Download PDF - Mahidol University

sc.mahidol.ac.th

Download PDF - Mahidol University

Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

www.elsevier.com/locate/cbpb

Isolation and characterization of cDNA encoding Argonaute,

a component of RNA silencing in shrimp (Penaeus monodon) ☆

Sasimanas Unajak a,b , Vichai Boonsaeng a , Sarawut Jitrapakdee a,b, ⁎

a Center of Excellence for Shrimp Biotechnology, Mahidol University, Bangkok 10400, Thailand

b Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Rd, Bangkok 10400, Thailand

Received 12 February 2006; received in revised form 28 June 2006; accepted 6 July 2006

Available online 13 July 2006

Abstract

We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila

melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated

molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that

exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D.

melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate

Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference

(RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in

intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2–3-fold increased during the early period of viral infection

but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction

of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.

© 2006 Elsevier Inc. All rights reserved.

Keywords: Argonaute; Differential display RT-PCR; Penaeus monodon; RNA interference; Gene silencing; Yellow head virus; Immune system; RNA virus

1. Introduction

Shrimp, like other crustaceans, do not possess an adaptive

immune response, but rely primarily on the innate immune system

including the hemolymph coagulation system (Yeh et al., 1998,

1999), production of anti-bacterial peptides such as penaedins

(Destoumieux et al., 1997), anti-lipopolysaccharide factor

(Somboonwiwat et al., 2005), and the melanization reaction

through the production of prophenol oxidase cascade system

(Perazzolo and Barracco, 1997; Söderhäll, 1999; Sritunyalucksana

et al., 1999) to respond to bacterial or fungal infections.

These mechanisms, in turn, prevent the spread of these pathogens

to the body. However, there are few reports on the defense

molecules produced by shrimp upon infection by viruses. Luo

☆ The nucleotide sequence reported in this paper has been submitted to

GenBank with accession number DQ343133.

⁎ Corresponding author. Tel.: +66 2 201 5460; fax: +66 2 354 7174.

E-mail address: scsji@mahidol.ac.th (S. Jitrapakdee).

et al. (2003) identified a group of genes that are differentially

expressed in shrimp infected with the white spot syndrome virus

(WSSV). One of them is the PmAV, an antiviral gene. This gene

encodes a 170-amino acid polypeptide with a C-type lectin-like

domain whose function remains to be elucidated. Using a similar

approach, a group of housekeeping genes has been isolated from

WSSV-resistant shrimp, and an interferon-like protein with

antiviral activity has been identified from hemocytes (He et al.,

2005).

Here we employed the differential display reverse transcriptase-PCR

technique to identify a group of genes in the lymphoid

organs of Penaeus monodon shrimp that respond to RNA virus

infection. Of particular interest, we identified a cDNA homologue

of human/Drosophila melanogaster Argonaute proteins.

The Argonaute protein family is a group of proteins that are

conserved from fission yeasts to human. Argonaute protein 1

(Ago1) and several other members of this protein family play

crucial role in stem cell differentiation or RNA interference

(RNAi). Members of this family consist of proteins with an N-

1096-4959/$ - see front matter © 2006 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2006.07.002


180 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

terminal PAZ and C-terminal PIWI domain. In D. melanogaster

and C .elegans, Ago1 and Ago2 proteins appear to play a key

role in double stranded RNA-induced post-transcriptional gene

silencing (Carmell et al., 2002). Here we also present evidence

that the expression of shrimp putative Argonaute is highly

expressed in lymphoid organ, and that the expression of this

gene is correlated with viral infection.

2. Materials and methods

2.1. Experimental animals and sample preparation

Shrimp (P. monodon)(20–25 g) from local farms in Thailand

were used as a source throughout the experiments. Each white

spot syndrome virus (WSSV)–or yellow head virus (YHV)–free

shrimp was examined by PCR using IQ2000 WSSV Detection

and Prevention System and IQ2000 YHV/GAV Detection and

Typing System (Farming IntelliGene Technology Corporation,

Taiwan). The virus-free shrimp were acclimatized in brackish

water for one day before being injected intramuscularly with

100 μL of 1:100 dilution of YHV suspension in LHM medium

(1.8 M NaCl, 61 mM CaCl 2 , 5 mM KCl, 15 mM MgCl 2 ,0.3mM

Na 2 H 2 PO 4 ).

