Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac

Abstract

Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis. Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis. Bacillus thuringiensis (Bt) 3The abbreviations used are: Bt, B. thuringiensis; APN, aminopeptidase-N; PBS, phosphate-buffered saline; Cry, crystalline inclusion protein; AP, alkaline phosphatase; dsRNA, double-stranded RNA; RNAi, RNA interference; HaAPN, H. armigera APN; DEPC, diethyl pyrocarbonate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; RT, reverse transcription. is a Gram-positive soil bacterium that produces crystalline inclusions in spore mother cells. These inclusions are composed of proteins known as Cry toxins that are toxic to the larvae of different insect orders and a few other invertebrates. Several strains of Bt are used as alternative insecticides to control the important pests of agricultural crops and also public health pests, especially mosquitoes (1Schnepf E. Crickmore N. Van Rie J. Lereclus D. Baum J. Feitelson J. Zeigler D.R. Dean D.H. Microbiol. Mol. Biol. Rev. 1998; 62: 775-806Crossref PubMed Google Scholar). Incorporation of genes encoding these insecticidal proteins into various crops resulted in widespread cultivation of these transgenic crops. Global cultivation of these Bt-protein expressing crops resulted in economic and environmental benefits. There have been intense efforts to understand the mode of action of these insecticidal proteins. The larvae feed on the insecticidal protein, which upon solubilization in the alkaline midgut of insect is further proteolytically processed to 66-kDa mature protein that binds to specific receptors at the midgut epithelium. The activated monomeric toxin molecules oligomerize and insert into the membrane resulting in the formation of pores. The formation of the insertion pores results in osmotic imbalance of the membrane epithelium leading to collapse of the intestine and finally the death of the insect (2DeMaagd R.A. Bravo A. Crickmore N. Trends Genet. 2001; 17: 193-199Abstract Full Text Full Text PDF PubMed Scopus (473) Google Scholar, 3Gould F. Annu. Rev. Entomol. 1998; 43: 701-726Crossref PubMed Scopus (1060) Google Scholar). Although there is an absolute agreement on the above mentioned scheme of mode of action, the identity of the receptor(s) to these insecticidal proteins and the precise mechanism of formation of the pore in the membrane are not very clear. Two membrane proteins, cadherin and aminopeptidase-N (APN), have been implicated as possible receptors to insecticidal proteins. The putative receptor molecules have been cloned, heterologously expressed, and have been shown to bind Cry1A proteins by ligand blot analysis (4Denolf P. Hendrickx K. VanDamme J. Jansens S. Peferoen M. Egheele D. VanRie J. Eur. J. Biochem. 1997; 248: 748-761Crossref PubMed Scopus (89) Google Scholar, 5Garczynski S.F. Adang M.J. Insect. Biochem. Mol. Biol. 1995; 25: 409-415Crossref Scopus (71) Google Scholar, 6Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (358) Google Scholar, 7Belfiore C.J. Vadlamudi R.K. Osman Y.A. Bulla Jr., L.A. Biochem. Biophys. Res. Commun. 1994; 200: 359-364Crossref PubMed Scopus (30) Google Scholar, 8Vadlamudi R.K. Weber E. Ji I. Ji T.H. Bulla Jr., L.A. J. Biol. Chem. 1995; 270: 5490-5494Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 9Nagamatsu Y. Toda S. Yagamuchi F. Ogo M. Kogure M. Nakamura M. Shibata Y. Katsumoto T. Biosci. Biotechnol. Biochem. 1998; 62: 718-726Crossref PubMed Scopus (84) Google Scholar, 10Nagamatsu Y. Koike T. Sasaki K. Yoshimoto A. Furukawa Y. FEBS Lett. 1999; 460: 385-390Crossref PubMed Scopus (117) Google Scholar, 11Yaoi K. Kadotani T. Kuwana H. Shinkawa A. Takahashi T. Iwahana H. Sato R. Eur. J. Biochem. 