Date on Master's Thesis/Doctoral Dissertation

8-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Perlin, Michael

Committee Co-Chair (if applicable)

Schultz, David

Committee Member

Schultz, David

Committee Member

Graham, James

Committee Member

Barati, Michelle

Committee Member

Running, Mark

Author's Keywords

Phytopathogen; Smut fungus; effector protein; microbotryum lychnidis-dioicae; Silene latifolia

Abstract

The smut fungal species Microbotryum lychnidis-dioicae is an obligate phytopathogen colonizing the plant host, Silene latifolia. A significant feature of M. lychnidis-dioicae infection is that the fungus can replace pollen on the anthers of susceptible host plants with fungal teliospores, thus earning the fungus the name: anther smut disease of flowers. The fungus synthesizes and secretes effector proteins into the cells of the plant host during infection, and the protein-protein interactions may interfere with and modify metabolism, plant development, and gene expression of the host to allow fungal colonization. Two potential fungal effector proteins, MVLG_06175 and MVLG_05122, were identified by genome sequence analysis and prior expression studies. The yeast-two hybrid screening was used to identify their potential plant protein interactors. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), while those interacting with MVLG_05122 were identified as COP9 signalosome subunit 5a and 5b (CSN5a/5b). CASPL2C1 might facilitate the polymerization of the Casparian strip by forming a protein scaffold at the endodermal cells where lignin deposits. CSN5a/5b could adjust the rate of protein ubiquitination and degradation by interacting with Cullin-RING E3 ubiquitin ligases (CRLs). CRLs is a large enzyme family labelling proteins with ubiquitin, and these proteins are subsequently recognized and degraded by the 26S proteasome. MVLG_06175 and MVLG_05122 were tagged by generating fusion proteins with mCherry or cyan fluorescent protein (CFP), respectively, and then transformed into the model host plant Arabidopsis thaliana. Images taken from a confocal microscopy showed that fluorescence signals of MVLG_06175 form clustered granules or punctate regions at the tips of trichomes on leaves and in root caps, while those of MVLG_05122 formed a clear band structure at the base of leaf trichomes. These results indicate that the fungal MVLG_06175 might affect the formation of the Casparian strip in the roots and the synthesis of phytochemicals in the trichomes of host plants, and MVLG_05122 might modify the development of trichomes. There were no significant phenotype changes in A. thaliana transformed with MVLG_05122, while A. thaliana transformed with MVLG_06175 showed statistically smaller rosette diameter and leaf quantity. It is significant that both effector proteins located to the trichomes on leaves of model transgenic plants. Trichomes are defensive structures of plants protecting against both biotic and abiotic stresses. These findings suggested that M. lychnidis-dioicae might apply the two effector proteins to alter host metabolism related to immune responses including the structure of trichomes in host plants.

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