Baculovirus Resistance in Codling Moth Is Virus Isolate-Dependent and the Consequence of a Mutation in Viral Gene Pe38

Baculovirus resistance in codling moth is virus isolate-dependent and the consequence of a mutation in viral gene pe38

Manuela M. Gebhardta, Karolin E. Eberlea,b, Pit Radtkeb, and Johannes A. Jehlea,b,1

aInstitute for Biological Control, Federal Research Center for Cultivated Plants, Julius Kühn-Institut, 64287 Darmstadt, Germany; and bDepartment of Phytopathology, Agricultural Service Center Palatinate (DLR Rheinpfalz), 67435 Neustadt an der Weinstrasse, Germany

Edited by Fred L. Gould, North Carolina State University, Raleigh, NC, and approved September 26, 2014 (received for review June 16, 2014) The baculovirus Cydia pomonella granulovirus (CpGV) is widely ap- Italy (6), Switzerland (2), and the Netherlands (2) (14). For the plied as a biocontrol agent of codling moth. After field resistance of codling moth strain CpR, which originated from a resistant field codling moth populations had been observed against the commer- population in south Germany, as well as the genetically ho- cially used Mexican (M) isolate of CpGV, infection experiments of lar- mogenous laboratory strain CpRR1, which derived from CpR, vae of the resistant codling moth strain CpRR1 showed that several the mode of inheritance was revealed to be incompletely domi- other naturally occurring CpGV isolates (I12, S, E2, and I07) from dif- nant, monogenic, and linked to the Z (sex) chromosome (15, 16). ferent geographic origins are still infectious to resistant CpRR1. Whole- A similar mode of inheritance was also observed in resistant genome sequencing and phylogenetic analyses of these geographic Czech and French codling moth populations arguing for a more CpGV variants revealed that their genomes share only a single com- or less universal mode of resistance in Europe (17, 18). These mon difference from that of CpGV-M, which is a mutation coding for data led to the hypothesis that a genetic adaptation of codling a repeat of 24 nucleotides within the gene pe38; this mutation results moth to CpGV-M infection had occurred and was selected for by in an additional repeat of eight amino acids that appears to be inserted the intensive use of products containing CpGV-M (15). to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abol- Insects manifest miscellaneous strategies to resist pathogens SCIENCES ished virus infection in codling moth cells and larvae, demonstrating but lack an adaptive immune system. Insect defense to viral

that it is an essential gene. When the CpGV-M deletion mutant was infections involves nonspecific factors such as physical barriers, APPLIED BIOLOGICAL repaired with pe38 from isolate CpGV-S, which originated from the enzymatic responses, and increasing ejection of infected midgut commercial product Virosoft and is infectious for the resistant codling cells as larvae age, as well as specific factors involving cellular and moth strain CpRR1, the repaired CpGV-M mutant was found to be fully humoral immunity (19). Physical barriers to infection include the pe38 infectious for CpRR1. Repair using from CpGV-M restored infec- perithropic membrane (PM), an ultrafilter for particles with size tivity for the virus in sensitive codling moth strains, but not in CpRR1. exclusion greater than 30 nm (20). Melanization of the cuticula Therefore, we conclude that CpGV resistance of codling moth is di- mediated by phenoloxidase enzymes is involved in the encapsu- rected to CpGV-M but not to other virus isolates. The viral gene lation reaction of pathogens, such as bacteria, fungi, or virus- pe38 is not only essential for the infectivity of CpGV but it is also the infected cells (21, 22). Developmental resistance, the decreasing key factor in overcoming CpGV resistance in codling moth. susceptibility of larvae with increasing age, is rather common, and is mediated by infected midgut cell-sloughing (23–25). Behavior Cydia pomonella granulovirus | codling moth | resistance | modifications also count among insect defense strategies (19). genome sequencing | mutation | resistance management Significance he codling moth Cydia pomonella L. is a worldwide occurring insect pest that infests apples, pears, and walnuts. The larvae T Registered in 34 countries worldwide, Cydia pomonella gran- of codling moth bore into the fruit and cause severe economic ulovirus (CpGV) is a highly important biological agent to con- damage if not controlled. A number of chemical and biological trol the codling moth in pome fruit production. Since 2005, 38 agents are available for the control of codling moth. One of codling moth field populations resistant to CpGV products the most efficient biological control agents (1–5) is the Cydia containing the Mexican isolate (CpGV-M) were discovered in pomonella granulovirus (CpGV), which belongs to the dsDNA Europe, and this was the first demonstrated field resistance virus family Baculoviridae (genus Betabaculovirus). CpGV was against commercial baculovirus products. By identifying and first discovered in Mexico (Mexican isolate, CpGV-M) in 1963 (6); sequencing different resistance-breaking CpGV isolates, it this isolate was later developed to commercial products now was found that resistance of codling moth is directed toward registered in 34 countries worldwide. The genome sequence of CpGV-M only. As shown by mutation experiments, CpGV-M is the in vivo cloned strain CpGV-M1 is ∼123 kbp and encodes for prone to resistance because of a 24-nucleotide repeat within 143 ORFs (7). Based on SNPs in highly conserved genes, dif- the viral gene pe38.Thus,pe38 can be used as a genetic ferent geographic CpGV isolates were classified into four ge- marker to identify resistance-breaking CpGV isolates for re- nome types, A–D (8). CpGV exhibits an extremely narrow host sistance management programs. range that is restricted to C. pomonella and a very few closely

