021.doi.org/10.1073/pnas.2110968118 j 1 ofPLANT BIOLOGYhypothesis by displaying that the ubiquitously expressed effector VdAve1 that is definitely secreted by the soil-borne fungal plant pathogen Verticillium dahliae acts as a bactericidal protein that promotes host colonization via the selective manipulation of host microbiomes by suppressing microbial antagonists (17, 18). Moreover, we demonstrated that VdAve1 in addition to a further antibacterial effector named VdAMP2 are exploited by V dahliae for . microbial competitors in soil and promote virulence of V dahliae . in an indirect manner (18). Collectively, these observations demonstrate that V dahliae dedicates part of its effector catalog . toward microbiota manipulation. Most likely, the V dahliae genome . encodes further effectors that act in microbiome manipulation. Evidently, bacterial and fungal evolution on land preceded land plant evolution. As a consequence, fungal pathogen effectors involved within the manipulation of (host-associated) microbial communities might have evolved from ancestors that served in microbial competition in terrestrial niches hundreds of millions of years ago before land plant evolution. Nevertheless, evidence for this hypothesis is presently lacking. V dahliae is an asexual xylem-dwelling Cathepsin K Purity & Documentation fungus that causes vas. cular wilt disease on a huge selection of plant species (19). The fungus survives in the soil inside the form of multicellular melanized resting structures, named microsclerotia, that provide protection against (a)biotic stresses and may persist in the soil for a lot of years (20). Microsclerotia represent the key inoculum source of V dahliae . in nature, and their germination is triggered by carbon- and nitrogen-rich exudates from plant roots (21). Following microsclerotia germination, fungal hyphae develop by way of the soil and rhizosphere toward the roots of host plants. Next, V dahliae col. onizes the root cortex and crosses the endodermis, from which it invades xylem vessels. As soon as the fungus enters those vessels, it types conidiospores which can be transported together with the water flow until they get trapped, for example, by vessel end walls. This CA Ⅱ review triggers germination in the conidiospores followed by penetration of cell walls, hyphal growth, and renewed sporulation, leading to systematic colonization with the plant (22). As soon as tissue necrosis commences and plant senescence occurs, host immune responses fade and V dahliae enters a saprophytic phase in . which it emerges from the xylem vessels to invade adjacent host tissues, that is accompanied by the production of microsclerotia. Upon littering and decomposition of plant tissues, these microsclerotia are released in to the soil (23).Results To determine effectors potentially acting in microbiome manipulation, we not too long ago queried the V. dahliae secretome for structural homologs of known antimicrobial proteins (AMPs), which led for the identification of 10 candidates such as the functionally characterized VdAMP2 (18). Amongst the remaining nine candidates, we now identified a compact cysteine-rich protein of four.9 kDa, which we name VdAMP3 (Ensembl: VDAG_JR2_ Chr3g05620a). As a first step within the characterization of VdAMP3, we assessed its predicted structure. Interestingly, VdAMP3 is predicted to adopt a cysteine-stabilized (CS) fold that’s also located in defensin-like proteins (Fig. 1A) (246). CS defensins represent a widespread and well-characterized family of antimicrobial proteins which can be presumed to share a single ancient origin inside the last common
