- Email: [email protected]
Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål)
Journal of Invertebrate Pathology 114 (2013) 255–257
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål) Ensi Shao a,1, Sijun Liu b, Li Lin a, Xiong Guan a,⇑ a b
Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian, PR China Department of Entomology, Iowa State University, Ames, IA 50011, USA
a r t i c l e
i n f o
Article history: Received 13 May 2013 Accepted 3 September 2013 Available online 8 September 2013 Keywords: Nilaparvata lugens Bacillus thuringiensis Cry1Ab toxin Gut proteases Proteolytic processing
a b s t r a c t To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella. Ó 2013 Elsevier Inc. All rights reserved.
1. Short Communication Bacillus thuringiensis (Bt) and Bt-crops are highly effective to control lepidopteran pests. However, the insecticidal activity of Bt to hemipteran pests is very limited (Schnepf et al., 1998). Alone with the widely planting of transgenic crops carrying Bt cry genes, target pest which is susceptive to Cry toxins will be effectively controlled (Wang et al., 2010). Bt-crops targeting piercing-sucking insect pests have yet to be developed (Chougule and Bonning 2012). The brown planthopper (BPH) is one of the most devastating insect pests in rice fields. Management of BPH has been mainly depended on the intensive use of chemical insecticides which could deteriorate the environment and was harmful to mammal (Sharma et al., 2000). Transgenic rice plants with Cry toxins are effective to control lepidopteran pests. However, transgenic rice varieties carrying Cry toxins showed no toxicity to BPH (Han et al., 2011). Toxicities of Bt toxins to hemipteran insects were previously tested such as in aphids (Cyt1Aa, Cry1Ac, Cry3Aa, Cry4Aa, Cry11Aa and cyt2Aa) (Porcar et al., 2009; Li et al., 2011; Chougule et al., 2013) and Lygus hesperus (TIC807) (Baum et al., 2012). In most of the tests, low to moderate toxicities were observed only when high doses of toxins were used. The reason of low insecticidal activity of Bt toxins in hemipteran insects is still unknown. ⇑ Corresponding author. Fax: +86 591 83789259. E-mail addresses: [email protected] (E. Shao), [email protected] (S. Liu), [email protected] (L. Lin), [email protected] (X. Guan). 1 Present address: 205 Binzhou Road, Fuzhou, Fujian 350008, PR China. 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.09.001
It has been shown that Cry1Ac toxin could be activated by gut proteases in pea aphids (Li et al., 2011). In addition, activated Cry1Ac could bind to aphid guts and the binding could be inhibited by GalNAC (Li et al., 2011). In lepidopteran insects, the intoxication pathway of Cry1A toxins includes protoxin activation, receptor binding, midgut cell membrane insertion and cell lysis (Bravo et al., 2005; de Maagd et al., 2001). Interruption of any of these steps may lead to a strongly decreased toxicity of Cry toxins in insects. Hence, understanding of the mechanisms responsible for the low toxicity of Cry toxins against hemipteran pests is important for improving the insecticidal activity of Cry toxins to BPH. In this study, we investigated proteolytic processing of Cry1Ab toxin by the proteases extracted from BPH gut. The cry1Ab gene was amplified from the plasmid p1AbPKK233-2 (obtained from Bacillus Genetic Stock Center, Ohio State University, Columbus, US) by PCR with a pair of cry1Ab specific primers (Forward: 50 -GGATCCATGGATAACAATCCGAACAT-30 ; Reverse: 50 -GTCGACT TACTATTCCTCCATAAGGAGTAATTC-30 ) containing a BamH I and a Sal I site in the forward and the reverse primers (underlined bases), respectively. The PCR fragments were double digested with the restriction enzymes BamH I and Sal I (TaKaRa, China), ligated into the plasmid pGEX-KG (GE healthcare, US) linearized with BamH I and Sal I (Guan and Dixon, 1991), and transformed into Escherichia coli BL21 strain (DE3). The GST-Cry1Ab fusion protein was isolated using a glutathione Sepharose column (GE healthcare, US) and the purified protein was recovered from the column after removal of the GST tag by thrombin digestion by following the instructions provided by the manufacturer. Protein purification resulted in a 133 kDa Cry1Ab protoxin (Fig. 1a and b). The purified Cry1Ab
