Date of Graduation

7-2020

Document Type

Thesis

Degree Name

Master of Science in Plant Pathology (MS)

Degree Level

Graduate

Department

Plant Pathology

Advisor

Martin J. Egan

Committee Member

Kenneth L. Korth

Second Committee Member

Yong Wang

Keywords

actin, appressoria, coronin, cytoskeleton, fungi, septin

Abstract

Rice blast disease, caused by the filamentous fungus Magnaporthe oryzae, destroys sufficient rice each year to feed 60 million people, and is a serious threat to global food security. A wheat-adapted lineage of M. oryzae now poses threat to global wheat production. Rice blast disease is currently controlled using limited fungicides, and the emergence of fungicide resistance within M. oryzae populations is a growing concern. There is a pressing need to identify new classes of fungicides to control the disease, which requires better understanding of the basic biology of the pathogen. To establish disease, M. oryzae forms a specialized dome shaped infection structure called an appressorium, which it uses to physically break into rice tissues. The timely formation of toroidal septin ring at the base of appressorium is required for successful infection of the rice plant. Septins belongs to a conserved family of GTP-binding proteins and self-assemble into hetero-oligomeric rods and filaments that form higher-order structures such as rings and gauzes at the cell cortex. Septin-mediated scaffolding of the filamentous actin (F-actin) network is needed for the emergence of a polarized penetration hypha from the base of the M. oryzae appressorium. Despite being highly conserved in eukaryotes, septins are absent in plants, which makes them a possible target for the development of novel fungicides. However, there is little understanding about how these septins rings form in time and subcellular space during appressorium morphogenesis. First, using quantitative 4D fluorescence imaging, we show that an incipient septin disc-like structure is dynamically remodeled into a toroidal ring, in a process requiring the formation of an F-actin contractile ring, during appressorium morphogenesis. We demonstrate that F-actin ring formation, and subsequent contraction of the incipient septin disc-like structure, requires melanin-dependent turgor pressure generation by the appressorium. Using a pharmacological approach, we demonstrate the requirement of an intact microtubule and F-actin cytoskeleton for the structural integrity of these incipient septin disc-like structures. Next, using an enzyme-catalyzed proximity dependent proteomics approach, we identify the actin modulating protein coronin (MoCrn1), as a putative septin proximal protein. Using a 4D fluorescence imaging approach, we demonstrate that MoCrn1 is transiently proximal to the septin cytoskeleton during appressorium development. Lastly, using a reverse genetic approach, we provide evidence that MoCrn1-mediated F-actin remodeling is required for normal septin ring formation. The findings of this research will provide new insight about cellular control of the septin organization in M. oryzae, a global cereal killer.

Video 1.mp4 (264 kB)
Video 2.mp4 (399 kB)
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Movie S5.mp4 (2365 kB)

Available for download on Wednesday, August 03, 2022

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