- We tested a synthetic bacterio-opsin (bO) gene derived from Halobacterium halobium for its effect on disease resistance in transgenic poplar hybrids, and assessed a genetic system for controlling expression of this gene. In tobacco the bO gene caused necrotic lesions similar to an induced hypersensitive response (HR), increased levels of several defense-related mRINAs, and enhanced resistance to pathogens (Mittler et al. 1995). Our goal was to test whether the bO gene could also promote the HR response and induce resistance against pathogens of poplar. Using Agrobacterium transformation, we produced 35 transgenic lines within three hybrid poplar clones. Expression of the bO gene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter and ) translational
enhancer, and the protein included a transit peptide to target it to the chloroplast.
Transgenic lines were confirmed through their ability to root on kanamycincontaining
medium, PCR amplification using bO-specific primers, and RNA blots probed with the bO gene. Necrotic lesions induced by the bO gene were seen predominantly on the lower and older leaves of greenhouse- and field-grown trees, similar to that reported in tobacco. Most lines displayed low to moderate numbers of lesions (mean lesion index=6.6, coefficient of variation=106%); however, one line had a very high lesion index (6-fold greater than the mean). Based on northern blots, bO expression among lines also varied widely (coefficient of variation=90%). We studied the effect of the bO gene on the expression of two types of defense-related genes, phenylalanine ammonia-lyase (PAL]), and two wound-inducible (win) chitinases, win6 and win8. Because of the low constitutive expression of the chitinases in poplar, their expression was induced by mechanical wounding prior to RINA extraction. No significant change in expression was found for any of these genes associated with the expression of the bO gene, including the line with very high bO expression. Resistance to different fungal pathogens was examined in the field and greenhouse after artificial inoculation. When field-grown trees were inoculated with fungal spores of Venturia leaf and shoot blight, bO-containing transgenics did not differ significantly from non-transgenic plants of the same clone (195-529). When clone 189-434, which is susceptible to the rust pathogen Melampsora occidentalis, was inoculated in the greenhouse, transgenic and control plants were also not significantly different in disease development. When all the lines were tested for susceptibility to two Septoria leaf and stem pathogens using in vitro inoculation, transgenics and controls were again similar in susceptibility. It was concluded that even though the bO gene was over-expressed and induced an HRmimic, lesion phenotype in some lines, it did not induce broad-spectrum defenserelated genes, and it failed to improve fungal disease resistance. To test a system that might be suitable for regulating expression of disease resistance transgenes in poplar, we studied the copper-based gene activation system reported to be effective in tobacco. Using both histochemical and fluorometric assays with a GUS reporter gene, we found that transgenic poplar leaves with both control (promoter-GUS only) and induction (promoter-GUS plus the copper
binding ACE] transcription factor) constructs displayed constitutive expression
without the addition of exogenous copper (basal medium concentration was 0.032 uM CuSO4). With the full induction construct, expression varied in a complex manner as a function of copper concentration (0 - 100 uM). It was concluded that because of the presence of endogenous transcription factors or inducing ions that compete with copper, the system is unlikely to be useful for regulating gene expression in poplar leaves.