Resistance to Bombyx mori nucleopolyhedrovirus via overexpression ...

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Arch Virol (2012) 157:1323–1328 DOI 10.1007/s00705-012-1309-8

ORIGINAL ARTICLE

Resistance to Bombyx mori nucleopolyhedrovirus via overexpression of an endogenous antiviral gene in transgenic silkworms Liang Jiang • Genhong Wang • Tingcai Cheng • Qiong Yang • Shengkai Jin • Gai Lu • Fuquan Wu Yang Xiao • Hanfu Xu • Qingyou Xia



Received: 25 November 2011 / Accepted: 13 March 2012 / Published online: 18 April 2012 Ó Springer-Verlag 2012

Abstract Transgenic technology is a powerful tool for improving disease-resistant species. Bmlipase-1, purified from the midgut juice of Bombyx mori, showed strong antiviral activity against B. mori nucleopolyhedrovirus (BmNPV). In an attempt to create an antiviral silkworm strain for sericulture, a transgenic vector overexpressing the Bmlipase-1 gene was constructed under the control of a baculoviral immediate early-1 (IE1) promoter. Transgenic lines were generated via embryo microinjection. The mRNA level of Bmlipase-1 in the midguts of the transgenic line was 27.3 % higher than that of the non-transgenic line. After feeding the silkworm with different amounts of BmNPV, the mortality of the transgenic line decreased to approximately 33 % compared with the nontransgenic line when the virus dose was 106 OB/larva. These results imply that overexpressing endogenous antiviral genes can enhance the antiviral resistance of silkworms.

Introduction Bombyx mori is an economically important insect for silk production. In many rural areas in China, India, Brazil, and

Electronic supplementary material The online version of this article (doi:10.1007/s00705-012-1309-8) contains supplementary material, which is available to authorized users. L. Jiang  G. Wang  T. Cheng  S. Jin  G. Lu  H. Xu  Q. Xia (&) State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China e-mail: [email protected] Q. Yang  F. Wu  Y. Xiao Sericulture and Farm Product Processing Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510610, China

other countries, sericulture is one of the main sources of income for farmers. However, several types of pathogens cause serious economic loss every year for sericulture. Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most serious of these pathogens, but no fundamental strategies have been established to cope with infections with this virus. Two virion phenotypes exist in the NPV life cycle: occlusion-derived virus (ODV) and budded virus (BV). ODV causes transmission between individuals, whereas BV causes systemic infection throughout the host [1, 2]. ODV particles are packaged in the viral polyhedral body, which is a highly symmetrical covalently cross-linked robust lattice [3]. Once the polyhedra are ingested by the host larvae per os, the primary infection is initiated upon the dissociation of the polyhedral bodies, and enveloped virions are released in the alkaline environment of the midgut juice [1, 4]. Subsequently, the infected hosts produce BV to cause a secondary infection. At the late stages of infection, some virions are occluded in polyhedral bodies, and after host death, the polyhedra are delivered to the natural environment [1, 4]. Usually, BmNPV invades silkworm larvae through the oral route in the case of ODV or the wound pathway in the case of BV. After infection with BmNPV per os, the virus is released into the midgut, where it is exposed to midgut juices containing some proteins with anti-BmNPV activity [5–8]. Bmlipase-1, a 29-kDa protein, first purified by Ponnuvel et al. [5] from the digestive juice of silkworm larvae, which had lipase activity, is representative of such proteins. ODV (860 ng/larva) treated with this protein (2.2 lg/larva) did not cause the death of fifth-instar larvae. Other proteins, such as B. mori serine protease-2 (BmSP-2) [6] and BmNox [7, 8], were also purified from the gut juice of silkworm larvae and showed strong antiviral activity against BmNPV.

