5.3.23
SEARCH FOR A PLANT GENE PROMOTER WHICH IS ACTIVATED BY A BROAD SPECTRUM OF PATHOGENS

JL JAKOBEK and PB LINDGREN

Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695, USA

Background and objectives
Transgenic plants expressing various antimicrobial compounds, viral coat proteins, or even functional antibodies have been shown to have increased resistance. Enhanced resistance has also been obtained by combining the transgenic expression of multiple defence genes. As the complexity and number of defence transgenes increases, the type of promoters, or regulatory regions, used to drive their expression will become increasingly important. The majority of these defence strategies utilize constitutive promoters. Constitutive expression is, however, problematic for the overall success of these strategies, for several reasons. For instance, the constitutive expression of numerous defence genes in a transgenic plant would necessarily result in an energy loss for the plant, which in turn would be expected to decrease yield. Additionally the constitutive expression of defence genes might speed up evolutionary variability and selection of the pathogen, resulting in the loss of durability of the transgenic resistance. There is also concern that over-expression of proteins with potential allergenicity could result in an increased health risk to consumers of the transgenic plant proteins. Plant promoters which are activated precisely when and where needed would be ideal for genetic engineering strategies for enhanced disease resistance. Therefore, the isolation and characterization of plant pathogen-inducible promoters suitable for transgenic defence engineering is highly desirable. Unfortunately, little information about pathogen-induced promoter (regulatory) regions is available. Our objective is to isolate and characterize plant promoters which are activated by a broad spectrum of plant pathogens.

Results and conclusions
We used differential screening methodologies to select specific cDNA clones which are pathogen induced from red kidney bean libraries. The selected clones were subjected to further rounds of screening to identify those which are rapidly induced by a broad spectrum of pathogens. From these initial screenings, and Northern analyses, a clone designated pic20, for pathogen-induced clone, was identified. Northern analyses demonstrated that pic20 transcript was rapidly and transiently induced in bean leaf tissue after infiltration with the compatible bacterium Pseudomonas syringae pv. phaseolicola, as well as the incompatible pathogen P.s. pv. tabaci. During both interactions, pic20 transcript was detected 2 h post-infiltration, with the maximum levels of pic20 transcript accumulation occurring approximately 8-10 h post-infiltration. By 12 h the level had dropped significantly, and it was no longer detected by the 24-h time point. This contrasts with the findings of others indicating that defence gene transcripts are induced more slowly in plants by compatible as compared with incompatible pathogens. The fact that pic20 is activated by P.s. pv. phaseolicola is also interesting because data from our laboratory indicate that this compatible strain produces an active suppressor of certain defence responses [1]. The pic20 transcript also accumulated in bean leaves after inoculation with tobacco necrosis virus, heat-killed bacteria, E. coli, as well as glutathione, an abiotic elicitor of defence genes. Preliminary data suggest that pic20 is not induced by osmotic or physical stress. We have never detected pic20 transcript in untreated control plants. We have isolated and sequenced this unique pathogen-induced bean cDNA. Homologues of pic20 are also found in other plants such as tomato. We will present data concerning pathogen induction of pic20 in bean and tomato, as well as efforts toward isolating and characterizing the promoter-regulatory region from tomato. We believe that these studies suggest that the promoter element from the pic20 gene could be used in genetic engineering strategies designed to enhance disease resistance of plants to a broad spectrum of pathogens.

References
1. Jakobek JL, Smith JA, Lindgren PB, 1993. Plant Cell 5, 57-63.