related tortricids (Lepidoptera) (9). Author contributions: M.M.G., K.E.E., P.R., and J.A.J. designed research; M.M.G., K.E.E., The development of resistance to baculovirus infection was and P.R. performed research; M.M.G., K.E.E., P.R., and J.A.J. analyzed data; and M.M.G. thought to be unlikely before 2005 (10, 11), which is when the and J.A.J. wrote the paper. first cases of resistant codling moth populations with a 1,000- to The authors declare no conflict of interest. 100,000-fold reduced susceptibility to commercial CpGV prod- This article is a PNAS Direct Submission. ucts containing the isolate CpGV-M were reported from organic Data deposition: The sequences reported in this paper have been deposited in the Gen- apple plantations in Germany and France, where CpGV prod- Bank database (accession nos. KM217573–KM217577). ucts had been intensively applied (12, 13). Since then, 38 apple 1To whom correspondence should be addressed. Email: [email protected]. plantations with CpGV resistance have been identified in Aus- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tria (2 orchards), Czech Republic (1), France (3), Germany (22), 1073/pnas.1411089111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1411089111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 AAAbd Abd A d Results 100 Assays of Different Naturally Occurring Isolates. After the isolate CpGV-I12 had been identified as the first resistance-breaking CpGV 80 isolate (30), we wondered, whether other geographic CpGV isolates bc would also differ in virulence against resistant and nonresistant 60 codling moth strains. Therefore, the susceptibilities of CpS and CpRR1 larvae were tested for the isolates CpGV-M, -I12, -S, -E2, and -I07 in laboratory assays using 1 × 103 occlusion bodies (OB) per 40 Mortality [%] Mortality milliliter, which corresponds to a mortality of ∼70–90% in CpS in a 14-d activity assay. In CpS, all selected CpGV isolates were able to 20 cause 70–90% mortality with no significant differences (t test, P < 0.05; Fig. 1). In contrast, that the CpRR1 strain was not a 0 susceptible to CpGV-M was clearly shown by the significant n/N 105/2M 114/3 103/3I12 71/2 67/2S 100/3 148/3E2 68/2 71/2I07 108/3 difference between the mortality caused by CpGV-M and all other tested isolates (t test, P < 0.05). CpRR1 was susceptible M I12 S E2 I07 to all other CpGV isolates, however, although at different Fig. 1. Mortality of CpS (white bars) or CpRR1 (gray bars) orally infected with levels ranging from 41% to 95% mortality. Thus, all isolates 1 × 103 OB/mL of the isolates CpGV-M, -I12, -S, -E2, or -I07. Mortality data were except CpGV-M were able to infect CpRR1. recorded 14 d postinfection and corrected for control mortality (<10% in untreated control). Error bars show SD. Columns marked by different letters Genome Sequencing and Phylogeny. When whole-genome sequenc- differ significantly (t test, P < 0.05; uppercase for CpS, lowercase for CpRR1). ing was conducted for the isolates CpGV-M, -I12, -S, -E2, and -I07, The tested virus isolates, the total number of tested individuals (n) and the sequence comparisons revealed highly conserved genomes with number of independent replicates (N) are given below the chart. similar sizes and GC contents (Table 1). The major difference between the genomes was the lack of four ORFs in CpGV-I07 (8). Feeding behaviors, e.g., can strongly affect the risk of insects for In addition, there were further SNPs and some sequence hetero- baculovirus infection as shown for Lymantria dispar L., which geneities, mainly in noncoding regions. The most striking and only exhibit heritable cadaver-avoidance behaviors (26). common difference among all resistance-breaking isolates and An alternative way for insects to get rid of virus-infected cells is via CpGV-M, however, was a repeat of 24 nt within ORF24. This programmed cell death—the apoptosis pathway (27). Baculoviruses, ORF codes for the gene pe38, which was described as an imme- however, are able to block apoptosis with the help of different virus diate-early expressed transactivator in AcMNPV (33). The encoded antiapoptotic proteins. In Autographa californica multiple repeated sequence encodes for the amino acid motif DTVD, nucleopolyhedrovirus (AcMNPV), these proteins—namely P35 and which appears to have been duplicated twice and is thus present P49—are inhibitors of the insect caspases. These inhibitors of apo- three times in CpGV-M; however, it is present only once in the ptosis (IAPs), thereby, ensure a permissive virus infection (28). isolates CpGV-I12, -S, -E2, and -I07 (Fig. 2). For codling moth, however, resistance based on the PM, the A phylogenetic analysis based on 35 core genes of the different midgut, or the immune system has been excluded for CpGV (16, CpGV isolates (Fig. 3) revealed that CpGV-M (genome type A) 29). Lack of CpGV DNA replication and a systemic resistance in is the most derived type among the virus isolates, whereas all five instars indicated an early block to virus replication in re- CpGV-I07 belongs to the phylogenetically oldest genome type C sistant codling moth individuals (29, 30). At the same time, it was of CpGV and is therefore ancestral to the other isolates. CpGV- observed that certain CpGV isolates, such as I12 or NPP-R1/R4, E2 appeared to be a mixture of genotypes, because it contained were able to infect larvae from resistant codling moth strains (30, the predominant genome type B (14) and a small portion of 31). Therefore, these naturally occurring CpGV isolates from SNPs typically for type A genomes. CpGV-S is