Elucidating the mechanism of baculovirus resistance (type I) in codling moth by identification of PE38-binding complex during the CpGV infection in Cp14R cells
Final Report Abstract
The codlng moth (Cydia pomonella) is the most important pest insect in pome fruit production worldwide. The larvae bore into unripe apples and pears and complete their development inside resulting in non-marketable fruits and significant economic losses. Different Cydia pomonella granulovirus (CpGV) preparations including resistance-breaking CpGV isolates have been successfully applied to reduce infestation and avoid fruit damage caused by susceptible and resistant (Type I-III) codling moth populations in pome fruit production in both organic and integrated pest management (IPM) plantations. The Type I resistance of codling moth to CpGV were found in several European countries, which requires to further explain the mechanism of the widely occurred Type I resistance in codling moth. Understanding the underlying resistance mechanisms is critical for the strategies of future pest management. Previous literatures reported that Inheritance of Type I CpGV resistance is dominant and linked to the Z chromosome. In addition, the viral pe38, the target gene that is responsible for breaking Type I resistance in codling moth, has been verified. The present project was aiming to identify the molecular basis of Type I CpGV resistance in codling moth by two approaches: (1) decoding the components of viral PE38 complex in CpGV infection in codling moth; (2) Z-chromosomal comparison between CpS (susceptible) and CpRR1_22 (Type I resistance). We applied Sleeping Beauty transposon system to establish a transformed Cp14R-pe38 cell line that can overcome the low expression of PE38 in CpGV-infected larvae. Transposon donor vector of pT2/HB-hsp-pe38-mCherry2-Zeocin has been constructed. Full plasmid sequence validated that hsp-pe38-mCherry2-Zeocin was correctly inserted into pT2/HB plasmid. Western Blotting showed that His-PE38-mCherry2 protein (75.13 kDa) could continually express in E. coli. The transformed Cp14R-pe38 cells were selected by Zeocin and validated by inverse-PCR, nested PCR and sequencing. These positive cell colonies with mCherry2 fluorescence activity were screened under the confocal microscope. This Cp14R-pe38 cell line is well-growing and can be continually passaged in laboratory to study the basic question how the codling moth blocks CpGV replication in the early stage of infection and how to reduce/eliminate Type I resistance and additional resistance cases by disrupting this infection pathway. We made the whole genome sequence of susceptible (CpS) larva and Type I resistant (CpRR1_22) larva on the MinION Mk1C (MC-110201, Oxford Nanopore Technologies, UK). We de novo assembled those reads using the Flye and canu software. Two genome sequences of CpS and CpRR1_22 were generated, which achieved the BUSCO score of 98.39% and 98.37%, respectively. The Z chromosomal sequences of CpS and CpRR1_22 were annotated using GeMoMa pipeline and were compared to identify the Type I resistance markers. There are 1350 genes shared by CpS and CpRR1_22, while 200 genes are specific to CpS and 156 genes are specific to CpRR1_22, of which are involved in codling moth against CpGV infection. These genetic differences can be developed as Type I resistance markers to investigate the occurrence of the resistant codling moth in field. Elucidating the mechanism of CpGV resistance to codling moth and identifying the Type I resistance markers can not only figure out a scientific issue, but also point out the way for sustainability of baculovirus formulation in pome fruit production.
Publications
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Chromosome-level comparison between susceptible and resistant codling moth strains to decoding the mechanism of CpGV resistance of codling moth [Conference oral presentation abstract], Annual Meeting of the Society for Invertebrate Pathology, Vienna, Austria..
Jiangbin Fan, Fang-Shiang Lim, Jö T. Wennmann, David Heckel, Petr Nguyen & Johannes A. Jehle
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Identification of the molecular basis of CpGV resistance in codling moth, Cydia pomonella [Conference poster presentation abstract], Annual Meeting of the Society for Invertebrate Pathology, Vienna, Austria.
Jiangbin Fan, Jö T. Wennmann & Johannes A. Jehle
