Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis

Accepted Manuscript Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis Zhi Zhou, Xiaopeng Yu, Jia Tang, Yunjie Zhu, Guangmei Chen, Liping Guo, Bo Huang PII:

S0145-305X(16)30353-6

DOI:

10.1016/j.dci.2017.01.009

Reference:

DCI 2792

To appear in:

Developmental and Comparative Immunology

Received Date: 18 October 2016 Revised Date:

6 January 2017

Accepted Date: 6 January 2017

Please cite this article as: Zhou, Z., Yu, X., Tang, J., Zhu, Y., Chen, G., Guo, L., Huang, B., Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora damicornis, Developmental and Comparative Immunology (2017), doi: 10.1016/ j.dci.2017.01.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Dual recognition activity of a rhamnose-binding lectin to pathogenic bacteria and zooxanthellae in stony coral Pocillopora

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damicornis

Zhi Zhou a *, Xiaopeng Yu a, Jia Tang a, Yunjie Zhu a, Guangmei Chen a, Liping Guo b, , Bo Huang a

a*

Key Laboratory of Tropical Biological Resources of Ministry of Education, Hainan

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a

b

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University, Haikou 570228, China

Beijing Normal University, Beijing 100875, China

Abstract

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Rhamnose-binding lectin (RBL) is a type of Ca2+-independent lectin with tandem repeat carbohydrate-recognition domain, and is crucial for the innate immunity in many invertebrates. In this study, the cDNA sequence encoding RBL in coral

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Pocillopora damicornis (PdRBL-1) was cloned. The PdRBL-1 protein shared highest

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amino acid sequence similarity (55%) with the polyp of Hydra vulgaris, and contained a signal peptide and two tandem carbohydrate-recognition domains in which all cysteine residues were conserved. Surface plasmon resonance method revealed that the recombinant PdRBL-1 protein bound to LPS and Lipid A, but not to LTA, β-glucan, mannose and Poly (I:C). Results also showed that it bonded with zooxanthellae using western blotting method, and that the boound protein was detectable only at concentrations higher than 102 zooxanthellae cell mL-1. When

ACCEPTED MANUSCRIPT recombinant PdRBL-1 protein was preincubated with LPS, lower amounts of protein bound to zooxanthellae compared to cells not preincubated with LPS. Furthermore, PdRBL-1 mRNA expression increased significantly at 12 h, and declined to the

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baseline at 24 h after heat stress at 31 ℃. These results collectively suggest that PdRBL-1 could recognize not only pathogenic bacteria but also symbiotic zooxanthellae, and that the recognition of zooxanthellae by PdRBL-1 could be

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repressed by pathogenic bacteria through competitive binding. This information

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allows us to gain new insights in the mechanisms influencing the establishment and maintenance of coral-zooxanthella symbiosis in coral P. damicornis.

1. Introduction

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Keywords: rhamnose-binding lectin, PAMPs, zooxanthellae, coral, symbiosis

Stony corals form complex mutualistic symbiosis with unicellular photosynthetic

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dinoflagellates (zooxanthellae), allowing them to proliferate and reproduce even in

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oligotrophic conditions (Rosic et al., 2014). Symbiotic zooxanthellae are harbored in the stony coral's endodermal cells which supply their hosts with organic carbon and oxygen. The organic carbon and oxygen generated from photosynthesis are served as the main source energy of entire coral reef ecosystems (Kavousi et al., 2015; Shinzato et al., 2014). In return, the coral host supplies carbon dioxide and indispensable inorganic nutrition for the symbiotic zooxanthellae and the protection against other grazers or predators (Hoegh-Guldberg et al., 2007). However, despite the apparent

ACCEPTED MANUSCRIPT significance of this symbiosis not only to the host but also to the entire coral reef ecosystems, many mechanisms underlying its establishment and maintenance remain to be explored.

