integrated analysis of natural glycans using a versatile pyrazolone-type heterobifunctional tag ANPMP.

Glycans mediate various biological processes through carbohydrate-protein interactions, and glycan microarrays have become indispensable tools for understanding these mechanisms. However, advances in functional glycomics are hindered by the absence of convenient and universal methods for obtaining natural glycan libraries with diverse structures from glycoconjugates. To address this challenge, we have developed an integrative approach that enables one-pot release and simultaneously capture, separation, structural characterization, and functional analysis of N/O-glycans. Using this approach, glycoconjugates are incubated with a pyrazolone-type heterobifunctional tag-ANPMP to obtain glycan-2ANPMP conjugates, which are then converted to glycan-AEPMP conjugates. We prepared a tagged glycan library from porcine gastric mucin, soy protein, human milk oligosaccharides, etc. Following derivatization by N-acetylation and permethylation, glycans were subjected to detailed structural characterization by ESI-MSn analysis, which revealed >83 highly pure glycan-AEPMPs containing various natural glycan epitopes. A shotgun microarray is constructed to study the fine details of glycan-bindings by proteins and antisera.

Bacillus proteolyticus OSUB18 triggers induced systemic resistance against bacterial and fungal pathogens in Arabidopsis.

Pseudomonas syringae and Botrytis cinerea cause destructive bacterial speck and grey mold diseases in many plant species, leading to substantial economic losses in agricultural production. Our study discovered that the application of Bacillus proteolyticus strain OSUB18 as a root-drench enhanced the resistance of Arabidopsis plants against P. syringae and B. cinerea through activating Induced Systemic Resistance (ISR). The underlying mechanisms by which OSUB18 activates ISR were studied. Our results revealed that the Arabidopsis plants with OSUB18 root-drench showed the enhanced callose deposition and ROS production when inoculated with Pseudomonas syringae and Botrytis cinerea pathogens, respectively. Also, the increased salicylic acid (SA) levels were detected in the OSUB18 root-drenched plants compared with the water root-drenched plants after the P. syringae infection. In contrast, the OSUB18 root-drenched plants produced significantly higher levels of jasmonyl isoleucine (JA-Ile) than the water root-drenched control after the B. cinerea infection. The qRT-PCR analyses indicated that the ISR-responsive gene MYC2 and the ROS-responsive gene RBOHD were significantly upregulated in OSUB18 root-drenched plants upon both pathogen infections compared with the controls. Also, twenty-four hours after the bacterial or fungal inoculation, the OSUB18 root-drenched plants showed the upregulated expression levels of SA-related genes (PR1, PR2, PR5, EDS5, and SID2) or JA-related genes (PDF1.2, LOX3, JAR1 and COI1), respectively, which were consistent with the related hormone levels upon these two different pathogen infections. Moreover, OSUB18 can trigger ISR in jar1 or sid2 mutants but not in myc2 or npr1 mutants, depending on the pathogen's lifestyles. In addition, OSUB18 prompted the production of acetoin, which was reported as a novel rhizobacterial ISR elicitor. In summary, our studies discover that OSUB18 is a novel ISR inducer that primes plants' resistance against bacterial and fungal pathogens by enhancing the callose deposition and ROS accumulation, increasing the production of specific phytohormones and other metabolites involved in plant defense, and elevating the expression levels of multiple defense genes.

Gestational diabetes mellitus affects the fucosylation and sialylation levels of N/O-glycans in human milk glycoproteins.

Gestational diabetes mellitus (GDM) has negative effects on mothers and offspring, which may be related to the glycosylation level of milk proteins. Here, the human milk N/O-glycome of healthy and GDM individuals was analyzed by HILIC-MS/MS. A total of 56 putative N-glycans were detected, among which 12 N-glycans were significantly different between GDM and healthy milk. A total of 25 putative O-glycans were detected, and 11 of them varied greatly between GDM and healthy milk, especially H1N1S1 and H2N2S1. Overall, the relative content of N/O-glycans in GDM milk was significantly lower than that of healthy milk. In GDM milk, fucosylated N-glycans present higher proportion, whereas the proportion of sialylated O-glycans were lower. These findings would provide a foundation for in-depth study on the structure-activity relationship of milk N/O-glycans and are expected to drive the design of infant formula for newborns.

