THE EFFECT OF A SIGNAL PEPTIDASE MUTATION ON PROTEIN SECRETION BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA

Author

Renata Belisario, University of KentuckyFollow

Author ORCID Identifier

https://orcid.org/0000-0002-5152-0386

Date Available

5-1-2025

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant Pathology

First Advisor

Dr. Lisa Vaillancourt

Abstract

Colletotrichum graminicola infects maize during several phases of plant growth, resulting in anthracnose leaf blight, stalk rot, and top die-back diseases. This ubiquitous fungus has significant destructive potential and causes millions of dollars of losses annually in North America. Colletotrichum graminicola is hemibiotrophic and initially invades living host cells via biotrophic primary infection hyphae before switching to necrotrophy and inducing host cell death, host cell-wall degradation, and lesion development.

An insertional mutation in the 3’UTR region of a gene (Cpr1), which encodes a homolog of the noncatalytic SPC22/23 subunit of the endoplasmic reticulum associated signal peptidase complex, completely abolished pathogenicity to maize leaves and stalks. The mutation is conditional, in that it has little or no effect on growth and development of the fungus in culture or in killed maize tissues. The Cpr1 mutant (MT) is interrupted early during infection of living host cells, and never shifts to necrotrophy, produces lesions, or sporulates. Although it germinates, enters the host plant, and produces primary infection hyphae, it fails to move beyond the first invaded cell. Interestingly, when the wild type (WT) strain is inoculated adjacent to (but not in contact with) the MT, it restores the ability of the MT to colonize beyond the first cell and establish a successful infection. This observation led to the hypothesis that the WT secretes pathogenicity proteins that promote colonization of living host cells, while the MT lacks this ability.

To address this hypothesis, I used several methods to evaluate the ability of the MT to produce and secrete effector proteins and cell wall degrading enzymes (CWDE), two important classes of secreted pathogenicity proteins. I visualized individual effector proteins and CWDE as fluorescent fusions and quantified their signals in WT, MT, and complemented mutant (C-MT) strains in vitro and in living or killed maize leaf sheaths. Additionally, hydrolase activity against plant cell wall polysaccharides in vitro was compared by using reducing-sugar assays and by quantifying degradation of chromogenic substrates.

Results indicated an overall significant reduction and delay in the production and secretion of effector fusion proteins by the MT compared with the WT and C-MT strains in living plant tissues. Furthermore, the MT strains exhibited different patterns of effector protein localization. In contrast, hydrolase activity against cell wall polysaccharides in vitro, and the accumulation and secretion of CWDE fusion proteins both in vitro and in planta, were mostly unaffected in the MT. This suggests that Cpr1 is involved in the secretion of some pathogenicity proteins, but that its role is not simply quantitative. My results indicate that Cpr1 has a more complex function in the regulation of protein secretion than was previously known, and that this function is particularly important for the early colonization of living host cells by C. graminicola. Since all fungi share the conserved CPR1 protein and use similar pathways to produce secreted pathogenicity proteins, further exploration of CPR1 and its function during plant infection and colonization could reveal potential universal targets for managing other fungal pathogens in addition to the important Colletotrichum species.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.229

Funding Information

This study was supported by the United States Department of Agriculture - National Institute of Food and Agriculture (Nos.: 2018-67013-28489 and 2020-70410-32901) in 2019 and 2020, respectively.

Recommended Citation

Belisario, Renata, "THE EFFECT OF A SIGNAL PEPTIDASE MUTATION ON PROTEIN SECRETION BY THE MAIZE ANTHRACNOSE PATHOGEN COLLETOTRICHUM GRAMINICOLA" (2024). Theses and Dissertations--Plant Pathology. 46.
https://uknowledge.uky.edu/plantpath_etds/46