Enhanced resistance to Septoria musiva in hybrid poplar transformed with a wheat oxalate oxidase gene or antimicrobial peptide genes

Septoria musiva is one of the most damaging fungal pathogens of hybrid poplar. The overall goal of this project was to enhance S. musiva resistance in hybrid poplar by genetic transformation. A wheat germin-like oxalate oxidase gene (pOxO), an antimicrobial peptide Ac-AMP1.2 gene (PCA1), and a transgene construct containing both a synthetic antimicrobial peptide ESF12 gene and Ac-AMP1.2 (pCWEA1) were individually introduced into the hybrid poplar clone “Ogy” (Populus x euramericana) by Agrobacterium -mediated transformation. The ESF12/Ac-AMP1.2 and a β-glucuronidase gene (pBI121) were also individually introduced into another hybrid poplar clone “NM6” (Populus nigra x P. maximowizii). The transgenes were successfully amplified from the genomic DNA of transformed plants by polymerase chain reaction (PCR) using transgene specific primers. Two pOxO-transformed “Ogy” lines were obtained and the encoded oxalate oxidase retained its ability to breakdown oxalic acid. In addition, two pCA1- and one pCWEA1-transformed “Ogy” lines and one pCWEA1- and one pBI121-transformed “NM6” lines were obtained. The coding regions of the Ac-AMPI.2 gene and the ESF12/Ac-AMP1.2 construct were fully transcribed in the transformed lines. In vitro pathogen resistance tests with S. musiva indicated that pOxO-, pCA1-, and pCWEA1-transformed “Ogy” plants showed significantly increased resistance as compared to the untransformed “Ogy”. A measurable decrease in the mean necrotic area in the pCWEA1-transformed “NM6” plants was observed, but this was not significantly different from the untransformed and pBI121-transformed “NM6” controls. An antimicrobial peptide ESF39A was designed to incorporate the synthetic antimicrobial ESF22B peptide, two tobacco etch virus (TEV) Nuclear Inclusion A protease (NIa) recognition sites, and a carboxyl-end hexahistidine tail. The antifungal and hemolytic activity of the ESF39A peptide was tested in vitro and the results showed that ESF39A maintained similar antifungal and hemolytic activity to the related peptide ESF22B. An optimal DNA sequence was constructed to encode the ESF39A peptide. This ESF39A gene also contained a signal sequence from the tobacco ap24 gene attached to the 5′ end of the transgene and a 5′-untranslated leader from tobacco mosaic virus RNA added in front of the ap24 signal sequence. The whole synthetic ESF39A gene was cloned into a TOPO-TA cloning vector and the correct sequence was confirmed by DNA sequencing.