Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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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 IDENTIFICATION AND CHARACTERIZATION OF NOVEL PLANT IMMUNE COMPONENTS USING THE ARABIDOPSIS-POWDERY MILDEW PATHOSYSTEM(2018) Zhang, Qiong; Xiao, Shunyuan; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Despite tremendous progress in the field of plant immunity in the past two decades, how plants mount spatiotemporally appropriate defenses against pathogens is still not well characterized. My thesis project took both forward and reverse genetic approaches to uncover novel mechanisms used by plants to fight against fungal pathogens, or exploited by fungi to adapt to host plants using the Arabidopsis-powdery mildew pathosystem. Through a reverse genetics approach, I found that two phospholipase D (PLD) genes PLDα1 and PLDδ play opposing roles in modulating basal, post-penetration resistance against mildew through a novel, yet-to-be characterized mechanism that is independent of EDS1/PAD4 (key immune components), salicylic acid (SA), and jasmonic acid (JA). Inspired by this finding, I designed and performed a large-scale forward genetic screen in the background of a super-susceptible Arabidopsis eds1-2pad4-1sid2-2 (eps) triple mutant. By screening EMS-mutagenized eps plants using powdery mildew species with different levels of adaptation on Arabidopsis, 5 susceptible to non-adapted PM (snap) and 18 compromised immunity yet poor infection (cipi) mutants have been identified. So far, this has led to the characterization of the MAP KINASE PHOSPHATASE1 (MKP1) gene, which is a negative regulator of PAMP-triggered immunity, and the MILDEW RESISTANCE LOCUS 2 (MLO2) gene, a susceptibility factor of powdery mildew, both of which act independently of EDS1, PAD4, and SA. Together, results from this work should contribute to a better understanding of the multi-layered plant immune system and powdery mildew’s host adaptation mechanisms.