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

Abstract

The baculovirus Cydia pomonella granulovirus (CpGV) is widely applied as a biocontrol agent of codling moth. After field resistance of codling moth populations had been observed against the commercially used Mexican (M) isolate of CpGV, infection experiments of larvae of the resistant codling moth strain CpRR1 showed that several other naturally occurring CpGV isolates (I12, S, E2, and I07) from different geographic origins are still infectious to resistant CpRR1. Whole-genome sequencing and phylogenetic analyses of these geographic CpGV variants revealed that their genomes share only a single common difference from that of CpGV-M, which is a mutation coding for a repeat of 24 nucleotides within the gene pe38; this mutation results in an additional repeat of eight amino acids that appears to be inserted to PE38 of CpGV-M only. Deletion of pe38 from CpGV-M totally abolished virus infection in codling moth cells and larvae, demonstrating that it is an essential gene. When the CpGV-M deletion mutant was repaired with pe38 from isolate CpGV-S, which originated from the commercial product Virosoft and is infectious for the resistant codling moth strain CpRR1, the repaired CpGV-M mutant was found to be fully infectious for CpRR1. Repair using pe38 from CpGV-M restored infectivity for the virus in sensitive codling moth strains, but not in CpRR1. Therefore, we conclude that CpGV resistance of codling moth is directed to CpGV-M but not to other virus isolates. The viral gene pe38 is not only essential for the infectivity of CpGV but it is also the key factor in overcoming CpGV resistance in codling moth.

Keywords: Cydia pomonella granulovirus; codling moth; genome sequencing; mutation; resistance; resistance management.

Fig. 1.

Mortality of CpS (white bars) or CpRR1 (gray bars) orally infected with 1 × 10³ OB/mL of the isolates CpGV-M, -I12, -S, -E2, or -I07. Mortality data were recorded 14 d postinfection and corrected for control mortality (<10% in untreated control). Error bars show SD. Columns marked by different letters differ significantly (t test, P < 0.05; uppercase for CpS, lowercase for CpRR1). The tested virus isolates, the total number of tested individuals (n) and the number of independent replicates (N) are given below the chart.

Fig. 2.

Sequence alignment of the gene pe38 (ORF24) of different CpGV 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. Distinguishable is the repeat of the amino acid motif DTVD in CpGV-M denoted by the roman numbers above (full alignment; Fig. S1).

Fig. 3.

Maximum-likelihood tree based on the predicted amino acid sequences of the 35 baculovirus core genes of five CpGV isolates and the corresponding genome types (given in brackets). The most closely related CpGV neighbor, Cryptophlebia leucotreta granulovirus (CrleGV), was used as outgroup. The optimal tree with the sum of branch length = 0.328 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.

Fig. 4.

Schematic overview of the construction of the different bacmids. Upper scheme illustrates the CpGV-M genome (sizing in kbp) with the position of the ORF24 (pe38) and the unique PacI restriction site. This restriction site was the target of the 8.6-kbp BacBsu36I cassette containing the kanamycin resistance (Kan^R) gene, LacZ with mini-attTn7 site, and mini-F replicon inserted to create bacCpGV. To generate the knockout mutant bacCpGV∆pe38_M, the pe38 gene was replaced by an ampicillin resistance (Amp^R) gene. To obtain the two recovery mutants bacCpGV∆pe38_M^pe38M::eGFP and bacCpGV∆pe38_M^pe38S::eGFP, either the fusion ORF pe38M::eGFP or pe38S::eGFP, together with the SV40 polyadenylation signal (PolyA) and a gentamycin resistance (Gm^R) gene as selection marker, was inserted into the mini-attTn7 by transposition.

Fig. 5.

Procedure of the production of bacmid-based occlusion bodies of CpGV pseudoviruses. Cp14R cells were transfected with different bacmid DNAs and then fed to susceptible CpS L4 larvae. Infected larvae exhibiting eGFP fluorescence were subjected to OB purification. After the virus identity was confirmed, it was propagated for further experiments in CpS. The resulting pseudovirus occlusion bodies were used for activity assays in CpS or CpRR1 larvae.

Fig. 6.

Mortality of codling moth larvae after 14 d in activity assay. (A) CpS larvae were fed either 3 μL Cp14R cell culture transfected with DNA of bacCpGV^hsp-eGFP (hsp-eGFP), the knockout mutant bacCpGV∆pe38_M (∆pe38_M), 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-M^hsp-eGFP (hsp-eGFP), CpGV-M∆pe38_M^pe38M::eGFP (pe38M), or CpGV-M∆pe38_M^pe38S::eGFP (pe38S). Control larvae were fed with water instead. Error bars show SD. Columns marked by different letters differ significantly (t test, P < 0.05). Mortality data of treatments were corrected for control mortality (<13% in untreated control). The total number of tested individuals (n) and the number of independent replicates (N) are given below the chart.

Fig. 7.

Schematic illustration of PE38 from different baculoviruses. The blue boxes indicate the RING-finger motifs, which all PE38 have in common. The white boxes indicate the leucine zipper motifs, if present. The red boxes indicates the DxxD motifs of CpGV. AcMNPV, Autographa californica multiple nucleopolyhedrovirus; BmNPV, Bombyx mori nucleopolyhedrovirus; ChocNPV, Choristoneura occidentalis nucleopolyhedrovirus; CpGV-M, CpGV containing the Mexican isolate; CpGV-S, CpGV containing the active ingredient of Virosoft; CrleGV, Cryptophlebia leucotreta granulovirus; PhopGV, Phthorimaea operculella granulovirus; PlxyNPV, Plutella xylostella nucleopolyhedrovirus; PrGV, Pieris rapae granulovirus.