proposed to different geographic origins were considered resistance-breaking represent a further genome type, termed type E. The observed isolates. Some of these isolates, meanwhile, replace CpGV-M in resistance of codling moth was isolate-specific and subjected commercial biocontrol agents in Europe and demonstrate the to CpGV-M (genome type A) only, rather than a general re- importance of identifying resistance-breaking CpGV variants sistance to CpGV. Because the 24-nt repeat in the ORF24 was (30–32) for managing CpGV-resistant codling moth populations; the solely common difference between CpGV-M and all however, their functional difference to CpGV-M allowing them tested CpGV isolates, it was hypothesized that pe38 is the to overcome CpGV resistance remained unknown. genetic determinant involved in overcoming CpGV resistance In this study we compared different naturally occurring geo- in CpRR1 larvae. graphic CpGV isolates representing all known CpGV genome types (8) in laboratory assays for their infectivity to susceptible (CpS) and Bacmid Construction and Pseudovirus Production. To elucidate the resistant (CpRR1) codling moth strains. We found that all tested potential role of pe38 in CpGV resistance, pe38 knockout and isolates—except CpGV-M—were able to overcome resistance in recovery mutants were constructed with the help of a CpGV CpRR1 larvae. Whole-genome sequencing of these isolates bacmid (34). This bacmid, bacCpGV, is based on the genome of revealed a single common difference in all resistance-breaking iso- CpGV-M1, an in vivo cloned genotype of CpGV-M, and con- lates, which was located in ORF24 (pe38). Therefore, occlusion tains an 8.6-kbp BAC cassette, including an attachment site for bodies of bacmid-based recombinant CpGV (in the following termed pseudoviruses) knockout and recovery mutants of pe38 were established and tested for their activity in susceptible CpS and re- Table 1. Genome comparison of the five virus isolates of CpGV sistant CpRR1 larvae. The recovery of infectivity of a CpGV-M– CpGV isolate Genome type Size, bp ORF GC, % Accession no. based pseudovirus harboring the pe38 of the resistance-breaking CpGV-S in CpRR1 larvae demonstrated the key function of pe38 in M A 123,529 142 45.3 KM217575 overcoming baculovirus resistance in codling moth. In their entirety, I12 D 124,269 142 45.2 KM217576 S E 123,193 140 45.3 KM217573 these investigations demonstrate the significance of using multiple + isolates of CpGV concurrently in field applications to avoid re- E2 B( A) 123,858 141 45.2 KM217577 I07 C 120,832 137 45.4 KM217574 sistance in codling moth populations.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1411089111 Gebhardt et al. Downloaded by guest on September 28, 2021 pe38S::eGFP I II III CpGV-MΔpe38M caused 73.4 ± 4.9% and 82.6 ± 15.1% *********** ********* mortality in CpS larvae, respectively (Fig. 6B); this confirmed MTEDDITKSVANDTVDDTVDDTVDDTIMR the functional recovery of PE38 in both constructs because the I12 T E D D I T K S V A N ------D T V D D T I M R Δ STEDDITKSVAN------DTVDDTIMR infectivity of the knockout mutant bacCpGV pe38M was ap- E2 TEDDITKSVAN------DTVDDTIM R parently restored for CpS. I07 T E D D I T K S V A N ------D T V D D T I M R However, when CpGV-Mhsp-eGFP (9.4 ± 13.0% mortality) and CpGV-MΔpe38 pe38M::eGFP (11.8 ± 12.4% mortality), both Fig. 2. Sequence alignment of the gene pe38 (ORF24) of different CpGV M carrying the CpGV-M type pe38, were fed to CpRR1 larvae, no isolates. Shown are the amino acid sequences from position 301 to 328 of < CpGV-M and from 301 to 320 of the other CpGV isolates, respectively. Dis- significant mortality (t test, P 0.05) compared with the un- tinguishable is the repeat of the amino acid motif DTVD in CpGV-M denoted treated control was observed (Fig. 6B), which implies that the pe38M::eGFP by the roman numbers above (full alignment; Fig. S1). recombinant CpGV-MΔpe38M , which was infectious for CpS, was not able to overcome resistance in CpRR1. pe38S::eGFP In contrast, CpGV-MΔpe38M induced mortality of the bacterial transposon Tn7 (mini-attTn7 site) (35) and a mini-F CpRR1 larvae of 94.6 ± 5.7% after 14 d. Thus, swapping of pe38 replicon for bacterial replication (Fig. 4), and was the basis for all ORF from the isolate CpGV-S into CpGV-M enabled the constructs used in this study. For generating the pe38 knockout recombinant virus to infect CpRR1 and confirmed the central role Δ bacmid bacCpGV pe38M, the Red/ET-mediated recombination of pe38 in overcoming resistance. In CpS larvae, the recovery mu- Δ pe38S::eGFP was used (36). Then, based on bacCpGV pe38M,tworecovery tant CpGV-MΔpe38M showed no significant difference in hsp-eGFP bacmids, carrying either the homologous pe38M ORF (from the mortality compared with the original CpGV-M and the pe38M::eGFP CpGV-M) or the heterologous pe38S ORF (from CpGV-S) were homologous recovery mutant CpGV-MΔpe38M (Fig. constructed by cloning these ORFs as eGFP fusion constructs 6B). The mortality caused by the pseudoviruses CpGV-Mhsp-eGFP, Δ pe38M::eGFP pe38S::eGFP into the modified pFastBac polyh vector (Fig. S2) (34), resulting CpGV-MΔpe38M ,andCpGV-MΔpe38M in pe38M::eGFP pe38S::eGFP in bacCpGVΔpe38M and bacCpGVΔpe38M , CpS or CpRR1 were comparable to the natural virus isolates respectively. The two recovery bacmids were transfected into cells CpGV-M or CpGV-S in CpS, and CpRR1 larvae and showed no of the C. pomonella cell line Cp14R (37), and after 20 d the significant differences in infectivity (t test, P < 0.05). transfected cells were fed to CpS larvae to produce bacmid based