For the bacterial challenge, a single colony of Vibrio harveyi

(114GL) grown on tryptic soy agar (supplemented with 1.5%

NaCl) (Difco) was inoculated in Müller–Hinton broth (Difco)

and incubated overnight at 30 °C with shaking. The next day, the

bacteria were washed 3 times with sterile phosphate buffer and

diluted to an optical density at 600 nm (OD 600 ) of 0.5. 100 μLof

bacterial suspension were injected intramuscularly into the live

shrimp and their lymphoid organs were collected at 6, 10 and

11 h post infection. The presence of V. harveyi in the infected

lymphoid organs after infection was examined by smearing the

lymphoid organ to the TCBS (thiosulphate, citrate, bile salts,

sucrose agar) agar (Difco). Plates were incubated for 18–24 h at

30 °C to examine the blue or blue-green colonies of V. harveyi.

2.2. RNA isolation

Pooled lymphoid organs from shrimp infected with YHV for

6, 12, 18, 24, 30, 48, 60 h or those infected with V. harveyi for 6,

10, 11 h and from the moribund shrimp were homogenized in

TRI reagent (Pacific science) and snap-frozen in liquid

nitrogen. Chloroform (0.2 vol) was added to the frozen samples.

The RNA was precipitated from an aqueous phase by adding

0.5 vol of chilled isopropanol, before being washed with 75%

(v/v) ethanol, dried and resuspended in 20 μLdiethylpyrocarbonate

(DEPC)-treated water. The RNA concentrations were

determined by spectrophotometry, and the integrity was verified

by 1% formaldehyde agarose gel electrophoresis. Contaminating

DNA in RNA samples was eliminated by digestion with the 10 U/

μL DNaseI (Amersham Bioscience) at 37 °C for 1 h.

2.3. Differential display RT-PCR (ddRT-PCR)

The Delta® Differential Display kit (BD Bioscience) was

used to identify the differentially expressed RNA transcripts.

Briefly, first stranded cDNAs were synthesized with Moloney

murine leukemia virus reverse transcriptase (MMLV) from 2 μg

of total RNA extracted from mock- or YHV-infected shrimp

using random hexamer primers. The PCR was performed in a

20-μL final volume containing 1 μM of each forward and

reverse primer, 50 μM of each dNTP, and 50 nM [α- 32 P]dATP

(specific activity of 3000 Ci/mmol; Amersham Bioscience) with

the supplied buffer. The thermal cycle consisted of an initial

cycle of 94 °C for 5 min, 40 °C for 5 min, 68 °C for 5 min,

followed by 5 cycles of 94 °C for 2 min, 40 °C for 5 min, 68 °C

for 5 min; 25 cycles of 94 °C for 1 min, 60 °C for 1 min, 68 °C

for 2 min; and 68 °C for 7 min. The reactions were terminated

by mixing with a denaturing loading dye (95% formamide/0.2%

bromophenol blue/0.2% xylene cyanol), and heated at 95 °C for

10 min and chilled. The samples were then subjected to 6%

(w/v) polyacrylamide −6 M urea gel electrophoresis for 6 h at

70 W. The gel was transferred to Whatman paper, dried and

autoradiographed for 48 h.

2.4. Identification of differential display products

To recover the differentially expressed cDNA bands, the

autoradiogram was aligned with the dried gel and the marked

fragments were excised and eluted in 50 μL sterile water before

heating at 100 °C for 5 min. The eluted DNA was re-amplified

in a 50 μL reaction mixture using the same set of primer used in

the ddRT-PCR. The reaction was performed with 8 μL of eluted

DNA, 1× PCR buffer (Advantage cDNA polymerase mix, BD

Bioscience), 50 μM of each dNTP, 1 μM each P and T primers

(supplied in the kit) and 1 unit of Advantage polymerase mix.

Cycling parameters were 94 °C, 2 min, 25 cycles of 94 °C,

1 min, 60 °C, 1 min, 68 °C, 2 min and 68 °C, 7 min. The PCR

products were purified using a QiaQuick gel extraction kit

(Qiagen) before cloning into pGEM®-T Easy vector (Promega).

The nucleotide sequences were determined in both directions by

automated sequencing. The nucleotide sequences were analyzed

against the GenBank database using the Basic Local

Alignment Search Tool (BLAST X) (Thompson et al., 1997).

2.5. RACE PCR

Rapid amplification of cDNA ends (RACE) was performed

using SMART RACE cDNA amplification Kit, (BD Bioscience).