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It has been suggested that the first step in establishing the symbiosis is the chemical recognition of the host and symbiont commonly mediated by lectin and glycan molecules (Iguchi et al., 2011; Kita et al., 2015; Wood-Charlson et al., 2006). For

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example, the lectin in the octocoral Sinularia lochmodes (SLL-2) and coral Acropora

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millepora (Millectin) binds with D-galactose and mannose in zooxanthella surface, respectively (Jimbo et al., 2013; Kvennefors et al., 2008). Specifically, the lectin in the coral Ctenactis echinata has been shown to bind with lactose, melibiose, and D-galactose, which results to the transformation of zooxanthellae from a flagellated

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motile form to a nonmotile coccoid form (equivalent to the symbiotic stage), and even suppress its growth (Jimbo et al., 2010). In S. lochmodes SLL-2, the transformation of zooxanthellae after its binding was attenuated by the presence of glycosidases or

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N-acetyl-D-galactosamine (Jimbo et al., 2013). Furthermore, trehalose has also been

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shown to function as a chemical attractant in the establishment of symbiosis in the coral Fungia scutaria (Hagedorn et al., 2015). Therefore, the recognition of the zooxanthellae by host coral as mediated by lectin plays a significant role in the establishment and maintenance of symbiosis, and thus, is indispensable. Lectins are generally important as pattern recognition receptors (PRRs) in invertebrates, which allows them to discriminate non-self from self through the binding to pathogen-associated molecular patterns (PAMPs) including glucan,

ACCEPTED MANUSCRIPT mannose, LPS among others (Arason, 1996; Hanington et al., 2010; Marques and Barracco, 2000; Yang et al., 2015). These lectins are therefore involved in the invertebrates’ innate immune responses, and act as the only defense against invading

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microorganisms (Loker et al., 2004; Wang and Wang, 2013). In arthropods, lectins are thought to be involved in suppressing inflammations due to viral and bacterial infections by inducing the production of antibiotics to prevent their proliferation

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(Lecchini et al., 2014; Wang et al., 2014). In mollusks, lectins can serve as opsonin to

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mediate the phagocytosis and encapsulation of invading pathogenic microorganisms (Yang et al., 2011). It has also been shown in cnidarians that lectins have agglutination activity against various bacteria but can be inhibited by some saccharides and glycoproteins (Imamichi and Yokoyama, 2010; Kvennefors et al., 2008). Coral lectin,

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like Millectin and tachylectin, have also been observed to participate in the immunity, suggesting the potential of coral lectins recognizing not only the symbiotic zooxanthellae but also the other pathogenic microorganisms in the endosymbiont.

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This potential function of coral lectins however remains to be further understood.

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Pocillopora damicornis, a coral mainly distributed in the tropical/subtropical area of the Indian and Pacific Oceans, belongs to Pocilloporidae. In the wake of global warming, increased sea surface temperature has resulted to the expulsion of the symbiotic zooxanthellae and the bleaching of stony corals. Because coral lectins play significant roles in the recognition and stabilization of zooxanthellae, understanding of their function would pave a way to further understand the underlying mechanisms in the establishment and maintenance of coral-zooxanthella symbiosis, such as in

ACCEPTED MANUSCRIPT bleaching events. In this study, we cloned the RBL gene in the coral P. damicornis (here as PdRBL-1), and used to investigate the affinity of PdRBL-1 to PAMPs and zooxanthellae. Then, we determined the effect of PAMPs on the zooxanthella

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recognition of PdRBL-1 and changes in its expression after heat stress. Results of this study will contribute to the further understanding of possible mechanisms underlying

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coral bleaching due to heat stress and the potential onset of pathogenic infections.

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2. Materials and methods 2.1. Coral collection and heat stress treatment

Coral P. damicornis colonies were collected from the coral reef in Wenchang, Hainan Province, China, and transferred and cultured in flow-through aquaria (ca. 100

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L) filled with filtered seawater (26 ℃ ) at Hainan University. Cultures were illuminated with four fluorescent bulbs (Philips T5HO Activiva Active 54 W) in a 12 h/12 h light-dark cycle for one month to acclimatize in laboratory conditions.

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Thirty coral nubbins were prepared from the clones of the same P. damicornis isolate.

