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Edited by Steven E. Lindow of the University of California, Berkeley, approved on October 29, 2021 (reviewed on June 14, 2021)

Microorganisms secrete a variety of molecules into their environment to mediate niche colonization. During the host’s entry, plant pathogenic microorganisms secrete effector proteins that promote disease development, many of which will relieve the host’s immune response. We recently demonstrated that plant pathogens also use effectors with antibacterial activity to manipulate beneficial plant microbiota to promote host colonization. Here, we show that the fungal pathogen Verticillium dahliae has selected an ancient antibacterial protein that may play a role in microbial competition in the terrestrial environment before the existence of terrestrial plants, as an effector that manipulates fungal competitors during host colonization. Therefore, we prove that the pathogen effector library contains antifungal proteins, and speculate that such effectors can be used to develop antifungal agents.

Microorganisms usually secrete too many molecules to promote niche colonization. Soil microorganisms are well-known producers of antimicrobial agents, and they are used to compete with microbial symbionts. In addition, phytopathogenic microorganisms secrete a variety of molecules into their environment to establish niches. After plants are colonized, microbial pathogens secrete so-called effector proteins to promote disease development. Although such effectors are generally considered to work only through direct host manipulation, we have recently reported that soil-borne, fungal, and xylem colonized vascular wilt pathogens Verticillium dahlia utilizes antibacterial effector proteins to manipulate beneficial host microbiota To promote host colonization. Since fungal evolution precedes the evolution of land plants, we now speculate that a subset of pathogen effectors involved in the manipulation of host microbiota evolved from ancient antibacterial proteins from the ancestors of terrestrial fungi. These proteins cause disease in plants Participate in microbial competition before sexual evolution. Here, we show that Vibrio dahlia has selected an ancient antibacterial protein as an effector, named VdAMP3, for fungal group manipulation in plants. We show that VdAMP3 is specifically expressed during the formation of resting structures in senescent mesophyll tissues to resist fungal niche competitors. Our findings indicate that the manipulation of the effector-mediated microbiome by plant pathogenic microorganisms is beyond the scope of bacteria and also involves eukaryotic members of the plant microbiome. Finally, we show that fungal pathogens can use the plant microbiome to manipulate effectors in a specific way in their life stages, and a subset of these effectors have evolved from the ancient antibacterial proteins of fungal ancestors, which may initially manipulate terrestrial organisms. Play a role in the group.

Microbes have been found in various ecological niches on our planet. In order to promote the establishment of microbial communities, microorganisms secrete a large number of molecules to manipulate each other. Many of these molecules exert antibacterial activity and are used to directly inhibit microbial symbionts in order to compete with them in the limited available nutrients and niche space. Antimicrobial agents secreted by microorganisms include a variety of molecules, including peptides and lyases, but also non-protein molecules such as secondary metabolites. Soil is one of the most biologically diverse and microbial competitive environments on the planet. The proliferation of microorganisms in the soil environment is usually limited by the availability of organic carbon (1), and soil microorganisms continue to compete for organic carbon. Therefore, many microorganisms living in saprophytic soil secrete effective antibacterial agents to promote niche protection or colonization. It is worth noting that these microorganisms are the main source of antibiotics that we use clinically (2, 3).

Like free-living microorganisms, microbial plant pathogens also secrete a large number of molecules into their environment to mediate niche colonization (4, 5). The research on the molecules secreted by microbial plant pathogens is largely limited to the background of the binary interaction between the pathogen and the host. In order to determine the disease, plant pathogenic microorganisms secrete a large number of so-called effectors, that is, various molecules that promote host colonization, which are generally considered to be mainly to relieve the regulation of the host's immune response (4, 6, 7). After the host is colonized, plant pathogens will encounter a large number of plant-related microorganisms, which together form a plant microbiota, which is a key factor for plant health. Beneficial plant-related microorganisms exist in all plant organs and help to reduce (a) biological stress (8⇓ ⇓ ⇓ ⇓ –13). Plants shape their microbiota and specifically attract beneficial microorganisms to suppress pathogens (14⇓ –16). Therefore, the plant microbiome can be considered as an exogenous exogenous layer that complements the endogenous innate immune system of plants. We previously assumed that plant pathogens not only use effectors to target components of host immunity and other aspects of host physiology to support host colonization, but also target the host microbiota to establish niche colonization (4, 5). We have recently provided experimental evidence for this hypothesis, demonstrating that the ubiquitously expressed effector VdAve1 secreted by the soil-borne fungal plant pathogen Verticillium dahliae acts as a bactericidal protein, and promotes it by selectively manipulating the host microbiome by inhibiting microbial antagonists Host colonization. 17, 18). In addition, we demonstrated that VdAve1 and a further antibacterial effector named VdAMP2 are used by Vibrio dahlia to compete with microorganisms in the soil and promote the virulence of Vibrio dahlia in an indirect manner (18). Collectively, these observations indicate that Vibrio dahlia uses part of its effector catalog for microbiota manipulation. It is possible that the genome of the dahlia encodes more effectors that play a role in the manipulation of the microbiome.

Obviously, the evolution of bacteria and fungi on land preceded the evolution of land plants. Therefore, the fungal pathogen effectors involved in manipulating (host-related) microbial communities may have evolved from the ancestors that participated in microbial competition in terrestrial niches before terrestrial plants evolved hundreds of millions of years ago. However, there is currently a lack of evidence to support this hypothesis.