The sequences of specific primer were designed for the 5′

or 3′ ends of cDNAs (Table 1). The PCR profile consisted of

5 cycles of 94 °C for 30 s, 72 °C for 1 min and 5 cycles of 94 °C for

30 s, 70 °C for 30 s, 72 °C for 1 min, followed by 25 cycles of

94 °C for 30 s, 68 °C for 30 s, 72 °C for 1 min and 72 °C for 7 min.

All RACE PCR products were cloned into pGEM® T-Easy vector

(Promega) and subjected to automated sequencing in both

directions.

2.6. Semi-quantitative reverse transcriptase polymerase chain

reaction

The expression of shrimp Argonaute (Ago1) and β-actin

genes in lymphoid organs was compared between the mock-


S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

181

Table 1

Primers used for the PCR

Primer

Sequence

162_3_S1

5′-GGAGCTGCTCATCCAGTTCTACAA-3′

162_5_AS1 5′-ACAAGAATGGAGTTGATGCCTCCC-3′

162_5_AS2 5′-AATTATAGTGAAGACAGTAACCTTTGTGCA-3′

162_5_AS3 5′-GGTATGTGTGTTTGTGTTCTTGTCCCACCT-3′

162_5_AS6 5′-CCTTGCGTGGACGTCTGTCAGGGGTAATAC-3′

162_R

5′-ATAGATCCTACTACGGCTGCA-3′

162_F

5′-AGGGGGAAACAGTTCTTCACA-3′

actinF

5′-TGACGGCCAGGTGATCACCA-3′

actinR

5′-GAAGCACTTCCTGTGAACGA-3′

and the YHV-infected shrimps using semi-quantitative RT-PCR

with specific primer set of 162F and 162R for Argonaute gene

or with primers actinF and actinR for β-actin gene (Table 1).

The RNAs were subjected to a one-step RT-PCR using

SuperScript III reverse transcriptase (Invitrogen) with

100 ng of RNA. The PCR amplified in a 25 μL-reaction

volume containing 1× PCR buffer, 1.6 mM MgSO 4 , 200 μM of

each dNTP, 0.2 μM of each forward and reverse primers and

1 μL of SuperScript III RT/Platinum® Taq Mix. Reverse

transcription was carried out at 50 °C for 30 min followed by

94 °C for 2 min. The cycling parameters were 25 cycles of 94 °C

for 30 s, 55 °C for 30 s, 72 °C for 30 s and 72 °C for 7 min. PCR

products were separated on 1.4% NuSieve 3:1 agarose (FMC

Bioproduct).

from this technique. One of the differentially expressed bands

(PCR1) when sequenced showed that it encoded a protein with

71.4% and 76.9% respective similarity to the Argonaute protein

from human (GenBank accession no. Q9UL78) and D.

melanogaster (GenBank accession no. BAA88078.1). This

band was down-regulated upon YHV-infection.

3.2. Cloning of putative Pm Ago1 cDNA

To further characterize this cDNA, we performed 5′- and 3′-

RACE PCR to obtain its 5′- and 3′-ends using cDNAs prepared

from YHV-infected shrimp. Upon amplification 502 bp (named

162_5_AS6), 762 bp (162_5_AS3) and 740 bp (162_5_AS1)

2.7. Phylogenetic tree construction

ClustalX (Thompson et al., 1997) was used to align amino

acid sequences and generate bootstrapping trees. GenBank

accession numbers are: Dme, D. melanogaster; DmeAgo1

(BAA88078), DmeAgo2 (Q9VUQ5), Gga, Gallus gallus;

GgaAgo2 (XP_418421), GgaAgo3 (NP_001026071),

GgaAgo4 (XP_417776), XlaAgo, Xenopus laevis

(AAH77863), Homo sapiens; HsaAgo1 (Q9UL78), HsaAgo2

(Q9UKV8), HsaAgo3 (Q9H9G7), HsaAgo4 (NP_060099)

AgaAgo, Anopheles gambiae; (Eaa00062), Dre, Danio rerio;

DreAgo1 (XP_699226), DreAgo3 (XP_696563), DreAgo4

(XP_691861), Ath, Arabidopsis thaliana, AthAgo1

(NP_175274), AthAgo4 (NP_565633), Mmu, Mus musculus;

MmuAgo1 (Q8CJG1), MmuAgo2 (Q8UKV8), MmuAgo3

(Q8CJF9), MmuAgo4 (Q8CJF8), Cel, Caenorhabditis elegans;

CelAlg1 (NP_510322.2), CelAlg2 (NP_871992.1).

