Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene
Current safety assessment of genetically modified crops requires detailed information about the insertion of the transgene and the effect of its expression on the biochemistry and physiology of the host plant. Whilst the intended effect of the transformation can be verified through phenotypic screening, molecular approaches are required to observe unintended effects. We investigated the molecular details of the integration of a polygalacturonase inhibiting protein 1 gene from Malus domestica (Mdpgip1), overexpressed in Solanum tuberosum (cv BP1) for enhanced resistance against Verticillium wilt. Genome walking studies of the selected AppA6 transformant revealed that the T-DNA containing the Mdpgip1 transgene under control of the CaMV 35S promoter was inserted into the genome without any non-T-DNA sequences from the pCAMBIA2300 vector. Sequence data indicate that the insertion of the Mdpgip1 transgene was in a gene-rich region of chromosome 1, adjacent to the photosystem Q B gene but without disruption of structural genes. Transcriptome-based cDNA-representational difference analysis revealed the distinctive expression of Mdpgip1 in the transgenic AppA6 line, verifying the intended effect. Protein extracts from the transgenic plants inhibited the activities of Verticillium dahliae polygalacturonases in in vitro studies, showing that the transgene is expressed to produce an active PGIP defense protein. cDNA-AFLP fingerprinting revealed genes that were differentially expressed, including genes encoding tryptophan/tyrosine permease, Ef-Tu domain and SKP1-like 1A proteins. qRT-PCR indicated that the Mdpgip1 transgene insertion resulted in increased expression in the AppA6 transgenic of the xyloglucan endotransglycosylase (xth) gene and an endogenous Stpgip1 gene. These unintended changes were either caused by the constitutive expression of the Mdpgip1 transgene or transformation-related somaclonal variation. The results indicate that the stable, single copy integration of the Mdpgip1 gene in the AppA6 transgenic line did not disrupt any structural genes but caused unintended effects that affected gene expression compared to the parental counterpart under the non-stressed experimental conditions investigated.
KeywordsAgrobacterium-mediated transformation Differential gene expression Genetically modified Genome walking PGIP Transgene insertion Unintended effects
The South African Agricultural Research Council (ARC), Department of Science and Technology (DST), AgriSETA, National Research Foundation (NRF) and Potato South Africa (PSA) are thanked for financial support. Drs. Inge Gazendam, Adri Veale and Prof. Dave Berger are thanked for their roles in generating the potato transformants. Dr. Christian Bachem (Potato Genome Sequencing Consortium) is thanked for assistance with the potato sequence information and Dr. Arnaud Thierry Djami-Tchatchou (University of Johannesburg) for guidance with the qRT-PCR work.
All authors conceptualised the research. LBTM performed the analyses. DO and IAD supervised the research and contributed to writing of the manuscript. All authors read and critically revised the manuscript.
Compliance with Ethical Standards
Conflict of Interest
LBTM and DO are staff members of the South African ARC. The authors declare that they have no competing interest regarding the publication of this article.
- AgBios (2008) GMO database. http://www.agbios.com/dbase.php
- Arendse MS, Dubery IA, Berger DK (1999) A polymerase chain reaction method for cloning gene homologues: isolation of a plant anti-fungal polygalacturonase-inhibiting protein gene. Electron J Biotechnol 2:152–159Google Scholar
- Cellini F, Chesson A, Colquhoun I, Constable A, Davies HV, Engel KH, Gatehouse AMR, Kӓrenlampi S, Kok EJ, Leguay J-J, Lehesranta S, Noteborn HPJM, Pedersen J, Smith M (2004) Unintended effects and their detection in genetically modified crops. Food Chem Toxicol 42:1089–1125CrossRefPubMedGoogle Scholar
- Department of Agriculture, Forestry and Fisheries (DAFF) (2013) Economic review of the South African Agriculture 2012/2013. www.daff.org.za
- James C (2010) Global status of commercialized biotech/GM crops. International Service for the Acquisition of Agri-Biotech Applications (ISAAA) Brief 42. www.isaaa.org/resources/publications/briefs/42/default.asp, Ithaca
- Joubert DA, Slaughter AR, Kemp G, Becker JVW, Krooshof GH, Bergmann C, Benen J, Pretorius IS, Vivier MA (2006) The grapevine polygalacturonase-inhibiting protein (VvPGIP1) reduces Botrytis cinerea susceptibility in transgenic tobacco and differentially inhibits fungal polygalacturonases. Transgenic Res 15:687–702CrossRefPubMedGoogle Scholar
- Kok EJ, Franssen-van Hal NL, Winnubst LN, Kramer EH, Dijksma WT, Kuiper HA, Keijer J (2007) Assessment of representational difference analysis (RDA) to construct informative cDNA microarrays for gene expression analysis of species with limited transcriptome information, using red and green tomatoes as a model. J Plant Physiol 164:337–349CrossRefPubMedGoogle Scholar
- König A, Cockburn A, Crecel RWR, Debruyne E, Grafstroem R, Hammerling U, Kimber I, Knudsen I, Kuiper HA, Peijnenburg AACM, Penninkis AH, Poulsen M, Schauzu M, Wal JM (2004) Assessment of the safety of food derived from genetically modified (GM) crops. Food Chem Toxicol 42:1047–1088CrossRefPubMedGoogle Scholar
- Maritz I (2002) Evaluation of polygalacturonase-inhibiting protein (PGIP)-mediated resistance against Verticillium dahliae, a fungal pathogen of potato. MSc Dissertation, University of Pretoria, Pretoria, South AfricaGoogle Scholar
- Motulsky H (1999) Analyzing data with GraphPad Prism: a companion to GraphPad Prism version 3. GraphPad Software, IncGoogle Scholar
- OECD (1993) Safety evaluation of foods derived by modern biotechnology: concepts and principles. Organisation for Economic Co-operation and Development, Paris http://www.oecd.org/dataoecd/57/3/1946129.pdf
- Oelofse D, Dubery IA, Meyer R, Arendse MS, Gazendam I, Berger DK (2006) Apple polygalacturonase inhibiting protein 1 expressed in transgenic tobacco inhibits polygalacturonases from fungal pathogens of apple and the anthracnose pathogen of lupins. Phytochemistry 67:255–263CrossRefPubMedGoogle Scholar
- Ruebelt MC, LIPP M, Reynolds TL, Schmuke JJ, Astwood JD, Dellapenna D, Engel K-H, Jany KD (2006) Application of two-dimensional gel electrophoresis to interrogate alterations in the proteome of genetically modified crops. 3. Assessing unintended effects. J Agric Food Chem 54:2169–2177CrossRefPubMedGoogle Scholar
- Shepherd LVT, McNicol JW, Razzo R, Taylor MA, Davies HV (2006) Assessing the potential for unintended effects in genetically modified potatoes perturbed in metabolic and developmental processes. Targeted analysis of key nutrients and anti-nutrients. Transgenic Res 15:409–425CrossRefPubMedGoogle Scholar
- Volpi C, Raiola A, Janni M, Gordon A, O’Sullivan DM, Favaron F, D’Ovidio R (2013) Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification. Plant Physiol Biochem 73:294–301CrossRefPubMedGoogle Scholar