V. dahliae is an asexual xylem fungus that can cause vascular blight in hundreds of plants (19). Fungi survive in the soil in the form of a multicellular melaninized static structure, called microsclerotia, which can provide protection against (a) biotic stress and can last for many years in the soil (20). Microsclerotia are the main source of inoculation of Vibrio dahlia in nature, and their germination is triggered by exudates rich in carbon and nitrogen from plant roots (21). After germination of the microsclerotia, the fungal hyphae grow to the roots of the host plant through the soil and rhizosphere. Next, the dahlia fungus colonizes the root cortex and penetrates the endothelial layer, invading the blood vessels of the xylem from the endothelial layer. Once the fungus enters these blood vessels, it will form conidia, and these spores will be transported with the water until they are captured, for example by the end walls of the blood vessels. This triggers the germination of conidia, which then penetrates the cell wall, grows hyphae, and re-spores, leading to systemic plant colonization (22). Once tissue necrosis begins and plant senescence occurs, the host immune response will be weakened, and Vibrio dahlia enters the saprophytic stage, at which stage it emerges from the xylem blood vessels and invades adjacent host tissues, accompanied by the production of micro sclerotia. After littering and decomposition of plant tissues, these micro sclerotia are released into the soil (23).

In order to determine the effectors that may play a role in microbiome manipulation, we recently searched the secretion group of dahlias to find structural homologues of known antibacterial proteins (AMP), thereby identifying 10 candidates, including the functionally characterized VdAMP2 (18). Among the remaining nine candidates, we have now identified a small cysteine-rich protein of about 4.9 kDa, which we named VdAMP3 (Ensembl: VDAG_JR2_Chr3g05620a). As the first step in the characterization of VdAMP3, we evaluated its prediction structure. Interestingly, VdAMP3 is expected to use cysteine-stabilized αβ (CSαβ) folding, which is also found in defensin-like proteins (Figure 1A) (24⇓ –26). CSαβ defensins represent a broad and well-characterized family of antibacterial proteins. It is speculated that they have a single ancient origin in the last common ancestor of animals, plants, and fungi that produced these proteins today (24⇓ ⇓ –27). However, it is important to note that many typical effectors of small cysteine-rich pathogens use AMP-like confirmation, and the tertiary structures of several AMP families are very similar to each other (27, 28). Therefore, structure prediction can easily lead to false positives classified as AMP or assigned to the wrong AMP family.

The V. dahliae effector VdAMP3 evolved from an ancient fungal protein. (A) VdAMP3 (left) is expected to use CSαβ defensin-like folding. The structure of the CSαβ defensin plectasin (right) of the fungus P. nigrella is included as a reference. The disulfide bonds that stabilize the antiparallel β-sheet and α-helix are highlighted in yellow. The positively and negatively charged amino acid residues are highlighted in blue and red, respectively. (B) The protein sequence alignment with CSαβ defensins plectasin and eurocin (E. amstelodami) supports the structure prediction of VdAMP3. (C) VdAMP3 homologues are widespread in the fungus kingdom. VdAMP3 is aligned with the protein sequence of a subset of homologues identified in higher (Ascomycota and Basidiomycota) and lower fungi (Mucerophyta and Zoopagomycota). The alignment shown in B and C shows the most conserved regions in the CSαβ defensin protein family, and was performed with HMMER and visualized with Espript3. The highly conserved cysteine ​​and glycine residues that contribute to the structure of CSαβ defensins are highlighted with yellow and red backgrounds, respectively. The number at the top of the alignment indicates the corresponding residue number of VdAMP3. The homologs shown in C were identified using blastP in the predicted proteome of each fungus included in the JGI 1000 Fungal Genome Project (32).

The structure of the CSαβ defensin or so-called cis-defensin is attributed to the highly conserved cis-disulfide bond, which establishes the interaction between the double-stranded or triple-stranded antiparallel β-sheet and the α-helix (25, 27) . In order to verify the prediction of VdAMP3 as a member of this ancient antibacterial protein family, we compared its amino acid sequence with the antibacterial CSαβ defensins plectasin and eurocin from the species Pseudoplectania nigrella and Eurotium amstelodami (previously known as Aspergillus amstelodami) (29 ⇓ –31). Although the biological relevance of these defensins to their respective fungi is unclear, their antibacterial activity and protein structure have been well characterized, which has led to their identification as true CSαβ defensins (29⇓ –31). Although the overall identity between the three proteins is quite low (25% to 40%), the protein sequence alignment shows that VdAMP3 contains six highly conserved cysteine ​​residues, which are considered to be important for CSαβ defensins. The structure is crucial (Figure 1B) (27). In order to further confirm that VdAMP3 belongs to the emerging picture of this specific protein family, and the detected similarity to plectasin and eurocin is not the result of astringent protein evolution, we inquired the predicted fungal proteome of the Joint Genome Institute (JGI) 1000 fungi The genome project of VdAMP3 homologs with higher sequence identity (32) includes a subset of protein alignments (Figure 1C). Interestingly, in addition to the homologs of Ascomycota and Basidiomycota, our sequence similarity search also revealed homologs of early divergent fungi from Mucor and Zoomycota [all previously classified Zygomycota (33)] (Figure 1C). Importantly, this difference is estimated to occur approximately 900 million years ago (34), indicating that it predates the evolution of the first land plants approximately 450 million years later (34⇓ ⇓ –37). Therefore, this analysis shows that VdAMP3 evolved from ancestral fungal genes before the emergence of terrestrial plants hundreds of millions of years ago.