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Twelve coral nubbins were incubated at 32 ℃, which we referred hereafter as the heat stress group, while another twelve coral nubbins remained at 26 ℃ and used as the control group. Subsequently, six nubbins were sampled in each group at 12 and 24 h of incubation, while nubbins at 0 h were considered as the blank group (no treatment). Each sample was stored in liquid nitrogen immediately for RNA extraction.

ACCEPTED MANUSCRIPT 2.2. RNA extraction, cDNA synthesis and qRT-PCR analysis of PdRBL-1 mRNA expression Total RNA was extracted from coral nubbins using Trizol reagent (Invitrogen)

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following the manufacturer’s protocol. The cDNA library was then constructed using Promega M-MLV kit, and diluted to 1:40 for the subsequent experiments. The qRT-PCR and 2-∆∆Ct method were employed to determine and estimate the mRNA

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expression levels of PdRBL-1 in coral nubbins under different temperature conditions

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as described in Zhou et al (Zhou et al., 2012). All the primers used were listed in Table 1 and the fragment of elongation factor (PdEF) was used as the endogenous control. Three replicates were tested for each sample or nubbin.

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2.3. Gene amplification and sequence characterization

Transcriptome libraries of the stony coral P. damicornis after ammonium stress were previously constructed and sequenced, and transcript assembly yielded 77,199

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coral-derived transcripts (Yuan et al., 2016). Based on BLAST results, one transcript

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(No. TR53818|c0_g1_i1) was homologous to the RBLs identified previously in other animals.

The whole-length cDNA sequence of PdRBL-1 was then obtained via rapid amplification of cDNA ends (RACE) method (Wang et al., 2012). The Expert Protein Analysis System was applied in the amino acid analysis (http://www.expasy.org/). The protein

domain

was

predicted

with

SMART

software

(http://smart.embl-heidelberg.de/). Multiple sequence alignment of PdRBL-1 with the

ACCEPTED MANUSCRIPT RBLs from other species (downloaded from GenBank) was employed by the ClustalW software.

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2.4. Preparation of purified recombinant PdRBL-1 protein The cDNA fragment encoding the mature peptide of PdPBL-1 was cloned and inserted into pEASY-E1 expression vector (TransGen, China). After transformation

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and screening for positive inserts using PCR, plasmids with correct inserts were

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isolated and transformed into E. coli BL21 (DE3)-Transetta (TransGen, China). Then, the positive transformants were isolated and protein expression was induced by adding IPTG at the final concentration of 0.5 mmol L-1. Finally, recombinant PdRBL-1 protein (here as rPdRBL-1) was purified by a Ni2+ chelating Sepharose

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column (Sangon Biotech).

2.5. PAMP binding assay based on surface plasmon resonance

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The binding activity of rPdRBL-1 to PAMPs was tested with surface plasmon

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resonance (SPR) method following the previous study (Xu et al., 2016). Analysis of ligand binding kinetics was performed at 24 ℃ on a BIAcore T200 SPR instrument (GE Healthcare). His-tag antibody was first immobilized onto CM5 chip, then 1.0 mg mL-1 PAMPs was injected including LPS (lipopolysaccharide. E. coli 0111:B4), Lipid A,

LTA (Staphylococcus

aureus),

β-Glucan

(Euglena

gracilis),

Mannose

(Saccharomyces cerevisiae) and Poly (I:C). After 5 min of dissociation, rPdRBL-1 and bound analytes were washed with glycine-HCl. Data was analyzed with BIAcore

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2.6. Zooxanthella binding assay of rPdRBL-1

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The recognition of rPdRBL-1 to zooxanthellae was tested via western blotting. Briefly, live zooxanthellae were first isolated from P. damicornis as described in Shaw et al (Shaw et al., 2012), and diluted with filtered seawater to the final concentrations

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of 10, 102, 103, 104 and 105 cell mL-1, which were resuspended with filtered seawater

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to a final volume of 300 µL. Then, 300 µL of 0.2 mg mL-1 rPdRBL-1 were added in each tube containing zooxanthellae. Bovine serum albumin (BSA) was used as the negative control. After 1 h of incubation at room temperature (24 ℃), the cells were pelleted by centrifugation at 1,600× g for 6 min to eliminate unbound protein. The

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zooxanthellae were washed with PBST for 3 times and resuspended in 40 µL of TBS before extraction and detection using SDS-PAGE. The protein was then transferred onto nitrocellulose membrane and blocked overnight at 4 ℃. After which, the

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membrane was successively incubated with first- and second- antibodies, and the

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band was visualized with ECL substrate reagents (Perkin Elmer).

