Universität zu Köln

Punt, Wilko ORCID: 0000-0003-4047-1117 (2026). Functional Insights into Antimicrobial Effectors Driving Niche Adaptation of the Soil-Borne Plant Pathogenic Fungus Verticillium dahliae. PhD thesis, Universität zu Köln.

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Abstract

Plants are colonized throughout their entire life by a complex community of microorganisms, termed the plant microbiota. Together, the plant and its microbiota form a functional unit, the holobiont, reflecting the idea that optimal plant performance depends on interactions with its microbial partners. While some microbes are vertically transmitted via seed endophytes, most are recruited from the environment, with soil as the primary source. The composition of the plant microbiota is shaped by both biotic and abiotic factors, including plant genotype, root exudates, environmental conditions, and agricultural practices. Members of these communities can interact with the plant in ways ranging from commensalism and mutualism to parasitism, and crucially, some can protect the host from pathogens, for example by secreting antibiotic compounds to antagonize the pathogen. In turn, emerging research has shown that plant pathogens deploy effector proteins not only to suppress host immune responses, but also to manipulate the host microbiota to their advantage and facilitate host colonization. This thesis investigates the roles of such antimicrobial effector proteins in plant pathogenic fungi, focusing on the broad host-range vascular wilt pathogen Verticillium dahliae and how these functions vary across environmental contexts. In this thesis I describe the development and application of a gnotobiotic system designed to thoroughly investigate the complex interactions among plants, their microbiota, and the fungal pathogen V. dahliae. I outline the establishment of reliable infection protocols within this sterile environment and demonstrate how a synthetic microbial community can effectively disrupt fungal disease progression. Importantly, the results show that specific antimicrobial effector proteins from V. dahliae contribute to fungal virulence in distinct ways. Some function in a microbiota-dependent context, while others have additional roles beyond manipulating the microbiota, suggesting a dual functionality of particular effectors. Further, I characterize a novel V. dahliae antimicrobial effector protein, called Av2. Building on in silico predictions suggesting antimicrobial properties of Av2, this thesis confirms its antimicrobial activity in vitro. By using a combination of microbiota sequencing, microbial co-cultivation assays, and experiments conducted within a gnotobiotic plant cultivation system, I demonstrate that Av2 serves as a microbiota-dependent virulence factor during host colonization. Specifically, these results show that Av2 is exploited to counteract the plant’s recruitment of antagonistic Pseudomonas species during host infection thereby facilitating successful fungal invasion. Additionally, in this thesis I explore underlying principles of microbiota assembly and how the antimicrobial effector protein Ave1, secreted by V. dahliae, affects pathogen virulence and microbial communities during infections of plants with diverse microbiota. I assembled an extensive collection of natural soil samples and showed, across three plant species, that root-associated bacterial and fungal communities are predominantly shaped by soil type, while the phyllosphere microbiota is largely determined by plant species. Utilizing this soil collection and microbiota profiling of V. dahliae-infected tomato plants, I reveal that the contribution of the antimicrobial effector Ave1 to fungal virulence varies depending on soil type. Although Ave1 consistently modifies host microbiota in all tested soils, the changes in microbial composition caused by the effector are strongly by the original soil’s microbial composition. These results indicate that while Ave1-driven manipulation of the microbiota is a general phenomenon, its effect on fungal virulence is shaped by the specific soil-derived microbial communities assembled by the plant. Collectively, the results presented in this thesis support the view that fungal antimicrobial effectors are important tools for establishment across diverse environments. These effectors however are not universally acting virulence determinants with the same function in every environment. Rather, they are key components of the fungal secretome whose contribution to niche establishment is tightly linked to the environmental and microbial conditions in which infections occur. Understanding these functions and the mechanisms underlying their variability may not only deepen our understanding of fungal niche adaptation but also inform the future development of more robust microbiota-based disease control strategies for agriculture.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Punt, Wilko wpunt@outlook.de https://orcid.org/0000-0003-4047-1117 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-795934
Date:	2026
Place of Publication:	KUPS
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Botanical Institute
Subjects:	Natural sciences and mathematics Agriculture
Uncontrolled Keywords:	Keywords Language Microbiota, Plant Pathology, Microbiology, English
Date of oral exam:	12 December 2025
Referee:	Name Academic Title Thomma, Bart Prof.Dr. Doehlemann, Gunther Prof.Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/79593