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fusion protein was quantified by the Bradford method using a Coomassie Protein Assay kit (Biomiga, China). Soluble fraction and membrane fraction of gut protease extracts were prepared from 500 pieces of BPH nymphs gut using the method described by Li et al. (2011). The protein concentration of each gut protease preparation was determined using the Bradford method. The proteolytic processing of Cry1Ab by BPH gut proteases was examined using the gut protease preparations with or without pretreatment with 3 mM EDTA and 3 mM cysteine to activate cysteine protease activities (Li et al., 2011). Cry1Ab or GST-Cry1Ab was mixed with either the soluble proteases or membrane-bound proteases in a ratio of either 2:1 (soluble proteases:Cry1Ab, w/w) or 5:1 (membrane-bound proteases:Cry1Ab, w/w) and incubated at 28 °C for 10 min (only for GST-Cry1Ab), 1, 4, 8 and 16 h respectively, with moderate shaking (40 rpm). Cry1Ab protoxins were also treated with cathepsin-L and trypsin (Li et al., 2011). Protein samples of each treatment described above were analyzed by western blot analysis using a polyclonal anti-Cry1Ab serum specific to Cry1Ab. To prepare Cry1Ab specific polyclonal antibodies, a New Zealand White rabbit was immunized with trypsin activated Cry1Ab protein with Freund’s adjuvant for 4 times. Results of Cry1Ab proteolytic hydrolysis were shown in Fig. 1. For all gut protease treatments, a 60 kDa final product was accumulated over time. A similar product was also observed from the samples of trypsin digestion, indicating the 60 kDa fragment was likely the activated Cry toxin. Several other bands also appeared during the incubation period for all gut protease treatments. These peptides were intermediate products from degradation of Cry1Ab or GST-Cry1Ab. Activation of cysteine proteases with 3 mM EDTA and 3 mM cysteine increased the proteolytic activities of the gut preparations, but cysteine protease activation is required for the activation of Cry1Ab by BPH gut proteases. Slightly difference was observed in the Cry1Ab degradation patterns between the treat-
ment with soluble and membrane-bound proteases (Fig. 1). The membrane-bound proteases seemed to completely hydrolyze Cry1Ab to the 60 kDa fragment after 16 h incubation, although two bands were produced in the samples processing without cysteine protease activators (Fig. 1a). Notably, a band slightly larger than the 60 kDa fragment was observed either in the samples of protoxins incubated with soluble proteases lacking cysteine protease activators (Fig. 1a) or GST-Cry1Ab processed with soluble proteases (Fig. 1b), suggesting either the protease activities in the soluble fraction were lower comparing to the membrane-bound proteases, or the protease components were different from the membranebound proteases. We next tested insecticidal activity of the Cry1Ab processed by BPH proteases. Twenty larvae of diamondback moth (DBM), Plutella xylostella, susceptible to Cry1Ab toxin, were fed with protoxin or Cry1Ab toxins processed with either trypsin or BPH proteases using a leaf residue bioassay procedure (Tabashnik et al., 1990) and larvae fed on PBS was conducted as a negative control. A similar test for assessment of Cry1Ab toxicity to BPH was also conducted. Protoxin or Cry1Ab toxins activated with different methods were mixed with D-97 artificial diet and fed to Nilaparvata lugens by a membrane feeding assay (Fu et al., 2001). The bioassay experiments were repeated 9 times and mortality from each treatment was recorded after 48 h. The bioassay results showed that the Cry1Ab activated by BPH gut proteases had similar normal toxicity as that of the protoxin and trypsin-activated toxins in DBM larvae (Table 1). No significant difference was observed between the LC50 of the trypsin-activated Cry1Ab (0.95 (0.83–1.06) lg/mL) and the Cry1Ab activated by the BPH proteases (1.01 (0.95–1.08) lg/mL) (one-way ANOVA, df = 18, F = 0.400, P = 0.535). However, Cry1Ab protoxin and the toxins activated by trypsin or cathepsin-L showed similar low toxicity in BPH. The LC50s of Cry1Ab protoxin, cathepsin-L treated and trypsin-treated toxins
kDa 170 130 95 72 55 43 EDTA and Cysteine (
)
EDTA and Cysteine (
Soluble proteases
)
a
EDTA and Cysteine (
)
EDTA and Cysteine (
