Multilayered synergistic regulation of phytoalexin biosynthesis by ethylene, jasmonate, and MAPK signaling pathways in Arabidopsis.

Camalexin, an indolic antimicrobial metabolite, is the major phytoalexin in Arabidopsis thaliana, and plays a crucial role in pathogen resistance. Our previous studies revealed that the Arabidopsis mitogen-activated protein kinases MPK3 and MPK6 positively regulate pathogen-induced camalexin biosynthesis via phosphoactivating the transcription factor WRKY33. Here, we report that the ethylene and jasmonate (JA) pathways act synergistically with the MPK3/MPK6-WRKY33 module at multiple levels to induce camalexin biosynthesis in Arabidopsis upon pathogen infection. The ETHYLENE RESPONSE FACTOR1 (ERF1) transcription factor integrates the ethylene and JA pathways to induce camalexin biosynthesis via directly upregulating camalexin biosynthetic genes. ERF1 also interacts with and depends on WRKY33 to upregulate camalexin biosynthetic genes, indicating that ERF1 and WRKY33 form transcriptional complexes to cooperatively activate camalexin biosynthetic genes, thereby mediating the synergy of ethylene/JA and MPK3/MPK6 signaling pathways to induce camalexin biosynthesis. Moreover, as an integrator of the ethylene and JA pathways, ERF1 also acts as a substrate of MPK3/MPK6, which phosphorylate ERF1 to increase its transactivation activity and therefore further cooperate with the ethylene/JA pathways to induce camalexin biosynthesis. Taken together, our data reveal the multilayered synergistic regulation of camalexin biosynthesis by ethylene, JA, and MPK3/MPK6 signaling pathways via ERF1 and WRKY33 transcription factors in Arabidopsis.

Phosphorylation of an ethylene response factor by MPK3/MPK6 mediates negative feedback regulation of pathogen-induced ethylene biosynthesis in Arabidopsis.

Plants under pathogen attack produce high levels of the gaseous phytohormone ethylene to induce plant defense responses via the ethylene signaling pathway. The 1-aminocyclopropane-1-carboxylate synthase (ACS) is a critical rate-limiting enzyme of ethylene biosynthesis. Transcriptional and post-translational upregulation of ACS2 and ACS6 by the mitogen-activated protein kinases MPK3 and MPK6 are previously shown to be crucial for pathogen-induced ethylene biosynthesis in Arabidopsis. Here, we report that the fungal pathogen Botrytis cinerea-induced ethylene biosynthesis in Arabidopsis is under the negative feedback regulation by ethylene signaling pathway. The ethylene response factor ERF1A is further found to act downstream of ethylene signaling to negatively regulate the B. cinerea-induced ethylene biosynthesis via indirectly suppressing the expression of ACS2 and ACS6. Interestingly, ERF1A is shown to also upregulate defensin genes directly and therefore promote Arabidopsis resistance to B. cinerea. Furthermore, ERF1A is identified to be a substrate of MPK3 and MPK6, which phosphoactivate ERF1A to enhance its functions in suppressing ethylene biosynthesis and inducing defensin gene expression. Taken together, our data reveal that ERF1A and its phosphorylation by MPK3/MPK6 not only mediate the negative-feedback regulation of the B. cinerea-induced ethylene biosynthesis, but also upregulate defensin gene expression to increase Arabidopsis resistance to B. cinerea.

Perception of the pathogen-induced peptide RGF7 by the receptor-like kinases RGI4 and RGI5 triggers innate immunity in Arabidopsis thaliana.

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.

Differential Phosphorylation of the Transcription Factor WRKY33 by the Protein Kinases CPK5/CPK6 and MPK3/MPK6 Cooperatively Regulates Camalexin Biosynthesis in Arabidopsis.