Online PGC-LC-ESI-MS/MS comparative analysis of variations in human milk O-glycopatterns from different secretor status.

O-glycome is one of the important components of glycoconjugates in human milk which is speculated to provide protective features similar to those observed in free oligosaccharides. The effects of maternal secretor status on free oligosaccharides and N-glycome in milk have been well researched and documented. Currently, milk O-glycome of secretors (Se+) and nonsecretors (Se-) was investigated through reductive ß-elimination combined with porous graphitized carbon-liquid chromatography-electrospray ionization-tandem mass spectrometry. A total of 70 presumptive O-glycan structures were identified, of which 25 O-glycans (including 14 sulfated O-glycans) were reported for the first time. Notably, 23 O-glycans exhibited significant differences between Se+ and Se- samples (p < 0.05). Compared to Se- group, the O-glycans of the Se+ group was two times more abundant in the total glycosylation, sialylation, fucosylation, and sulfation (p < 0.01). In conclusion, approximately one-third of the milk O-glycosylation was influenced by maternal FUT2-related secretor status. Our data will lay a foundation for the study of O-glycans structure-function relationship.

Human milk whey glycoprotein N-glycans varied greatly among different maternal secretor status.

Human milk glycans are complex carbohydrates, which play a pivotal role in infant health and neonatal development. Maternal secretor status is known to affect free oligosaccharides in milk. Here, the milk N-glycome of secretor (Se+) and nonsecretor (Se-) individuals was qualitatively and quantitatively analyzed by hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry. The total glycosylation, fucosylation, and sialylation of N-glycans was three times higher in the Se+ group compared to the Se- group (p < 0.001) per equal volume of milk. Importantly, 52 out of 63 N-glycans-including the eight most abundant ones-differed greatly between Se+ and Se- individuals (p < 0.05). Moreover, nine N-glycans (H5N3F1, H6N3, H3N5F1, H5N5F1, H5N5F1S1, H5N4F3S1, H6N4F2S1, H6N5F4S1, and H8N7S1) were >10 times more abundant in Se+ milk than in Se- milk. These findings lay a glycomics-basis for designing personalized nutrition supplements for infants.

Comparative Analysis of Human Milk Glycosphingolipids from Different Secretor Mothers Using HILIC-MS/MS.

Glycosphingolipids participate in brain development, intestinal tract maturation, and defense against gut pathogens. Here, we performed a qualitative and quantitative comparison of milk glycosphingolipids from secretors and nonsecretors. Hydrophilic interaction chromatography-electrospray ionization-tandem mass spectrometry was employed, along with an internal standard, to resolve the complications presented by the fact that glycosphingolipids are structurally diverse, varying in glycan composition and ceramide. In total, 101 glycosphingolipids were detected, of which 76 were reported for the first time, including fucose-modified neutral glycosphingolipids. Seventy-eight glycosphingolipids differed significantly between secretor and nonsecretor milk (p < 0.05), resulting in higher levels of certain neutral species (p < 0.001) but lower levels of fucose-modified monosialylated and disialylated species in secretor mothers (p < 0.01). In both milk types, the most abundant glycosphingolipids were of the monosialylated type, followed by disialylated, neutral, and trisialylated ones. Notably, fucose-modified monosialylated glycosphingolipids accounted for the highest proportion.

The natural pyrazolotriazine pseudoiodinine from Pseudomonas mosselii 923 inhibits plant bacterial and fungal pathogens.

Natural products largely produced by Pseudomonads-like soil-dwelling microorganisms are a consistent source of antimicrobial metabolites and pesticides. Herein we report the isolation of Pseudomonas mosselii strain 923 from rice rhizosphere soils of paddy fields, which specifically inhibit the growth of plant bacterial pathogens Xanthomonas species and the fungal pathogen Magnaporthe oryzae. The antimicrobial compound is purified and identified as pseudoiodinine using high-resolution mass spectra, nuclear magnetic resonance and single-crystal X-ray diffraction. Genome-wide random mutagenesis, transcriptome analysis and biochemical assays define the pseudoiodinine biosynthetic cluster as psdABCDEFG. Pseudoiodinine biosynthesis is proposed to initiate from guanosine triphosphate and 1,6-didesmethyltoxoflavin is a biosynthetic intermediate. Transposon mutagenesis indicate that GacA is the global regulator. Furthermore, two noncoding small RNAs, rsmY and rsmZ, positively regulate pseudoiodinine transcription, and the carbon storage regulators CsrA2 and CsrA3, which negatively regulate the expression of psdA. A 22.4-fold increase in pseudoiodinine production is achieved by optimizing the media used for fermentation, overexpressing the biosynthetic operon, and removing the CsrA binding sites. Both of the strain 923 and purified pseudoiodinine in planta inhibit the pathogens without affecting the rice host, suggesting that pseudoiodinine can be used to control plant diseases.

