RNAi screening identifies a new Toll from shrimp that restricts WSSV infection through activating Dorsal to induce antimicrobial peptides

The function of Toll pathway defense against bacterial infection has been well established in shrimp, however how this pathway responds to viral infection is still largely unknown. In this study, we report the Toll4-Dorsal-AMPs cascade restricts the white spot syndrome virus (WSSV) infection of shrimp. A total of nine Tolls from Litopenaeus vannamei namely Toll1-9 are identified, and RNAi screening in vivo reveals the Toll4 is important for shrimp to oppose WSSV infection. Knockdown of Toll4 results in elevated viral loads and renders shrimp more susceptible to WSSV. Furthermore, Toll4 could be a one of upstream pattern recognition receptor (PRR) to detect WSSV, and thereby leading to nuclear translocation and phosphorylation of Dorsal, the known NF-κB transcription factor of the canonical Toll pathway. More importantly, silencing of Toll4 and Dorsal contributes to impaired expression of a specific set of antimicrobial peptides (AMPs) such as anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to interact with several WSSV structural proteins. Taken together, we therefore identify the Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some specific AMPs. Author summary The TLR pathway mediated antiviral immune response is well identified in mammals, yet, Toll pathway governing this protection in invertebrates remains unknown. In the present study, we uncover that a shrimp Toll4 from a total of nine Tolls in L. vannamei confers resistance to WSSV thought inducing the NF-κB transcription factor Dorsal to inspiring the production of some antimicrobial peptides (AMPs) with antiviral activity. The anti-LPS-factor (ALF) and lysozyme (LYZ) family are identified as the Toll4-Dorsal pathway targeted genes with the ability to interact with viral structural proteins in response to WSSV infection. These results suggest that the Toll receptor induces the expression of AMPs with antiviral activity could be a general antiviral mechanism in invertebrates and Toll pathway established antiviral defense could be conserved during evolution.

anti-LPS-factor (ALF) and lysozyme (LYZ) family, which exert potent anti-WSSV activity. 48 Two AMPs of ALF1 and LYZ1 as representatives are demonstrated to have the ability to 49 interact with several WSSV structural proteins. Taken together, we therefore identify the 50 Toll4-Dorsal pathway mediates strong resistance to WSSV infection by inducing some 51 specific AMPs.

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Author summary：The TLR pathway mediated antiviral immune response is well identified 54 in mammals, yet, Toll pathway governing this protection in invertebrates remains unknown. 55 In the present study, we uncover that a shrimp Toll4 from a total of nine Tolls in L. vannamei 56 confers resistance to WSSV thought inducing the NF-κB transcription factor Dorsal to 57 inspiring the production of some antimicrobial peptides (AMPs) with antiviral activity. The

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Cloning, sequence analysis and phylogenetic tree of shrimp nine Tolls. 140 In order to identify all candidate Toll genes from shrimp (L. vannamei), we carried out  144 vannamei were obtained, of which the Toll1, Toll2 and Toll3 were perfectly identical to 145 previous reported LvToll1, LvToll2 and LvToll3,respectively (21). We next cloned the 146 full-length cDNA sequences of other Tolls by using rapid amplification cDNA ends 147 (RACE)-PCR method, and we subsequently designated these Tolls from Toll1 to Toll9. Their 148 sequences of Toll1-9 in FASTA format were available in Supplement Data S1. Functional shrimps following WSSV infection) ( Figure 2D). Therefore, we focused our attention special 186 on the Toll4 mediated mechanism underlying resistance to WSSV in this study. To further 187 confirm the above screening result, with in vivo RNAi again, a higher lethality was observed 188 in the silencing of Toll4 shrimps followed by WSSV infection when compared to that of the 189 control group ( Figure 2E). To investigate whether the increased lethality rates of Toll4 190 silenced shrimps was due to decreased resistance or tolerance to WSSV, we analyzed the viral 191 levels by absolute q-PCR in several tissues including hepatopancreas, gill and muscle at 48 192 hpi. We observed that shrimps with knockdown of Toll4 had elevated viral replication levels 193 in all the three tissues than control shrimps ( Figure 2F). The obviously lower viral levels   Figure 5C). In addition to 265 nuclear translocation, the activation of Dorsal could be manifested by phosphorylation on 266 some specific amino acids. We found that shrimp Dorsal contains a considerable conserved 267 region, which displays a significant degree of sequence similarity to a comparison region of 268 its mammalian counterparts. In this region, human p65 NF-κB factor contains a Ser276, 269 corresponding to Ser342 of shrimp Dorsal, which can be phosphorylated upon many stresses with different pathogens strongly suggest that Toll4 mediated Dorsal activation is WSSV 338 specific, which indicate that Toll4 could play a vital role in recognizing WSSV infection.

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Taken together, our data suggest that Toll4 is a key factor for sensing WSSV and mediating 340 downstream Dorsal activation, and thereby inducing some specific AMPs production.   Figures 10A and 10B). In the His tagged ALF1 pull-down assay with six WSSV 376 structural proteins (GST tag), we observed that ALF1 precipitated VP19, VP26, VP28, 377 wsv134 and wsv321 by SDS-PAGE with coomassie blue staining ( Figure 10C, lanes 1, 3, 4, 378 5 and 6, respectively). However, His tagged ALF1 did not interact with GST tagged VP24, 379 which indicates that the interaction of between ALF1 and other four structural proteins is 380 specific, but not related to the His and GST tags. We further confirmed this result by western 381 blotting with GST tag antibody, which is in good agreement with that of coomassie blue 382 staining ( Figure 10D). In the His tagged LYZ1 pull-down assay, we found that VP26, VP28, 383 wsv134 and wsv321 were enriched ( Figure 10E, lanes 3, 4, 5 and 6, respectively), and an 384 identical result was observed by western blotting ( Figure 10F). To further identify the above 385 results, six WSSV structural proteins with GST tag was used in a GST pull-down assay with 386 purified His tagged ALF1 or LYZ1 followed by SDS-PAGE with coomassie blue staining and 387 western blotting with His antibody, respectively. As shown in Figures 10G and 10H Figure 10K).   PGN and LPS (46). These data suggest that one type of shrimp Toll could recognize more 475 than one PAMP. In the present study, we observed that Dorsal activation and translocation to 476 the nucleus is dependent on Toll4 in response to WSSV infection, but not other tested 477 pathogens, which indicate that Toll4 could also be a specific PRR to detect WSSV in a 478 manner similar to Drosophila Toll7. Unravelling how Toll4 senses WSSV in the future will 479 be important to understand antiviral immunity in shrimp.

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The production of antimicrobial peptides (AMPs) is commonly considered to be an 481 evolutionarily conserved mechanism of the innate immune response and has been extensively   In order to obtain the cDNA sequence of all candidate Toll genes from shrimp, the amino 573 acid (aa) sequences of the Tolls and TLRs from Drosophila and human (DmToll1-9 and 574 HsTLR1-10) were collected and used as query sequences for in silico searches of L. 575 vannamei transcriptome data (34) using local TBLASTN alignment tool with E-value cutoff 576 of 1e -5 . Nine assembled EST sequences were identified as having high homology to the Toll 577 family genes. Gene-specific primers (Supplement Table 1) were designed for 5′ and 3′ rapid 578 amplification of cDNA ends (RACE) PCR to obtain the 5′ and 3′ end of L. vannamei Toll 579 genes. In brief, total RNA was extracted from pooled tissues of L. vannamei gill, hemocyte   Table 1 Table 1). Germany) specific primers (Supplement Table 1