pseudoviruses (Fig. 5). Controls included the same setup with Discussion SCIENCES hsp-eGFP bacCpGVΔpe38M and bacCpGV (34), which contains an For nearly 20 y, commercial CpGV products in Europe were enhanced GFP (eGFP) under the control of the Drosophila heat- solely based on the Mexican isolate CpGV-M. The preponderant APPLIED BIOLOGICAL shock promoter (hsp). This two-step approach, transfection in occurrence of resistance of codling moth to the virus isolate vitro followed by infection in vivo, was necessary to produce CpGV-M in organic apple plantations, where the application of sufficient inoculum for further in vivo activity tests because CpGV products was the main and often only measure to control Cp14R cells are extremely slow-growing and produce only codling moth, is no doubt the result of mutation and selection, the a very limited number of occlusion bodies. guiding principle of Darwinian evolution in insect populations (15, 29, 30). Studies showing that naturally occurring geo- Infectivity of Bacmids in CpS Larvae. If pe38 is essential for the onset graphic CpGV isolates, such as I12, NPP-R1/R4, and others, of the CpGV infection, it was postulated, that the knockout mutant were still infectious to resistant codling moth populations and Δ bacCpGV pe38M should not be infectious for either the permissive led to the development and registration of improved resistance- cell line Cp14R or the susceptible CpS larvae, and, therefore, breaking CpGV products (18, 30–32). Consequently, the un- no viable virus would be produced. To test this hypothesis, derstanding of resistance to CpGV and resistance-breaking by Δ Cp14R cells were first transfected with either bacCpGV pe38M CpGV are of particular importance for codling moth control hsp-eGFP or with bacCpGV (positive control), or mock trans- and may serve as a model for other baculoviruses used in insect fected (negative control) and then used as inoculum for peroral pest control. infection of CpS larvae. As shown in Fig. 6A, feeding mock- Here, we demonstrate that all known CpGV genome types (B– transfected Cp14R cells to fourth instar CpS did not cause E), except for type A (CpGV-M), are able to overcome CpGV significant mortality (3.0 ± 4.3%)after14d,andneitherdid resistance. Resistance to CpGV is, therefore, isolate-dependent Δ ± cells transfected with bacCpGV pe38M (1.0 1.4% mortality). and specific for CpGV-M; as such, it cannot be generalized In contrast, the cells transfected with the control construct bacCpGVhsp-eGFP caused significant mortality of 96.8 ± 5.5% after 14 d (t test, P < 0.05), simultaneously confirming the 24 nt repeat in pe38 knockout of pe38 and revealing the absolute importance of pe38 88 CpGV-M (A) for infectivity in codling moth larvae. 82 CpGV-I12 (D) 58 Recombinant Pseudovirus Activity Assay in CpS and CpRR1. To analyze CpGV-S (E) Δ pe38M::eGFP the viability of the recovery mutants bacCpGV pe38M CpGV-E2 (B) and bacCpGVΔpe38 pe38S::eGFP, Cp14R cells were transfected M CpGV-I07 (C) with bacmid DNA and cells were harvested as aforementioned. When the cell pellets were fed to fourth instar CpS and the larvae 0.0005 were reared for 8–10 d, small spots of eGFP fluorescence became ~0.3 CrleGV visible, indicating ongoing infection in CpS. Larvae expressing eGFP were collected and occlusion bodies from single larvae were Fig. 3. Maximum-likelihood tree based on the predicted amino acid purified, checked for their identity by partially sequencing and sequences of the 35 baculovirus core genes of five CpGV isolates and the DNA restriction endonuclease analysis, before a larger virus stock corresponding genome types (given in brackets). The most closely related was propagated in CpS larvae. The obtained occlusion bodies con- CpGV neighbor, Cryptophlebia leucotreta granulovirus (CrleGV), was used as pe38M::eGFP outgroup. The optimal tree with the sum of branch length = 0.328 is shown. taining recombinant pseudoviruses of CpGV-MΔpe38M Δ pe38S::eGFP The percentage of replicate trees in which the associated taxa clustered and CpGV-M pe38M were then used for activity assays together in the bootstrap test (500 replicates) is shown next to the branches. in susceptible CpS and resistant CpRR1 larvae (Fig. 5). After 14 d The tree is drawn to scale, with branch lengths in the same units as those of pe38M::eGFP of infection, both pseudoviruses CpGV-MΔpe38M and the evolutionary distances used to infer the phylogenetic tree.