)
Membrane-bound proteases
kDa
kDa
170 130 95 72 55
72 55
43
Soluble proteases
b
Membrane-bound proteases
c
Fig. 1. In vitro processing of Cry1Ab toxin (a and b) and interaction with brown planthopper, BPH midgut (c). For the in vitro processing, Cry1Ab protoxin was incubated with the soluble proteases or membrane-bound proteases for 1 h, 4 h, 8 h and 16 h, respectively. Processed Cry1Ab products after incubation with soluble or membrane-bound gut proteases were separated by SDS–PAGE gel electrophoresis and Cry1Ab proteins were detected by Western blot with a rabbit polyclonal Cry1Ab serum as the primary antibody and an anti-rabbit-IgG, HRP conjugated, as the secondary antibody. Cry1Ab activated by BPH gut proteases was incubated with the BBMV from BPH and DBM guts in binding buffer (PBS, 0.1% BSA, 0.1% Tween 20, pH 7.6) at 28 °C for 1 h and then centrifuged to pull down the Cry1Ab bound to the BBMV. The BBMV pellets bound with Cry1Ab were washed three times. The detection of BBMV-bound Cry1Ab proteins was the same as that of in vitro processed Cry1Ab proteins. (a) In vitro processing of Cry1Ab protoxin by proteases in BPH guts; (b) in vitro processing of GST-Cry1Ab fusion protein by pre-activated proteases in the soluble and the membrane fraction of BPH guts; and (c) pulldown assay of Cry1Ab bound to the gut BBMV from BPH and DBM.
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E. Shao et al. / Journal of Invertebrate Pathology 114 (2013) 255–257 Table 1 Susceptibility of BPH and DBM to Cry1Ab protoxin and activated toxins.
DBM BPH
Protoxin (95% CI) (LC50 lg/ml)
Trypsin-processed (95% CI) (LC50 lg/ml)
Cathepsin L-processed (95% CI) (LC50 lg/ml)
BPH gut proteases processed (95% CI) (LC50 lg/ml)
0.88 (0.74–1.02) 190.23 (136.67–243.80)
0.95 (0.83–1.06) 173.49 (128.07–218.92)
N/A 180.32 (125.09–235.55)
1.01 (0.95–1.08) N/A
CI, confidence interval. LC50, 50% lethal concentration the LC50 value and their 95% fiducial limits were assessed by probit analysis using excel and the online statistical tools (http://www.xuru.org/st/ DS.asp).
in BPH were 180.32 (125.09–235.55) lg/mL, 173.49 (128.07– 218.92) lg/mL and 190.23 (136.67–243.80) lg/mL, respectively, which was more than 200-fold higher than the LC50 in diamondback moth (0.88 lg/mL) (Table 1). There was no statistical difference among toxicity of protoxin, cathepsin-L activated and trypsin-activated toxins in BPH (Table 1). To test the affinity of the activated Cry1Ab to midgut BBMVs from BPH and DBM, BBMVs of N. lugens and P. xylostella were prepared using the well established preparation method by Wolfersberger et al. (1987). Pull-down assay of Cry1Ab binding to gut BBMV (either from BPH or DBM) was conducted as previously described by Pérez et al. (2005). The BBMV-bound Cry1Ab toxin was detected by western blot analysis. Results showed that the 60 kDa BPH gut protease-activated toxin could bind to the midgut BBMV from DBM, while its binding to the BBMV from BPH was minimally detectable (Fig. 1c). Results from this study, together with previous works on aphids, demonstrated that some Cry toxins could be activated in the midgut of hemipteran insects with sap sucking mouthparts. However, activated Cry1Ab toxins bind to the midgut BBMV of BPH to a much less extent in comparison with the binding to the midgut BBMV from DBM. The similar phenomenon was observed in pea aphids (Li et al., 2011) which is consistent with low toxicity in BPH and other hemipteran insects. Inhibition of either trypsin or cysteine proteases should be performed in further study to determine which protease plays more significant role in Cry1Ab toxin activation in BPH gut. On the other hand, modification of Bt toxins to increase binding of the toxin to the midgut of target insects is an effective approach to improve its insecticidal activity and expand its insecticidal spectrum (Pardo-López et al., 2009; Chougule et al., 2013). Cry receptors APN, alkaline phosphates (ALP) and cadherin in the midgut of hemipteran pests have been identified (Cristofoletti et al., 2006; Liu et al., 2012). Future studies to understand the interactions between Cry functional domains and potential receptors on the gut epithelium of hemipteran insects will help developing novel Cry toxins targeting hemipteran pests.
