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. &nbsp

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage–specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage-specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota

An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial coinhabitants. Also, plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne, fungal, xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life stage-specific manner and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota

Plant pathogen effector proteins as manipulators of host microbiomes?

Opinion piec

Microbiome manipulation by a soil-borne fungal plant pathogen using effector proteins

During colonization of their hosts, pathogens secrete effector proteins to promote disease development through various mechanisms. Increasing evidence shows that the host microbiome plays a crucial role in health, and that hosts actively shape their microbiomes to suppress disease. We proposed that pathogens evolved to manipulate host microbiomes to their advantage in turn. Here, we show that the previously identified virulence effector VdAve1, secreted by the fungal plant pathogen Verticillium dahliae, displays antimicrobial activity and facilitates colonization of tomato and cotton through the manipulation of their microbiomes by suppressing antagonistic bacteria. Moreover, we show that VdAve1, and also the newly identified antimicrobial effector VdAMP2, are exploited for microbiome manipulation in the soil environment, where the fungus resides in absence of a host. In conclusion, we demonstrate that a fungal plant pathogen uses effector proteins to modulate microbiome compositions inside and outside the host, and propose that pathogen effector catalogues represent an untapped resource for new antibiotics. A secreted protein effector from the fungal pathogen Verticillium dahliae has bactericidal properties. It allows the pathogen to modify the root microbiome in tomato and cotton, specifically eliminating plant-protective bacteria, to increase its own virulence

The soil-borne white root rot pathogen Rosellinia necatrix expresses antimicrobial proteins during host colonization.

Rosellinia necatrix is a prevalent soil-borne plant-pathogenic fungus that is the causal agent of white root rot disease in a broad range of host plants. The limited availability of genomic resources for R. necatrix has complicated a thorough understanding of its infection biology. Here, we sequenced nine R. necatrix strains with Oxford Nanopore sequencing technology, and with DNA proximity ligation we generated a gapless assembly of one of the genomes into ten chromosomes. Whereas many filamentous pathogens display a so-called two-speed genome with more dynamic and more conserved compartments, the R. necatrix genome does not display such genome compartmentalization. It has recently been proposed that fungal plant pathogens may employ effectors with antimicrobial activity to manipulate the host microbiota to promote infection. In the predicted secretome of R. necatrix, 26 putative antimicrobial effector proteins were identified, nine of which are expressed during plant colonization. Two of the candidates were tested, both of which were found to possess selective antimicrobial activity. Intriguingly, some of the inhibited bacteria are antagonists of R. necatrix growth in vitro and can alleviate R. necatrix infection on cotton plants. Collectively, our data show that R. necatrix encodes antimicrobials that are expressed during host colonization and that may contribute to modulation of host-associated microbiota to stimulate disease development