2.7. LPS blockage of rPdRBL-1 binding activity to zooxanthellae The blockage assay of rPdRBL-1 was carried out to explore whether LPS could influence the binding of rPdRBL-1 to zooxanthellae. In the LPS positive (LPS+) groups, 300 µL of 0.2 mg mL-1 rPdRBL-1 was incubated with LPS (0.2 mg mL-1) for 15 min at room temperature (24 ℃) with gentle shaking. This was followed by the

ACCEPTED MANUSCRIPT addition of 300 µL of 2 × 103 cell mL-1 zooxanthellae, the mixture was incubated for 1 h. In the LPS negative (LPS-) groups, zooxanthellae and rPdRBL-1 were incubated without the addition of LPS. After centrifugation at 1,600× g for 6 min, the harvested

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zooxanthellae were washed three times with PBST. The mixture was then spun down at 1,600× g for 6 min to eliminate the unbound protein. Success rate of the binding of rPdRBL-1 to zooxanthella cells was determined using western blotting technique as

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previously mentioned in Section 2.6. BSA was also used as the negative control, while

2.8. Statistical analysis

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PBS instead of PdRBL-1 was added in the blank group.

All values obtained were presented as means ± SD and significant differences were

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tested using one-way ANOVA and a Student-Newman-Keus (S-N-K) comparison.

0.05).

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3. Results

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Letters (a/b/c, etc.) were marked in figures to illustrate the statistical significance (p <

3.1. The molecular features of PdRBL-1 The whole-length sequence of PdRBL-1 cDNA was of 1065 bp (Accession No. KU882099 in GenBank), which contained 235 amino acids when translated with a molecular mass of 26.80 KDa and theoretical isoelectric point of 8.80. SignalP software analysis predicted a signal peptide of 19 amino acid residues at the N-terminus of PdRBL-1. In addition, PdRBL-1 contained two tandem galactose

ACCEPTED MANUSCRIPT binding lectin domains (Gal_Lectin domain, PF02140, from Ile38 to Cys121, from Leu143 to Cys227), which were its carbohydrate-recognition domain (CRD). The amino acid sequence of PdRBL-1 shared highest similarity of 55% with that

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from Hydra vulgaris (XP_002166914), and 51% for both oyster Crassostrea gigas (XP_011428526) and Halyomorpha halys (XP_014291623). It was less similar with Sinocyclocheilus

anshuiensis

(XP_016360799)

and

Astyanax

mexicanus

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(XP_007234690), with only 48% shared similarities. Furthermore, multiple sequence

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alignments of CRD in PdRBL-1 and RBLs from C. gigas and Oncorhynchus mykiss (BAA92257) revealed all seven conserved cysteine residues (Fig. 1).

3.2. The temporal mRNA expression of PdRBL-1 after heat stress

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The expression level of PdRBL-1 mRNA was upregulated after heat stress (Fig. 2). PdRBL-1 expression in the heat stress group increased dramatically at 12 h (2.16-fold, p < 0.05), and declined to the initial level at 24 h (1.50-fold, p > 0.05). Furthermore,

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no significant difference was observed between the blank and control groups.

3.3. Purification and PAMP binding activity of rPdRBL-1 After bacterial transformation and IPTG induction, the bacterial lysate was successfully separated and a distinct band of purified rPdRBL-1 was observed with a molecular mass of about 28 kDa (Fig. 3). This was adjusted to 0.5 mg mL-1 and used for the binding assay. Results of the SPR assay revealed that LPS and Lipid A displayed apparent

ACCEPTED MANUSCRIPT resonance signal and rapid association with rPdRBL-1. However, no resonance signals were observed for other PAMPs including LTA, β-glucan, mannose and Poly (I:C) (Fig. 4), which indicated that rPdRBL-1 exhibited high binding affinity solely

3.4. The binding activity of rPdRBL-1 to zooxanthellae

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with LPS and Lipid A.