As a first step in determining the potential role of VdAMP3 in the biology of dahlia infection, we assessed whether we can find evidence of VdAMP3 expression during host colonization. Analysis of the transcriptome data sets previously generated by different dahlia strains during the colonization of multiple hosts did not reveal the plant expression of VdAMP3 (17, 38⇓ –40). However, in the transcriptome analysis of the Dahlia V. dahliae strain XS11 grown in vitro, the strong induction of this effector gene was reported during the formation of microsclerotia (24). To verify this finding, we analyzed the in vitro expression of VdAMP3 in V. dahliae strain JR2. To this end, the dahlia conidia were spread on a nitrocellulose membrane placed on top of a solid minimal medium, and the fungal material was harvested before microsclerotia formation after 48 hours of incubation and after microsclerotia formation started after 96 hours of incubation. The expression of VdAMP3 was determined at two time points by real-time PCR and the expression of the Chr6g02430 gene, which encodes a putative cytochrome P450 enzyme as a marker of micronucleus formation (24, 41). Consistent with the observation results of Dahlia strain XS11 (24), no VdAMP3 expression was detected at 48 hours, at this time Chr6g02430 was also not expressed and no visible microsclerotia formation was observed on the growth medium (Figure 2A). However, the induction of VdAMP3 and Chr6g02430 was observed after 96 hours of incubation. At this time, the formation of microsclerotia on the growth medium also became obvious (Figure 2A). Overall, these data indicate that the expression of VdAMP3 is consistent with the in vitro microsclerotia formation of the JR2 strain of Vibrio dahliae.

VdAMP3 is specifically expressed in hyphae cells that develop into microsclerotia. (A) The expression of VdAMP3 and the microsclerotium development marker gene Chr6g02430, relative to the family gene VdGAPDH cultured for 48 and 96 hours in vitro (n = 3). (B) Expression of VdAve1, VdAMP3 and Chr6g02430 in Nicotiana benthamiana leaves at 7 to 22 dpi (n = 5). (C) Expression of VdAve1, VdAMP3 and Chr6g02430 in tissues of Nicotiana benthamiana harvested at 22 dpi after 8 days incubation in a sealed plastic bag (n = 3). (D) Micronucleus formation of pVdAMP3::eGFP reporter gene mutant detected after 7 days of cultivation in Czapek Dox medium. The typical microsclerotia chain (42, 43) is indicated by arrows. (E) Bright field images of various Dahlia cell types after culturing in Czapek Dox for 7 days, including hyphae (*), expanded hyphae cells developed into micro sclerotia (‡) and mature micro sclerotia cells (#) . (F) The GFP signal of the image shown in E, indicating the activity of the VdAMP3 promoter, is only detected in swollen hypha cells that develop into micronuclei. (G) The superposition of E and F.

Although the previous transcriptome analysis failed to detect the plant expression of VdAMP3, we realized that these analyses were mainly performed at the infection stage, when the fungus was still restricted to the blood vessels in the xylem and the microsclerotia had not yet begun to form. Therefore, the expression of VdAMP3 in plants may be missed. Therefore, we inoculated tobacco with dahlias and determined the expression of VdAMP3 in leaves and petioles sampled at different time points, and showed different disease phenotypes, ranging from asymptomatic 7 days after inoculation (dpi) to 22 dpi When completely necrotic. As expected, strong induction of the previously characterized VdAve1 effector gene was detected at 7 dpi and 14 dpi (Figure 2B) (17, 18). However, in contrast, even at the latest point in time when the leaf tissue was completely necrotic, no expression of VdAMP3 was recorded (Figure 2B). Importantly, the expression of Chr6g02430 was not detected at any of these time points (Figure 2B), indicating that microsclerotia have not yet begun to form in these tissues. In fact, visual inspection of necrotic plant tissue collected at 22 dpi did not reveal the presence of microsclerotia. To induce the formation of microsclerotia, tobacco plants inoculated with V. dahliae harvested at 22 dpi were sealed in plastic bags and cultured in the dark to increase the relative humidity and simulate the conditions that occur during tissue decomposition in the soil. Interestingly, after 8 days of incubation, the first microsclerotia can be observed and the induction of VdAMP3 and Chr6g02430 can be detected (Figure 2C). It is worth noting that, compared with their expression in vitro, the induction of these two genes in plants is significantly weaker (Figure 2A). However, this may be due to the fact that the proportion of synchronously developing microsclerotia in the total cell population of dahlias is much smaller, and also because the time window from conidia germination to hyphae growth to microsclerotia formation is much smaller in vitro than in plants. many. In general, our research results show that the expression of VdAMP3 in plants is consistent with the formation of microsclerotia, similar to our observations in vitro. In addition, our data indicate that VdAMP3 expression is mainly dependent on the developmental stage of Vibrio dahlia, rather than host factors such as tissue necrosis.

In order to more accurately determine the location of VdAMP3 expression and improve our understanding of how Vibrio dahlia can benefit from effector expression during micronucleus formation, we have generated a reporter strain of Vibrio dahlia expressing eGFP under the control of the VdAMP3 promoter . Interestingly, the microscopic analysis of the reporter strain in the in vitro microsclerotium formation stage (Figure 2D) showed that VdAMP3 was expressed by expanded hyphae cells, which acted as the primordia and subsequently developed into microsclerotia, but not by It is expressed by adjacent hyphal cells or recently developed microsclerotia cells. Figure 2 EG). This highly specific expression of VdAMP3 indicates that the effector protein may promote the formation of microsclerotia in decaying host tissues. In view of its presumed antibacterial activity, VdAMP3 may participate in the antagonistic activity against opportunistic decomposing organisms in the microbial competition niche.

