FLAGELLIN SENSING 2 (FLS2) encodes a pattern recognition receptor that perceives bacterial flagellin. While putative FLS2 orthologs are broadly conserved in plants, their functional characterization remains limited. Here, we report the identification of orthologs in cucumber (Cucumis sativus) and melon (C. melo), named CsFLS2 and CmFLS2, respectively. Homology searching identified CsFLS2, and virus-induced gene silencing (VIGS) demonstrated that CsFLS2 is required for flg22-triggered ROS generation. Interestingly, genome re-sequencing of melon cv. Lennon and subsequent genomic PCR revealed that Lennon has two CmFLS2 haplotypes, haplotype I encoding full-length CmFLS2 and haplotype II encoding a truncated form. We show that VIGS-mediated knockdown of CmFLS2 haplotype I resulted in a significant reduction in both flg22-triggered ROS generation and immunity to a bacterial pathogen in melon cv. Lennon. Remarkably, genomic PCR of CmFLS2 revealed that 68% of tested commercial melon cultivars possess only CmFLS2 haplotype II: these cultivars thus lack functional CmFLS2. To explore evolutionary aspects of CmFLS2 haplotype II occurrence, we genotyped the CmFLS2 locus in 142 melon accessions by genomic PCR and analyzed 437 released sequences. The results suggest that CmFLS2 haplotype II is derived from C. melo subsp. melo. Furthermore, we suggest that the proportion of CmFLS2 haplotype II increased among the improved melo group compared with the primitive melo group. Collectively, these findings suggest that the deleted FLS2 locus generated in the primitive melo subspecies expanded after domestication, resulting in the spread of commercial melon cultivars defective in flagellin recognition, which is critical for bacterial immunity.

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2-1 and glecrk-i.2-2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2-1 and glecrk-i.2-2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.

Temporospatial regulation of immunity components is essential for properly activating plant defense response. Flagellin-sensing 2 (FLS2) is a surface-localized receptor that recognizes bacterial flagellin. The immune function of FLS2 is compromised in early stages of shoot development. However, the underlying mechanism for the age-dependent FLS2 signaling is not clear. Here, we show that the reduced basal immunity of juvenile leaves against Pseudomonas syringae pv. tomato DC3000 is independent of FLS2. The flg22-induced marker gene expression and reactive oxygen species activation were comparable in juvenile and adult stages, but callose deposition was more evident in the adult stage than the juvenile stage. We further demonstrated that microRNA156, a master regulator of plant aging, does not influence the expression of FLS2 and FRK1 (Flg22-induced receptor-like kinase 1) but mildly suppresses callose deposition in juvenile leaves. Our experiments revealed an intrinsic mechanism that regulates the amplitude of FLS2-mediated resistance during aging. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phloem is a critical tissue for transport of photosynthates and extracellular signals in vascular plants. However, it also represents an ideal environment for pathogens seeking access to valuable host nutrients. Although many vascular pathogens induce economically relevant crop damage, there is still little known about the mechanisms by which immune signaling operates through the phloem. An existing phosphoproteomic dataset was mined to identify proteins that were both phosphorylated in response to the defense-elicitor flagellin (flg22) and expressed in vascular cells. A single candidate, OCTOPUS (OPS), is polarly associated with the plasma membrane of sieve element cells and has been characterized as an inhibitor of brassinosteroid insensitive-2 in promotion of brassinosteroid-related phytohormone signaling. The observation that OPS is differentially phosphorylated in response to flg22 led us to the examine whether OPS may also regulate flg22-induced immune signaling. Two independent alleles of ops exhibited enhanced immunity outputs across multiple signaling branches of PAMP-triggered immunity (PTI), constitutively and in response to flg22 treatment. Together with our observation that interactions between OPS and brassinosteroid insensitive-2 were disrupted by induction of salicylic acid and depletion of brassinosteriod, these data support a model whereby OPS modulates brassinolide and immune signaling to control downstream responses. We present OPS as a novel addition to the list of proteins with documented roles in PAMP-PTI signaling. These results further indicate that immune signaling in the phloem may be a significant and unique component of the host detection and response to pathogens in vascular plants.

A prophage tail-like protein (Bg_9562) of Burkholderia gladioli strain NGJ1 possesses broad-spectrum antifungal activity, and it is required for the bacterial ability to forage over fungi. Here, we analyzed whether heterologous overexpression of Bg_9562 or exogenous treatment with purified protein can impart disease tolerance in tomato. The physiological relevance of Bg_9562 during endophytic growth of NGJ1 was also investigated. Bg_9562 overexpressing lines demonstrate fungal and bacterial disease tolerance. They exhibit enhanced expression of defense genes and activation of mitogen-activated protein kinases. Treatment with Bg_9562 protein induces defense responses and imparts immunity in wild-type tomato. The defense-inducing ability lies within 18-51 aa region of Bg_9562 and is due to sequence homology with the bacterial flagellin epitope. Interaction studies suggest that Bg_9562 is perceived by FLAGELLIN-SENSING 2 homologs in tomato. The silencing of SlSERK3s (BAK1 homologs) prevents Bg_9562-triggered immunity. Moreover, type III secretion system-dependent translocation of Bg_9562 into host apoplast is important for elicitation of immune responses during colonization of NGJ1. Our study emphasizes that Bg_9562 is important for the endophytic growth of B. gladioli, while the plant perceives it as an indirect indicator of the presence of bacteria to mount immune responses. The findings have practical implications for controlling plant diseases.

