REPOSITORY > RESULTS

Abstract

Plant pathogens are responsible for substantial economic losses. Understanding the exact mechanisms of plant response to pathogens is required for designing stress resilient plant varieties. One of our aims was to decipher a plant immune regulatory network through characterisation of the promoters in different potato genotypes. These could in the next stage be used for designing specific sensors of immune signalling which would allow precise monitoring of spatiotemporal dynamics of this process. Nucleotide alignments of the promoter sequences from selected genes involved in plant hormonal signalling showed high variability between, but also within the analysed genotypes. Accordingly, in silico analysis of the promoter variants resulted in the identification of diverse pathogen-inducible regulatory elements in the promoters of the selected genes. The activities of the promoter variants with the most promising combination of regulatory elements were analysed in transient and stable expression set-ups after promoters were fused with fluorescent proteins. To prepare a sensor which will enable simultaneous following of the activity of several promoters, different combinations of fluorescent proteins under different promoters first need to be introduced into the plant genome. As cloning multiple DNA fragments is still one of the main technological challenges in plant synthetic biology, we developed Plant X-tender, an extension of multigene assembly strategy AssemblX, based on overlap-depended cloning methods and rare-cutting restriction enzymes. Plant X-tender consists of a set of plant expression vectors and the protocols for the most efficient cloning into the novel vector set. It covers different empty backbones with different selection markers to allow full design flexibility and includes ccdB counterselection to allow straightforward and highly efficient transfer of multigene constructs into the novel vector set. We have further validated the developed procedure for plant multigene expression in a transient set-up. In the third part of this thesis, we studied the involvement of cell death in hypersensitive response (HR)-conferred resistance to viral infection. We showed that potato virus Y can be detected outside the cell death zone in Ny-1-mediated HR in potato plants (cv. Rywal), observed as individual infected cells or clusters of infected cells outside the cell death zone. We confirmed that the cells at the border of the cell death zone harbour a viable virus that is able to reinitiate the infection. We further studied the dynamics of cell death zone expansion and occurrence of infected cells outside it. We compared the response of Rywal plants to their transgenic counterparts, impaired in salicylic acid accumulation, where the lesions develop but the spread of the virus is not restricted. We showed that the virus is detected outside the cell death zone in all developmental stages of HR lesions. We observed rapid lesion expansion and even faster virus spread in salicylic acid-depleted plants. On the other hand, the majority of analysed lesions slowly expand also in HR-conferred resistance opening the possibility that the infected cells are eventually engulfed by cell death zone. Thus we suggest that the HR-associated cell death is separated from the resistance mechanisms which lead to potato virus Y restriction in Ny-1 genetic background and propose that HR should be regarded as a process where the dynamics of events is crucial for the effectiveness of viral arrest.