3. Results

3.1. Differential display of normal and infected shrimp gene

expression

We employed differential display PCR from the cDNAs

prepared from normal and infected shrimp with 90 pairs of

arbitrary primers supplied with the differential display PCR kit.

Approximately, 161 bands showed differential expression

between the normal and the YHV-infected shrimp. Fig. 1

shows an example of the differentially displayed bands obtained

Fig. 1. Representative ddRT-PCR generated from PCR with different sets of

primers shown in Table 1. Total RNAs were isolated from the lymphoid organs

of mock- or YHV-infected shrimp collected at 6 h post infection. Arrows

indicate differentially expressed transcripts. M1 is a 100 bp DNA marker; C,

mock-infected shrimp; V, YHV-infected shrimp. T and P indicate the name of

arbitrary and anchor primers used in each reaction.


182 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

PCR products were obtained from 5′-RACE while a 776 bp

PCR product (163_3_S1) was obtained from 3′-RACE. These 4

RACE clones together with PCR1 clone were overlapped and

spanned the entire coding region of the putative cDNA. The full

length of the cDNA comprised 3178 nucleotides including the

5′-untranslated region, coding sequence, 3′-untranslated region

and poly(A) tail (Fig. 2). The longest open reading frame

commenced at nucleotide 115 and ended at nucleotide 2931,

encoding a putative 939-amino acid polypeptide with calculated

molecular weight (Mr) of 104 kDa and pI of 9.49. Using a

potential initiation codon at position 115, 62 amino acid

residues at the N-terminus showed relatively low homology to

argonaute proteins in the database, while a second potential

initiation codon located at nucleotide 309 gave much higher

similarity. Therefore, it is unclear whether the authentic

initiation codon is located at nucleotide position 115 or 309.

The amino acid sequence inferred from nucleotide position 309

to the stop codon showed 73.8% identity with D. melanogaster

Fig. 2. Nucleotide and deduced amino acid sequences of shrimp Ago1 cDNA. (GenBank accession no. DQ343133). A, schematic diagram to show the strategy for isolating

the full length shrimp Ago1 cDNA. PCR1 fragment was initially isolated by the differential display PCR while the other four overlapping fragments, 162_5_AS6,

162_5_AS3, 162_5_AS1 and 162_3_S1, spanning an entire coding region of Pm Ago1 cDNAwere isolated by 5′-and3′-RACE PCR. Each fragment was named after the

primers used in the PCR amplification. B, nucleotide and inferred amino acid sequences of Pm Ago1. Nucleotide sequences are numbered while the amino acid number is in

bold type. Amino acids are shown in a single capital letter.


S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

183

Ago1 protein (GenBank accession no. BAA88078.1), and

68.4% with human translation initiation factor 2C1 (eIF2C1),

also called Argonaute-1 (GenBank accession no. Q9UL78). The

highest homologies were observed between residues 280–401

and 569–897, that correspond to the PAZ and the PIWI domains

of the mammalian and Drosophila proteins, as shown in Fig. 3.

Fig. 3. Alignment of the amino acid sequences of shrimp Ago1 and other species' Ago. The amino acid sequence of Pm Ago1 was aligned with sequences as follows: Aga,

A. gambiae (GenBank accession no. EAA00062), Dme, D. melanogaster (GenBank accession no. BAA88078), Hsa, Human; (GenBank accession no. Q9UL78), Mmu, M.

musculus (GenBank accession no. Q8CJG1), Xla, X. laevis (GenBank accession no. AAH77863), using ClustalW. Gaps were introduced to maximize the alignment. The

highly conserved residues are shown as white text against shaded background. PAZ and PIWI domains are shown by open boxes.


184 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

Interestingly, P. monodon Argonaute contains an extra 27

amino acid (NGSTTQGQSASDGSRPRQLTFARTAHD) in the

PIWI domain. This insertion is not present in Argonaute

proteins of other species.

3.3. Evolution of Argonaute protein of P. monodon

Phylogenetic analysis was performed to characterize the

evolutionary relationships of the Pm Ago and other invertebrate

and invertebrate Argonaute protein subtypes using the CLUS-

TAL W and PHYLIP programs. As shown in Fig. 4, three

clusters in the phylogenetic tree were identified which included

group 1 (X. laevis Ago1, D. rerio Ago1, Pm Ago1, D.

melanogaster Ago1 and 2, A. gambiae Ago, C. elegans Ago1

and 2, A. thaliana Ago1 and 4), group 2 (G. gallus Ago2, M.

musculus Ago2 and H. sapiens Ago2) and group 3 (G. gallus

Ago3 and 4, M. musculus Ago 1, 3, D. rerio Ago3 and 4, H.

sapiens Ago 1, 3 and 4). As Pm Ago1 appears to be clustered into

group 1 and is more closely related to the insect rather than

vertebrate Argonautes, we therefore suggest that Pm Ago belongs

to Ago1 subfamily.