Camalexin is a major phytoalexin that plays a crucial role in disease resistance in Arabidopsis (Arabidopsis thaliana). We previously characterized the regulation of camalexin biosynthesis by the mitogen-activated protein kinases MPK3 and MPK6 and their downstream transcription factor WRKY33. Here, we report that the pathogen-responsive CALCIUM-DEPENDENT PROTEIN KINASE5 (CPK5) and CPK6 also regulate camalexin biosynthesis in Arabidopsis. Chemically induced expression of constitutively active CPK5 or CPK6 variants was sufficient to induce camalexin biosynthesis in transgenic Arabidopsis plants. Consistently, the simultaneous mutation of CPK5 and CPK6 compromised camalexin production in Arabidopsis induced by the fungal pathogen Botrytis cinerea Moreover, we identified that WRKY33 functions downstream of CPK5/CPK6 to activate camalexin biosynthetic genes, thereby inducing camalexin biosynthesis. CPK5 and CPK6 interact with WRKY33 and phosphorylate its Thr-229 residue, leading to an increase in the DNA binding ability of WRKY33. By contrast, the MPK3/MPK6-mediated phosphorylation of WRKY33 on its N-terminal Ser residues enhances the transactivation activity of WRKY33. Furthermore, both gain- and loss-of-function genetic analyses demonstrated the cooperative regulation of camalexin biosynthesis by CPK5/CPK6 and MPK3/MPK6. Taken together, these findings indicate that WRKY33 functions as a convergent substrate of CPK5/CPK6 and MPK3/MPK6, which cooperatively regulate camalexin biosynthesis via the differential phospho-regulation of WRKY33 activity.

Ligand-induced monoubiquitination of BIK1 regulates plant immunity.

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens1-3. Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants4. The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

Phosphoregulation of Ca2+ Influx in Plant Immunity.

A rapid influx of calcium into the cytosol represents a hallmark of plant immune responses. Recent studies in arabidopsis (Tian et al.) and rice (Wang et al.) reveal that pathogen-responsive receptor-like cytoplasmic kinases phosphorylate and activate calcium-permeable cyclic nucleotide-gated channels to trigger calcium influx, filling a missing link between pathogen perception and calcium signaling.

The Arabidopsis Pleiotropic Drug Resistance Transporters PEN3 and PDR12 Mediate Camalexin Secretion for Resistance to Botrytis cinerea.

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.

Proteolytic Processing of SERK3/BAK1 Regulates Plant Immunity, Development, and Cell Death.

Plants have evolved many receptor-like kinases (RLKs) to sense extrinsic and intrinsic cues. The signaling pathways mediated by multiple Leucine-rich repeat (LRR) RLK (LRR-RLK) receptors require ligand-induced receptor-coreceptor heterodimerization and transphosphorylation with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1)/SOMATIC EMBRYOGENESIS RECEPTOR KINASES family LRR-RLKs. Here we reveal an additional layer of regulation of BAK1 via a Ca2+-dependent proteolytic cleavage process that is conserved in Arabidopsis (Arabidopsis thaliana), Nicotiana benthamiana, and Saccharomyces cerevisiae The proteolytic cleavage of BAK1 is intrinsically regulated in response to developmental cues and immune stimulation. The surface-exposed Asp (D287) residue of BAK1 is critical for its proteolytic cleavage and plays an essential role in BAK1-regulated plant immunity, growth hormone brassinosteroid-mediated responses, and cell death containment. BAK1D287A mutation impairs BAK1 phosphorylation on its substrate BOTRYTIS-INDUCED KINASE1 (BIK1), and its plasma membrane localization. Intriguingly, it aggravates BAK1 overexpression-triggered cell death independent of BIK1, suggesting that maintaining homeostasis of BAK1 through a proteolytic process is crucial to control plant growth and immunity. Our data reveal that in addition to layered transphosphorylation in the receptor complexes, the proteolytic cleavage is an important regulatory process for the proper functions of the shared coreceptor BAK1 in diverse cellular signaling pathways.

The Monocot-Specific Receptor-like Kinase SDS2 Controls Cell Death and Immunity in Rice.

Programmed cell death (PCD) plays critical roles in plant immunity but must be regulated to prevent excessive damage. The E3 ubiquitin ligase SPL11 negatively regulates PCD and immunity in plants. We show that SPL11 cell-death suppressor 2 (SDS2), an S-domain receptor-like kinase, positively regulates PCD and immunity in rice by engaging and regulating SPL11 and related kinases controlling defense responses. An sds2 mutant shows reduced immune responses and enhanced susceptibility to the blast fungus Magnaporthe oryzae. Conversely, SDS2 over-expression induces constitutive PCD accompanied by elevated immune responses and enhanced resistance to M. oryzae. SDS2 interacts with and phosphorylates SPL11, which in turn ubiquitinates SDS2, leading to its degradation. In addition, SDS2 interacts with related receptor-like cytoplasmic kinases, OsRLCK118/176, that positively regulate immunity by phosphorylating the NADPH oxidase OsRbohB to stimulate ROS production. Thus, a plasma membrane-resident protein complex consisting of SDS2, SPL11, and OsRLCK118/176 controls PCD and immunity in rice.