In order to determine whether VdAMP3 actually exerts antibacterial activity, we tried to produce VdAMP3 heterologously in yeast Pichia pastoris and E. coli, but these attempts failed, indicating that the effector protein has potential antibacterial activity. Therefore, the chemical synthesis of VdAMP3 was carried out. Next, we cultivated a set of randomly selected bacterial isolates with effector proteins and monitored their growth in vitro. Concentrations of VdAMP3 as high as 20 µM resulted in no or only marginal bacterial growth inhibition (SI appendix, Figure 1). Similar determinations of fungal isolates showed that incubation with 5 µM VdAMP3 has significantly affected the growth of filamentous fungi Alternaria brasicicola and Cladosporium cucumerinum and yeast P. pastoris and Saccharomyces cerevisiae (Figure 3A and B). This finding indicates that VdAMP3 is more active against fungi than against bacteria. Importantly, the thorough heat treatment involving boiling VdAMP3 eliminated its antifungal activity (SI appendix, Figure 2), indicating that the specificity of this activity depends on its correct three-dimensional confirmation.

VdAMP3 is an antifungal protein that contributes to the biomass accumulation of dahlia in the decaying host foliar. (A) Photomicrograph of fungal isolate grown in 5% PDB supplemented with 5 µM VdAMP3 or ultrapure water (Milli-Q). VdAMP3 can impair the growth of A. brasicicola, C. cucumerinum, P. pastoris and S. cerevisiae. The photos were taken after 24 hours of incubation (A. brasicicola, C. cucumerinum and S. cerevisiae) or 64 hours (P. pastoris). (B) ImageJ (unpaired two-sided Student's t-test; n = 4) was used to quantify the fungal growth shown in A. (C) VdAMP3 does not contribute to the establishment of Verticillium wilt in tobacco. The photo shows a representative phenotype of a N. benthamiana plant infected with wild-type dahlia (WT), VdAMP3 deletion (ΔVdAMP3), and two complementary (Comp) mutants at 14 dpi. (D) The relative dahlia biomass in the above-ground tobacco tissues determined by real-time PCR. Different letter labels represent significant differences (one-way analysis of variance and Tukey's post-hoc test; P <0.05; n ≥ 27. (E) After 28 days of incubation in a plastic bag, the representative phenotype of tobacco plants is shown in C (F) The relative dahlia biomass in tobacco tissues of Ben's tobacco, as shown in E. Letters represent significant differences (one-way analysis of variance and Tukey's post-hoc test; P <0.05; n ≥ 27).

Considering its antifungal activity and the highly controlled timely and local expression of VdAMP3, we tested whether the application of exogenous VdAMP3 would have a negative impact on the mycelial growth of dahlias. Interestingly, the incubation of dahlia with 5 µM VdAMP3 significantly affected its growth (SI appendix, Figure 3A and B). However, it needs to be realized that this effector protein is produced when most of the fungus's hyphae lose their function, because the host tissue has senescence and quickly decomposes, and the fungus will produce micro sclerotia for long-term survival. Next, in order to verify whether the growth or development of Vibrio dahlia is affected by VdAMP3, we generated a VdAMP3 deletion mutant (SI Appendix, Figure 4), and we cultured it in vitro with wild-type (WT) Vibrio dahlia. As expected, the absence of VdAMP3 does not accelerate the growth of fungi (SI Appendix, Figure 3C), confirming that the effector gene does not impair fungal development in the life stage before the formation of microsclerotia. In addition, the absence of VdAMP3 will not damage the ability of dahlias to form a static structure, nor will they damage their ability to infect new plants and cause diseases (SI appendix, Figure 3 CE). Next, we aimed to determine whether the antifungal activity of VdAMP3 contributes to the development of Verticillium wilt. To this end, tobacco plants were inoculated with Dahlia WT and VdAMP3 complementation and deletion mutants (SI appendix, Figure 4). Consistent with our inability to detect expression in the early stages of infection, the use of real-time PCR to quantitatively analyze the disease phenotype and the biomass of Vibrio dahlia did not reveal the contribution of VdAMP3 to host colonization for up to 2 weeks after inoculation (Figure 3C and D). Considering the cell type-specific expression of VdAMP3 in the developing micronucleus, we speculate that when the fungus emerges from the xylem and colonizes the mesophyll, the effector protein contributes to the establishment of the niche of Vibrio dahlia during the senescence of the host plant. To test this hypothesis, we performed additional disease tests using Dahlia WT and VdAMP3 deletion mutants, and sealed Nicotiana benthamiana plants in plastic bags after harvest to stimulate tissue decomposition and microsclerotia formation. Interestingly, when we visually inspected the plants after 4 weeks of cultivation, we detected scattered dark mycelium on the surface of the plant colonized by Dahlia Dahlia WT, which is a typical feature of the small sclerotia of Dahlia (SI Appendix, Figure 5). Strikingly, we did not find such plaques on plants colonized by VdAMP3 deletion mutants, indicating that Vibrio dahlia relies on the antifungal activity of VdAMP3 to form microsclerotia in the decaying host leaf. It should be noted that an experimental setup relies on the appearance of apparently unpredictable micro sclerotia plaques on the surface of decaying plant parts. These micro sclerotia are colonized by various opportunistic microbial groups that weaken the defenses of plants. While seizing the opportunity to multiply, it is feasible to standardize and quantify the formation of microsclerotia. In addition, this setup does not allow the evaluation of deeper microsclerotia formation in decaying tissues. Instead, we used real-time PCR to quantify the biomass of dahlias. As expected, compared with Dahlia WT and complementary mutants (Figure 3 E and F), we detected a significant reduction in the biomass of VdAMP3 deletion mutants, confirming that VdAMP3 is indeed essential in the formation of plant micronuclei It is probably by acting on the self-protection of other microorganisms.