Acknowledgments We thank Institute of Plant Protection, Chinese Academy of Agricultural Sciences providing polyclonal anti-Cry1Ab serum for this research. We also thank Guillaume Tetreau for critical reading and editing of the manuscript. This study is supported by Key Projects of Fujian Provincial Department of Science and Technology (Grant Nos. 2011N5003 and 2013N0003), National Natural Science Foundation of Fujian Province (Grant No. 2013J01079), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20093515110010), National Advanced Technology Research and Development Program of China (863 Program) (Grant No.
2011AA10A203) and National Natural Science Foundation of China (Grant No. 31071745). References Baum, J.A., Sukuru, U.R., Penn, S.R., Meyer, S.E., Subbarao, S., Shi, X., Flasinski, S., Heck, G.R., Brown, R.S., Clark, T.L., 2012. Cotton plants expressing a hemipteranactive Bacillus thuringiensis crystal protein impact the development and survival of Lygus hesperus (Hemiptera: Miridae) nymphs. J. Econ. Entomol. 105 (2), 616– 624. Bravo, A., Gill, S., Soberón, M., 2005. Bacillus thuringiensis mechanisms and use. Compr. Mol. Insect Sci. 6, 175–205. Chougule, N.P., Bonning, B.C., 2012. Toxins for transgenic resistance to hemipteranpests. Toxins 4 (6), 405–429. Chougule, N.P., Li, H., Liu, S., Linz, L.B., Narva, K.E., Meade, T., Bonning, B.C., 2013. Retargeting of the Bt toxin Cyt2Aa against hemipteran insect pests. Proc. Natl. Acad. Sci. USA 211 (21), 8465–8470. Cristofoletti, P.T., Mendonça de Sousa, F.A., Rahbé, Y., Terra, W.R., 2006. Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells. FEBS. J. 273 (24), 5574–5588. de Maagd, R.A., Bravo, A., Crickmore, N., 2001. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17, 193–199. Fu, Q., Zhang, Z., Hu, C., Lai, F., Sun, Z., 2001. A chemically defined diet enables continuous rearing of the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae). Appl. Entomol. Zool. 36 (1), 111–116. Guan, K.L., Dixon, J.E., 1991. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal. Biochem. 192 (2), 262. Han, Y., Xu, X., Ma, W., Yuan, B., Wang, H., Liu, F., Wang, M., Wu, G., Hua, H., 2011. The influence of transgenic cry1Ab/cry1Ac, cry1C and cry2A rice on non-target planthoppers and their main predators under field conditions. Agr. Sci. China 10 (11), 1739–1747. Li, H., Chougule, N.P., Bonning, B.C., 2011. Interaction of the Bacillus thuringiensis delta endotoxins Cry1Ac and Cry3Aa with the gut of the pea aphid, Acyrthosiphon pisum (Harris). J. Invertebr. Pathol. 107, 69–78. Liu, S., Chougule, N.P., Vijayendran, D., Bonning, B.C., 2012. Deep sequencing of the transcriptomes of soybean aphid and associated endosymbionts. PLoS One 7 (9), e45161. Pardo-López, L., Muñoz-Garay, C., Porta, H., Rodríguez-Almazán, C., Soberón, M., Bravo, A., 2009. Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Peptides 30 (3), 589–595. Pérez, C., Fernandez, L.E., Sun, J., Folch, J.L., Gill, S.S., Soberón, M., Bravo, A., 2005. Bacillus thuringiensis subsp. israelensis Cyt1Aa synergizes Cry11Aa toxin by functioning as a membrane-bound receptor. P. Natl. Acad. Sci. USA 102 (51), 18303. Porcar, M., Grenier, A.M., Federici, B., Rahbé, Y., 2009. Effects of Bacillus thuringiensis d-Endotoxins on the Pea Aphid (Acyrthosiphon pisum). Appl. Environ. Microb. 75 (14), 4897–4900. Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D., Dean, D., 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. R. 62 (3), 775–806. Sharma, H.C., Sharma, K.K., Seetharama, N., Ortiz, R., 2000. Prospects for using transgenic resistance to insects in crop improvement. Electron. J. Biotechnol. 3 (2), 21–22. Tabashnik, B.E., Cushing, N.L., Finson, N., Johnson, M.W., 1990. Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera: Plutellidae). J. Econ. Entomol. 83 (5), 1671–1676. Wang, Y., Zhang, G., Du, J., Liu, B., Wang, M., 2010. Influence of transgenic hybrid rice expressing a fused gene derived from cry1Ab and cry1Ac on primary insect pests and rice yield. Crop. Prot. 29 (2), 128–133. Wolfersberger, M., Luethy, P., Maurer, A., Parenti, P., Sacchi, F., Giordana, B., Hanozet, G., 1987. Preparation and partial characterization of amino acid transporting brush border membrane vesicles from the larval midgut of the cabbage butterfly (Pieris brassicae). Comp. Biochem. Phys. A 86 (2), 301–308.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Proteolytic processing of Bacillus thuringiensis toxin Cry1Ab in rice brown planthopper, Nilaparvata lugens (Stål) Ensi Shao a,1, Sijun Liu b, Li Lin a, Xiong Guan a,⇑ a b
Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Fujian Agriculture and Forestry University, 350002 Fuzhou, Fujian, PR China Department of Entomology, Iowa State University, Ames, IA 50011, USA
a r t i c l e
i n f o
Article history: Received 13 May 2013 Accepted 3 September 2013 Available online 8 September 2013 Keywords: Nilaparvata lugens Bacillus thuringiensis Cry1Ab toxin Gut proteases Proteolytic processing
a b s t r a c t To understand the low toxicity of Cry toxins in planthoppers, proteolytic activation of Cry1Ab in Nilaparvata lugens was studied. The proteolytic processing of Cry1Ab protoxin by N. lugens midgut proteases was similar to that by trypsin activated Cry1Ab. The Cry1Ab processed with N. lugens midgut proteases was highly insecticidal against Plutella xylostella. However, Cry1Ab activated either by trypsin or the gut proteases of the brown planthopper showed low toxicity in N. lugens. Binding analysis showed that activated Cry1Ab bound to brush border membrane vesicles (BBMV) from N. lugens at a significantly lower level than to BBMV from P. xylostella. Ó 2013 Elsevier Inc. All rights reserved.