Regulation of Arabidopsis brassinosteroid receptor BRI1 endocytosis and degradation by plant U-box PUB12/PUB13-mediated ubiquitination.

Plants largely rely on plasma membrane (PM)-resident receptor-like kinases (RLKs) to sense extracellular and intracellular stimuli and coordinate cell differentiation, growth, and immunity. Several RLKs have been shown to undergo internalization through the endocytic pathway with a poorly understood mechanism. Here, we show that endocytosis and protein abundance of the Arabidopsis brassinosteroid (BR) receptor, BR INSENSITIVE1 (BRI1), are regulated by plant U-box (PUB) E3 ubiquitin ligase PUB12- and PUB13-mediated ubiquitination. BR perception promotes BRI1 ubiquitination and association with PUB12 and PUB13 through phosphorylation at serine 344 residue. Loss of PUB12 and PUB13 results in reduced BRI1 ubiquitination and internalization accompanied with a prolonged BRI1 PM-residence time, indicating that ubiquitination of BRI1 by PUB12 and PUB13 is a key step in BRI1 endocytosis. Our studies provide a molecular link between BRI1 ubiquitination and internalization and reveal a unique mechanism of E3 ligase-substrate association regulated by phosphorylation.

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsisthaliana: the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling.

Differential Regulation of Two-Tiered Plant Immunity and Sexual Reproduction by ANXUR Receptor-Like Kinases.

Plants have evolved two tiers of immune receptors to detect infections: cell surface-resident pattern recognition receptors (PRRs) that sense microbial signatures and intracellular nucleotide binding domain leucine-rich repeat (NLR) proteins that recognize pathogen effectors. How PRRs and NLRs interconnect and activate the specific and overlapping plant immune responses remains elusive. A genetic screen for components controlling plant immunity identified ANXUR1 (ANX1), a malectin-like domain-containing receptor-like kinase, together with its homolog ANX2, as important negative regulators of both PRR- and NLR-mediated immunity in Arabidopsis thaliana ANX1 constitutively associates with the bacterial flagellin receptor FLAGELLIN-SENSING2 (FLS2) and its coreceptor BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). Perception of flagellin by FLS2 promotes ANX1 association with BAK1, thereby interfering with FLS2-BAK1 complex formation to attenuate PRR signaling. In addition, ANX1 complexes with the NLR proteins RESISTANT TO PSEUDOMONAS SYRINGAE2 (RPS2) and RESISTANCE TO P. SYRINGAE PV MACULICOLA1. ANX1 promotes RPS2 degradation and attenuates RPS2-mediated cell death. Surprisingly, a mutation that affects ANX1 function in plant immunity does not disrupt its function in controlling pollen tube growth during fertilization. Our study thus reveals a molecular link between PRR and NLR protein complexes that both associate with cell surface-resident ANX1 and uncovers uncoupled functions of ANX1 and ANX2 during plant immunity and sexual reproduction.

Ligand-Induced Receptor-like Kinase Complex Regulates Floral Organ Abscission in Arabidopsis.

Abscission is a developmental process that enables plants to shed unwanted organs. In Arabidopsis, the floral organ abscission is regulated by a signaling pathway consisting of the peptide ligand IDA, the receptor-like kinases (RLKs) HAE and HSL2, and a downstream MAP kinase (MAPK) cascade. However, little is known about the molecular link between ligand-receptor pairs and intracellular signaling. Here, we report that the SERK family RLKs function redundantly in regulating floral organ abscission downstream of IDA and upstream of the MAPK cascade. IDA induces heterodimerization of HAE/HSL2 and SERKs, which transphosphorylate each other. The SERK3 residues mediating its interaction with the immune receptor FLS2 and the brassinosteroid receptor BRI1 are also required for IDA-induced HAE/HSL2-SERK3 interaction, suggesting SERKs serve as co-receptors of HAE/HSL2 in perceiving IDA. Thus, our study reveals the signaling activation mechanism in floral organ abscission by IDA-induced HAE/HSL2-SERK complex formation accompanied by transphosphorylation.

