An investigation into the role of deubiquitinating enzymes in plant disease resistance.
PhD thesis, University of Glasgow.
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The importance of the ubiquitin-proteasome pathway in eukaryotic cellular regulation has become increasingly apparent during the last decade. In plants, regulated degradation by the ubiquitin/26S proteasome has been implicated in diverse signalling events including embryogenesis, hormone signalling and disease resistance. Ubiquitin moieties are ligated to target proteins through the sequential activities of E1, E2 and E3 enzymes leading either to proteasomal degradation or other regulatory outcomes in the cell. It is now established that ubiquitination is a reversible process and that removal of ubiquitin from target proteins by deubiquitinating enzymes (also termed ubiquitin proteases) can also serve a regulatory function. Deubiquitinating enzymes are proteases with specificity for the isopeptide linkages formed during ubiquitin ligation events.
Current understanding of deubiquitinating enzyme function in plants is relatively limited and the aim of this project was to establish novel findings in this emerging field. This study reports an extensive analysis of the deubiquitinating enzymes in the Arabidopsis thaliana genome and functional characterisation of two closely related Arabidopsis Ubiquitin Specific Proteases: AtUBP12 and AtUBP13 and their respective orthologs in the solanaceous plants tobacco (Nicotiana tabacum) and Nicotiana benthamiana.
Previous work suggested the potential involvement of NbUBP12 in disease resistance, in this study, established methodologies in Arabidopsis, tobacco and Nicotiana benthamiana were applied to investigate this possibility. Transcript induction studies in Arabidopsis reported the induction of both AtUBP12 and AtUBP13 by avirulent Pseudomonas and exogenously applied Salicylic acid (SA). Pathology assays in single allele Arabidopsis ubp12 and ubp13 mutants reported no alteration in resistance against virulent and avirulent strains of Pseudomonas, raising the possibility that AtUBP12 and AtUBP13 are functionally redundant. Investigations into redundancy between AtUBP12 and AtUBP13 were conducted using transgenic RNAi based cosupression and the isolation of genetic crosses between ubp12 and ubp13 mutant alleles. Collectively these approaches provide the first report that AtUBP12 and AtUBP13 are functionally redundant and are required for normal plant development with homozygous ubp12 ubp13 double mutants exhibiting a seedling lethal phenotype. Phenotypic analysis of ubp12 and ubp13 mutants indicated that functional redundancy between these genes was not complete with the novel observation of early flowering in ubp12 alleles under both long and short day photoperiods. Short day early flowering in ubp12 mutants was accompanied by the development aerial rosettes and suggests the crucial involvement of deubiquitination in the floral transition.
The cDNA sequence of the tobacco AtUBP12 ortholog NtUBP12 was determined and utilised for VIGS based NbUBP12 gene silencing studies during disease resistance signalling in N. benthamiana. Loss of function studies indicated that NbUBP12 functions as a negative regulator of hypersensitive cell death (HR) induced by the Cladisporium fulvum elicitor Avr9 and R gene independent viral resistance against TMV. These findings represent the first reported link between deubiquitination and plant disease resistance. Respective cDNAs for AtUBP12 and NtUBP12 were cloned and expressed to demonstrate the function of their gene products by in vitro ubiquitin protease activity assays. Ubiquitin protease activity of UBP12 was directly implicated in C.fulvum Avr9 elicited cell death during tobacco transient overexpression assays. This experimental approach confirmed that UBP12 activity negatively regulates the Avr9 elicited HR with overexpression of AtUBP12 causing HR suppression and the corresponding AtUBP12 C208S active site mutant conferring a dominant negative HR promotion effect.
Overall the presented data reports several novel insights which implicate Arabidopsis UBPs: AtUBP12 and AtUBP13 in plant development and suggests they also may stabilise common substrates which regulate disease resistance. AtUBP12 is also specifically implicated as a floral suppressor and in vitro assays have demonstrated that AtUBP12 and NtUBP12 encode functional ubiquitin proteases. In solanaceous plants, UBP12 activity negatively regulates the defence associated HR and virus resistance.
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