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As shown in Fig. 5, the positive bands of rPdRBL-1 increased with cell

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concentration of zooxanthellae, except for 10 cell mL-1, revealing its lower limit of detection. In addition, there was no detected distinct positive signal in both negative control (BSA) and blank control (loading buffer). These results indicated that

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PdRBL-1 bond with zooxanthellae in a concentration-dependent manner.

3.5. The LPS blockage of rPdRBL-1’s binding activity to zooxanthellae Based on Fig. 6, the binding activity of rPdRBL-1 to zooxanthellae was suppressed

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significantly in the LPS+ groups for both live and dead zooxanthellae, indicating that

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LPS was capable of binding to rPdRBL-1 and antagonizing its recognition of the zooxanthellae. In addition, the recognition ability of dead zooxanthellae seemed to be weaker compared with the live cells.

4. Discussion Rhamnose-binding lectins (RBLs) belong to a group of lectins that bind specifically to rhamnose and galactose, which do not require Ca2+ for their binding activity. The

ACCEPTED MANUSCRIPT recognition to exogenous harmful pathogens and beneficial zooxanthellae is very important for stony corals since it is the first step in establishing and later maintaining the obligatory coral-zooxanthella symbiosis. Coral lectin has been reported to take

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part in the non-self recognition activities (Jimbo et al., 2010; Kvennefors et al., 2008; Wood-Charlson et al., 2006), but the detailed underlying mechanisms remain unclear. In this study, sequence similarity analysis revealed that the PdRBL-1 gene amplified

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from the stony coral P. damicornis shared high similarities with RBLs from other

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species. Further, PdRBL-1 contained one signal peptide and two tandem Gal_Lectin domains (carbohydrate-recognition domain), while all cysteine residues were also completely conserved in the two tandem Gal_Lectin domains of PdRBL-1, which were the typical features of RBL structure (Hosono et al., 1999). The presence of

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signal peptide in PdRBL-1 revealed that its potential functionality after being released from coral cells. These suggest that PdRBL-1 was a homologue of rhamnose-binding lectin in coral P. damicornis.

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RBL can serve as a significant PRR in the innate immune systems in other animals

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(Tateno et al., 2002). To understand whether PdRBL-1 could recognize the potential pathogenic microorganism, its binding ability to PAMPs was determined first through SPR method. Results revealed that the rPdRBL-1 could bind LPS and Lipid A, but not LTA, β-glucan, mannose nor Poly (I:C). Similar results were also observed in the RBL in Steelhead Trout Oncorhynchus mykiss except that it binds LTA and not Lipid A (Tateno et al., 2002). Interestingly, we had also demonstrated that PdRBL-1 could recognize gram-negative pathogenic bacteria through the recognition of PAMPs. This

ACCEPTED MANUSCRIPT has significant implications in the understanding of how corals recognize and inhibit infections of pathogenic bacteria like Vibrio coralliilyticus, which is a known pathogen of coral P. damicornis (Ben-Haim et al., 2003a; Ben-Haim et al., 2003b).

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The binding would activate the immune response against the invading pathogenic bacteria by agglutinating the bacteria through the PdRBL-1 and mediating phagocytosis (Cammarata et al., 2014; Franchi et al., 2011).

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As the other non-self organisms in corals, the concentration and state of symbiotic

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zooxanthellae are under the dynamic regulation of their hosts (Jones and Yellowlees, 1997). We determined the binding activity of rPdRBL-1 to zooxanthellae to understand its function in the establishment and maintenance of coral symbiosis. Our results demonstrated that the binding ability of rPdRBL-1 to zooxanthellae was

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concentration-dependent. Although there were similar reports that lectin was detected on the surface of symbiotic zooxanthellae in A. millepora and S. lochmodes (Jimbo et al., 2000; Kvennefors et al., 2008), it was the first attempt to quantify the binding of

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coral lectin to symbiotic zooxanthellae. The detectable rPdRBL-1 even under