1. Short Communication Bacillus thuringiensis (Bt) and Bt-crops are highly effective to control lepidopteran pests. However, the insecticidal activity of Bt to hemipteran pests is very limited (Schnepf et al., 1998). Alone with the widely planting of transgenic crops carrying Bt cry genes, target pest which is susceptive to Cry toxins will be effectively controlled (Wang et al., 2010). Bt-crops targeting piercing-sucking insect pests have yet to be developed (Chougule and Bonning 2012). The brown planthopper (BPH) is one of the most devastating insect pests in rice fields. Management of BPH has been mainly depended on the intensive use of chemical insecticides which could deteriorate the environment and was harmful to mammal (Sharma et al., 2000). Transgenic rice plants with Cry toxins are effective to control lepidopteran pests. However, transgenic rice varieties carrying Cry toxins showed no toxicity to BPH (Han et al., 2011). Toxicities of Bt toxins to hemipteran insects were previously tested such as in aphids (Cyt1Aa, Cry1Ac, Cry3Aa, Cry4Aa, Cry11Aa and cyt2Aa) (Porcar et al., 2009; Li et al., 2011; Chougule et al., 2013) and Lygus hesperus (TIC807) (Baum et al., 2012). In most of the tests, low to moderate toxicities were observed only when high doses of toxins were used. The reason of low insecticidal activity of Bt toxins in hemipteran insects is still unknown. ⇑ Corresponding author. Fax: +86 591 83789259. E-mail addresses: [email protected] (E. Shao), [email protected] (S. Liu), [email protected] (L. Lin), [email protected] (X. Guan). 1 Present address: 205 Binzhou Road, Fuzhou, Fujian 350008, PR China. 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.09.001
It has been shown that Cry1Ac toxin could be activated by gut proteases in pea aphids (Li et al., 2011). In addition, activated Cry1Ac could bind to aphid guts and the binding could be inhibited by GalNAC (Li et al., 2011). In lepidopteran insects, the intoxication pathway of Cry1A toxins includes protoxin activation, receptor binding, midgut cell membrane insertion and cell lysis (Bravo et al., 2005; de Maagd et al., 2001). Interruption of any of these steps may lead to a strongly decreased toxicity of Cry toxins in insects. Hence, understanding of the mechanisms responsible for the low toxicity of Cry toxins against hemipteran pests is important for improving the insecticidal activity of Cry toxins to BPH. In this study, we investigated proteolytic processing of Cry1Ab toxin by the proteases extracted from BPH gut. The cry1Ab gene was amplified from the plasmid p1AbPKK233-2 (obtained from Bacillus Genetic Stock Center, Ohio State University, Columbus, US) by PCR with a pair of cry1Ab specific primers (Forward: 50 -GGATCCATGGATAACAATCCGAACAT-30 ; Reverse: 50 -GTCGACT TACTATTCCTCCATAAGGAGTAATTC-30 ) containing a BamH I and a Sal I site in the forward and the reverse primers (underlined bases), respectively. The PCR fragments were double digested with the restriction enzymes BamH I and Sal I (TaKaRa, China), ligated into the plasmid pGEX-KG (GE healthcare, US) linearized with BamH I and Sal I (Guan and Dixon, 1991), and transformed into Escherichia coli BL21 strain (DE3). The GST-Cry1Ab fusion protein was isolated using a glutathione Sepharose column (GE healthcare, US) and the purified protein was recovered from the column after removal of the GST tag by thrombin digestion by following the instructions provided by the manufacturer. Protein purification resulted in a 133 kDa Cry1Ab protoxin (Fig. 1a and b). The purified Cry1Ab
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fusion protein was quantified by the Bradford method using a Coomassie Protein Assay kit (Biomiga, China). Soluble fraction and membrane fraction of gut protease extracts were prepared from 500 pieces of BPH nymphs gut using the method described by Li et al. (2011). The protein concentration of each gut protease preparation was determined using the Bradford method. The proteolytic processing of Cry1Ab by BPH gut proteases was examined using the gut protease preparations with or without pretreatment with 3 mM EDTA and 3 mM cysteine to activate cysteine protease activities (Li et al., 2011). Cry1Ab or GST-Cry1Ab was mixed with either the soluble proteases or membrane-bound proteases in a ratio of either 2:1 (soluble proteases:Cry1Ab, w/w) or 5:1 (membrane-bound proteases:Cry1Ab, w/w) and incubated at 28 °C for 10 min (only for GST-Cry1Ab), 1, 4, 8 and 16 h respectively, with moderate shaking (40 rpm). Cry1Ab protoxins were also treated with cathepsin-L and trypsin (Li et al., 2011). Protein