OsWRKY97, an Abiotic Stress-Induced Gene of Rice, Plays a Key Role in Drought Tolerance.

Drought stress is one of the major causes of crop losses. The WRKY families play important roles in the regulation of many plant processes, including drought stress response. However, the function of individual WRKY genes in plants is still under investigation. Here, we identified a new member of the WRKY families, OsWRKY97, and analyzed its role in stress resistance by using a series of transgenic plant lines. OsWRKY97 positively regulates drought tolerance in rice. OsWRKY97 was expressed in all examined tissues and could be induced by various abiotic stresses and abscisic acid (ABA). OsWRKY97-GFP was localized to the nucleus. Various abiotic stress-related cis-acting elements were observed in the promoters of OsWRKY97. The results of OsWRKY97-overexpressing plant analyses revealed that OsWRKY97 plays a positive role in drought stress tolerance. In addition, physiological analyses revealed that OsWRKY97 improves drought stress tolerance by improving the osmotic adjustment ability, oxidative stress tolerance, and water retention capacity of the plant. Furthermore, OsWRKY97-overexpressing plants also showed higher sensitivity to exogenous ABA compared with that of wild-type rice (WT). Overexpression of OsWRKY97 also affected the transcript levels of ABA-responsive genes and the accumulation of ABA. These results indicate that OsWRKY97 plays a crucial role in the response to drought stress and may possess high potential value in improving drought tolerance in rice.

Real-Time Detection of Reactive Oxygen Species Production in Immune Response in Rice with a Chemiluminescence Assay.

Reactive oxygen species (ROS) play vital roles in a variety of biological processes, including the sensing of abiotic and biotic stresses. Upon pathogen infection or challenge with pathogen-associated chemicals (pathogen-associated molecular patterns [PAMPs]), an array of immune responses, including a ROS burst, are quickly induced in plants, which is called PAMP-triggered immunity (PTI). A ROS burst is a hallmark PTI response, which is catalyzed by a group of plasma membrane-localized NADPH oxidases-the RBOH family proteins. The vast majority of ROS comprise hydrogen peroxide (H2O2), which can be easily and steadily detected by a luminol-based chemiluminescence method. Chemiluminescence is a photon-producing reaction in which luminol, or its derivative (such as L-012), undergoes a redox reaction with ROS under the action of a catalyst. This paper describes an optimized L-012-based chemiluminescence method to detect apoplast ROS production in real-time upon PAMP elicitation in rice tissues. The method is easy, steady, standardized, and highly reproducible under firmly controlled conditions.

How a single receptor-like kinase exerts diverse roles: lessons from FERONIA.

FERONIA (FER) is a member of the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein subfamily, which participates in reproduction, abiotic stress, biotic stress, cell growth, hormone response, and other molecular mechanisms of plants. However, the mechanism by which a single RLK is capable of mediating multiple signals and activating multiple cellular responses remains unclear. Here, we summarize research progress revealing the spatial-temporal expression of FER, along with its co-receptors and ligands determined the function of FER signaling pathway in multiple organs. The specificity of the FER signaling pathway is proposed to operate under a four-layered mechanism: (1) Spatial-temporal expression of FER, co-receptors, and ligands specify diverse functions, (2) Specific ligands or ligand combinations trigger variable FER signaling pathways, (3) Diverse co-receptors confer diverse FER perception and response modes, and (4) Unique downstream components that modify FER signaling and responses. Moreover, the regulation mechanism of the signaling pathway- appears to depend on the interaction among the ligands, RLK receptors, co-receptors, and downstream components, which may be a general mechanism of RLKs to maintain signal specificity. This review will provide a insight into understanding the specificity determination of RLKs signaling in both model and horticultural crops.