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.

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

Asymmetric localization of PIN proteins controls directionality of auxin transport and many aspects of plant development. The Arabidopsis mutant, unhinged-1 (unh-1), has defects to leaf veins and other root and shoot phenotypes. I identify UNH as the Arabidopsis VPS51 homologue, a member of the Arabidopsis GARP complex, and show that UNH interacts with VPS52, another member of the complex. I also show that UNH co-localizes with SYP61, a trans Golgi network marker. The GARP complex in yeast and metazoans retrieves vacuolar sorting receptors to the TGN and is important in sorting proteins for lysosomal degradation. PIN1 expression in the margin of unh-1 leaves is expanded and the unh leaf phenotype is suppressed by pin1 mutation, supporting the idea that the phenotype results from expanded PIN expression. My results suggest the UNH is important to restrict expression of PIN1 within the margin, likely by targeting PIN1 to the lytic vacuole. / xv, 100 leaves : ill. ; 29 cm

In the following study, I have investigated aberrant integration events of the maize Activator/Dissociation ( Ac/Ds) family of transposable elements (TEs) in Arabidopsis. The purpose of the study was twofold: (i) to identify sequence modifications associated with aberrant transposition that are informative regarding the mechanism of Ac/Ds transposition; and (ii) to extend our understanding of the mechanisms by which class II TEs can influence genome structure. This work focuses on a large inversion identified on chromosome II. A lone Ds element comprises one breakpoint of the inversion and the second breakpoint is composed solely of Arabidopsis sequences. The analysis of the sequence modifications present at both breakpoints indicates that the event was precipitated by the abortive transposition of Ds. This is the first event of its kind identified for an Ac/Ds and the event defines a novel mechanism by which these TEs can induce change within a genome. Further, the presence of deletions at both termini of the implicated Ds suggests that the transposition of Ac/Ds involves fully excised intermediates. To obtain further support for this model, a population of Arabidopsis individuals harboring Ds excision events was screened for reintegrated elements. Several integrations were analyzed at the sequence level and compared to wild-type integration sites. While no genome rearrangements were detected, a number of integrations display small deletions within both the Ds termini and the DNA flanking the elements. These results are consistent with the presence of fully excised Ac/Ds intermediates. Further, the results suggest that dissolution of the transposase active complex at different points in the transposition process will result in the formation of distinct aberrant transposition products. During the characterization of the inversion, a novel Arabidopsis TE family, FARE, was identified. The FARE elements are foldback transposons, a heterogeneous and poorly characteri

TCP genes comprise a large family of genes that have been implicated in a diverse range of plant developmental pathways ranging from lateral branching (Doebley et al, 1997) to organ symmetry (Luo et al, 1999) and leaf curvature (Nath et al, 2003; Palatnik et al, 2003). I studied three closely related Arabidopsis TCP genes, one of which was recovered in an enhancer trap screen to identify downstream targets of the regulator of inflorescence architecture, BREVIPEDICELLUS (Douglas and Riggs, 2005). The enhancer trap marker line served as a reporter for TCP15 expression. Data mining has revealed a possible link between TCP15 and the hormone auxin. Using the DR5::GUS molecular reporter for auxin accumulation I found that TCP15 and the related TCP14 genes limit auxin maxima in seedling and reproductive tissues and that auxin transport is necessary for correct TCP15 expression. The closely related TCP8 gene was found to regulate leaf shape as demonstrated by decreased leaf index values. The rounder leaves of tcp8 plants also exhibited increased adaxial trichome and stomatal densities resulting in significantly decreased spacing between adjacent cells. tcp8 leaves showed increased serration density suggesting that TCP8 limits marginal outgrowth. Vein patterning was also perturbed in the mutants. Vein loops were rounder and smaller, and decreased loop subdivision indicated that vein patterning is retarded in the mutant. TCP8 evokes organ-specific effects on vascular patterning as mutant rosette leaves showed increased vascular complexity, whereas mutant cauline leaves showed decreased vein complexity. These results suggest that TCP8 is necessary for correct leaf development. The Arabidopsis genome contains 24 TCP genes, many of which have not been characterized. Studies of these genes will lead to the identification of additional factors necessary to control plant architecture and enable us to optimize plant growth and yield using genetic engineering.

Telomeres form a nucleoprotein cap at the end of eukaryotic chromosomes. The telomere protein constituents repress the DNA damage response (DDR) and facilitate maintenance of terminal sequences by a specialized ribonucleoprotein complex called telomerase. In turn, factors involved in the DDR guarantee telomerase acts only in telomere homeostasis, and not at double-strand breaks (DSBs). Thus, the three pathways surrounding telomeres display incredible overlap and are immensely complex. Here, I report a novel regulatory pathway that limits telomerase action during DNA damage. Duplication of the telomerase RNA subunit (TER) in Arabidopsis has given rise to a TER that is not required for telomere homeostasis. Indeed, this TER, termed TER2, is a competitive inhibitor of TER1 RNP complexes. Exposure to genotoxic agents results in TER2 upregulation and a subsequent inhibition of telomerase activity. Using data from the 1,001 Arabidopsis genomes project, I determine that the TER duplication and inhibitory nature of TER2 is likely derived from a transposon-like element within TER2. This element is found throughout Brassicaceae, with at least 32 members in Arabidopsis lyrata. These findings highlight the complex and diverse mechanisms by which an organism will regulate telomerase action. Here I characterize two members of the A. thaliana POT1 gene family. Contrary to POT1a, these proteins appear to have derived unique ways to perform their roles in chromosome-end protection. POT1b may protect telomeres as part of a TER2 telomerase RNP complex, as telomere defects only appear in the absence of both POT1b and TER2. POT1c is also appears to provide for chromosome end protection and appears to compete with POT1a to regulate telomerase access to the G-overhang. Together, these proteins represent part of a critical telomere capping complex distinct from CST. Additionally, I describe a means for elucidating factors that regulate telomere addition at DSBs. This incredibly detrimental process, termed de novo telomere formation (DNTF), is toxic, and thus this work describes the first in depth characterization of DNTF in multicellular eukaryotes. In summary, my work describes several novel regulatory and protective mechanisms for keeping telomeres and DSBs distinct.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Michigan State University. Cell and Molecular Biology Program, 2006. / Title from PDF t.p. (viewed on Nov. 20, 2008) Includes bibliographical references. Also issued in print.