SUNY College of Environmental Science and Forestry, Syracuse, NY 13210, USA

Background and objectives
Field testing for host resistance to leaf and stem canker pathogens has indicated the need for enhanced production of hybrid poplar with a multi-layered, durable defence system. The overall objective of this study was to produce a stable, rooted transgenic hybrid poplar which expressed antimicrobial peptides at levels inhibitory to pathogen invasion. Specific objectives of this study included: design and synthesis of antimicrobial peptides; determination of the minimum inhibitory concentrations (MIC) lethal to fungal and bacterial pathogens; construction of 'synthetic' genes based on the peptide sequence; transformation of host plant tissue (hybrid poplar) with these peptide genes; assay for expression of encoded peptides in transgenic hybrid poplar; and demonstration of pathogen inhibition in transgenic host tissue under controlled conditions.

Materials and methods
Synthetic 'ESF' peptides [1] were designed and their structures predicted using the Protean module within the Lasergene program (DNASTAR Inc., Madison, WI). The amino acid sequences were synthesized by Genosys Biotechnologies (The Woodlands, TX). Three fungi, Septoria musiva, Fusarium oxysporum f.sp. lycopersici and Cryphonectria parasitica and two bacteria, Pseudomonas syringiae and strain Bo542, were assayed against dilutions (0-250 µM) of the ESF peptides, Magainin II and negative controls. Data were analysed using multivariate analysis of variance within a general linear models procedure (SAS Institute, Inc., Cary, NC).

Design of the gene encoding ESF12 (18 amino acids) included a Kozak translational initiation sequence, plant preferred codons, and minimal mRNA secondary structure. The pCEA1 vector contained ESF12 and AcAMP1.2 (AcAMP1 analog) subcloned into the plant expression vector, pBI121 (Clontech Laboratories, Inc., Palo Alto, CA). The pCWEA1 vector was constructed with the wound-inducible promoter WIN3.12 [2] upstream of the ESF12/AcAMP1.2 gene construct. An A. tumefaciens-mediated system (strain LBA4404 transformed with pCEA1, pCWEA1 or a control) was used to transform Populus x euramericana OGY (Vic Steenackers, Poplar Research Center, Geraardsbergen, Belgium). To confirm transformation, extracted DNA was subjected to polymerase chain reaction (PCR) and Southern hybridization.

To quantitate expression of the ESF12 and AcAMP1.2 peptides, known masses of fresh transgenic hybrid poplar leaf tissue were extracted in buffer containing protease inhibitors and anti-phenolics. The polyclonal IgG fraction of antibodies raised to either ESF12 or AcAMP1.2 was used in ELISA assays. Total nanograms of peptide were estimated from quadratic regression curves of standards and compared using Tukey’s standardized range test.

Results and conclusions
72 plants from 50 independent pCEA1 transformation events rooted. Transformation with pCWEA1 yielded five independent explant lines surviving on antibiotic selective media for 1 year. Six of the rooted pCEA1 transformants are stable after growing in a non-selective, artificial soil medium for up to 13 months. PCR and Southern analysis of total genomic DNA confirmed the presence of the entire two-gene construct. Detectable ESF12 for one transgenic line statistically grouped with the highest standard control. Another line showed similarly high AcAMP1.2 expression. These results demonstrate that Agrobacterium-mediated transformation can produce transgenic OGY hybrid poplars which express small antimicrobial peptides at significantly high levels.

1. Powell WA, Catranis CM, Maynard CA, 1995. Molecular Plant-Microbe Interactions 8, 792-794
2. Hollick JB, Gordon MP, 1993. Plant Molecular Biology 22, 561-572.