3.4. Expression of Pm Ago1 in various tissues

To determine whether Pm Ago1 mRNA was expressed in a

tissue-specific manner, we performed a semi-quantitative RT-

PCR of mRNA in various tissues including gill, heart,

hemocytes, hepatopancreas, intestine, pleopod, lymphoid

organ and muscle of normal shrimp. As shown in Fig. 5A, the

expression of Pm Ago1 mRNA showed tissue-specific expression.

It is highly expressed in lymphoid organ, expressed at

a low level in hemocytes, intestine, pleopod and muscle, but

was barely detectable in gill, heart and hepatopancreas.

3.5. Expression of Pm Ago1 in YHV infected lymphoid organ

We next examined whether the expression of Pm Ago1 is

affected by an RNA virus. We used semi-quantitative RT-PCR

to detect the expression level of Pm Ago1 mRNA in lymphoid

organ of shrimp infected with YHV at various time points. As

shown in Fig. 5B and C, the expression level of Pm Ago1 was

increased to 3-fold at 24 h post infection. However, the level of

Pm Ago1 mRNA rapidly declined at 30 h and onward,

becoming undetectable in the moribund shrimp (60 h). In

contrast the expression of Pm Ago1 in mock-infected shrimp

was unchanged at various time points. Infection of shrimp with

the white spot syndrome virus (WSSV) yielded similar results

i.e. the level of Pm Ago1 mRNA was decreased at the late stage

of infection, becoming undetectable in moribund shrimp (data

not shown).

We next investigated whether the alteration in the level of Pm

Ago1 mRNA expression is specific to the viral infection. We

Fig. 4. Phylogenetic analysis of Argonaute proteins. Multiple alignment was constructed by ClustalX (version 1.83) (Thompson et al., 1997). Treeview was used in tree

construction. GenBank accession numbers are: Dme, D. melanogaster; DmeAgo1 (GenBank accession no. BAA88078), DmeAgo2 (GenBank accession no.

Q9VUQ5), Gga, G. gallus; GgaAgo2 (GenBank accession no. XP_418421), GgaAgo3 (GenBank accession no. NP_001026071), GgaAgo4 (GenBank accession no.

XP_417776), XlaAgo, X. laevis (GenBank accession no. AAH77863), Has, H. sapiens; HsaAgo1 (GenBank accession no. Q9UL78), HsaAgo2 (GenBank accession

no. Q9UKV8), HsaAgo3 (GenBank accession no. Q9H9G7), HsaAgo4 (GenBank accession no. NP_060099) AgaAgo, A. gambiae; (GenBank accession no.

Eaa00062), Dre, Danio rerio; DreAgo1 (GenBank accession no. XP_699226), DreAgo3 (GenBank accession no. XP_696563), DreAgo4 (GenBank accession no.

XP_691861), Ath, A. thaliana, AthAgo1 (GenBank accession no. NP_175274), AthAgo4 (GenBank accession no. NP_565633), Mmu, M. musculus; MmuAgo1

(GenBank accession no. Q8CJG1), MmuAgo2 (GenBank accession no. Q8UKV8), MmuAgo3 (GenBank accession no. Q8CJF9), MmuAgo4 (GenBank accession

no. Q8CJF8), Cel, C. elegans; CelAlg1 (GenBank accession no. NP_510322.2), CelAlg2 GenBank accession no. (NP_871992.1).


S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

185

4. Discussion

Fig. 5. Expression of Pm Ago1 in different tissues of shrimp. (A) Total RNA

from various tissues of normal shrimp were subjected to semi-quantitative RT-

PCR. These samples were subjected to RT-PCR using specific primers for Pm

Ago1 or β-actin genes. 1, gill; 2, heart; 3, hemocytes; 4, hepatopancreas; 5,

intestine; 6, pleopod; 7, lymphoid organ; 8, muscle; 9, negative control. (B)