The dominant negative ARM domain uncovers multiple functions of PUB13 in Arabidopsis immunity, flowering, and senescence.

Regulating the intensity and duration of immune responses is crucial to combat infections without deleterious side effects. Arabidopsis FLS2, the receptor for bacterial flagellin, activates immune signalling by association with its partner BAK1. Upon flagellin (flg22) perception, the plant U-box E3 ubiquitin ligases PUB12 and PUB13 complex with FLS2 in a BAK1-dependent manner, and ubiquitinate FLS2 for protein degradation, thereby down-regulating flagellin signalling. Domain deletion analysis indicates that the ARM domain of PUB13 interacts with the FLS2-BAK1 complex and is phosphorylated by BAK1. Overexpression of the PUB13 ARM domain alone inhibits flg22-induced FLS2-PUB13 association and PUB12/13-mediated FLS2 ubiquitination and degradation in Arabidopsis, suggesting that ectopic expression of the ARM domain in planta generates a dominant negative effect via blocking the ubiquitination activity. Similar to the pub12pub13 double mutant, transgenic plants expressing the PUB13 ARM domain display enhanced immune responses compared with wild-type plants. Moreover, PUB13ARM transgenic plants and the pub12pub13 mutant are more sensitive to stress-induced leaf senescence accompanied by elevated expression of stress-induced senescence marker genes. The resemblance between PUB13ARM transgenic plants and the pub12pub13 mutant provides genetic evidence that ectopic expression of the PUB ARM domain serves as an alternative approach to dissect the overlapping functions of closely related PUB genes.

Ubiquitination of plant immune receptors.

Ubiquitin is a highly conserved regulatory protein consisting of 76 amino acids and ubiquitously expressed in all eukaryotic cells. The reversible ubiquitin conjugation to a wide variety of target proteins, a process known as ubiquitination or ubiquitylation, serves as one of the most important and prevalent posttranslational modifications to regulate the myriad actions of protein cellular functions, including protein degradation, vesicle trafficking, and subcellular localization. Protein ubiquitination is an ATP-dependent stepwise covalent attachment of one or more ubiquitin molecules to target proteins mediated by a hierarchical enzymatic cascade consisting of an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme, and E3 ubiquitin ligase. The plant plasma membrane resident receptor-like kinase Flagellin Sensing 2 (FLS2) recognizes bacterial flagellin and initiates innate immune signaling to defend against pathogen attacks. We have recently shown that two plant U-box E3 ubiquitin ligases PUB12 and PUB13 directly ubiquitinate FLS2 and promote flagellin-induced FLS2 degradation, which in turn attenuates FLS2 signaling to prevent excessive or prolonged activation of immune responses. Here, we use FLS2 as an example to describe a protocol for detection of protein ubiquitination in plant cells in vivo and in test tubes in vitro. In addition, we elaborate the approach to identify different types of ubiquitin linkages by using various lysine mutants of ubiquitin. The various in vivo and in vitro ubiquitination assays will provide researchers with the tools to address how ubiquitination regulates diverse cellular functions of target proteins.

The Pseudomonas syringae effector HopF2 suppresses Arabidopsis immunity by targeting BAK1.

Pseudomonas syringae delivers a plethora of effector proteins into host cells to sabotage immune responses and modulate physiology to favor infection. The P. syringae pv. tomato DC3000 effector HopF2 suppresses Arabidopsis innate immunity triggered by multiple microbe-associated molecular patterns (MAMP) at the plasma membrane. We show here that HopF2 possesses distinct mechanisms for suppression of two branches of MAMP-activated MAP kinase (MAPK) cascades. In addition to blocking MKK5 (MAPK kinase 5) activation in the MEKK1 (MAPK kinase kinase 1)/MEKKs-MKK4/5-MPK3/6 cascade, HopF2 targets additional component(s) upstream of MEKK1 in the MEKK1-MKK1/2-MPK4 cascade and the plasma membrane-localized receptor-like cytoplasmic kinase BIK1 and its homologs. We further show that HopF2 directly targets BAK1, a plasma membrane-localized receptor-like kinase that is involved in multiple MAMP signaling. The interaction between BAK1 and HopF2 and between two other P. syringae effectors, AvrPto and AvrPtoB, was confirmed in vivo and in vitro. Consistent with BAK1 as a physiological target of AvrPto, AvrPtoB and HopF2, the strong growth defects or lethality associated with ectopic expression of these effectors in wild-type Arabidopsis transgenic plants were largely alleviated in bak1 mutant plants. Thus, our results provide genetic evidence to show that BAK1 is a physiological target of AvrPto, AvrPtoB and HopF2. Identification of BAK1 as an additional target of HopF2 virulence not only explains HopF2 suppression of multiple MAMP signaling at the plasma membrane, but also supports the notion that pathogen virulence effectors act through multiple targets in host cells.