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Two disease-control methods have been proven effective in plants: knocking down expression of genes of the viral pathogens in transgenic hosts by RNAi and overexpressing the anti-pathogen genes in hosts by transgenic technology. Disease resistance can be enhanced in plants by overexpressing anti-pathogen genes [9, 10]. Transgenic tobacco shows strong resistance to bacterial pathogens when the bactericidal peptide sarcotoxin IA from insects is overexpressed [9]. A transgenic carrot line (P23) with the rice cationic peroxidase OsPrx114 also exhibits a strong resistance to necrotrophic pathogens [10]. In animals, however, overexpression of antiviral genes via transgenic technology has only been applied in mice to increase pathogen resistance [11, 12]. Overexpression of murine interleukin-10 significantly stimulates the resistance of mice to cerebral ischemia [11]. The phenotypes of mitochondrial diseases caused by mutations are ameliorated by introducing a transgene encoding the mitochondrial transcription factor A (Tfam) into mice [12]. Silkworm is a typical model for the order Lepidoptera and is an economically important insect [13–16]. Resistance to BmNPV can be boosted via the down-regulation of BmNPV genes by RNAi in transgenic silkworms [17, 18]. When the exogenous gene lef-1 was knocked down by RNAi, virus proliferation in transgenic silkworm was only partially reduced, but the mortality caused by infection with BmNPV did not decline [17]. Silencing of the ie-1 gene induced resistance to baculoviruses in transgenic silkworms [18]. To date, there are no reports on increased pathogen resistance via overexpression of anti-pathogen genes in insects. The present study is the first to apply transgenic strategies in activating strong anti-BmNPV activity in the gut juice of silkworm by overexpressing Bmlipase-1.

Materials and methods Silkworm strain and virus The Dazao strain of B. mori was maintained at the Gene Resource Library of Domesticated Silkworm (Southwest University, China). The silkworm was reared on fresh mulberry leaves under standard conditions. The Dazao larvae were infected with BmNPV (Guangdong strain, China) per os, and OBs were then harvested from haemolymph from these larvae.

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basic transgenic vector with a 3 9 P3-EGFP-sv40 gene as a reporter marker in which EGFP expression is driven by the 3 9 P3 promoter and occurs in the compound eyes and nervous tissues of the silkworm [20], to generate the transgenic vector piggyBac [IE1P-Bmlipase-1-SV40-3 9 p3 EGFP afm] (abbreviated as pb-LI). Germline transformation of silkworm To generate nondiapausing embryos, Dazao embryos were incubated at 15 °C after acidic treatment. The larvae were fed mulberry leaves under standard conditions. Their embryos were nondiapaused. Mixtures of pb-LI plasmid (400 ng/lL) and helper vector pHA3PIG (400 ng/lL) were injected into the nondiapausing Dazao embryos within 2 h to 3 h after oviposition [19, 20]. The G1 embryos were screened for EGFP-positive expression in ocelli under a fluorescent microscope (Olympus) [20], and the transgenic moths were sibling-mated to generate an offspring in each EGFP-positive G1 brood. Two transgenic lines were screened, which were named LI-A and LI-B for the subsequent detection experiments. Southern blot and insertion site analysis The genomic DNAs of LI-A and LI-B were extracted from G1 male moths. A total of 20 lg of the genomic DNA of LI-A, LI-B, and non-transgenic Dazao (Nm) was fully digested with Kpn I, for which a single site was present in the insert, for 10 h at 37 °C, and the fragments were separated via electrophoresis in a 0.9 % agarose gel and transferred onto a nylon filter. The EFGP fragment was PCR-amplified from piggyBac [3 9 p3 EGFP afm] using the primers F (50 -CCACAAGTTCAGCGTGTCCG-30 ) and R (50 -AGTTCACCTTGATGCCGTTCTT-30 ). The PCR product was labeled using DIG-High Prime reagent (Roche), and hybridization was detected and recorded using an Image Scanner III (GE). A total of 20 lg of genomic DNA of LI-A and LI-B was fully digested with Hae III for 10 h at 37 °C and then purified and self-ligated with solution I (TaKaRa). The ligated product was PCR-amplified with the transposonspecific primers left F (50 -ATCAGTGACACTTACCGCA TTGACA-30 ) and left R (50 -TGACGAGCTTGTTGGTG AGGATTCT-30 ), and the PCR product was cloned and sequenced.

Vector construction qPCR analysis of Bmlipase-1 The Bmlipase-1 gene was amplified from the midgut cDNA of Dazao. The termination signal SV40 was cloned from the piggyBac [3 9 p3 EGFP afm] vector. The IE1 promoter was excised from the pBSII-IE1-orf vector. The three fragments were added to piggyBac [3 9 p3 EGFP afm], which is a

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Different developmental stages of LI-A, LI-B, and Nm as well as their midguts were harvested, frozen immediately in liquid nitrogen, and stored at -80 °C until RNA extraction. Total RNA was extracted using a Total RNA