1997; 246: 652-657Crossref PubMed Scopus (89) Google Scholar). Although functional proof for cadherin as a Cry1A protein receptor has been demonstrated by cytolysis of insect cells expressing lepidopteran cadherin genes on exposure to Cry1Ab/Cry1Aa (10Nagamatsu Y. Koike T. Sasaki K. Yoshimoto A. Furukawa Y. FEBS Lett. 1999; 460: 385-390Crossref PubMed Scopus (117) Google Scholar, 12Tsuda Y. Nakatani F. Hashimoto K. Ikawa S. Matsuura C. Fukada T. Sugimoto K. Himeno M. Biochem. J. 2003; 369: 697-703Crossref PubMed Scopus (49) Google Scholar, 13Hua G. Jurat-Fuentes J.L. Adang M.J. J. Biol. Chem. 2004; 279: 28051-28056Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), a similar characterization of APN-Cry1A interaction has not been investigated yet. The only evidence that lepidopteran APN is a functional receptor to Cry1A comes from elegant studies by Gill and Ellar (14Gill M. Ellar D.J. Insect. Mol. Biol. 2002; 11: 619-625Crossref PubMed Scopus (101) Google Scholar). They expressed APN receptor of Manduca sexta in Drosophila and demonstrated that the transgenic fly acquired sensitivity to insecticidal protein Cry1Ab, a bioactive protein against M. sexta. We have earlier demonstrated the functional relevance of APN of Spodoptera litura to Cry1C protein (15Rajagopal R. Sivakumar S. Agrawal N. Malhotra P. Bhatnagar R.K. J. Biol. Chem. 2002; 277: 46849-46851Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar) by direct gene silencing strategy using RNAi. In spite of these efforts, a complete molecular characterization of the receptor to Cry1Ac protein in the major polyphagus pest Helicoverpa armigera is lacking. The insect H. armigera (family, Noctuidae; order, Lepidoptera) is the most important insect pest affecting a wide range of economically important crops. The insecticidal protein Cry1Ac is extremely effective against this insect. We have reported earlier the identification and cloning of aminopeptidases from this insect and have demonstrated HaAPN1 interaction with all the three Cry1A family of proteins (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar). Now we demonstrate the specificity and functional interaction of Cry1Ac protein with HaAPN1 by insect cell line (Sf21)-based assays and by specific RNAi both in cell lines and in whole insects. Affinity studies by surface plasmon resonance also revealed tight binding of Cry1Ac with HaAPN1. Membrane Preparation—The Sf21 cells infected with HaAPN1 virus (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar) at a multiplicity of infection of 10 were harvested after 65 h of infection. The cells were washed twice in buffer 10 10 and cell in and the at for 10 at The further at for at The of washed twice in buffer and finally in the in and at further Ligand of cells expressing HaAPN1 with from cells were as earlier (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar). The were in and a the membrane in in PBS, were with activated Cry1Ac toxin for The blot washed with and with Cry1Ac for three with PBS, were with alkaline The blot with after three with of the from Sf21 cells expressing HaAPN1 were at for h with in buffer The were by for at The from the HaAPN1 protein by and with The of the by the using in the protein and for of earlier (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar). a at a of The amount of at after at The using a of at The from Sf21 cells were and used as a of HaAPN1 The with and of to used to the protein. the were for and on HaAPN1 were and against and The protein using to The protein further using that with buffer mentioned The of HaAPN1 on and the purified HaAPN1 were using The a of HaAPN1 protein and used for interaction analysis with Cry1Ac protein by The purified protein also by using (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar). of by the interaction Cry1Ac protein and surface plasmon resonance on BIAcore The purified in 10 and on cell at a of on the surface of a by to the buffer and used the the cell surface with to The at different buffer and at a of 10 for both and of were by for at to a an protein used as an at a of and The were and using analysis The were to a binding to of Cry1Ac Cry1Ac toxin as inclusion by the reported earlier by Dean D.