Gebhardt et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 pe38 PacI (39). The mortality data of the different constructed pseudo- CpGV 25 50 75 100 viruses (Fig. 6B) and CpGV-M or CpGV-S (Fig. 1), respectively, pe38 BacBsu36I cassette did not show any significant differences in the efficacy of the bacCpGV pseudoviruses compared with the natural virus isolates. Because 25 50 75 100 125 all produced pseudoviruses were infectious to CpS larvae but R Amp BacBsu36I cassette only the recovery mutant with pe38 from CpGV-S was able to bacCpGV∆pe38M 25 50 75 100 125 infect CpRR1, it is unequivocally demonstrated that pe38 is essential for the infection cycle of CpGV and that the lack of mini-attTn7 the eight amino acid repeat in PE38 is apparently sufficient to overcome CpGV resistance in CpRR1. Interestingly, the BacBsu36I cassette PacI KanR LacZ mini-F replicon PacI mortality of CpRR1 caused by the different naturally occurring resistance-breaking isolates varied and was significantly

pe38M::eGFP pe38M::eGFP PolyA GmR bacCpGV ∆pe38M lower for CpGV-I12 compared with CpGV-S, -E2, and -I07; or this suggests that beyond the resistance-overcoming role of bacCpGV ∆pe38 pe38S::eGFP pe38S::eGFP PolyA GmR M PE38, further genomic factor(s) must contribute to the virulence of these isolates. Fig. 4. Schematic overview of the construction of the different bacmids. Upper pe38 scheme illustrates the CpGV-M genome (sizing in kbp) with the position The gene is not present in all baculoviruses, and with only of the ORF24 (pe38) and the unique PacI restriction site. This restriction 26% amino acid identity it is poorly conserved (33). Homologs of site was the target of the 8.6-kbp BacBsu36I cassette containing the the pe38 gene predominantly occur in the group I alphabaculo- kanamycin resistance (KanR) gene, LacZ with mini-attTn7 site, and mini-F viruses (40) as well as in some betabaculoviruses (33, 41, 42). For replicon inserted to create bacCpGV. To generate the knockout mutant AcMNPV, it was shown that pe38 is an important factor for virus Δ bacCpGV pe38M,thepe38 gene was replaced by an ampicillin resistance formation. The knockout of pe38 dramatically reduced virulence R Δ pe38M::eGFP (Amp ) gene. To obtain the two recovery mutants bacCpGV pe38M when applied orally to Heliothis virescens larvae (40). In contrast Δ pe38S::eGFP and bacCpGV pe38M , either the fusion ORF pe38M::eGFP or to bacCpGVΔpe38 , where no occlusion bodies or budded pe38S::eGFP, together with the SV40 polyadenylation signal (PolyA) and M R viruses were produced, the production of occlusion bodies and a gentamycin resistance (Gm ) gene as selection marker, was inserted Δ into the mini-attTn7 by transposition. budded viruses of AcMNPV pe38 was still possible. Additionally, bypassing the midgut by injecting AcMNPVΔpe38 budded viruses directly into the hemocoel of H. virescens larvae resulted to other CpGV genome types. Genome sequencing of differ- in similar efficacy of the knockout mutant (40) compared with ent CpGV isolates showed that all resistance-breaking isolates wild-type budded viruses, underscoring that pe38 is indeed lacked the repeat of 24 nt in the gene pe38 (ORF24) as the only important but not essential for AcMNPV. In contrary, injection common difference to CpGV-M. This sequence repeat results in of budded viruses of CpGV-M into the hemocoel of CpS or the triplication of a predicted DTVD (or related permutations) CpRR1 larvae revealed that only susceptible larvae became amino acid motif in PE38 of CpGV-M. Because this repeat is the infected when bypassing the midgut. Thus, the mode of action only common difference between all resistance-breaking CpGV of PE38 in CpGV must be somehow different to that in isolates and CpGV-M, it is most plausible to assume that the AcMNPV (29) because the resistance of CpRR1 is still present absence of this repeat is responsible for their infectivity to re- when bypassing the midgut. sistant codling moth. The pe38 gene of AcMNPV codes for a 38-kDa nuclear protein The phylogenetic analysis of CpGV genomes confirmed the (43) that transactivates (along with products of other immediate- previously established phylogeny and genome classification of early genes) early gene expression (43, 44). PE38 of AcMNPV CpGV (8). The phylogenetic tree clearly placed CpGV-I07 as basal branch of the CpGV isolates and determines the C-type genomes as ancestral genome types. The newly incorporated genome type E Bacmid DNA: Transfection (CpGV-S) is grouped between genome type B (CpGV-E2) and the bacCpGVhsp-eGFP Feeding genome types A and D, which belong to the youngest CpGV ge- bacCpGV∆pe38M bacCpGV∆pe38 pe38M::eGFP “ ” M nome types. Because CpGV-M is the only and youngest genome bacCpGV∆pe38 pe38S::eGFP harboring the 24-nt repeat in pe38, we conclude that an addition M was made to CpGV-M rather than a deletion to the other isolates. Cp14R CpS Interestingly, the virus isolate CpGV-R5, which is able to overcome resistance in a French-selected resistant codling moth strain, also Purfication from lacks the 24-nt addition in pe38 (31, 38). single larva and We successfully deleted pe38 of CpGV by mutating the bacmid OB propagation bacCpGV, which derived from an in vivo cloned genotype of CpGV-M (34). Because the knockout of pe38 in CpGV totally CpS Activity Purification abolishes viral infection, it can be assumed that the observed repeat of the eight amino acids in CpGV-M preserves the testing functionality of the protein but may modify the mode of action. Pseudoviruses: The recovery of bacCpGVΔpe38M with either pe38M::eGFP or CpRR1 CpGV-Mhsp-eGFP pe38S::eGFP inserted into the mini-attTn7 site of the BAC cas- pe38M::eGFP CpGV-M∆pe38M CpS pe38S::eGFP sette demonstrated that the functionality of pe38 is independent CpGV-M∆pe38M of its position in the genome. For virus propagation, only larvae that showed eGFP fluorescence were chosen. Because eGFP was Fig. 5. Procedure of the production of bacmid-based occlusion bodies of N-terminally fused to PE38, we can conclude that (i) pe38 is CpGV pseudoviruses. Cp14R cells were transfected with different bacmid DNAs and then fed to susceptible CpS L4 larvae. Infected larvae exhibiting expressed in vitro and in vivo, and (ii) fusion to eGFP has no eGFP fluorescence were subjected to OB purification. After the virus identity detectable influence on protein-folding properties or function of was confirmed, it was propagated for further experiments in CpS. The PE38, because the formation of the chromophore of eGFP is resulting pseudovirus occlusion bodies were used for activity assays in CpS or linked to the correct folding of the N-terminal–tagged protein CpRR1 larvae.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1411089111 Gebhardt et al. Downloaded by guest on September 28, 2021 A B AcMNPV B A A Ab PlxyNPV 100 100 BmNPV

