Effector-mediated microbiome manipulation by the soil-borne fungal plant pathogen Verticillium dahliae

To facilitate disease establishment, plant pathogenic microbes secrete a wide diversity of effectors that promote host colonization through a multitude of mechanisms. Typically, effectors are considered to be small cysteine-rich in planta-secreted proteins, most of which are thought to be involved in the deregulation of host immune responses or in the manipulation of other aspects of host physiology. Consequently, effector proteins are almost exclusively studied in the context of binary plant-pathogen interactions. However, plants associate with numerous microbes that collectively form their microbiota. It is becoming increasingly evident that plant microbiomes, i.e. the microbes and their genomes in their environment, are an important determinant for plant health. Moreover, plants actively shape their microbiome compositions to suppress potential pathogens.

In Chapter 1 we hypothesize that microbial plant pathogens manipulate plant microbiomes through the secretion of particular effector proteins with antimicrobial activity to promote disease establishment on their hosts. Furthermore, the organism that was studied to address this hypothesis, namely the soil-borne broad host-range fungal plant pathogen Verticillium dahliae, is introduced.

Chapter 2 provides an opinion manuscript in which we elaborate on the hypothesis that plant pathogens secrete effector proteins to manipulate host microbiomes. Additionally, we propose a number of strategies that can be exploited to identify such effector proteins.

In Chapter 3 we show that the previously identified V. dahliae virulence effector VdAve1 is a protein with selective antibacterial activity that facilitates colonization of tomato and cotton through the manipulation of their microbiomes by suppressing bacteria with antagonistic activity towards V. dahliae. Moreover, we show that VdAve1, and also the newly identified antimicrobial effector VdAMP2, are exploited for microbiome manipulation in the soil, where the fungus resides in absence of a host. Thus, we provide evidence for the hypothesis that fungal plant pathogens utilize effector proteins to modulate microbiome compositions inside and outside the host, and propose that pathogen effector catalogs represent an untapped resource for novel antibiotics.               

In vitro antimicrobial activity assays uncovered that VdAve1 inhibits growth of various bacterial species, including the Gram-positive bacterium Bacillus subtilis. By subjecting B. subtilis to transcriptome profiling and forward genetic analyses, we reveal in Chapter 4 that similar processes operate in B. subtilis in response to VdAve1 as in defense against lysozyme. Furthermore, we show that teichoic acids play a prominent role in tolerance against the detrimental activity of the VdAve1 effector protein. Collectively, the data in this chapter suggest that VdAve1 may act as a lysozyme.

Lysozymes are antimicrobial enzymes that target the bacterial cell wall polymer peptidoglycan by hydrolyzing the β-1,4 glycosidic bonds between the N-acetylmuramic acid and N-acetylglucosamine subunits. Although lysozymes are ubiquitous in a diversity of organisms, as they are found in animals and in viruses, they are extremely rare in fungi and have not been described in plants. VdAve1 shares no homology with known hydrolytic enzymes and is not predicted to carry any enzymatic domain. In Chapter 5, we show that VdAve1 is able to hydrolyze peptidoglycan, thereby uncovering the effector as a novel type of lysozyme. In addition to its hydrolytic activity, we show that VdAve1, like many previously described lysozymes, also exerts non-enzymatic antimicrobial activity that involves direct cell membrane perturbation, which is mediated by an arginine- and lysine-rich peptide that is embedded within the protein. Likely, these two activities complement each other as the peptidoglycan hydrolase activity of VdAve1 is likely to facilitate the access of the cationic peptide to the bacterial cell membrane. Importantly, V. dahliae originally acquired VdAve1 through horizontal gene transfer from plants, where VdAve1 homologs are ubiquitous and annotated as plant natriuretic peptides (PNPs), several members of which have been characterized as stress-related proteins with homeostatic roles in the regulation of ion fluxes, stomatal movement and fluid circulation affecting plant biological activities such as photosynthesis and respiration. Based on our findings, we propose that these PNPs are the plant lysozymes that have remained enigmatic thus far.

Following systemic host colonization, V. dahliae produces multicellular melanized resting structures, called microsclerotia, in the decaying tissues of its hosts. After host tissue decomposition, these resting structures are released into the soil where the pathogen can survive for many years. In Chapter 6 we describe the identification and characterization of the defensin-like V. dahliae effector protein VdAMP3. We show that VdAMP3 has antimicrobial activity and that VdAMP3 is specifically expressed in hyphal sections that develop into microsclerotia, suggesting that V. dahliae exploits VdAMP3 to protect microsclerotia formation. Accordingly, we show that VdAMP3 contributes to V. dahliae biomass accumulation in decaying host tissue. Hence, our findings demonstrate that V. dahliae employs VdAMP3 to protect its microsclerotia and corroborate the hypothesis that V. dahliae exploits different antimicrobial effector proteins at different stages of its life cycle.

Finally, Chapter 7 discusses the results obtained in this thesis and provides an outlook for the anticipated roles of fungal plant pathogen effector proteins in microbiome manipulation in a broader context. Moreover, potential implications and applications of our finding that plant pathogens exploit effectors for microbiome manipulation are discussed with respect to plant disease control and the development of novel antibiotics.