Resistance of transgenic silkworms to BmNPV infection

Mini Kit (Watson), and digested with 20 U of RNase-free DNase I (Promega). A total of 2 lg of treated RNA was reverse-transcribed in a 25-lL reaction system using M-MLV reverse transcriptase (Promega). One lL of cDNA was used for qPCR reactions with Bmlipase-1 primers Bmlip-1qRT F (50 -ACAAATGGCAATGTCAACTCTA TC-30 ) and Bmlip-1qRT R (50 -CCACGCCAGTCTACAA CAATAA-30 ) on an ABI Prism 7000 sequence detection system (Applied Biosystems) using an SYBR Premix Ex Taq Kit (TaKaRa) following the manufacturer’s instructions. The housekeeping gene BGIBMGA003186-TA was used as an internal control to standardize the variance among the different templates. Each assay was performed three times. Mortality analysis in transgenic silkworm The mortality of LI-A, LI-B, and Nm after oral inoculation with wild-type BmNPV with 106 OB/larva and another dose at the newly exuviated fourth instar (LD50 of Nm is 106 OB/ larva at the fourth instar) were investigated. Each line was infected three or more repeats, and each repeat included 100 larvae. A fresh mulberry leaf was cut into round pieces 1 cm in diameter and then coated with OBs. Newly exuviated fourth-instar larvae that ate a whole piece of leaf with OBs were selected for continuous rearing. For each line, noinfection repeats were done in triplicate, and every repeat had 100 larvae. Cumulative survival rates were calculated daily from the time of infection to the moth stage.

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from ten treated larvae were used as templates for qPCR detection of the BmNPV GP41 gene (F, 50 -CGTAGTAGT AGTAATCGCCGC-30 ; R, 50 -AGTCGAGTCGCGTCG CTTT-30 ) [21]. Each assay was performed three times. The DNA templates (20 ng) were PCR-amplified using BmNPV GP41 primers using an ABI Prism 7000 sequence detection system. BmGAPDH (F, 50 -CATTCCGC GTCCCTGTTGCTAAT-30 ; R, 50 -GCTGCCTCCTTGA CCTTTTGC-30 ) was used as a reference gene to standardize the variance among the different templates.

Results Construction of the overexpression plasmid and screening transgenic silkworms

Total DNA was obtained from the larvae of LI-A and Nm at 24 h and 48 h post-infection. DNA samples extracted

The Bmlipase-1 gene was cloned from the midgut cDNA template of Dazao, and the overexpression vector pb-LI (Fig. 1A) was constructed using the transgenic vector piggyBac [3 9 p3 EGFP afm], in which Bmlipase-1 expression was driven by an IE1 promoter. Microinjection was performed with mixtures of the pb-LI vector and helper plasmid pHA3PIG in nondiapausing Dazao embryos. The numbers of injected embryos and hatched larvae were 266 and 146, respectively. The G0 moths were mated with each other or backcrossed to Nm moths to produce G1 offspring. Ten G1 broods were obtained and screened for EGFP, and EGFP-positive G1 moths from the same broods were sibling-mated to generate offspring. In total, two transgenic lines (LI-A and LI-B) were obtained for the subsequent assays. LI-A and LI-B were sibling-mated to generate offspring for each generation.

Fig. 1 Transgenic vector construction and screening of transgenic silkworm lines. (A) Schematic of the pb-LI vector. piggyBac [3 9 p3 EGFP afm] is a basic transgenic vector with a 3 9 P3-EGFP-sv40 gene as a reporter marker. PL and PR represent its left and right terminal inverted repeats, respectively. IE1P, IE1 promoter; Bmlipase-1, coding sequence of the Bmlipase-1 gene; SV40, polyadenylation signal. (B) Southern blot analysis to determine the DNA copy number in the LI transgenic silkworm. The genomic DNAs of

transgenic lines LI-A, LI-B, and Nm were extracted. Samples of genomic DNA were fully digested with Kpn I, followed by Southern blot using one EGFP probe. Lanes 1, 2, and 3 show the samples from LI-A, LI-B, and Nm, respectively. (C) Analysis of insertion sites of LI-A and LI-B. Inverse PCR was performed to detect the insertion site. Genomic DNAs of LI-A and LI-B were fully digested with Hae III and then self-ligated. PCR-amplified products with transposonspecific primers were cloned and sequenced