80 80 ChocNPV CpGV-M

60 60 CpGV-S

PrGV 40 40 Mortality [%] CrleGV AA a a 20 20 PhopGV

Fig. 7. Schematic illustration of PE38 from different baculoviruses. The blue 0 0 boxes indicate the RING-finger motifs, which all PE38 have in common. The n/N 85/3 114/3 96/3 83/4 146/5 85/4 129/496/4 236/6 white boxes indicate the leucine zipper motifs, if present. The red boxes indicates the DxxD motifs of CpGV. AcMNPV, Autographa californica mul- mock hsp-eGFP ∆pe38M hsp-eGFP pe38M pe38S tiple nucleopolyhedrovirus; BmNPV, Bombyx mori nucleopolyhedrovirus; ChocNPV, Choristoneura occidentalis nucleopolyhedrovirus; CpGV-M, CpGV Bacmid DNA Pseudoviruses containing the Mexican isolate; CpGV-S, CpGV containing the active in- gredient of Virosoft; CrleGV, Cryptophlebia leucotreta granulovirus; Fig. 6. Mortality of codling moth larvae after 14 d in activity assay. (A) PhopGV, Phthorimaea operculella granulovirus; PlxyNPV, Plutella xylostella CpS larvae were fed either 3 μL Cp14R cell culture transfected with DNA hsp-eGFP nucleopolyhedrovirus; PrGV, Pieris rapae granulovirus. of bacCpGV (hsp-eGFP), the knockout mutant bacCpGVΔpe38M (Δpe38M), or mock-transfected Cp14R (mock). Control larvae were fed with water instead. (B) Larvae of CpS (white bars) or CpRR1 (gray bars) were fed with 1 × 103 OB per larvae of the pseudoviruses CpGV-Mhsp-eGFP (hsp-eGFP), cells of resistant CpRR1 could be able to initiate cell death and the pe38M::eGFP pe38S::eGFP insect could prevent the spread of virus. This mechanism would CpGV-MΔpe38M (pe38M), or CpGV-MΔpe38M (pe38S). Control larvae were fed with water instead. Error bars show SD. Columns partly explain why eGFP-tagged CpGV-M seems to be able to infect

marked by different letters differ significantly (t test, P < 0.05). Mortality CpRR1 marginally, although no significant levels of DNA SCIENCES data of treatments were corrected for control mortality (<13% in untreated replication or budded virus production were detectable in CpRR1 control). The total number of tested individuals (n) and the number of in- larvae (29), because it is hampered and apparently overpowered APPLIED BIOLOGICAL dependent replicates (N) are given below the chart. by the host defense systems; this is indicated by the small fluorescent eGFP spots in the midgut and fat body of infected CpRR1 larvae (29). Recent microarray studies of CpRR1 infected with augments IE1-induced apoptosis in Sf9 cells by an unknown CpGV-M revealed transcripts for all temporal gene classes from mechanism, but the expression of PE38 alone had no effect immediate-early to very late genes of CpGV-M in CpRR1, but at (45). The pe38 of CpGV is most likely also an early transcribed significantly reduced and delayed rates followed by a breakdown gene, because it contains an early but no late baculovirus pro- of the infection after 4–5 d (48). A full or partial suppression of moter motif. The presumption that resistance to CpGV is CpGV-M infection in CpRR1 is also suggested by the observation somehow related to early gene expression was previously made that heterozygous males (ZRZS)fromCpRR1× CpS crossings are when it was found that resistance was (i)midgutreceptorin- only 100–1,000× less susceptible to CpGV-M than CpS larvae, dependent, (ii)systemic,and(iii) linked to an early inter- whereas resistant females (ZRW) or homozygous males (ZRZR)of ruption of the infection process causing a blockage of viral CpRR1 exhibit a 10,000–100,000× lower susceptibility to CpGV-M DNA replication (29). (15). Although heterozygous ZRZS males survived a discrimina- PE38 from different baculoviruses contain an N-terminal RING- tive concentration of CpGV-M, they did not pupate but died due finger domain (Fig. 7), a specialized type of zinc finger, which is – – to virus infection with a delay of 1 2 wk, arguing for an ongoing probably involved in mediating protein protein interactions. RING- infection pressure by CpGV-M and a collapsing cell defense in finger domains are often found in proteins, which are involved in heterozygous males (15). viral replication or signal transduction. This motif seems, therefore, Which genetic adaptation(s) on the insect level occurred that characteristic for PE38. Furthermore, many but not all PE38 con- codling moth developed resistance to CpGV-M is still unclear, tain C-terminal leucine zipper motifs (43), which are often re- but we demonstrate unambiguously that PE38 is essential for sponsible for the dimerization of DNA binding proteins. Mutations the viral infection cycle of CpGV in codling moth larvae, and of the leucine zipper motif inhibit the ability of PE38 of AcMNPV that the repeat of 24-nt coding for two DTVD motifs in PE38 of to augment the IE1-induced apoptosis (45), but the precise function CpGV-M renders this isolate to be prone to CpGV resistance. of PE38 in the infection cycle of AcMNPV is still not elucidated. Because the nucleotide sequence of pe38 can be used as a mo- In CpGV-M, a triplication of the C-terminal sequence motif lecular marker for the identification of resistance-breaking CpGV DTVD or related permutations occurred. These sequence motifs are isolates, this finding will play a decisive role in further efforts to specific for CpGV (Fig. 7). The motifs DxVD or DxxD are common find new CpGV isolates against resistant codling moth pop- for proteins that are substrates of caspases. Caspases, which contain ulations. The current picture of CpGV resistance and overcoming also RING-finger motifs (45) such as PE38, are important for ap- of resistance, however, may be even more complex, as it is pro- optosis (46). Baculoviruses benefit from inhibiting virus-induced host posed by the observed virulence differences of resistance-breaking cell apoptosis by either substrate inhibitors or IAPs to enhance their isolates. Elucidating the genetic factors that determine the viru- own multiplication (47). As a homologous PE38 of AcMNPV, PE38 lence for insect larvae under natural infection conditions will, of CpGV is expected to transactivate viral genes as well as interact however, be crucial for an efficient management of resistant and with specific host components. Thus, PE38 of CpGV must have nonresistant field populations of insect pests that shall be con- a further functional domain at its C terminus; alternatively, the trolled by using baculovirus control agents. function of the N-terminal RING-finger or leucine zipper may be structurally impaired by this mutation. Therefore, it could be possi- Materials and Methods ble that the repeat of the putative recognition motif may prevent the Viruses, Insects, and Cell Lines. Five different naturally occurring geographic correct binding of interacting partners. It is conceivable that host isolates of CpGV (M, I12, S, E2, and I07) were used as described in ref. 8. Either