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar) from a the Cry1Ac gene The amount of the toxin by the inclusion on The inclusion the toxin were in and activated with at a for at This activated toxin purified by and with in This Cry1Ac protein and with in a to a of of cells were and at in and were as a to in of cells were harvested from a and to a at cells the cells for were infected with HaAPN1 (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar) at a multiplicity of infection of for a of the virus and the cells were with and at the and the cell washed twice with PBS, and with of activated Cry1Ac insecticidal protein in at The cells were also with P. to as a The cells were with the proteins for a of after which were a The of cells in a in to the cells in the an of for and three of from the midgut of H. armigera larvae reported by (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google Scholar). of by using the and The of H. armigera in and used for the of a the gene for of P. in used as earlier P. A. A. Agrawal N. Bhatnagar R.K. Mol. Microbiol. 2002; PubMed Scopus Google Scholar). The were by using and reverse The purified and used as for after the in by P. A. A. Agrawal N. Bhatnagar R.K. Mol. Microbiol. 2002; PubMed Scopus Google Scholar). The and RNA were used to RNA and from the dsRNA, of RNA and RNA were to 65 and by h by for at The with and with in the of at The washed with and in to a of RNAi in the cells were infected with HaAPN1 at an multiplicity of infection of for the and the cells were washed twice with of not to the cells. These cells were with 10 of of in of and for h at of to and the cells were at for were harvested from for RNA transcript analysis and blot The other of cells processed for toxin as earlier for toxin of cells. These were of H. armigera in an with a of a of and 10 h of The larvae were on the H. J. Google Scholar). larvae were used for the of of of into the larvae 10 to a used for this The larvae with of as of the RNA from the midgut using to The purified from the RNA by using and and used as for reverse of and the cells were harvested and processed as the earlier A. Malhotra P. Bhatnagar R.K. Biochem. Res. Commun. 2004; PubMed Scopus Google Scholar). The cell in and on to insect midgut in were and their protein were by the the membrane for in buffer in and washed three with have a on the amount of protein the were on a and processed as The blot with of for The were with of for and washed three with in on the membrane for with PBS, the blot with in alkaline Cry1Ac on H. armigera of Cry1Ac toxin were as reported earlier by Dean D.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The amount of the toxin by the inclusion on from to in 10 and on The and in a of a H. armigera on and to the toxin for h the to a after and the by analysis using the larvae were for and the three were for larvae of H. armigera for of RNAi RNA from the cells using the and to using the and the mentioned earlier and used for the cloning of the were used for the The and analysis in in of of and reverse of used as the to the of and The by using the and were used to the levels of a in control and larvae to the analysis Res. 2001; Scopus Google Scholar). of HaAPN1-expressing Sf21 with Cry1Ac expressed in the membrane of cells infected with a interaction with Cry1Ac protein on ligand blot analysis There an in with an in HaAPN1 that the interaction specific and of membrane of Sf21 cells expressing HaAPN1 with revealed a interacting protein corresponding to the of HaAPN1. The functional of expression of HaAPN1 further investigated by insect cells expressing HaAPN1 to various of insecticidal protein Cry1Ac. of cells expressing HaAPN1 with Cry1Ac of cells displayed and and were not in Sf21 that the expressing HaAPN1 directly with the insecticidal protein, and as a are The of cells with after Cry1Ac in different of cells were were and The cells with an protein to Cry1Ac, not of HaAPN1-expressing cells after with of activated Cry1Ac toxin for a of h are the of of and three an of cells to the of cells in a of to the of R.A. D. J. 1998; PubMed Scopus Google Scholar). The were by a using the in the range of for of cells with of cells with from other at with the are not different from with different are different from other from other at with the are not different from with different are different from other from other at with the are not different from with different are different from other from other at with the are not different