A highly polymorphic effector protein promotes fungal virulence through suppression of plant-associated Actinobacteria

Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in the absence of the corresponding immune receptor Ve1. We identify a multiallelic V. dahliae gene displaying c. 65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L), which shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve locus, does not recognize VdAve1L. Additionally, we demonstrate that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum, that is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens

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

Microbiota manipulation through the secretion of effector proteins is fundamental to the wealth of lifestyles in the fungal kingdom

Fungi are well-known decomposers of organic matter that thrive in virtually any environment on Earth where they encounter wealths of other microbes. Some fungi evolved symbiotic lifestyles, including pathogens and mutualists, that have mostly been studied in binary interactions with their hosts. However, we now appreciate that such interactions are greatly influenced by the ecological context in which they take place. While establishing their symbioses, fungi not only interact with their hosts but also with the host-associated microbiota. Thus, they target the host and its associated microbiota as a single holobiont. Recent studies have shown that fungal pathogens manipulate the host microbiota by means of secreted effector proteins with selective antimicrobial activity to stimulate disease development. In this review, we discuss the ecological contexts in which such effector-mediated microbiota manipulation is relevant for the fungal lifestyle and argue that this is not only relevant for pathogens of plants and animals but also beneficial in virtually any niche where fungi occur. Moreover, we reason that effector-mediated microbiota manipulation likely evolved already in fungal ancestors that encountered microbial competition long before symbiosis with land plants and mammalian animals evolved. Thus, we claim that effector-mediated microbiota manipulation is fundamental to fungal biology. Recent findings show that fungal pathogens secrete effector molecules to manipulate the host microbiota to stimulate disease development; here, we argue that fungal effector-mediated microbiota manipulation occurs beyond plant pathogens, is likely widespread in the fungal kingdom and occurs in many contexts

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?

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