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relatively low cell count of zooxanthellae revealed its sensitive and strong recognition activity. The recognition could have been mediated by the interactions between the Gal_Lectin domains in PdRBL-1 and the rhamnose/galactose on the surface of symbiotic zooxanthellae. These were consistent with previous reports that α-mannose/α-glucose and α-galactose residues on the zooxanthella surface served as the recognition ligands for the lectin in coral F. scutaria (Wood-Charlson et al., 2006), while rhamnose was identified in the mucus of zooxanthellae (Wild et al., 2010). The

ACCEPTED MANUSCRIPT recognition function of PdRBL-1 might be of similar physiological significance with the lectins in C. echinata and S. lochmodes, which could assist coral to arrest zooxanthellae and transform their state from motile to non-motile coccoid forms

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(Jimbo et al., 2013; Jimbo et al., 2010). The binding activity of PdRBL-1 to PAMPs and zooxanthellae collectively suggested that rhamnose-binding lectin PdRBL-1 had the power to not only distinguish the exogenous pathogenic bacteria, but also to

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recognize, arrest and stabilize zooxanthellae, which was of great importance for the

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establishment and maintenance of coral-zooxanthella symbiosis in stony coral P. damicornis.

Since both harmful pathogenic bacteria and beneficial zooxanthellae could be recognized by PdRBL-1, the more interesting question is how these two recognition

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activities interact especially when the coral is suffering from pathogenic bacterial infections. To solve this problem, the binding activity of rPdRBL-1 to zooxanthellae was determined with the preincubation of LPS, which resulted to significant reduction

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in the adherence of rPdRBL-1 to zooxanthellae. This indicated that both pathogenic

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bacteria and zooxanthellae could compete for PdRBL-1, and that the preferential binding object of PdRBL-1 might depend on the relative concentrations of the types of non-self organisms. When the concentration of pathogenic bacteria was much higher than that of the zooxanthellae, more PdRBL-1 bound pathogenic bacteria after the disassociation from zooxanthellae. Less bonded PdRBL-1 might not stabilize symbiotic zooxanthellae, resulting to potential bleaching and expulsion of symbionts. Such is the case of observed in the infection of the pathogen V. coralliilyticus that led

ACCEPTED MANUSCRIPT to reduced symbiotic zooxanthella concentration, even bleaching, and the significant expression change of lectin genes in coral P. damicornis (Ben-Haim et al., 2003b; Vidal-Dupiol et al., 2011). PdRBL-1 had weaker affinity to dead zooxanthellae

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compared with the living ones. This could be mainly due to the zooxanthella fixation and resulting protein crosslinks, which declined the polysaccharides exposed on the cell walls of zooxanthellae. Thus, less PdRBL-1 protein would bind to dead

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zooxanthellae compared with live zooxanthellae. The other potential reason could be

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the response of live zooxanthellae to LPS, but more studies are needed to support our hypothesis. These results demonstrated that the arresting and stabilization of the rhamnose-binding lectin PdRBL-1 on zooxanthellae could be repressed by the invading pathogenic bacteria through the competitive binding, and that PdRBL-1 is an

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important molecule in maintaining coral-zooxanthella symbiosis and inhibiting pathogens because of its dual recognition function. To further understand the potential effects of environmental factors on PdRBL-1

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function, changes in its mRNA expression was monitored at different times after

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incubation in elevated temperatures. It was significantly upregulated at 12 h, and decreased to the baseline at 24 h after heat stress, demonstrating that the expression of PdRBL-1 was induced during the early stage of heat stress. Such dramatic alterations of lectin expression after heat stress were also observed in coral A. millepora (Bellantuono et al., 2012; Rodriguez-Lanetty et al., 2009). Because the thermal-tolerant coral A. millepora was equipped with higher expression level of a mannose-binding lectin (Bellantuono et al., 2012), the upregulated expression of

ACCEPTED MANUSCRIPT PdRBL-1 could then be an adaptive response to heat stress. It has been suggested that more PdRBL-1 would stabilize symbiotic zooxanthellae through the strong recognition and repression of expulsion of zooxanthellae in the early stage of heat

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stress, which could potentially explain why P. damicornis is relatively resilient to heat stress. Results of the present research indicated that the recognition function of

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coral-zooxanthella symbiosis in coral P. damicornis.

