Date on Master's Thesis/Doctoral Dissertation

12-2025

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biology

Degree Program

Biology, PhD

Committee Chair

Perlin, Michael

Committee Member

Schultz, David

Committee Member

Running, Mark

Committee Member

Hwangbo, Dae-Sung

Committee Member

Barati, Michelle

Author's Keywords

fungi; effectors; biotrophic

Abstract

Host-pathogen interactions in smut fungi are characterized by effector proteins and their role in infection. The goal of this work is to better understand these interactions by further characterizing an effector from Microbotryum lychnidis-dioicae and heterologously expressing this and other effectors in heterologous species to see if the range of these effectors can play a role in pathogenicity when expressed in distantly and closely related fungal species. These effectors have been attuned over time to their host plant but have homologous interactors in other plant species. In this study, Microbotryum lychnidis-dioicae, Sporisorium reilianum, and Ustilago maydis, were all used to try and understand effector dynamics when expressed in non-native environments. Also, understanding effectors on a molecular level is important in how the pathogen communicate with their host. This work first tries to further characterize an effector from M. lychnidis-dioicae, MVLG_01732. Previous in vivo studies used yeast-2 hybrid (Y2H) to elucidate protein-protein interactions of MVLG_01732. This study revealed putative interactors Cellulose Synthase Interactive Protein-1 (CSI1) and Calcium Lipid Binding protein. To further characterize these interactions, heterologously expressed MVLG_01732 in A. thaliana was performed for localization and co-immunoprecipitation studies. These results showed that the protein localizes to cell boundaries in the leaf and trichome of the plant. Scanning electron microscopy (SEM) was also done on the anthers which showed a collapsed appearance of the pollen, consistent with CSI-1 knockouts. The pull-down assay also confirmed interaction of MVLG_01732 with AtCLB and CSI1, further confirming these interactions. This work also examines how effectors act when expressed in heterologous fungal systems. The aim was to express these effectors in closely related and distantly related species to investigate if these effectors can increase pathogenicity or modulate development when expressed in another species, as many effectors act upon similar targets. I looked at the effector Suppressor of Apical Dominance-1 (SAD1) from Sporisorium reilianum, and MVLG_01732, and MVLG_04107 from M. lychnidis-dioicae. SAD1 was expressed in U. maydis, a very close relative of S. reilianum, and a fungus that shares the same host. When SAD1 was expressed in U. maydis there was a trend toward fungi producing medium galls (tumors) when compared to wild-type U. maydis. Even though there was no significant height difference between the mutant and WT fungus, the fungus expressing SAD1 had a more significant height difference when compared to uninfected plants, which possibly shows some change in plant development. Conversely, when MVLG_01732 was expressed in a distantly related species S. reilianum, there was a moderate increase in disease severity. The results were subtle, but there was a trend toward ears containing < 50% spores in plants infected with transgenic fungi when compared to plants infected with WT S. reilianum. Finally, we expressed the effector MVLG_04107 from M. lychnids-dioicae in a closely related sister species M. silenes-dioicae. We identified an effector cassette in both of these species that share homology between the effectors contained within the cassette. We identified MVLG_04107 was the effector that diverged most from the ortholog MvSd_12496. We hypothesized that these small changes in effector may be a way the effector plays a role in host specificity and the result of evolving to interact with its host. When MVLG_04107 is expressed in MvSd, there was no significant change in infection symptoms when compared to plants infected with MvSd WT. Overall, this dissertation was to examine dynamics of fungal effectors within and out of their native species. This is still an ongoing discussion and area of research to see where these effectors diverge and if they play a role in speciation on a molecular level, how they interact with their host. The findings show that there can be subtle influence on infection when non-native effectors are expressed in new systems. It also reveals that effector dynamics are complicated and temporal regulation and other cellular processes may need to be involved for full effect of effector activity.

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