Developmental and Comparative Immunology 35 (2011) 105–114 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci Litopenaeus vannamei tumor necrosis factor receptor-associated factor 6 (TRAF6) responds to Vibrio alginolyticus and white spot syndrome virus (WSSV) infection and activates antimicrobial peptide genes Pei-Hui Wang a , Ding-Hui Wan a , Zhi-Hua Gu a , Xie-Xiong Deng a , Shao-Ping Weng a , Xiao-Qiang Yu c , Jian-Guo He a,b,∗ a State Key Laboratory of Biocontrol/MOE Key Laboratory of Aquatic Product Safety, School of Life Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, People’s Republic of China b School of Marine Sciences, Sun Yat-sen University, 135 Xingang Road West, Guangzhou 510275, People’s Republic of China c Division of Cell Biology and Biophysics, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, MO 64110, USA article info Article history: Received 10 July 2010 Received in revised form 29 August 2010 Accepted 30 August 2010 Available online 17 September 2010 Keywords: Litopenaeus vannamei LvTRAF6 Antimicrobial peptide genes WSSV Vibrio alginolyticus Toll signal pathway abstract Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a key signaling adaptor protein not only for the TNFR superfamily but also for the Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) super- family. To investigate TRAF6 function in invertebrate innate immune responses, Litopenaeus vannamei TRAF6 (LvTRAF6) was identified and characterized. The full-length cDNA of LvTRAF6 is 2823 bp long, with an open reading frame (ORF) encoding a putative protein of 594 amino acids, including a RING- type Zinc finger, two TRAF-type Zinc fingers, a coiled–coil region, and a meprin and TRAF homology (MATH) domain. The overall amino acid sequence identity between LvTRAF6 and other known TRAF6s is 22.2–33.3%. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvTRAF6 could activate the promoters of antimicrobial peptide genes (AMPs), including Drosophila Attacin A and Drosomycin, and shrimp Penaeidins. Real-time quantitative PCR (qPCR) indicated that LvTRAF6 was constitutively expressed in various tissues of L. vannamei. After Vibrio alginolyticus and white spot syndrome virus (WSSV) challenge, LvTRAF6 was down-regulated, though with different expression patterns in the intes- tine compared to other tissues. After WSSV challenge, LvTRAF6 was up-regulated 2.7- and 2.3-fold over the control at 3 h in gills and hepatopancreas, respectively. These results indicated that LvTRAF6 may play a crucial role in antibacterial and antiviral responses via regulation of AMP gene expression. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Since the 1970s, Penaeid shrimp culture has developed rapidly and become a major economic activity throughout the world. Litopenaeus vannamei, the primary farmed shrimp species in China and Southern Asia, suffer from infection by two main shrimp Abbreviations: TRAF6, tumor necrosis factor receptor-associated factor 6; WSSV, white spot syndrome virus; TNFR, tumor necrosis factor receptor; IL-1, interleukin-1 receptor; TLR, Toll-like receptor; ORF, open reading frame; MATH, meprin and TRAF homology; qPCR, real-time quantitative PCR; AMPs, antimicrobial peptides; PAMPs, pathogen-associated molecular patterns; IRAK4, IL-1 receptor-associated kinase- 4; IKK, IB kinase; MAPK, mitogen-activated protein kinases; JNK, Jun-N-terminal kinases; IRF, IFN regulatory factor; LB, Luria broth; RACE, rapid amplification of cDNA end; S2, Drosophila Schneider 2; FBS, fetal bovine serum; ALFs, antilipopolysaccha- ride factors. ∗ Corresponding author at: State Key Laboratory of Biocontrol/MOE Key Labora- tory of Aquatic Product Safety, School of Life Sciences, People’s Republic of China. Tel.: +86 20 39332988; fax: +86 20 39332849. E-mail address: lsshjg@mail.sysu.edu.cn (J.-G. He). pathogens, white spot syndrome virus (WSSV) and Vibrio spp. (Wang et al., 2010; Yang et al., 2007). A better understanding of immune responses to WSSV and Vibrio spp. infection is nec- essary to design strategies for prevention and control of shrimp diseases. Recently, new Toll receptors have been indentified in L. vannamei, Fenneropenaeus chinensis, Penaeus monodon and Marsu- penaeus japonicus (Mekata et al., 2008; Yang et al., 2008; Yang et al., 2007). Silencing of LvToll was found to increase mortality and reduce pathogen clearance after V. harveyi challenge, but not WSSV challenge (Wang et al., 2010), and it was shown that LvToll was not involved in the anti-WSSV response (Labreuche and O’Leary, 2009). Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is a crucial signal transducer of Toll-like receptor (TLR) signal pathway that is conserved from Drosophila to humans, though this canonical Toll signaling pathway is absent in nema- todes (Kim and Ausubel, 2005; Leulier and Lemaitre, 2008; Qiu et al., 2009). TRAFs are intracellular signal transducers for a number of immune receptor superfamilies, including TNFR superfamily and Interleukin-1 receptor/Toll-like receptor (IL-1/TLR) family mem- 0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2010.08.013