samples of each treatment described above were analyzed by western blot analysis using a polyclonal anti-Cry1Ab serum specific to Cry1Ab. To prepare Cry1Ab specific polyclonal antibodies, a New Zealand White rabbit was immunized with trypsin activated Cry1Ab protein with Freund’s adjuvant for 4 times. Results of Cry1Ab proteolytic hydrolysis were shown in Fig. 1. For all gut protease treatments, a 60 kDa final product was accumulated over time. A similar product was also observed from the samples of trypsin digestion, indicating the 60 kDa fragment was likely the activated Cry toxin. Several other bands also appeared during the incubation period for all gut protease treatments. These peptides were intermediate products from degradation of Cry1Ab or GST-Cry1Ab. Activation of cysteine proteases with 3 mM EDTA and 3 mM cysteine increased the proteolytic activities of the gut preparations, but cysteine protease activation is required for the activation of Cry1Ab by BPH gut proteases. Slightly difference was observed in the Cry1Ab degradation patterns between the treat-
ment with soluble and membrane-bound proteases (Fig. 1). The membrane-bound proteases seemed to completely hydrolyze Cry1Ab to the 60 kDa fragment after 16 h incubation, although two bands were produced in the samples processing without cysteine protease activators (Fig. 1a). Notably, a band slightly larger than the 60 kDa fragment was observed either in the samples of protoxins incubated with soluble proteases lacking cysteine protease activators (Fig. 1a) or GST-Cry1Ab processed with soluble proteases (Fig. 1b), suggesting either the protease activities in the soluble fraction were lower comparing to the membrane-bound proteases, or the protease components were different from the membranebound proteases. We next tested insecticidal activity of the Cry1Ab processed by BPH proteases. Twenty larvae of diamondback moth (DBM), Plutella xylostella, susceptible to Cry1Ab toxin, were fed with protoxin or Cry1Ab toxins processed with either trypsin or BPH proteases using a leaf residue bioassay procedure (Tabashnik et al., 1990) and larvae fed on PBS was conducted as a negative control. A similar test for assessment of Cry1Ab toxicity to BPH was also conducted. Protoxin or Cry1Ab toxins activated with different methods were mixed with D-97 artificial diet and fed to Nilaparvata lugens by a membrane feeding assay (Fu et al., 2001). The bioassay experiments were repeated 9 times and mortality from each treatment was recorded after 48 h. The bioassay results showed that the Cry1Ab activated by BPH gut proteases had similar normal toxicity as that of the protoxin and trypsin-activated toxins in DBM larvae (Table 1). No significant difference was observed between the LC50 of the trypsin-activated Cry1Ab (0.95 (0.83–1.06) lg/mL) and the Cry1Ab activated by the BPH proteases (1.01 (0.95–1.08) lg/mL) (one-way ANOVA, df = 18, F = 0.400, P = 0.535). However, Cry1Ab protoxin and the toxins activated by trypsin or cathepsin-L showed similar low toxicity in BPH. The LC50s of Cry1Ab protoxin, cathepsin-L treated and trypsin-treated toxins
kDa 170 130 95 72 55 43 EDTA and Cysteine (
)
EDTA and Cysteine (
Soluble proteases
)
a
EDTA and Cysteine (
)
EDTA and Cysteine (
)
Membrane-bound proteases
kDa
kDa
170 130 95 72 55
72 55
43
Soluble proteases
b
Membrane-bound proteases
c
Fig. 1. In vitro processing of Cry1Ab toxin (a and b) and interaction with brown planthopper, BPH midgut (c). For the in vitro processing, Cry1Ab protoxin was incubated with the soluble proteases or membrane-bound proteases for 1 h, 4 h, 8 h and 16 h, respectively. Processed Cry1Ab products after incubation with soluble or membrane-bound gut proteases were separated by SDS–PAGE gel electrophoresis and Cry1Ab proteins were detected by Western blot with a rabbit polyclonal Cry1Ab serum as the primary antibody and an anti-rabbit-IgG, HRP conjugated, as the secondary antibody. Cry1Ab activated by BPH gut proteases was incubated with the BBMV from BPH and DBM guts in binding buffer (PBS, 0.1% BSA, 0.1% Tween 20, pH 7.6) at 28 °C for 1 h and then centrifuged to pull down the Cry1Ab bound to the BBMV. The BBMV pellets bound with Cry1Ab were washed three times. The detection of BBMV-bound Cry1Ab proteins was the same as that of in vitro processed Cry1Ab proteins. (a) In vitro processing of Cry1Ab protoxin by proteases in BPH guts; (b) in vitro processing of GST-Cry1Ab fusion protein by pre-activated proteases in the soluble and the membrane fraction of BPH guts; and (c) pulldown assay of Cry1Ab bound to the gut BBMV from BPH and DBM.