qPCR analysis of virus after viral inoculation

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Identifying the insertion site of transgenic lines The genomic DNAs of the transgenic lines (LI-A and LI-B) and Nm were extracted from G1 male moths. The copy number of the inserts was determined by Southern blot using an EGFP probe. The results showed that each transgenic line produced a band that hybridized with the EGFP probe, and the control Nm did not yield a hybridization signal (Fig. 1B). The hybridization signal for LI-A was higher than that for LI-B, but that does not mean that the copy number was higher for LI-A. To confirm the above result, inverse PCR was performed to detect the insertion site of the LI transgenic lines. The PCR-amplified products with transposon-specific primers and templates of ligated product were cloned and sequenced. The results indicate that copies of LI-A and LI-B were present in chromosome 12 and chromosome 26, respectively (Fig. 1C). Bioinformatic analysis showed that the insertion sites were in an intergenic region. The nearest genes to the left and right of the insertion site in LI-A were BGIBMGA010307 and BGIBMGA010299, which were 24 kb and 22 kb away, respectively. There were no expressed sequence tags (ESTs) or predicted functions for these two genes in the silkworm genome database (SilkDB). The nearest genes to the left and right of the insertion site in LI-B were BGIBMGA010751 and BGIBMGA010750, which were 45 kb and 175 kb away, respectively. BGIBMGA010751 and BGIBMGA010750 have an NAC domain and a homeobox domain, respectively. Expression profile of Bmlipase-1 in transgenic lines To test whether the expression of Bmlipase-1 was up-regulated in the LI lines, the cDNA templates of the hatched silkworm, first-instar molt, fourth-instar larvae, fourth

Fig. 2 qPCR analysis of Bmlipase-1 in transgenic and non-transgenic silkworms. (A) Expression of Bmlipase-1 in larvae and pupae of LI and Nm. RNA of hatched silkworm, first-instar molt, fourth-instar larvae, fourth-instar molt, fifth-instar larvae, and pupae of LI-A, LI-B, and Nm were extracted and then reverse-transcribed to cDNA. cDNA templates were used for qPCR reactions with Bmlipase-1 primers.

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instar molt, fifth-instar larvae, and pupae of LI-A, LI-B, and Nm were used in the qPCR test. The results of the qPCR analysis showed that the expression levels of Bmlipase-1 in LI lines were significantly higher than in Nm at the larvae and pupa stages. The expression levels of Bmlipase-1 in LI-A (47.4 %, 76.5 %, and 36.6 %) and LIB (42.2 %, 98.6 %, and 30.6 %) were significantly higher than those in Nm from the fourth instar, fourth-instar molt, and fifth instar, respectively (Fig. 2A). The expression levels of Bmlipase-1 were further analyzed in the midguts of LI-A, LI-B, and Nm via qPCR. The results revealed that Bmlipase-1 in LI-A (27.3 % and 7.5 %) and in LI-B (31.0 %, 3.1 %) were higher than those of Nm in the midguts of the fourth-instar (significantly) and the fifthinstar larvae, respectively (Fig. 2B). These results indicate that the LI transgenic lines can significantly enhance the transcription of Bmlipase-1. Measurement of BmNPV resistance of LI lines To investigate the resistance of LI lines to BmNPV infection, newly exuviated fourth-instar larvae of LI-A, LIB, and Nm were infected with BmNPV per os using 106 OB/larva. The OBs were daubed onto a piece of 1-cmdiameter fresh mulberry leaf; larvae that ate a whole piece of leaf with OBs were selected for the subsequent assay. Nm and the two LI lines were infected with BmNPV two and three repeats, respectively. Triplicate no-infection repeats were also carried out for each line. Each repeat consisted of 100 larvae. Cumulative survival rates were calculated daily until the moth stage. For most of the larvae that died, death occurred mainly just before the fourthinstar molt (96 h to 120 h postinfection). Most of the latemolt larvae died, but most of the newly exuviated fifthinstar larvae survived in the subsequent stages. The results

(B) Expression of Bmlipase-1 in the midguts of LI and Nm. RNA of the midguts of fourth- and fifth-instar larvae of LI and Nm were extracted and then reverse-transcribed to cDNA, which were used for qPCR reactions. Bars represent standard deviation. Statistically significantly differences: * P \ 0.05, ** P \ 0.01

Resistance of transgenic silkworms to BmNPV infection

Fig. 3 Survival curve of LI after oral inoculation with 106 OB/larva. Three silkworm lines, LI-A, LI-B, and Nm Dazao, were infected with BmNPV per os using a dose of 106 OB/larva for the newly exuviated fourth-instar larva. The OBs were daubed on pieces of fresh mulberry leaf; the larvae that ate a whole piece of leaf with OBs were selected for the subsequent rearing. PID means post-infection day. Cumulative survival rates are shown for each day from the time of infection to the moth stage. PID 1–4 is fourth instar, PID 5 is fourth-instar molt, PID 6–12 is fifth instar, PID 13–21 is pupa stage, and PID 22 is moth stage. The mortality rate for Nm is given as the average of two repeated infections; the mortality rates for LI-A and LI-B are the average of triplicate infections. LI-A, LI-B, Nm are the infected lines, LI-A(C), LI-B(C), Nm(C) are the untreated lines (control). Each repeat consisted of 100 larvae. Bars represent standard deviation. P represents a statistically significantly different value