Through crosstalk, FLAGELLIN SENSITIVE 2 (FLS2) and RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) are involved in regulating the homeostasis of cellular reactive oxygen species (ROS) and are linked to the metabolic response of plants toward both biotic and abiotic stress. In the present study, we examined the metabolome of Arabidopsis seedlings under drought and salt conditions to better understand the potential role of FLS2 and RBOHD-dependent signaling in the regulation of abiotic stress response. We identified common metabolites and genes that are regulated by FLS2 and RBOHD, and are involved in the response to drought and salt stress. Under drought conditions, D-aspartic acid and the expression of associated genes, such as ASPARAGINE SYNTHASE 2 (ASN2), increased in both fls2 and robed/f double mutants. The accumulation of amino acids, carbohydrates, and hormones, such as L-proline, D-ribose, and indoleacetaldehyde increased in both fls2 and rbohd/f double mutants under salt conditions, as did the expression of related genes, such as PROLINE IMINOPEPTIDASE, PHOSPHORIBOSYL PYROPHOSPHATE SYNTHASE 5, and NITRILASE 3. Collectively, these results indicate that the FLS2-RBOHD module regulates plant response to drought and salt stress through ROS signaling by adjusting the accumulation of metabolites and expression of genes related to metabolite synthesis.

Plant receptor kinases are key transducers of extracellular stimuli, such as the presence of beneficial or pathogenic microbes or secreted signaling molecules. Receptor kinases are regulated by numerous post-translational modifications.1,2,3 Here, using the immune receptor kinases FLS24 and EFR,5 we show that S-acylation at a cysteine conserved in all plant receptor kinases is crucial for function. S-acylation involves the addition of long-chain fatty acids to cysteine residues within proteins, altering their biochemical properties and behavior within the membrane environment.6 We observe S-acylation of FLS2 at C-terminal kinase domain cysteine residues within minutes following the perception of its ligand, flg22, in a BAK1 co-receptor and PUB12/13 ubiquitin ligase-dependent manner. We demonstrate that S-acylation is essential for FLS2-mediated immune signaling and resistance to bacterial infection. Similarly, mutating the corresponding conserved cysteine residue in EFR suppressed elf18-triggered signaling. Analysis of unstimulated and activated FLS2-containing complexes using microscopy, detergents, and native membrane DIBMA nanodiscs indicates that S-acylation stabilizes, and promotes retention of, activated receptor kinase complexes at the plasma membrane to increase signaling efficiency.

In eukaryotes, dynamins and dynamin-related proteins (DRPs) are high-molecular weight GTPases responsible for mechanochemical fission of organelles or membranes. Of the six DRP subfamilies in Arabidopsis thaliana, AtDRP1 and AtDRP2 family members serve as endocytic accessory proteins in clathrin-mediated endocytosis. Most studies have focused on AtDRP1A and AtDRP2B as critical modulators of plant pattern-triggered immunity (PTI) against pathogenic, flagellated Pseudomonas syringae pv. tomato DC3000 bacteria and immune signaling in response to the bacterial flagellin peptide flg22. Much less is known about AtDRP2A, the closely related paralog of AtDRP2B. AtDRP2A and AtDRP2B are the only classical, or bona fide, dynamins in Arabidopsis, based on their evolutionary conserved domain structure with mammalian dynamins functioning in endocytosis. AtDRP2B but not AtDRP2A is required for robust ligand-induced endocytosis of the receptor kinase FLAGELLIN SENSING2 for dampening of early flg22 signaling. Here, we utilized Arabidopsis drp2a null mutants to identify AtDRP2A as a positive contributor to effective PTI against P. syringae pv. tomato DC3000 bacteria, consistent with reduced PATHOGEN RELATED1 (PR1) messenger RNA accumulation. We provide evidence that AtDRP2A is a novel modulator of late flg22 signaling, contributing positively to PR1 gene induction but negatively to polyglucan callose deposition. AtDRP2A has no apparent roles in flg22-elicited mitogen-activated protein kinase defense marker gene induction. In summary, this study adds the evolutionary conserved dynamin AtDRP2A to a small group of vesicular trafficking proteins with roles as non-canonical contributors in immune responses, likely due to modulating one or both the localization and activity of multiple different proteins with distinct contributions to immune signaling. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.