257
E. Shao et al. / Journal of Invertebrate Pathology 114 (2013) 255–257 Table 1 Susceptibility of BPH and DBM to Cry1Ab protoxin and activated toxins.
DBM BPH
Protoxin (95% CI) (LC50 lg/ml)
Trypsin-processed (95% CI) (LC50 lg/ml)
Cathepsin L-processed (95% CI) (LC50 lg/ml)
BPH gut proteases processed (95% CI) (LC50 lg/ml)
0.88 (0.74–1.02) 190.23 (136.67–243.80)
0.95 (0.83–1.06) 173.49 (128.07–218.92)
N/A 180.32 (125.09–235.55)
1.01 (0.95–1.08) N/A
CI, confidence interval. LC50, 50% lethal concentration the LC50 value and their 95% fiducial limits were assessed by probit analysis using excel and the online statistical tools (http://www.xuru.org/st/ DS.asp).
in BPH were 180.32 (125.09–235.55) lg/mL, 173.49 (128.07– 218.92) lg/mL and 190.23 (136.67–243.80) lg/mL, respectively, which was more than 200-fold higher than the LC50 in diamondback moth (0.88 lg/mL) (Table 1). There was no statistical difference among toxicity of protoxin, cathepsin-L activated and trypsin-activated toxins in BPH (Table 1). To test the affinity of the activated Cry1Ab to midgut BBMVs from BPH and DBM, BBMVs of N. lugens and P. xylostella were prepared using the well established preparation method by Wolfersberger et al. (1987). Pull-down assay of Cry1Ab binding to gut BBMV (either from BPH or DBM) was conducted as previously described by Pérez et al. (2005). The BBMV-bound Cry1Ab toxin was detected by western blot analysis. Results showed that the 60 kDa BPH gut protease-activated toxin could bind to the midgut BBMV from DBM, while its binding to the BBMV from BPH was minimally detectable (Fig. 1c). Results from this study, together with previous works on aphids, demonstrated that some Cry toxins could be activated in the midgut of hemipteran insects with sap sucking mouthparts. However, activated Cry1Ab toxins bind to the midgut BBMV of BPH to a much less extent in comparison with the binding to the midgut BBMV from DBM. The similar phenomenon was observed in pea aphids (Li et al., 2011) which is consistent with low toxicity in BPH and other hemipteran insects. Inhibition of either trypsin or cysteine proteases should be performed in further study to determine which protease plays more significant role in Cry1Ab toxin activation in BPH gut. On the other hand, modification of Bt toxins to increase binding of the toxin to the midgut of target insects is an effective approach to improve its insecticidal activity and expand its insecticidal spectrum (Pardo-López et al., 2009; Chougule et al., 2013). Cry receptors APN, alkaline phosphates (ALP) and cadherin in the midgut of hemipteran pests have been identified (Cristofoletti et al., 2006; Liu et al., 2012). Future studies to understand the interactions between Cry functional domains and potential receptors on the gut epithelium of hemipteran insects will help developing novel Cry toxins targeting hemipteran pests.
Acknowledgments We thank Institute of Plant Protection, Chinese Academy of Agricultural Sciences providing polyclonal anti-Cry1Ab serum for this research. We also thank Guillaume Tetreau for critical reading and editing of the manuscript. This study is supported by Key Projects of Fujian Provincial Department of Science and Technology (Grant Nos. 2011N5003 and 2013N0003), National Natural Science Foundation of Fujian Province (Grant No. 2013J01079), Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20093515110010), National Advanced Technology Research and Development Program of China (863 Program) (Grant No.
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