In order to study whether the effects of VdAMP3 are limited to Nicotiana benthamiana or whether these effects also extend to other hosts, we inoculated Arabidopsis plants with dahlia WT and VdAMP3 deletion mutants. Consistent with our observations on Ben's tobacco, the absence of VdAMP3 does not affect the establishment of verticillium wilt in Arabidopsis (SI appendix, Figure 6A and B). However, 3 weeks after inoculation, the quantification of the biomass of Vibrio dahlia in the aboveground tissues of Arabidopsis showed that in the absence of VdAMP3, the accumulation of Vibrio dahlia was reduced (SI appendix, Figure 6C). Therefore, the impact of VdAMP3 is not limited to a single host.

Since in vitro antibacterial activity analysis indicated that fungi are the main target of VdAMP3, we speculate that Vibrio dahlia uses VdAMP3 to inhibit fungal competitors from decomposing host tissues to protect the formation of resistance structures. In order to characterize the microbial communities associated with the decomposition of Nicotiana benthamiana and determine the effect of VdAMP3 on these microbial communities, we characterized the leaf microbiota of freshly simulated Nicotiana benthamiana plants and decaying plants infected with Dahlia WT or VdAMP3 deletion by shotgun Metagenomic sequencing, mutants incubated in plastic bags. Consistent with the main role of fungi in decomposing dead plant material (44⇓ ⇓ ⇓ –48), we detected Dahlia V. compared with healthy simulated treatment plants (Figure 4A and B). These changes were accompanied by a decrease in alpha diversity in the decaying leaf space (Figure 4C). In addition, principal coordinate analysis (PCoA) based on the Bray-Curtis difference (β diversity) revealed a clear separation between the microbiota of healthy plants and rotting plants (Figure 4D). PCoA also revealed a weak but possibly related separation of the microbiota colonized by Vibrio dahlia WT and VdAMP3 deletion mutants, indicating that the secretion of VdAMP3 manipulates the microbiome composition (Figure 4D). Interestingly, when we compared the abundance of identified microbial genera between the microbiomes colonized by V. dahliae WT and the VdAMP3 deletion mutant, we detected fungi (10.1%) significantly higher than bacteria (3.8%) ( Figure 4E) (SI Appendix, Tables 1 and 2). Interestingly, although the number of bacterial genera that showed increased or decreased abundance in the presence of VdAMP3 was more or less equal, the vast majority of differentially abundant fungal genera (82.1%) were inhibited in the presence of VdAMP3 (Figure 3). 4F). In addition, although no consistent suppression of bacterial genera from the same category was detected, we specifically determined the suppression of differentially abundant fungal genera from yeast or Clostridium in the presence of VdAMP3 (Figure 4G and H). Therefore, these observations indicate that Dahlia VdAMP3 mainly functions as an antifungal effector protein, and its selective activity mainly affects the fungal community in the decaying host leaf.

VdAMP3 manipulates the fungal community in the leaf of Nicotiana benthamiana that is decomposing. (A and B) The leaf decay of N.benthamiana caused by V. dahliae is related to the decrease of bacteria and the increase of fungus abundance. The relative abundance of bacteria (A) and fungi (B) in the leaf space of decaying N.benthamiana plants of WT V. dahliae (WT) or VdAMP3 deletion mutants (14 dpi and 28 days after incubation), excluding V. dahliae In plastic bags) and in the leaf borders of fresh tobacco plants (simulated). The letters represent significant differences in total bacterial/fungal abundance between the three treatments (one-way analysis of variance and Tukey's post-hoc test; P <0.05; n = 3). (C) The decay of N.benthamiana plants induced by V. dahliae affects the alpha diversity of the leaf space. The figure shows the mean Shannon index ± SD; letters represent significant differences (one-way analysis of variance and Tukey's post hoc test; P <0.05; n = 3). (D) PCoA based on Bray-Curtis difference (β diversity) reveals the separation of the microbiome based on three different treatments. (E) The differential abundance analysis of the microbial genera between the microbiomes colonized by V. dahliae WT and the VdAMP3 deletion mutant showed that the secretion of VdAMP3 significantly affects the proportion of fungi than the secretion of bacteria (two-tailed Fisher's exact test). (F) Among the differentially abundant microbial genera, significantly more fungi show reduced abundance in the presence of VdAMP3 compared to bacteria (two-tailed Fisher's exact test). (G and H) An overview of the different genera of bacteria (G) and fungi (H). And VdAMP3 deletion mutants (Wald test, P adjustment <0.05 and P <0.05, n = 3). In the presence of VdAMP3 (ie by Vibrio dahlia WT), the differentially rich fungal genera from yeast or Sordariomycetes are always suppressed.

In order to further confirm that the inhibition of yeast and Clostridium is a direct result of VdAMP3 activity, we incubated the fungal species belonging to the inhibited genus with the effector to determine their sensitivity. Consistent with the previously observed sensitivity of yeasts P. pastoris and S. cerevisiae, Saccharomycete species Cyberlindnera jadinii, Debaromyces vanrijiae, Rhodotorula bogoriensis and Meyerozyma amylolytica also showed significantly reduced growth in the presence of VdAMP3 and BA (Figure 5). ). Similarly, the growth of Sordariomycetes Cordyceps militaris and Trichoderma viride was inhibited by effectors (Figure 5A and B). Therefore, these findings support the observed suppression of Saccharomycetes and Sordariomycetes in the tobacco leaf-stem fungal community as a direct result of VdAMP3 activity.