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PdRBL-1 could be employed to resist environmental changes and maintain the

Acknowledgements

The authors were grateful to all the laboratory members for continuous technical advice and helpful discussion. This research was supported by the scientific research

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foundation (kyqd1554) and Midwestern construction projects (ZXBJH-XK006) of Hainan University, and grants from the Natural Science Foundation of Hainan

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ACCEPTED MANUSCRIPT Figure legend Fig. 1. Multiple sequences alignment of carbohydrate recognition domains of PdRBL-1 and RBLs from Crassostrea gigas (EKC40275) and Oncorhynchus mykiss

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(BAA92257) deposited in GenBank. The black shadow region indicated positions where all sequences share the same amino acid residue. Similar amino acids were

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indicated the conserved cysteine residue.

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shaded in grey. Gaps were indicated by dashes to improve the alignment. The asterisk

Fig. 2. Temporal expression of PdRBL-1 mRNA detected by real-time PCR after heat stress for 0, 12, and 24 h. PdEF gene was used as an internal control to calibrate the cDNA template for all the samples. Each value was shown as mean ± SD (N= 6), and

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bars with different letters were significantly different (p < 0.05). Six biological replicates were tested for each group, and three technical replicates for each sample or

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nubbin.

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Fig. 3. SDS-PAGE analysis of rPdRBL-1. Lane M: protein molecular standard (kDa). Lane 1: negative control for rPdRBL-1 (without induction). Lane 2: IPTG induced rPdRBL-1. Lane 3: purified rPdRBL-1.

Fig. 4. PAMP binding activity assay of rPdRBL-1 using surface plasmon resonance method. LPS and Lipid A displayed strong resonance signal and rapid association with rPdRBL-1, while LTA, β-glucan, mannose and Poly (I:C) exhibited no resonance

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Fig. 5. Binding activity of rPdRBL-1 to zooxanthellae of different concentrations.

buffer was used in the blank control group.

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BSA (bovine serum albumin) was employed as the negative control, while loading

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Fig. 6. The LPS blockage of rPdRBL-1’s binding activity to zooxanthellae. BSA

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(bovine serum albumin) was employed as the negative control, while loading buffer

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was used in the blank control group.

ACCEPTED MANUSCRIPT Table 1 Sequences of the primers used in the experiment. Sequence (5'-3')

Sequence information

P1 (forward)

ACAATGAGGATGTACTTAGTGGTTCTC

PdRBL-1 primer for 3RACE

P2 (forward)

TCAGTCACATCCACCCTTCTTC

PdRBL-1 primer for 3RACE

P3 (reverse)

GTCTCCGTATTCTGCGTTGTTG

PdRBL-1 primer for 5RACE

P4 (reverse)

GGAGCGGTCTGTTCTTCCATAG

PdRBL-1 primer for 5RACE

P5 (forward)

CGAAGAAGGGTGGATGTGACTG

Real-time PdRBL-1 primer

P6 (reverse)

GCTTCACGAAGTTACCTTACCCAC

Real-time PdRBL-1 primer

P7 (forward)

CGCTGGCAAAGTGACAAAGG

Real-time PdEF primer

P8 (reverse)

CAGACTTGCGATGAAATAGATAGGA

P9 (forward)

TGGCCTACACAAGAAGTAAGAAGC

P10 (reverse)

TCAGTCACATCCACCCTTCTTC

PdRBL-1 recombinant primer

M13-47

CGCCAGGGTTTTCCCAGTCACGAC

pMD19-T vector primer

RV-M

GAGCGGATAACAATTTCACACAGG

pMD19-T vector primer

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Primer

Real-time PdEF primer

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PdRBL-1 recombinant primer

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ACCEPTED MANUSCRIPT PdRBL-1 bound LPS and Lipid A, but not LTA, β-glucan, mannose and Poly (I:C). PdRBL-1 bound zooxanthellae in a concentration-dependent manner. The PdRBL-1’s binding to zooxanthellae was repressed by LPS.

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The expression of RdRBL-1 mRNA was upregulated by heat stress.