Time course expression of Pm Ago1 mRNA at 0, 6, 12, 18, 24, 30, 48 and 60 h

(moribund) upon infection by YHV (upper panel) or mock-infected (lower

panel). (C) The abundance of Pm Ago1 mRNA of YHV-infected shrimp at

above time points is normalized with β-actin and is shown as relative gene

expression+standard deviations of three independent experiments. The relative

gene expression detected at 0 h was arbitrarily set as 1. In each experiment 3

shrimp were infected at each time point and the lympoid organs were combined

for RNA extraction. (D) Expression of Pm Ago1 in lymphoid organs of shrimp

infected with V. harveyi. Normal shrimp were infected with 0.5 OD of V. harveyi

and the lymphoid organs were collected at 6 and 19 h post infection. Total RNA

samples were extracted from lymphoid organs and subjected to RT-PCR using

specific primers for Pm Ago1 or β-actin genes.

infected shrimp with bacterial pathogen, V. harveyi, and

followed the expression of Pm Ago1 mRNA at different time

points. As shown in Fig. 5D, bacterial infection did not cause

the reduction of Pm Ago1 mRNA seen throughout the infection

as in the case of viral infection. These data suggest that the rapid

fall of Pm Ago1 mRNA towards the late phase of infection is

specific to the virus but not the bacteria.

It is known that crustaceans, including shrimp, lack the humoral

immune response against invading pathogen(s), but contain the

innate immune system which is an ancient defense mechanism.

This includes phagocytic activity, encapsulation and the release of

the prophenoloxidase system, production of antibacterial peptides

and a protease inhibitor. All of these mechanisms are mediated by

circulating hemocytes in the hemolymph. Recent studies showed

that the lymphoid organ is a major organ which contains

exocytosed, granular cells that have phagocytosed foreign

materials, particularly viruses, suggesting that the lymphoid

organ constitutes a major site for penaeid antiviral defense (Hasson

et al., 1999; Anggraeni and Owens, 2000). Using subtraction

hybridization and differential hybridization, He et al. (2005) were

able to isolate a group of housekeeping genes including cofilin,

translational control tumor protein (TCTP), hepatic lectin and

interferon-like protein that are up-regulated in the WSSV resistant

shrimp. These gene products were suggested to play important roles

in the antiviral process. However, the biological activities of these

gene products with respect to an antivirus mechanism in shrimp

remain unknown. Luo et al. (2003) isolated an antiviral gene using

differential display PCR, similar to our approach. They isolated

PmAV as an important defense molecule with antiviral activity.

Taken together, it appears that shrimp utilize an innate immune

system via the network of various defense molecules to protect

itself from viral infection. We have undertaken a differential display

approach to identify a cDNA encoding the Argonaute protein from

P. monodon shrimp as one of various genes that are differentially

expressed during viral infection.

The Argonaute protein family is composed of highly conserved

proteins whose functions are implicated in the mechanism of RNA

interference, and the development and maintenance of stem cell

fate determination. Here, we report the cloning and characterization

of cDNA encoding a putative Argonaute protein from a

crustacean for the first time. The inferred amino acid sequence of

thefulllengthPmAgo1cDNAshowedhighsequencesimilarities

to Ago1/eukaryotic initiation factor 2C1 (eIF2C1), a member of

the Argonaute protein family, and provided the first evidence that

the cDNA clone we isolated is indeed a member of this protein

family. Secondly, the putative Pm Ago1 possesses a molecular

weight and pI similar to the eIF2C1 of higher eukaryote species,

i.e. they form a conserved family of ∼100 kDa highly basic

proteins, characterized by an N-terminal PAZ domain and a C-

terminal PIWI domain (Carmell et al., 2002). Structural

determinations by nuclear magnetic resonance and X-ray

crystallography demonstrate that the PAZ domain of Ago1 and

Ago2 of D. melanogaster contains a nucleic acid binding domain

while the PIWI domain is less well characterized. Recently, the

structure of the PIWI domain of the archaebacterium, Pyrococcus

furiosis, has been shown to adopt an RNase H fold, similar to the

RNase H from E. coli (Lingel and Izaurralde, 2004). In fission

yeasts, only one isoform of Ago has been identified and this

protein plays a crucial role in meiotic gene silencing (Volpe et al.,

2002). However, in multicellular organisms, this gene family has

increased up to 24 members as in the case of C. elegans (Carmell

et al., 2002). In D. melanogaster, Ago1 and Ago2 are required for


186 S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

an efficient RNAi system (Williams and Rubin, 2002; Pal-Bhadra

et al., 2002). There is only one member of the Argonaute family,

Ago1 present in the protozoan parasite, Trypanosoma brucei and

this protein is indeed essential for RNAi (Shi et al., 2004).