Molecular cloning and characterization of two novel hepcidins from orange-spotted grouper, Epinephelus coioides.

Orange-spotted grouper, Epinephelus coioides is one of the most important economic species of marine-cultured fish in China and Southeast Asia countries. However, very little information of the innate immune mechanisms against microbial pathogens is available in grouper, Epinephelus sp. Hepcidin, as an antimicrobial peptide (AMP), is a very important component in the innate immune system and widespread in fish. In this study, two novel types of hepcidin gene (designated EC-hepcidin1 and EC-hepcidin2) were cloned from E. coioides. They consist of open reading frames (ORFs) of 267 bp and 263 bp encoding the putative peptides of 88 and 87 amino acids, respectively. The homologous identity of deduced amino acid sequences between EC-hepcidin1 and EC-hepcidin2 is up to 79%, and predicted mature regions of both them have four cysteines residues. Genomic DNAs of both EC-hepcidin1 and EC-hepcidin2 consist of three exons and two introns. RT-PCR results showed that EC-hepcidin1 transcript was most abundant in liver and less in stomach. However, the transcript of EC-hepcidin2 was only detected in liver. The expressions of both EC-hepcidins were up-regulated by microbial and viral challenges, and iron overload, respectively, and EC-hepcidin1 was more responsive. The growth of Gram-negative bacterium of Vibrio vulnificus and Gram-positive bacterium of Staphylococcus aureus was inhibited by synthetic EC-hepcidins, and EC-hepcidin1 displayed stronger antimicrobial activity. The replication of Singapore grouper iridovirus (SGIV) was inhibited in the EC-hepcidin1 and EC-hepcidin2 over-expressed stable transfected fish cell lines (GS/pcDNA-Hep1, GS/pcDNA-Hep2) indicative of the antiviral activity of EC-hepcidins. These data should offer important information on the antimicrobial and antiviral roles of EC-hepcidins, and will be help to the better understanding of molecular mechanisms of grouper innate immunity.

Molecular cloning, characterization and expression analysis of a C-type lectin (Ec-CTL) in orange-spotted grouper, Epinephelus coioides.

C-type lectins play crucial roles in pathogen recognition, innate immunity, and cell-cell interactions. In this study, a new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of Ec-CTL was composed of 840 bp with a 651 bp open reading frame (ORF) that encodes a 216-residue protein. The deduced amino acid sequence of Ec-CTL possessed all conserved features crucial for the fundamental structure, such as the four cysteine residues (Cys(71), Cys(152), Cys(167), Cys(175)) involved in the formation of disulphide bridges and the potential Ca(2+)/carbohydrate-binding sites. Ec-CTL contains a signal peptide and a single carbohydrate recognition domain (CRD). The genomic DNA of the gene consists of three exons and two introns. Ec-CTL showed high similarity of 54% to the C-type lectin of killifish Fundulus heteroclitus. Ec-CTL mRNA is predominately expressed in liver and skin, and lower expressed in kidney, intestine, heart, brain and spleen. The expression of Ec-CTL was differentially up-regulated in orange-spotted grouper challenged with Saccharomyces cerevisiae, Vibrio vulnificus, Staphyloccocus aureus and Singapore grouper iridovirus (SGIV). Recombinant mature Ec-CTL (rEc-CTL) was expressed in E. coli BL21, purified and characterized as a typical Ca(2+)-dependent carbohydrate-binding protein possessing hemagglutinating activity. It bound to all examined bacterial and yeast strains, and aggregated with S. cerevisiae, V. vulnificus and S. aureus in a Ca(2+)-dependent manner.