Struktur und Funktion des GR1-Gens aus Arabidopsis thaliana (L.) Heynh
Arabidopsis thaliana shows race-specific interactions with the obligate biotrophic oomycete Hyaloperonospora parasitica. Thus, accession Wassilewskija (Ws-0) is resistant to the isolates WELA and NOCO. The gr1 mutant was isolated from a T-DNA population of Ws-0 plants showing susceptibility towards the WELA isolate, however, retaining resistance towards the NOCO isolate. Since it seemed that the T-DNA insertion into the GR1 gene was the cause for the loss of resistance against the WELA isolate, it was the aim of this thesis project to characterize the GR1 gene and the gr1 mutant and to study the function of the gene and protein. Towards characterization of the GR1 gene, sites of transcription initiation and termination, typical elements of the GR1 promoter, and the translation initiation site should be identified. Using 5'RACE it was shown that the first of two ATGs at the 5'-end serves as the translation initiation site. Moreover, the likely transcription initiation site was localized 118bp upstream of the first ATG. Another 35bp upstream of the transcription initiation site there is an AT-rich region which could serve as the TATA-box. A potential CAAT-box, as a second regulatory element, was found 90bp upstream. Using 3'RACE, it was shown that there is no strictly defined end of transcription but that the transcripts end in AT-rich regions. Towards characterization of the gr1 mutant the precise position of the T-DNA insertion should be localized. Accordingly, the left-border of the T-DNA was found to be integrated 1843bp downstream of the ATG and additional 50bp non-GR1 sequence was found and showed to be present in frame. The right-border was localized in the third exon 1921bp downstream of the ATG. Since the left- and right-border have integrated at different sites in the gr1 mutant a deletion of 78bp in the third exon became apparent. To test, whether the mutated gene is responsible for the susceptibility-phenotype of the gr1 mutant to Hyaloperonospora parasitica, the gr1 mutant should be complemented with a wild-type GR1 construct. Thus, the coding sequence of GR1 under the control of the native promoter was used. Microscopic analysis of independent transgenic lines infected with the WELA isolate revealed no complementation of the susceptible phenotype even though with RT-PCR expression of the artificial GR1 gene in the transgenic plants could be shown. Another way to test whether GR1 has a role in defence against the WELA isolate would be the identification of an independent gr1 mutant. Screening a population of the "Arabidopsis Knockout Facility at the University of Wisconsin-Madison" a T-DNA insertion in the second intron could be found. However, this T-DNA was spliced normally and thus this mutant didn't yield new results. With the help of gene silencing, it was attempted to repress or reduce expression of GR1 in Ws-0 plants transformed with RNAi constructs, in order to recapitulate the susceptible phenotype of the gr1 mutant. Even though RT-qPCR showed that GR1 transcript levels in particularly two transgenic lines were reduced by 250-500fold, no reproduction of the susceptible phenotype could be observed after WELA infection. RT-PCR showed that in the gr1 mutant the first to the third exon is transcribed until the T-DNA insertion including the 50 non-GR1 nucleotides. To investigate whether gr1 might a "gain-of-function" mutant, in which the aberrant peptide might interfere with the function of GR1, two experimental strategies have been chosen. On the one hand, the gr1 mutant was transformed with RNAi constructs. On the other hand wild-type Ws-0 plants were transformed with a construct mimicking the aberrant gr1 transcript. The transgenic gr1 lines were still susceptible to WELA infection. Thus, a reversion of the "gain-of-function" effect to a resistant phenotype could not be observed, even though RT-qPCR showed a reduction of transcript levels up to 500-1000fold. The transgenic Ws-0 lines didn't show a change in phenotype and were all resistant to WELA infection. Thus, a "gain-of-function" effect a phenocopy of the gr1 mutant could not be observed, even though expression of the aberrant transgene could be shown with RT-PCR. The discovery of two nearly perfect 14-3-3 binding sites in GR1, a systematic test of yeast-two-hybrid interaction of the GR1 protein with the Arabidopsis 14-3-3 proteins was initiated. However, in the yeast-two-hybrid assay, no interaction between GR1 and the 14-3-3 proteins could be detected, even though protein expression in the yeast cells could be shown. In order to obtain positive yeast-two-hybrid interaction results, the twelve 14-3-3 proteins from Arabidopsis were systematically tested for interaction with each other. According to our data, 14-3-3 proteins from Arabidopsis don't show homodimerization but heterodimerization with specific isoforms. In summary it can be concluded from the present work that GR1 probably does not play a role in the race-specific resistance of Arabidopsis thaliana accession Wassilewskija (Ws-0) to the WELA isolate of Hyaloperonospora parasitica.
info:eu-repo/classification/ddc/570, Ackerschmalwand, Resistenzgen, Pathogene Pilze, Biowissenschaften, Biologie, Arabidopsis thaliana, Rassenspezifische Resistenz, 14-3-3 Proteine
Networked Digital Library of Theses and Dissertations
Publikationsserver der RWTH Aachen University