However, in A. gambiae, five members of Argonaute proteins

have been identified but only Ago2 and Ago3 are involved in

RNAi (Hoa et al., 2003). In human, up to 8 Argonaute proteins

have been reported. Although the function of each isoform of

human Agonaute protein is not yet clear, some family members

have been shown to be associated with the DICER protein of the

RNAi system (Sasaki et al., 2003; Liu et al., 2004).

As the lymphoid organ is one of the important antiviral tissues,

our finding that Pm Ago1 mRNA is highly expressed in this tissue

suggests that Pm Ago1 may play a defense role against viral

infection. An RNAi-like mechanism against viral infection in two

species of shrimp i.e. Liptopenaeus vannamei and P. monodon

has recently been reported (Robalino et al., 2004; Tirasophon

et al., 2005; Yodmuang et al., 2006). More recently, sequencespecific

antiviral protection or silencing of an endogenous shrimp

gene can be achieved after administration of the cognate double

stranded RNAs, suggesting that an RNAi-like mechanism exists

in shrimp (Robalino et al., 2005). In this present study, an

increased level of Pm Ago1 mRNA at 24 h post infection suggests

that an RNAi mechanism may operate efficiently during the early

stages of infection. However, down-regulation of Pm Ago1

mRNA during the late or towards the final stage (moribund) of

infection may reflect the failure of an RNA silencing system of the

host to inhibit viral replication. Robalino et al. (2004) have shown

that the efficiency of an RNA silencing system to suppress viral

infection in the shrimp L. vannamei can be overwhelmed by viral

load. The production of silencing suppressors from viruses via

transcriptional repression of key components and/or effector

molecules of RNA silencing during the late stage of infection

(Lecellier and Voinnet, 2004) may contribute to this observation.

Our report on the cloning of a cDNA encoding the putative Pm

Ago1 and determination of its expression during viral infection

provides supporting evidence to suggest that the RNA silencing

does occur in shrimp. Whether our reported Pm Ago1 is the only

member of this family that plays a crucial role in RNA interference

in shrimp, remains to be elucidated by a knock-down experiment.

Acknowledgement

The authors thank Mr. K. Manopwisedjaroen for technical

assistance with V. harveyi detection. We thank Professors T.W.

Flegel and J.C. Wallace, for critically reading the manuscript.

This work was partly supported by Mahidol University Grant.

S.U. was supported by the Royal Golden Jubilee Ph.D. program

(Grant No.PHD/0098/2546) from the Thailand Research Fund.

References

Anggraeni, M., Owens, L., 2000. The haemocytic origin of lymphoid organ spheroid

cells in the penaeid prawn Penaeus monodon.Dis.Aquat.Org.40,85–92.

Carmell, M.A., Xuan, Z., Zhang, M.Q., Hannon, G.J., 2002. The Argonaute

family: tentacles that reach into RNAi, developmental control, stem cell

maintenance, and tumorigenesis. Genes Dev. 16, 2733–2742.

Destoumieux, D., Bulet, P., Loew, D., Van Dorsselaer, A., Rodriguez, J.,

Bachere, E., 1997. Penaeidins, a new family of antimicrobial peptides

isolated from the shrimp Penaeus vannamei (Decapoda). J. Biol. Chem.

272, 28398–28406.

Hasson, K.W., Lightner, D.V., Mohney, L.L., Redman, R.M., White, B.M.,

1999. Role of lymphoid organ spheroids in chronic Taura syndrome virus

(TSV) infections in Penaeus vannamei. Dis. Aquat. Org. 38, 93–105.

He, N., Qin, Q., Xu, X., 2005. Differential profile of genes expressed in

hemocytes of White Spot Syndrome Virus-resistant shrimp (Penaeus

japonicus) by combining suppression subtractive hybridization and

differential hybridization. Antivir. Res. 66, 39–45.

Hoa, N.T., Keene, K.M., Olson, K.E., Zheng, L., 2003. Characterization of RNA

interference in an Anopheles gambiae cell line. Insect Biochem. Mol. Biol.

33, 949–957.

Lecellier, C., Voinnet, O., 2004. RNA silencing: no mercy for viruses? Immunol.

Rev. 198, 285–303.

Lingel, A., Izaurralde, E., 2004. RNAi: finding the elusive endonuclease. RNA

10, 1675–1679.

Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.-J.,

Hammond, S.M., Joshua-Tor, L., Hannon, G.J., 2004. Argonute 2 is the

catalytic engine of mammalian RNAi. Science 305, 1436–1441.

Luo, T., Zhang, X., Shao, Z., Xu, X., 2003. PmAV, a novel gene involved in

virus resistance of shrimp Penaeus monodon. FEBS Lett. 551, 53–57.

Pal-Bhadra, M., Bhadra, U., Birchler, J.A., 2002. RNAi related mechanisms

affect both transcriptional and posttranscriptional transgene silencing in

Drosophila. Mol. Cell 9, 315–327.

Perazzolo, L.M., Barracco, M.A., 1997. The prophenoloxidase activating

system of the shrimp Penaeus paulensis and associated factors. Dev. Comp.

Immunol. 21, 385–395.

Robalino, J., Browdy, C.L., Prior, S., Metz, A., Parnell, P., Gross, P., Warr, G.,

2004. Induction of antiviral immunity by double-stranded RNA in a marine

invertebrate. J. Virol. 78, 10442–10448.

Robalino, J., Bartlett, T., Shepard, E., Prior, S., Jaramillo, G., Scura, E.,

Chapman, R.W., Gross, P.S., Browdy, C.L., Warr, G.W., 2005. Doublestranded

RNA induces sequence-specific antiviral silencing in addition to

nonspecific immunity in a marine shrimp: convergence of RNA interference

and innate immunity in the invertebrate antiviral response? J. Virol. 79,

13561–13571.

Sasaki, T., Shiohama, A., Minoshima, S., Shimizu, N., 2003. Identification of

eight members of the Argonaute family in the human genome. Genomics 82,

323–330.

Shi, H., Djikeng, A., Tschudi, C., Ullu, E., 2004. Argonaute protein in the early

divergent eukaryote Trypanosoma brucei: control of small interfering RNA

accumulation and retroposon transcript abundance. Mol. Cell. Biol. 24,

420–427.

Söderhäll, K., 1999. Invertebrate immunity. Dev. Comp. Immunol. 3, 263–266.

Somboonwiwat, K., Marcos, M., Tassanakajon, A., Klinbunga, S., Aumelas, A.,

Romestand, B., Gueguen, Y., Boze, H., Moulin, G., Bachere, E., 2005.

Recombinant expression and anti-microbial activity of anti-lipopolysaccharide

factor (ALF) from the black tiger shrimp Penaeus monodon. Dev.

Comp. Immunol. 29, 841–851.

Sritunyalucksana, K., Cerenius, L., Söderhäll, K., 1999. Molecular cloning and

characterization of prophenoloxidase in the black tiger shrimp, Penaeus

monodon. Dev. Comp. Immunol. 23, 179–186.

Thompson, J., Gibson, T., Plewniak, F., Jeanmougin, F., Higgins, D., 1997. The

CLUSTAL_X windows interface: flexible strategies for multiple sequence

alignment aided by quality analysis tools. Nucleic Acids Res. 25,

4876–4882.

Tirasophon, W., Roshorm, Y., Panyim, S., 2005. Silencing of yellow head virus

replication in penaeid shrimp cells by dsRNA. Biochem. Biophys. Res.

Commun. 334, 102–107.

Volpe, T.A., Kidner, C., Hall, I.M., Teng, G., Grewal, S.I., Martienssen, R.A.,

2002. Regulation of heterochromatic silencing and histone H3 lysine-9

methylation by RNAi. Science 297, 1833–1837.

Williams, R.W., Rubin, G.M., 2002. ARGONAUTE1 is required for efficient

RNA interference in Drosophila embryos. Proc. Natl. Acad. Sci. U. S. A. 99,

6889–6894.


S. Unajak et al. / Comparative Biochemistry and Physiology, Part B 145 (2006) 179–187

187

Yeh, M., Chen, Y.L., Tsai, I.-H., 1998. The hemolymph clottable proteins of

tiger shrimp, Penaeus monodon, and related species. Comp. Biochem.

Physiol., B 121, 169–176.

Yeh, M., Huang, C., Leu, J., Lee, Y.C., Tsai, I.-H., 1999. Molecular cloning and

characterization of a hemolymph clottable protein from tiger shrimp

(Penaeus monodon). Eur. J. Biochem. 266, 624–633.

Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W., Panyim, S.,

2006. YHV-protease dsRNA inhibits YHV replication in Penaeus

monodon and prevents mortality. Biochem. Biophys. Res. Commun.

341, 351–356.

More magazines by this user
Similar magazines