Gebhardt et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 the Cydia pomonella cell line Cp14R (37) or larvae of virus susceptible (CpS) Bacmid Construction, Pseudovirus Production, and Activity Testing of Constructs. Dif- and resistant (CpRR1) strains of C. pomonella was used (15) for virus prop- ferent knockout and rescue bacmids and derivative pseudoviruses were constructed agation and activity testing of bacmids and pseudoviruses as described in SI with the help of a CpGV bacmid (34) as detailed in SI Materials and Methods. Materials and Methods. ACKNOWLEDGMENTS. The authors thank Doreen Winstanley and Sally hsp-eGFP Virus Genome Sequencing and Phylogenetic Analysis. Sanger sequencing (I12) Hilton for providing the bacmids bacCpGV and bacCpGV ,andLoy Volkman (University of California, Berkeley) for critical reading of and or 454 pyrosequencing (M, S, E2, and I07) was used. Sequence annotation was valuable comments on the manuscript. Support for this work was pro- done using Lasergene Software; phylogenetic analysis was performed using vided by European Commission Grant 032857 and Deutsche Forschungs- MEGA5 (for details, see SI Materials and Methods). gemeinschaft Grant Je245/8-1.

1. Glen DM, Payne CC (1984) Production and field evaluation of codling moth granulosis 24. Kirkpatrick BA, Washburn JO, Volkman LE (1998) AcMNPV pathogenesis and de- virus for control of Cydia pomonella in the United Kingdom. Ann Appl Biol 104(1): velopmental resistance in fifth instar Heliothis virescens. J Invertebr Pathol 72(1): 87–98. 63–72. 2. Huber J, Dickler E (1976) Das Granulosevirus des Apfelwicklers: Seine Erprobung für 25. Teakle RE, Jensen JM, Giles JE (1986) Age-related susceptibility of Heliothis punctiger die biologische Schädlingsbekämpfung [The granulosis virus of the codling moth: to a commercial formulation of nuclear polyhedrosis virus. J Invertebr Pathol 47(1): Testing its potency for biological control]. Zeitschrift für Angewandte Entomologie 82–92. 82(1-4):143–147. German. 26. Parker BJ, Elderd BD, Dwyer G (2010) Host behaviour and exposure risk in an insect- 3. Falcon LA, Kane WR, Bethell RS (1968) Preliminary evaluation of a granulosis virus for pathogen interaction. J Anim Ecol 79(4):863–870. control of codling moth. J Econ Entomol 61(5):1208–1213. 27. Kerr JF, Wyllie AH, Currie AR (1972) Apoptosis: A basic biological phenomenon with 4. Jaques RP, et al. (1981) Apple orchard tests on the efficacy of the granulosis virus of wide-ranging implications in tissue kinetics. Br J Cancer 26(4):239–257. the codling moth, Laspeyresia pomonella. Entomophaga 26(2):111–117. 28. Clem RJ (2001) Baculoviruses and apoptosis: The good, the bad, and the ugly. Cell 5. Jaques RP, Laing JE, Laing DR, Yu DSK (1987) Effectiveness and persistence of the Death Differ 8(2):137–143. granulosis virus of the codling moth Cydia pomonelIa (L.) (Lepidoptera: Olethreuti- 29. Asser-Kaiser S, Radtke P, El-Salamouny S, Winstanley D, Jehle JA (2011) Baculovirus dae) on apple. Can Entomol 119(12):1063–1067. resistance in codling moth (Cydia pomonella L.) caused by early block of virus repli- – 6. Tanada Y (1964) A granulosis virus of the codling moth, Carpocapsa pomonella cation. Virology 410(2):360 367. (Linnaeus) (Olethreutidae, Lepidoptera). J Insect Pathol 6:378–380. 30. Eberle KE, Asser-Kaiser S, Sayed SM, Nguyen HT, Jehle JA (2008) Overcoming the 7. Hilton S, Winstanley D (2007) Identification and functional analysis of the origins of resistance of codling moth against conventional Cydia pomonella granulovirus – DNA replication in the Cydia pomonella granulovirus genome. J Gen Virol 88(Pt 5): (CpGV-M) by a new isolate CpGV-I12. J Invertebr Pathol 98(3):293 298. 1496–1504. 31. Berling M, et al. (2009) Cydia pomonella granulovirus genotypes overcome virus re- 8. Eberle KE, Sayed S, Rezapanah M, Shojai-Estabragh S, Jehle JA (2009) Diversity and sistance in the codling moth and improve virus efficiency by selection against resistant – evolution of the Cydia pomonella granulovirus. J Gen Virol 90(Pt 3):662–671. hosts. Appl Environ Microbiol 75(4):925 930. 9. Cross JV, et al. (1999) Biocontrol of pests of apples and pears in northern and central 32. Zingg D (2008) Madex Plus and Madex I12 overcome virus resistance of codling moth. Available at orgprints.org/13705/1/256-260.pdf. Accessed June 15, 2014. Europe: 1. Microbial agents and nematodes. Biocontrol Sci Technol 9(2):125–149. 33. Luque T, Finch R, Crook N, O’Reilly DR, Winstanley D (2001) The complete sequence of 10. Moscardi F (1999) Assessment of the application of baculoviruses for control of the Cydia pomonella granulovirus genome. J Gen Virol 82(Pt 10):2531–2547. Lepidoptera. Annu Rev Entomol 44:257–289. 