No description available.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity.

Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1. To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well. mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity. mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance. Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

Mitochondrial function is dependent upon mitochondrial protein import (MPI), a complex process that transports nuclear-encoded proteins into mitochondria. Little is known about MPI in neurons. We examined the effects of Resveratrol (RSV), a polyphenolic antioxidant compound from grapes, on MPI in a neuronal cell model, differentiated PC12 cells. RSV (50µM, 24h) increased levels of mtGFP, a nuclear encoded mitochondrially targeted green fluorescent protein, and mtHsp70, a physiological mitochondrial heat shock protein, in mitochondria. In addition RSV also increased levels of Tom20, a key translocase of the outer mitochondrial membrane. The RSV mediated increases in mitochondrial proteins were independent of increases in mitochondrial mass or changes in intramitochondrial degradation. RSV also reduced mitochondria membrane potential and decreased basal levels of reactive oxygen species. Taken together, these findings show that RSV increases MPI and that this effect may be an important mechanism in the reported neuroprotective effects of RSV.

No description available.

The PhD thesis is composed of three publications on genomic, metabolic, and cellular integration between the host and its symbionts in the tripartite nested mealybug system. The articles revealed a path to an intimate endosymbiosis that can be compared to what we think happened before (and to some extent after) bacterial ancestors of key eukaryotic organelles, mitochondria and plastids, became highly integrated into their host cells. I argue that these much younger symbioses may tell us something about how the mitochondria and plastids came to be, at the very least by revealing what types of evolutionary events are possible as stable intracellular relationships proceed along the path of integration.

No description available.

The presented thesis includes three related projects, that are linked by a common interest in the evolution of eukaryotic organelles and machineries that import proteins into these compartments. The first project considers the possibility of peroxisomes (eukaryotic organelles known in aerobic organisms) being conserved in two related anaerobic protists: a free-living amoeba Mastigamoeba balamuthi and a parasite Entamoeba histolytica. The most important hint for the presence of peroxisomes was the discovery of proteins that are homologous to known components of the peroxisomal protein import machinery. The second project aims to characterize the unknown protein translocase of the inner membrane (TIM) in the mitosomes (extremely reduced mitochondria) of an anaerobic protozoan Giardia intestinalis. We have discovered an important subunit of the mitosomal translocase (Tim44), which usually tethers the Hsp70/PAM (presequence translocase-associated motor) complex to the TIM translocon. The last project shows that the protein translocase of the outer mitochondrial membrane in trypanosomatids is related to a typical eukaryotic channel Tom40. This finding is important because the absence of Tom40 was previously considered an ancestral feature of trypanosomatids.