Cell Biology & Molecular Genetics Theses and Dissertations
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Item INVESTIGATING MECHANISMS UNDERLYING MLO’S ROLE AS A HOST FACTOR ESSENTIAL FOR PATHOGENESIS OF POWDERY MILDEW FUNGI(2024) Bloodgood, David; Xiao, Shunyuan; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Loss-of-function mutations in Mildew Locus O (MLO) family genes confer broad-spectrum resistance to powdery mildew (PM) fungi in various plant species. mlo-mediated resistance is invariably coupled with increased defense responses and early leaf senescence; hence the molecular basis of mlo-mediated resistance remains unresolved. A saturated genetic screen in the background of an Arabidopsis triple mutant where three essential immune components, EDS1, PAD4 and SID2 are mutated, led to the identification of five allelic mutations in MLO2, each of which results in compromised immunity yet poor infection (cipi) to PM. Further CRISPR-targeted mutagenesis of two functional homologs, MLO6 and MLO12 in a cipi mutant background result in complete lack of infection from PM fungi. The sextuple mutant, eds1pad4sid2mlo2mlo6mlo12 (epsm3) showed no early leaf senescence, ROS accumulation or expression of defense genes, indicating that MLO2, MLO6 and MLO12 are bona fide host susceptibility factors for PM. Expression of MLO2-GFP as a transgene in epsm3 restores susceptibility to PM and MLO2-GFP focally accumulates at the fungal penetration site. Thus, restoration of susceptibility to PM in the epsm3 background can be used as a sensitive reporter to assess whether other MLO family members share a conserved molecular function when expressed in leaf epidermal cells. The Barley MLO and Arabidopsis MLO7 enabled PM pathogenesis whereas MLO1, MLO3 and MLO4 could not, suggesting the existence of two distinct classes of MLO family members. Sequence alignment identified three conserved amino acid residues in the C terminal calmodulin-binding domain of MLO2, and MLO7, which are absent in MLO1, MLO3 and MLO4. This observation suggests that the C-terminal domain of MLO proteins could contribute to their functional divergence. Creation and functional assays of chimeric MLO2/MLO1 proteins by swapping their C terminal domains revealed that the C terminus determines the localization pattern of MLO proteins. The Feronia (FER) receptor-like kinase is required for localization of MLO7 in synergid cells; however, CRISPR-targeted mutagenesis of FER did not disrupt the localization of MLO2 to the fungal penetration site. Based on the results described above, it can be inferred that MLO2 localization to and possible stabilization of the plasma membrane at the fungal penetration site is essential for allowing PM fungi to penetrate the host cell and subsequently differentiate the haustorium. Further multiplexed CRISPR mutagenesis of other gene families suggests that SYP121 and SYP122, two closely related SNARE genes play essential roles in focal accumulation of MLO2 at the fungal penetration site.Item BOLT, AN AP2/ERF TRANSCRIPTION FACTOR, REGULATES ABIOTIC STRESS AND DEFENSE RESPONSES IN ARABIODPSIS THALIANA(2016) Bouten, Roxane; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Biotic and abiotic stresses negatively affect plant growth and development, hence decrease productivity. Many AP2/ERF family transcription factors in plants have important roles in stress response signaling although most have not yet been studied. Here I show that expression of an Arabidopsis thaliana AP2/ERF family member, which I call BOLT, is regulated by a MAPK pathway that includes MEKK1, MKK1, MKK2, and MPK4, and has roles in both biotic and abiotic stress response as well influencing growth and development. In this thesis, I examined BOLT’s gene expression pattern and protein localization, using GUS and YFP reporter genes respectively, measured its expression in response to biotic and abiotic stress and plant hormones using RT-qPCR, examined phenotypes by generating overexpressing and RNAi lines, and analyzed its effect on downstream gene expression using a microarray at time points after inducing BOLT expression. I found that BOLT is expressed in various plant tissues and the protein localizes to nuclear bodies as demonstrated in onion epidermal cells. Knockdown (RNAi) plants exhibit greater drought tolerance and are larger than wild type under low light conditions, while the overexpressors exhibit a dramatic early flowering phenotype and are small and weak