34. Hilton S, Kemp E, Keane G, Winstanley D (2008) A bacmid approach to the genetic 11. Cory JS, Meyers JH (2003) The ecology and evolution of insect baculoviruses. Annu Rev manipulation of granuloviruses. J Virol Methods 152(1-2):56–62. Ecol Evol Syst 34:239–272. 35. Arciszewska LK, Drake D, Craig NL (1989) Transposon Tn7. cis-Acting sequences in 12. Fritsch E, Undorf-Spahn K, Kienzle J, Zebitz CPW, Huber J (2005) Apfelwickler Granulo- transposition and transposition immunity. J Mol Biol 207(1):35–52. virus: Erste Hinweise auf Unterschiede in der Empfindlichkeit lokaler Apfelwickler- 36. Zhang Y, Buchholz F, Muyrers JPP, Stewart AF (1998) A new logic for DNA engi- Populationen [Codling moth granulovirus: First evidence of differences in the sensitivity neering using recombination in Escherichia coli. Nat Genet 20(2):123–128. of local codling moth populations]. Nachrichten des Deutschen Pflanzenschutzdienstes 37. Winstanley D, Crook NE (1993) Replication of Cydia pomonella granulosis virus in cell 57:29–34. German. cultures. J Gen Virol 74(Pt 8):1599–1609. 13. Sauphanor B, et al. (2006) Carpocapse des pommes. Cas de résistance au virus de la 38. Graillot B, et al. (2013) Sequence analysis of CpGV-R5 isolate, able to efficiently granulose en verges biologique [Codling moth: Cases of resistance to granuloviruses control CpGV-M resistant insects: Relation between biological activity and genome. in organic orchards]. Phytoma—La Défense des Végétaux 590:24–27. French. IOBC WPRS Bull 90:195–199. 14. Schmitt A, et al. (2013) The occurrence and distribution of resistance of codling moth 39. Waldo GS, Standish BM, Berendzen J, Terwilliger TC (1999) Rapid protein-folding – to Cydia pomonella granulovirus in Europe. J Appl Entomol 137(9):641 649. assay using green fluorescent protein. Nat Biotechnol 17(7):691–695. 15. Asser-Kaiser S, et al. (2007) Rapid emergence of baculovirus resistance in codling 40. Milks ML, Washburn JO, Willis LG, Volkman LE, Theilmann DA (2003) Deletion of pe38 – moth due to dominant, sex-linked inheritance. Science 317(5846):1916 1918. attenuates AcMNPV genome replication, budded virus production, and virulence in 16. Asser-Kaiser S, Heckel DG, Jehle JA (2010) Sex linkage of CpGV resistance in a het- heliothis virescens. Virology 310(2):224–234. erogeneous field strain of the codling moth Cydia pomonella (L.). J Invertebr Pathol 41. Escasa SR, Lauzon HAM, Mathur AC, Krell PJ, Arif BM (2006) Sequence analysis of the – 103(1):59 64. Choristoneura occidentalis granulovirus genome. J Gen Virol 87(Pt 7):1917–1933. 17. Zichova T, Stara J, Kundu JK, Eberle KE, Jehle JA (2013) Resistance to Cydia pomonella 42. Ferrelli ML, et al. (2012) Genome of Epinotia aporema granulovirus (EpapGV), a poly- granulovirus follows a geographically widely distributed inheritance type within organotropic fast killing betabaculovirus with a novel thymidylate kinase gene. BMC – Europe. BioControl 58(4):525 534. Genomics 13:548. 18. Berling M, Sauphanor B, Bonhomme A, Siegwart M, Lopez-Ferber M (2013) A single 43. Krappa R, Knebel-Mörsdorf D (1991) Identification of the very early transcribed ba- sex-linked dominant gene does not fully explain the codling moth’s resistance to culovirus gene PE-38. J Virol 65(2):805–812. granulovirus. Pest Manag Sci 69(11):1261–1266. 44. Kovacs GR, Guarino LA, Summers MD (1991) Novel regulatory properties of the IE1 19. Narayan K (2004) Insect defense: Its impact on microbial control of insect pests. Curr and IE0 transactivators encoded by the baculovirus Autographa californica multi- Sci 86(6):800–814. capsid nuclear polyhedrosis virus. J Virol 65(10):5281–5288. 20. Richards AG, Richards PA (1977) The peritrophic membranes of insects. Annu Rev 45. Prikhod’ko EA, Miller LK (1999) The baculovirus PE38 protein augments apoptosis Entomol 22:219–240. induced by transactivator IE1. J Virol 73(8):6691–6699. 21. Reeson AF, Wilson K, Gunn A, Hails RS, Goulson D (1998) Baculovirus resistance in the 46. Fuentes-Prior P, Salvesen GS (2004) The protein structures that shape caspase activity, noctuid Spodoptera exempta is phenotypically plastic and responds to population specificity, activation and inhibition. Biochem J 384(Pt 2):201–232. density. Proc Biol Sci 265(1407):1787–1791. 47. Clem RJ (2007) Baculoviruses and apoptosis: A diversity of genes and responses. Curr 22. Ashida M, Brey PT (1997) Molecular Mechanisms of Immune Responses in Insects, eds Drug Targets 8(10):1069–1074. Brey PT, Hultmark D (Chapman and Hall, London), pp 135–172. 48. Schneider D, Jehle JA (2014) Temporal transcription analysis of Cydia pomonella 23. Briese DT (1986) The Biology of Baculoviruses, eds Granados RR, Federici BA (CRC, granulovirus in the midgut of codling moth by using microarray analysis. J Plant Boca Raton, FL), pp 237–263. Dis Prot 121(2):96–97.

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