VdAMP3 has a negative effect on yeast and Sordariomycetes. (A) Photomicrograph of fungal isolate grown in 5% PDB supplemented with 5 µM VdAMP3 or ultrapure water (Milli-Q). VdAMP3 can damage the growth of D. vanrijae, M. amylolytica, C. jadinii, R. bogoriensis, C. militaris and T. viride. The photos were taken after 10 (D. vanrijae and C. jadinii), 24 (M. amylolytica and R. bogoriensis), or 30 (C. militaris and T. viride) hours of cultivation. (B) ImageJ (unpaired two-sided Student's t-test; n = 4) was used to quantify the fungal growth shown in A.

The cell-type-specific expression of VdAMP3, combined with its role in the operation of the fungal biome, strongly suggests that VdAMP3 is used to resist fungal niche competitors in plants to protect the formation of microsclerotia in dahlias. In order to test whether VdAMP3 is indeed essential for the formation of Dahlia microsclerotia in the presence of other fungi, we co-cultured Dahlia WT and VdAMP3 deletion and complementary mutants with D. vanrijiae and M. amylolytica. Once the microsclerotia formation of V. dahliae WT became apparent (Figure 6A), we quantified the number of static structures formed by different V. dahliae genotypes. As expected, compared with the complementary mutants of Dahlia WT and the two fungi, we detected that the microsclerotia formed by the VdAMP3 deletion mutants were significantly reduced, confirming that Dahlias rely on the antifungal activity of VdAMP3 to form microsclerotia. In the case of a competitor in a specific fungal niche (Figure 6 B and C). In addition, in order to confirm that this activity is not only related to the presence of a single microbial interactor, but also to promote the formation of microsclerotia when the fungal community exists, we conducted similar experiments using two synthetic communities, except for D. vanrijiae and M. amylolytica, Also includes filamentous fungus C. militaris or yeast C. jadinii plus filamentous fungus T. viride. Also in these experiments, compared with Dahlia WT and complementary mutants, we detected a significant reduction in microsclerotia formed by VdAMP3 deletion mutants (Figure 6B and C). In general, these findings support the view that Vibrio dahlia uses the antifungal activity of VdAMP3 to protect the formation of its resting structure by resisting fungal niche competitors in the mesophyll tissue of the aging host.

In the presence of fungal niche competitors, VdAMP3 contributes to the formation of Dahlia microsclerotia. (A) In D. vanrijae (6 dpi), M. amylolytica (6 dpi), including D. vanrijae, M. amylolytica and C. militaris (6 dpi), or including D. vanrijae, M. amylolytica, C. jadinii Synchronization system with T. viride (9 dpi). (B) VdAMP3 contributes to the formation of Dahlia sclerotia in the presence of other fungal species. Representative micrographs showing V. dahliae WT, VdAMP3 deletion mutant (ΔVdAMP3) and two complementary mutants (Comp) cultured in the presence of fungal species/communities, such as A. (C) Micrographs formed by V. dahliae The number of sclerotia in the presence of fungal species or communities (one-way analysis of variance and Tukey’s post-hoc test; P <0.05; n = 4).

Microorganisms secrete a large number of molecules to promote niche colonization (4). Free-living microorganisms are well-known producers of antimicrobial agents, and they are secreted to compete with microbial symbionts in the microbial community. Microbial plant pathogens secrete a variety of so-called effector molecules during the host's entry, many of which are small cysteine-rich proteins, which can relieve the host's immune response to promote colonization (4, 6, 7). When studying the vascular blight fungus Dahlia dahlia, we recently demonstrated that plant pathogens not only use effector proteins to promote disease establishment through direct host manipulation, but also manipulate the plant microbiota through antibacterial activity (18). Considering that the emergence of fungi on the earth precedes the evolution of land plants, we speculate that a subset of pathogen effectors involved in the manipulation of the host microbiota may have evolved from antibacterial proteins, which originally appeared in the first land plants It plays a role in the competition of microorganisms in the terrestrial niche with plants. Pathogenic evolution. Here, we proved that the soil-borne fungal plant pathogen V. dahliae has selected an ancient antibacterial protein as the effector of plant flora manipulation to protect the formation of its static structure. Therefore, our research results indicate that the phytopathogenicity in fungi is not only related to the evolution of novel effectors that manipulate plants or their related microbial communities, but also related to the co-selection of previously evolved secreted proteins that originally served Alternative lifestyles, such as saprophysis, act as effectors that promote host colonization. In addition, our research results show that the effector-mediated manipulation of microbial plant pathogens on the plant microbiota is not limited to bacterial targets, but also extends to eukaryotic microorganisms.

The functional characterization of VdAMP3 shows that during the formation of microsclerotia in dahlia, the effector evolved to play a specific role in the life stage in the operation of the microbiome. Recently, we described the characteristics of the first microbiome manipulation effectors secreted by Vibrio dahlia, VdAve1 and VdAMP2 (18). VdAve1 is a ubiquitously expressed bactericidal effector, which promotes the colonization of dahlia hosts by inhibiting microbial antagonists to selectively manipulate the host microbiota in the roots and xylem. In addition, VdAve1 is also expressed in soil biomes, where it also contributes to niche colonization. Interestingly, VdAMP2 is only expressed in the soil, and has the same antibacterial activity as VdAve1, which helps to establish a niche. Interestingly, VdAMP2 and VdAve1 show different activity profiles, so they may complement each other for optimal soil colonization. In decaying host tissues, neither VdAve1 nor VdAMP2 is expressed, but VdAMP3 expression occurs. In general, our findings on VdAve1, VdAMP2, and VdAMP3 indicate that Vibrio dahlia uses a large portion of its effector protein catalog for microbiome manipulation, and that each of these effector proteins is in a life stage-specific manner kick in.

