Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene

Abstract

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.

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Acknowledgments

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.

Authors’ Contributions

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.

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Correspondence to Ian A. Dubery.

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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.

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Fig. S1

A diagrammatic representation of the plasmid map of the pCAMBIA2300-appgip1A recombinant used to design the required gene-specific oligonucleotides (GSOs) for genome walking (GW). Oligonucleotides designed from the Malus domestica polygalacturonase inhibiting protein 1 (Mdpgip1) gene were designated Mdpgip1 GSOs, oligonucleotides designed from the CaMV 35S promoter were designated CaMV GSOs and oligonucleotides designed from the neomycin transferase II (nptII) gene were designated Border GSOs (Tables S1-S3). (DOCX 39 kb)

Fig. S2

a cDNA-RDA: PCR amplification of the tester and driver hybridisation to produce the Second Difference Product (DP2) using N Dpn24 oligonucleotide. b cDNA-RDA: PCR amplification of the tester and driver hybridisation to produce the Third Difference Product (DP3) using J Dpn24 oligonucleotide. (PDF 159 kb)

Fig. S3

Inhibition of Verticillium dahliae polygalacturonases (PGs) by MdPgip1 expressed in the AppA6 transgenic potato. The linear range activity of PG dilution series determined using the reducing sugar assay. (DOCX 31 kb)

Fig. S4

AFLPs: An autoradiography visualisation of genes differentially expressed between the untransformed BP1 and Mdpgip1 transgenic AppA6 potato transcriptomes obtained using cDNA-AFLP fingerprinting. (PDF 1.11 mb)

Table S1

Characteristics of oligonucleotides used during genome walking (GW 1) using a method adapted from Siebert et al. (1995). These oligonucleotides include the protocol specific AP1 and AP2, DNA Walking SpeedUp™ kit GSO1 previously designed, and oligonucleotides used during isolation and screening of the Mdpgip1 gene. (DOCX 15 kb)

Table S2

Characteristics of oligonucleotides used during genome walking (GW 2) using the DNA Walking SpeedUp™ kit. These oligonucleotides include the designed gene-specific oligonucleotides (GSOs) and the kit supplied DNA Walking-Annealing Control Primers (DW-ACPs). (DOCX 15 kb)

Table S3

Characteristics of oligonucleotides used during genome walking (GW 3) using the APAgene™ GOLD Genome Walking kit. These oligonucleotides include the kit-based specific DRT A, DRT B, DRT C, DRT D, UAP-N1 and UAP-N2. The Mdpgip1 GSOs were designed from the Mdpgip1 gene, the nptII GSOs were designed from the nptII gene and the CaMV GSOs were designed using the pCAMBIA2300-based CaMV 35S promoter. (DOCX 15 kb)

Table S4

Characteristics of oligonucleotides and probes used during real time quantitative reverse transcriptase PCR (qRT-PCR) to determine differentially expressed genes between the Mdpgip1 transgenic AppA6 and untransformed BP1 potato transcriptomes. (DOCX 15 kb)

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Matsaunyane, L.B.T., Oelofse, D. & Dubery, I.A. Detailed Molecular Characterisation of the Transgenic Potato Line, AppA6, Modified with the Apple (Malus domestica) Polygalacturonase Inhibiting Protein 1 (pgip1) Gene. Potato Res. 59, 129–147 (2016). https://doi.org/10.1007/s11540-016-9316-x

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Keywords

  • Agrobacterium-mediated transformation
  • Differential gene expression
  • Genetically modified
  • Genome walking
  • PGIP
  • Transgene insertion
  • Unintended effects