under low light. Gene expression analysis suggests BOLT regulates genes involved in photosynthesis, hormone biosynthesis and signaling, and defense, many of which are also regulated in the MAPK pathway. Increased BOLT expression downregulates two discreet systems, cyclic electron flow and glycine cleavage, components of photosynthesis and photorespiration, respectively, which are two systems that are important under low light conditions. Taking these results together, I conclude that BOLT functions downstream of a stress responsive MAPK pathway and regulates a variety of growth- and stress-related genes necessary to balance growth and defense in response to biotic or abiotic stresses, or low light conditions.Item Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo(2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.Item Insights into the regulation of ethylene receptor signaling by RTE1(2008-10-10) rivarola, maximo lisandro; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is an important regulator of plant growth, development and responses to environmental stresses. The higher plant Arabidopsis thaliana perceives ethylene through five homologous receptors, which negatively regulate ethylene responses. The molecular mechanism by which these receptors signal to their next downstream component remains elusive. Genetic analyses have shown that the RTE1 locus is a positive regulator of ETR1. RTE1 encodes a novel protein of unknown molecular function, and is conserved in plants, animals and some protists. The goal of this research was to analyze the mechanisms involved in the regulation of ethylene receptor signaling by RTE1 and to enhance our understanding of the conserved cellular role of RTE1. Here we tested hypotheses for how RTE1 affects ETR1 and is specific to only ETR1, not the other ethylene receptor isoforms. We show that ETR1 and RTE1 gene expression patterns partially overlap and that the ETR1 receptor co-localizes with RTE1 within the cell. Moreover, RTE1 has no effect on ETR1 protein abundance or subcellular localization suggesting other mechanisms to regulate ETR1. We provide supporting evidence that RTE1 affects ETR1 signaling by restoring signaling of a non-functional ETR1 in an rte1 null through changes in ETR1 conformation(s). We next addressed the question of RTE1 specificity to ETR1. We discovered that ETR1 is surprisingly distinct from the other four ethylene receptor genes; in that RTE1-dependent mutations only confer insensitivity in ETR1 and not in the other ethylene receptors when the same mutations are introduced. In contrast, the RTE1-independent ETR1 insensitive mutations do give insensitivity in the closest receptor to ETR1, ERS1. Furthermore, we uncover that the ethylene binding domains are not completely interchangeable between ETR1 and ERS1. Our data point to a model in which RTE1 specifically promotes ETR1 signaling via conformational changes in a unique way that does not occur in other ethylene receptors. These findings highlight the importance and uniqueness of ETR1 signaling conformation(s) with respect to the other ethylene receptors, as well as advance our knowledge of RTE1 at the molecular and cellular level.Item MOLECULAR CHARACTERIZATION OF INTERACTIONS BETWEEN TMV REPLICASE PROTEIN AND AUXIN RESPONSIVE PROTEINS: IMPLICATIONS IN DISEASE DEVELOPMENT(2006-11-25) Padmanabhan, Meenu Sreedevi; Culver, James; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, these findings suggest that TMV induced disease symptoms can partially be explained by the ability of the virus to disrupt the functioning of interacting Aux/IAA proteins within susceptible hosts. The significance of such interactions is yet to be determined but it appears that they may be advantageous to the virus while infecting tissues that are in a developmentally static stage.Item The Importance of Sorting Calcium in Plant Cells: Uncovering the Roles of A Sarcoplasmic/Endoplasmic Reticulum-Like Calcium ATPase(2006-11-29) Li, Xiyan; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The spatial and temporal dynamics of intracellular Ca2+ in response to environmental and hormonal cues underscore the importance of Ca2+ transport during plant growth and development. The Arabidopsis thaliana genome predicts multiple genes encoding Ca2+ transporters, though the biological roles of most are unknown. Here I determine the function of AtECA3 which represents the first plant P-type 2A ATPase resembling mammalian sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA). AtECA3 (At1g10130) expressed in a yeast mutant lacking its endogenous Ca2+ pumps functionally substitutes for the defective Ca2+-ATPases. AtECA3-dependent yeast growth is blocked by thapsigargin, a specific SERCA inhibitor. The results suggest that AtECA3 is a cation pump with specificity for Ca2+ and Mn2+, and that AtECA3 enhances yeast growth on Ca2+-depleted medium or on medium with high Mn2+ by sequestrating Ca2+ or Mn2+, respectively, into endomembrane compartments. AtECA3 is expressed in pollen grains as revealed by promoter::GUS analyses and a green fluorescence protein (GFP)-tagged to AtECA3 labels endomembranes at the pollen tip. In vitro tube growth of wild-type pollen is enhanced by 10 mM Ca2+, and inhibited by thapsigargin, suggesting that AtECA3 supports tube elongation by sorting intracellular Ca2+ to appropriate compartments. This idea is supported by genetic evidence, where three T-DNA insertional mutants show 33% reduction in pollen tube length. This defect lowers sperm transmission shown as segregation distortion and decreased seed set. AtECA3 is also expressed in vascular tissues of young roots and leaves, and the GFP-tagged protein colocalizes with two Golgi markers. Three millimolar Ca2+ stimulate root growth of wild-type but not of mutants, indicating that Ca2+ accumulation in Golgi lumen is critical for growth. Root growth of eca3-4, but not of wild-type, is hypersensitive to 50 μM Mn2+. Thus loading Mn2+ into Golgi lumen by AtECA3 supports root growth. Intriguingly, mutant roots show 80% increase in apoplastic peroxidase suggesting that secretory activities became deregulated. In conclusion, I provide molecular evidence for a Golgi Ca2+/Mn2+ pump in plants. Ca2+ and Mn2+ accumulation into Golgi/secretory compartments by AtECA3 and perhaps cation release from these stores affect secretory activities critical for root growth, pollen tube elongation and male fertility.Item LARSON directly represses AGAMOUS during early flower organogenesis in Arabidopsis thaliana(2004-04-30) Bao, Xiaozhong; Liu, Zhongchi; Cell Biology & Molecular GeneticsHow cells in a multicellular organism assume their developmental fates and form distinct patterns is a fundamental biological question. To address this question, I studied genetic and molecular regulation of Arabidopsis flower organ formation and identity determination. Specifically, how the expression of floral meristem and floral organ identity gene AGAMOUS (AG) was regionalized during flower organogenesis. A novel AG repressor LARSON (LSN) was previously isolated in a genetic screen. lsn loss-of-function mutations caused precocious expression of AG in the inflorescence meristem and ectopic expression of AG in sepal primordia, resulting in partial homeotic transfomation of late inflorescences into floral meristems and strong homeotic transformation of first whorl sepals into carpels. LSN encoded a homeodomain protein that directly bond to AG cis-regulatory elements in vitro. The cis-regulatory elements were conserved in 17 Brassicaceae species. LSN was expressed in a subset of cells located in the peripheral zones of inflorescence and floral meristems. LSN expression was significantly reduced in the sepal and petal primordia in wild-type flowers, indicating that repression of AG in the sepals and petals was independent of LSN transcription. LSN might establish epigenetic AG repression in the ancestral cells in the peripheral zone to specify the identities of descendant cell types in the floral organs. Therefore, floral organ identities were not only dependent upon gene expression in the organs, but were also dependent upon the histories of the cell development. Genetic and molecular analyses showed that LSN acted upstream of a putative repression complex, which, I proposed, was involved in the maintenance of AG repression in flowers. The putative repressive complex consistes of APETALA1 (AP1), LUNIG (LUG) and SEUSS (SEU) known to encode flower specific repressors of AG. Mutations in these three genes enhanced the lsn phenotypes. However, none of their proteins interacted with LSN in yeast. Instead, AP1, SEU, and LUG might form a protein complex. Genetic and molecular analyses suggested that the AG-repressive functions of the putative complex depended upon LSN activity in the peripheral zone of floral meristem. The AG-repressive function of LSN in the inflorescence meristem was independent of AP1/SEU/LUG putative complex.