The specific development of the life stage of the antimicrobial effectors VdAve1 and VdAMP3 secreted in plants is well reflected by their antimicrobial activity and the microbiota of the niche in which they act. Contrary to the previous V. dahliae transcriptome analysis that repeatedly identified VdAve1 as one of the most highly expressed effector genes in plants (17, 38⇓ –40), we detected that the effector genes were suppressed when decomposing tobacco tissues (Figure 1) B and C). Characterization of the antibacterial activity exerted by VdAve1 It was previously found that this protein only affects bacteria and not fungi (18). Because of their ability to produce a variety of hydrolytic enzymes, fungi are the main decomposers of plant debris on earth (44). The leaf space of plants includes a variety of fungi (49⇓ -51). What is important is that these fungi are the first way to come into contact with decaying substances when plants are senescence. Once the host's immune response is weakened, they can act opportunistically. Therefore, compared with healthy plants, we detected an increase in the abundance of fungi in the leaf space of the decomposing Nicotiana benthamiana plant diseased by the dahlia tree (Figure 4B). The observed inhibition of VdAve1 and subsequent induction of VdAMP3 in niches where Vibrio dahlia encounters more fungal competition emphasizes that Vibrio dahlia tailors its microbiome manipulation effector expression according to the various microbiota encountered at different times the opinion of. Life stage. Along these lines of thinking, it is easy to speculate that during the process of soil satting, Vibrio dahlia uses antibacterial effect proteins to resist other eukaryotic competitors, including soil parasites, such as fungus-eating nematodes or protists. However, there is currently a lack of evidence to support this hypothesis.

Antimicrobial resistance of bacteria and fungi poses an increasing threat to human health. Possibly, the microbiome manipulation effector is a valuable source for the identification and development of new antimicrobial agents that can be used to treat microbial infections. It can be said that we found that the microbiome manipulation effectors secreted by plant pathogens also contain antifungal proteins, which opens up opportunities for the identification and development of antifungal drugs. Most fungal pathogens in mammals are saprophytes, which usually thrive in soil or decaying organic matter, but may cause diseases in patients with weakened immune functions​​ (52⇓ –54). Azoles are an important class of antifungal agents used to treat fungal infections in humans. Unfortunately, agricultural practices include spraying azoles in large quantities to control fungal plant pathogens, as well as azoles that are widely used in personal care products, UV stabilizers, and aircraft preservatives, such as those that cause azole resistance in opportunistic pathogens. Enhance mammals in the evolutionary environment (52, 55). For example, azole-resistant Aspergillus fumigatus strains are ubiquitous in agricultural soils and decomposing crop waste, where they thrive as saprophytes (56, 57). Therefore, mammalian fungal pathogens, such as Aspergillus fumigatus, include niche competitors for fungal plant pathogens. Therefore, we speculate that, like V. dahliae, other phytopathogenic fungi may also carry effective antifungal proteins in their effector catalogs, helping to compete with these fungi for niche. Possibly, the identification of such effectors will help develop new antifungal agents.

The in vitro culture of Vibrio dahlia strain JR2 used to analyze the expression of VdAMP3 and Chr6g02430 was as described above (24). In addition, in plant expression analysis, total RNA was isolated from individual leaves or whole Nicotiana benthamiana plants harvested at different time points after the inoculation of dahlia roots. To induce microsclerotia formation, Nicotiana benthamiana plants were harvested at 22 dpi and incubated in a sealed plastic bag (volume = 500 mL) for 8 days before RNA isolation. Use Maxwell 16 LEV Plant RNA Kit (Promega) for RNA isolation. As mentioned earlier, use the primers listed in Table 3 (17) in the SI Appendix for real-time PCR.

VdAMP3 deletion and complementation mutants and eGFP expression mutants were generated using the primers listed in Table 3 (18) of the SI Appendix as described above. To generate the VdAMP3 complementary construct, the VdAMP3 coding sequence was amplified and cloned into pCG (58) along with flanking sequences (approximately 0.9 kb upstream and approximately 0.8 kb downstream). Finally, this construct was used for Agrobacterium tumefaciens-mediated transformation of Vibrio dahlia, as described previously (59). The in vitro growth and microsclerotia production of VdAMP3 deletion mutants were tested and quantified, as previously described (18).

The bacterial isolates were grown on lysogenic broth agar at 28 °C. Select a single colony and grow it in low-salt lysogenic broth (LB) (10 g/L tryptone, 5 g/L yeast extract, and 0.5 g/L sodium chloride) at 28°C overnight, and at the same time Oscillate at 200 rpm. The overnight culture was resuspended in fresh low-salt LB supplemented with 20 μM VdAMP3 or ultrapure water (Milli-Q) to an optical density (OD) 600 = 0.025. As mentioned earlier, CLARIOstar microplate reader (BMG Labtech) was used to quantify in vitro growth (18).

