Engineering disease resistance in plants

The genetic engineering of plants for increased pathogen resistance has engaged researchers and companies for decades. Until now, thenumberof crops with genetically engineered disease resistance traits which have entered the market are limited to products displaying virus and insect resistance. Development and registration of plants with increased broad-spectrum resistance to bacteria or fungi have failed due to limited efficacy or poor agronomic properties. This is largely due to the high diversity of pathogens that are able to attack plants, the strategies they use and the complexity of the plant signaling networks underlying natural resistance mechanisms. Generally, plant resistance is composed of multiple defence layers, jointly able to resist the majority of pathogens. Induced resistance responses are often the final layer of defence. At this stage, plants employ an active defence mechanism to stop pathogen invasion. Upon attempted pathogen ingress, plants activate distinct defence pathways to prevent the pathogen from causing disease. These active defence responses are very effective, refined and are based on very specific recognition mechanisms. In the case of gene-for-gene resistance,plant R -genes areable to provide resistance to pathogens carrying the matching Avr -gene. This natural occurring system can be turned into a genetic engineering strategy by transferring an Avr -gene to a plant containing the matching R -gene and by placing the Avr -gene under control of a pathogen-responsive promoter, the system is only activated when the plant is attacked by pathogens. Crucial for this approach is the pathogen-inducible promoter since activation of this system in the absence of pathogens can be very detrimental to plant growth and yield (Chapter 1). Apart from this particular approach, many other biotechnological applications to increase pathogen resistance in crop plants also depend on the availability of such pathogen-responsive promoters. For example, the expression of antimicrobial proteins or enzymes that synthesize antimicrobial compounds can have a negative effect on plant vigour and yield as well and therefore conditional expression of thetransgene,only in the presence of pathogens is key. Chapter 3 and 4 describe the isolation of novel plant pathogen-inducible promoters and the characterization of these promoters in transgenic plants. Different approaches can be applied to identify appropriate pathogen-inducible promoters. In Chapter 3 we have reported the results of a promoter tagging approach to identify promoters that are induced upon pathogen attack in Arabidopsis thaliana . One candidate out of 500 screened tagging lines displayed the desired phenotype. The tagged promoter was cloned in front of a reporter gene and was shown to be functional in transgenic Arabidopsis and Brassica napus plants. The isolated promoter sequence appeared to be linked to a predicted serine threonine kinase gene in the reverse orientation. Expression of the kinase messenger RNA was shown to be upregulated in response to pathogen infection and SA treatment, similar to the activity of the isolated promoter and the original tagging line.Furthermore, two novel plant promoters were isolated from genes that display a local response after pathogen infection (Chapter 4). These promoters were fused to the Uid A reporter gene as well and transformed into potato. Characteristics of these newly isolated promoters were compared with two well-studied plant pathogen-inducible promoters isolated from the genes encoding Vitis stilbene synthase 1 ( Vst1)and potato Glutathione-S transferase 1 ( Gst1). Twenty transgenic lines of each promoter- Uid A fusion were analysed using conventional histochemical staining and real time RT-PCR analysis to visualize spatial, kinetic and dynamic properties of these promoters. All promoter- Uid A fusions were shown to be responsive to an oomycete pathogen in the crop plant potato and displayed differential pathogen-responsive properties with respect to localization, timing, level and frequency of induction.Apart from the pathogen-inducible promoter, properties of the pathogen-derived Avr -gene are of importance as well. It is expected that the development of an HR stimulates strong transcriptional changes. In Chapter 5, the ability of the Cladosporium fulvum Avr9 avirulence protein to induce transcriptional changes in potato was investigated. Multiple expression profiling experiments using cDNA microarrays were performed to follow the expression profile of approximately 10.000 potato genes in response to Avr9 application. Avr9 protein preparations were manually infiltrated in leaves of potato plants that were either untransformed or transformed with the complementary tomato R -gene, Cf-9 . At least 510 potato gene elements on the array have been shown to respond to Avr9 infiltration in potato. These genes were designated Solanum tuberosum Avr9-responsive genes (STAR genes). In Cf-9 expressing plants, strong shifts in gene expression were observed. Concurrent with an apparent induction of many defence and stress-associated genes, genes related to primary metabolism and photosynthesis were repressed. Limited gene expression alteration was observed in untransformed potato plants after infiltration with the Avr9 protein.An alternative approach to engineer disease resistance in plants includes the expression of antimicrobial proteins. Chapter 6 describes the identification of novel plant derived antimicrobial proteins in extracts from salicylic acid treated lettuce and sunflower leaves. Proteins of approximately 60 kDa in both extracts were found to be responsible for the observed antimicrobial activity. Further characterization of these proteins and cloning of the respective cDNAs revealed close homology to various plant oxidases. Dissection of the enzymatic activity of the proteins revealed them to be carbohydrate oxidases with broad substrate specificity and with hydrogen peroxide as one of the reaction products. Characterization of the mode of action of these proteins revealed that the hydrogen peroxide produced was responsible for the observed antimicrobial activity. Tobacco plants overexpressing Ha-CHOX displayed increased resistance to Pectobacterium carotovorum subsp. carotovorum and the resistance level was proportional to the Ha-CHOX enzymatic activity, which exemplifies the utility of these enzymes to engineer disease resistance. Finally in chapter 7, different strategies are discussed that plants employ to resist pathogen infection and the strategies that are currently under development to engineer disease resistance in plants.The applied research described in this thesis has provided novel components, tools and knowledge which will contribute to the quality and efficacy of genetically modified crops with increased pathogen resistance and to a better understanding of the mechanisms plants employ to recognize and resist microbial pathogens.