from with different are different from other cells to from other at with the are not different from with different are different from other from other at with the are not different from with different are different from other from other at with the are not different from with different are different from other in a RNAi of and on Cry specificity of HaAPN1-expressing cells and insecticidal protein Cry1Ac by the expression of HaAPN1. after infection with were for h with of HaAPN1. The of transcript by and the amount of expressed protein by blot analysis using after the expression of HaAPN1 and protein reduced in cells as with control cells and cells with to The transcript reduction to on cells were similar reduction in the HaAPN1 protein expression after of Cry1Ac insecticidal protein on Sf21 cells resulted in a interaction with the insecticidal protein expression of HaAPN1 directly correlated with reduced of cells of cells were after Cry1Ac toxin as with cells in HaAPN1-expressing cells. of Cry1Ac on H. armigera the specificity of interaction HaAPN1 and Cry1Ac and also the of this interaction on Cry1Ac protein to H. armigera we first to the of Cry1Ac on H. armigera larvae 10 that were with of on the 10 of were to of Cry1Ac toxin from to The results revealed that the only after toxin at the of of different of Cry1Ac on larvae of H. armigera larvae were used for the of Cry1Ac toxin were in and on a of The in a and larvae were in and to feed on the for the larvae were on a on the after toxin and the by analysis using the and to The as the of Cry1Ac insecticidal protein larvae were for and three The at different the at 10 and 10 in the range of for after 10 after 10 in a clearly on the of HaAPN1 as a receptor for Cry1Ac, we to a for the larvae to after and this a of the The larvae 10 after with of were to for resulted in no and on the 10 on the toxin Cry1Ac, from to this the and to the as the larvae to of Cry protein. after of resulted in a of the larvae The as of Cry1Ac insecticidal protein RNAi of on H. armigera and on Cry1Ac identified the range for Cry1Ac on larvae of H. we these larvae with of of HaAPN1. of midgut RNA of after a reduction in transcript levels of with control with of the HaAPN1 protein expression in these revealed an reduction of HaAPN1 expression in with control and We the of larvae to Cry1Ac. These larvae were for and on the were to different of Cry1Ac, and in the control and larvae a in only of the larvae that were with at the larvae that were with of the toxin and only against in of Cry1Ac on larvae of H. armigera Two after the larvae were with Cry1Ac and after h the larvae were on a larvae were used and the assays were on different on the after toxin The were by and their at with a and are different from after 10 in a and of HaAPN1 cells HaAPN1 as a protein solubilization for were used to the protein in a of cell CHAPS, and and also to the protein in the a at the of to the protein in protein for and toxin the from the protein using an and the to that the protein and The by this protein is in by and similar to the results earlier with whole cells expressing HaAPN1 (16Rajagopal R. Agrawal N. Selvapandiyan A. Sivakumar S. Ahmad S. Bhatnagar R.K. Biochem. J. 2003; 370: 971-978Crossref PubMed Google of of of HaAPN1 protein from cells The APN with protein by HaAPN1-expressing Sf21 cell The for by the of after of of the protein with of The at of processed Sf21 cell from HaAPN1-expressing cell The of by an of of an of three with in in the range of in in the of APN in the of APN HaAPN1 from Sf21 in a The HaAPN1 protein purified using which resulted in HaAPN1 and and This further to and using and HaAPN1 in which were and to finally resulting in a HaAPN1 The purified protein used for toxin interaction and Affinity of Cry1Ac and HaAPN1 binding of Cry1Ac toxin protein to purified the receptor on a and the toxin receptor formation by surface plasmon binding of Cry1Ac with HaAPN1 The for Cry1Ac toxin protein and HaAPN1 are in The of Cry1Ac as a we also the binding of HaAPN1 with an protein to Cry1Ac, There no binding of to no interaction of HaAPN1 receptor protein against Cry1Ac in a H. armigera