The fungal isolate was grown on potato dextrose agar (PDA) at 22°C. For yeast, select a single colony and grow it overnight at 28°C in 0.05×Potato Dextrose Broth (PDB) while shaking at 200 rpm. The overnight culture was resuspended in fresh 5% PDB supplemented with ultrapure water (Milli-Q), 5 μM VdAMP3 or 5 μM VdAMP3 to OD600 = 0.01, and incubated in a PCR thermal cycler at 95°C for 16 hours. Alternatively, for filamentous fungi, collect spores from PDA and suspend them in 5% PDB supplemented with 5 μM VdAMP3 or ultrapure water (Milli-Q) to a final concentration of 104 spores/mL. Next, aliquot 200 μL of the fungal suspension into a transparent 96-well flat-bottomed polystyrene tissue culture plate. The plate was incubated at 28°C, and fungal growth was imaged using SZX10 stereo microscope (Olympus) and EP50 camera (Olympus).

The inoculation and incubation of Nicotiana benthamiana plants were carried out as described above. Use the primers listed in Table 3 (60) of the SI Appendix for subsequent genomic DNA isolation and dahlia biomass quantification as described above. After 4 weeks of incubation in the dark at room temperature in a plastic bag, samples of decaying tobacco leaf space colonized by Dahlia WT and VdAMP3 deletion mutants were collected. The leaf space of a fresh 3-week-old N. benthamiana plant was included as a control. All samples were quick-frozen in liquid nitrogen, ground with a mortar and pestle, and DNeasy PowerSoil Kit (Qiagen) was used to isolate genomic DNA. Use TruSeq DNA Nano kit (Illumina) to prepare sequencing library, and perform paired-end 150-bp sequencing on Utrecht Sequencing Facility's Illumina NextSeq500 platform.

The sequencing data is processed as follows. The ATLAS metagenomic workflow uses the default parameters of the configuration file (61) for quality control of reads, adaptor pruning, and removal of Nicotiana benthamiana reads. Use metaSPAdes (k-mer sizes used: 21, 33, and 55) to combine and assemble reads from different samples to obtain a single metagenomic cross-assembly (62). Subsequently, the cross-assembled contigs were classified using the contig annotation tool and classified by genus (63). Use Burrows-Wheeler Aligner Maximum Exact Match (64) to map individual sample readings to merged contigs. Next, use SAMtools (65) version 1.10 to convert the mapping file to bam format, and convert the reads mapped to a single contigs to the "reads per million" of a single sample. The generated classification table and abundance table are then converted into phyloseq(66) objects (version 1.30.0) in R (version 3.6.1) to facilitate the analysis of the microbiome. The determination of α diversity (Shannon index) and β diversity (Bray-Curtis dissimilarity) are as described above (66, 67). The DESeq2 extension of phyloseq is used to identify diverse microbial genera (68). To do this, a parametric model is applied to the data, and a negative binomial Wald test is used to test the abundance of significant differences.

The fungal isolate grows on the PDA at room temperature. For D. vanrijiae, M. amylolytica, and C. jadinii, a single colony was selected and grown overnight in 5% PDB at 28°C while shaking at 200 rpm. Overnight cultures of D. vanrijiae and M. amylolytica were resuspended in fresh 5% PDB to OD600 = 0.0001. For the synthetic community, D. vanrijiae, M. amylolytica, and C. jadinii were resuspended to OD600 = 0.001, and the spores of Cordyceps militaris and green psyllids were collected from the PDA and resuspended to 104 spores/mL. Next, equal volumes of various fungal suspensions were mixed to obtain two synchronized bacteria: (A) D. vanrijae, M. amylolytica and C. militaris and (B) D. vanrijae, M. amylolytica, C. jadinii and T. viride, store it in 5% PDB at -20 °C and supplement with 10% glycerol (weight/volume). To use, thaw the synchronizer at room temperature and dilute it 10 times (A) or 25 times (B) in fresh 5% PDB, and then mix them with Vibrio dahlia. For this purpose, conidia and VdAMP3 deletion and complementation mutants of Dahlia strain JR2 were harvested from PDA plates and diluted in ultrapure water (Milli-Q) to a final concentration of 104 or 105 conidia/mL. Next, 150 μL of the fungal suspension and 150 μL of the dahlia conidia suspension (104 conidia/mL for culturing with Synchronous A or Mycoplasma amylovora, 105 conidia/mL for culturing Synchronous B or D. vanrijae) 24-well flat-bottomed polystyrene tissue culture plate in clarification. Finally, after 6 to 9 days of incubation at 22°C, the fungal growth was imaged using an SZX10 stereo microscope (Olympus) with an EP50 camera (Olympus). The number of micro sclerotia formed by different dahlia strains was quantified by counting.

The metagenomics data has been deposited in the GenBank database of the National Center for Biotechnology Information, and the BioProject accession number is. PRJNA728211 (69) (https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA728211).

BPHJT is supported by the Earth and Life Science Research Committee of the Netherlands Organization for Scientific Research (NWO). BPHJT recognizes the funding provided by the Alexander von Humboldt Foundation within the framework of the position of Professor Alexander von Humboldt granted by the German Federal Ministry of Education. In addition, the German Research Foundation (German Research Foundation) is based on the German Excellence Strategy-EXC 2048 /1 Provide support for research—Project ID: 390686111. Thanks to Utrecht University Medical Center, Utrecht University Hubrecht Institute and the Utrecht Sequencing Facility funded by the Netherlands X-omics Initiative (NWO Project 184.034.019) for providing sequencing services.

Author contributions: research on the design of NCS, GCP and BPHJT; research on NCS, GCP and GCMvdB; data analysis of NCS, GCP, MFS and BPHJT; NCS and BPHJT wrote this paper.

The author declares no competing interests.

This article is directly contributed by PNAS.

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