shown in Fig. 3 indicate that the LI transgenic lines have sustainable and significant protection ability. The survival rates of Nm, LI-A, and LI-B were 50 %, 83 %, and 75 %, respectively. Interestingly, the mortality of LI-A and LI-B was 33 % and 25 % lower, respectively, than that of Nm. Almost all untreated silkworms survived and pupated normally. The mortality statistics indicate that the resistance activity of LI-A was significantly higher than that of Nm. This finding is attributed to the expression level of Bmlipase-1 in LI-A, which was significantly higher than that in Nm (Figs. 2A, B, 3). qPCR analysis of virus after BmNPV infection To analyze the resistance of the LI lines to BmNPV further, the amount of BmNPV after infection was measured in the transgenic lines and Nm. The LI lines and Nm were inoculated with equal amounts of virus per os at the newly exuviated fourth-instar larval stage. Total DNA was extracted from ten treated larvae of LI-A and Nm at 24 h and 48 h post-infection, respectively. The amount of BmNPV DNA was measured using qPCR with GP41 primers. The average for Nm was set to 100 % at each time point, and the value of LI-A was standardized against Nm. The amount of virus DNA accumulated in LI-A was threefold lower than in Nm at 2 days postinfection (Fig. 4), suggesting that LI-A inhibits the proliferation of BmNPV.

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Fig. 4 qPCR analysis of BmNPV in LI-A and Nm DZ after viral inoculation. For the newly exuviated fourth-instar larvae, equal amounts of virus were used for inoculation. Total DNA was extracted from ten treated LI-A and Nm larvae at 24 and 48 h postinfection. GP41 primers were used for qPCR analysis of BmNPV. The average for Nm was set to 100 % at each time point, and the value of LI-A was standardized against Nm. Bars represent standard deviation. Statistically significantly difference: ** P \ 0.01

Discussion Ponnuvel et al. [5] purified Bmlipase-1 from the digestive juice of B. mori larvae and revealed that the protein has a strong anti-BmNPV activity. In the current study, an overexpression vector of Bmlipase-1 was constructed, driven by the IE1 promoter. Two transgenic silkworm lines, LI-A and LI-B, were selected for resistance analysis. The LI lines showed increased levels of Bmlipase-1 expression and enhanced resistance to BmNPV. In the current study, higher mortality was observed when the silkworm was infected with a higher dose of BmNPV, whereas the mortality differences between the transgenic line and Nm after oral inoculation with a semilethal dose of BmNPV were obvious (Fig. 3, Supplemental Figs. 1-3). Therefore, anti-BmNPV activity was enhanced by overexpressing a single antiviral gene in a transgenic silkworm. LI lines can block the invasion of BmNPV to the midgut, inhibit the proliferation of BmNPV, and enhance resistance to this virus by increasing the expression of Bmlipase-1. Endogenous Bmlipase-1 is a midgut-specific protein [5, 15]. The IE1 promoter is active in almost all tissues of silkworms. Thus, Bmlipase-1 showed no obvious increase in the midgut of the transgenic silkworm compared to individual larvae (Fig. 2A and B). As expected, the use of transgenics produced insects with increased Bmlipase-1 levels and enhanced anti-BmNPV ability when the midgut-specific promoter was used in commonly used silkworm species. BmNPV is a major pathogen of silkworms and infects silkworm larvae mainly per os. Overexpressing the antiviral genes is the most promising method to improve resistance. RNAi of viral genes by transgenic technology is

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also an effective strategy. However, transgenic RNAi can be achieved only after the virus infects silkworm cells, with the cost of destruction of host cells by the virus to some extent. A combination of both strategies may be more effective than using them individually. In summary, by overexpressing an endogenous antiviral gene, transgenic silkworm lines with powerful resistance to BmNPV were successfully obtained. The transgenic antiBmNPV line may be applied in the sericultural industry to decrease the mortality of silkworm larvae. This is the first report on resistance to disease via overexpression of an anti-pathogen gene in insects, and this can pave the way for future disease control in other insects. Acknowledgments This work was funded by the National Basic Research Program of China (No. 2012CB114600), the National HiTech Research and Development Program of China (No. 2011AA100306), the National Natural Science Foundation of China (No. 30901054), the Production, Education & Research of Guangdong, and the Ministry of Education (No. 2010A090200079).

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