is the most important agricultural insect pest in the as is widespread in and of and and and important crops and and The insecticidal protein Cry1Ac by the bacterium B. thuringiensis is the most of all the insecticidal proteins by this against the polyphagus insect pest H. armigera. of the in for insect pests this the transgenic lines in the this protein, and has very in the of H. resulting in reduction in C. Global of Scholar). is very for to understand the mechanism by which Cry1Ac H. armigera. on midgut membrane of lepidopteran to the putative receptor for Cry1Ac have reported to aminopeptidases have been and expressed from M. and H. armigera and have been shown to with Cry1A toxins (4Denolf P. Hendrickx K. VanDamme J. Jansens S. Peferoen M. Egheele D. VanRie J. Eur. J. Biochem. 1997; 248: 748-761Crossref PubMed Scopus (89) Google Scholar, 5Garczynski S.F. Adang M.J. Insect. Biochem. Mol. Biol. 1995; 25: 409-415Crossref Scopus (71) Google Scholar, 6Knight P.J.K. Crickmore N. Ellar D.J. Mol. Microbiol. 1994; 11: 429-436Crossref PubMed Scopus (358) Google Scholar). this we have different aminopeptidases from H. HaAPN1 and and have shown that both these proteins with Cry1Ac. have been demonstrated by ligand blot analysis a of the which not A. S. Dean D.H. Adang M.J. Microbiol. 2002; PubMed Scopus Google Scholar). direct functional of putative receptor has also been by various other cell lines have earlier been used to Bt insecticidal protein membrane insertion and formation M.J. M. J.L. R. J. Biol. 1995; PubMed Scopus Google Scholar, R. D. R. J.L. J. 1994; Scopus Google Scholar). of insect cell lines have also been for their to different Cry proteins K. A. Scholar, J. 1994; PubMed Scopus Google Scholar), and that cells very to and with Cry1C R.A. D. J. 1998; PubMed Scopus Google Scholar). The of using insect cells expressing putative receptor proteins and the of the protein to as a receptor to Bt toxin has been shown by (10Nagamatsu Y. Koike T. Sasaki K. Yoshimoto A. Furukawa Y. FEBS Lett. 1999; 460: 385-390Crossref PubMed Scopus (117) Google Scholar). They have been to demonstrate and of cells expressing cadherin protein of B. after with Drosophila cell line expressing M. sexta cadherin gene and with G. Jurat-Fuentes J.L. Adang M.J. J. Biol. 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Biochem. 2001; PubMed Scopus Google Scholar, F. J.L. Dean D.H. Mol. Microbiol. PubMed Scopus Google Scholar). The for in this for HaAPN1 with Cry1Ac to which is the for protein studies in the range of the binding of Cry1Ac to HaAPN1 to a very tight binding interaction to the very of the We are by the in the and reported from other insect possible the different Although we have used a for solubilization from other studies have as In both the has been as a for of In the the have all that not the of and that the not and at different on the APN The of to the that the APN in this has been heterologously expressed and earlier studies on APN purified from membrane of the insect midgut F. J.L. Dean D.H. Mol. Microbiol. PubMed Scopus Google Scholar). RNA interference has an effective and important to the functional relevance of various proteins and genes in an in in various RNAi by results in of the N. A. Malhotra P. Bhatnagar R.K. Microbiol. Mol. Biol. 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Entomol. 1999; Scopus Google Scholar), larvae of H. armigera are not to Although of RNAi into the with a with on H. armigera resulted in transcript reduction not to the insect for after resulted in no in 10 and of against Cry1Ac of of in H. armigera at the as as the with These results with results with Sf21 cells expressing HaAPN1 reduced to Cry1Ac after RNAi. these results demonstrate a functional for HaAPN1 in the of the insecticidal protein Cry1Ac to H. not of other Cry at the there are cadherin as a receptor to Cry1Ac in F. 2001; PubMed Scopus Google Scholar). is that both protein for Cry1Ac and the of the midgut of insect are a of interacting A. I. J. C. J. R. M. Gill M. Biophys. 2004; PubMed Scopus Google Scholar). the insecticidal protein into these receptors is a of intense We and by with the BIAcore with