A fungal member of the Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a GH25 lysozyme

Much like the ‘good bacteria’ that live in our guts, many microscopic organisms can co-exist with and even benefit the plants they live on. For instance, the yeast Moesziomyces bullatus ex Albugo (MbA for short) can shield the leaves of its plant host against white rust, a disease caused by the organism Albugo laibachii. Studies have started to unveil how the various microbes at the surface of leaves interact and regulate their own community, yet the genetic mechanisms at play are less well-known.

To investigate these processes, Eitzen et al. examined the genes that were switched on when MbA cells were in contact with A. laibachii on a leaf. This experiment revealed a few gene candidates that were then deleted, one by one, in MbA cells. As a result, a gene emerged as being key to protect the plant from white rust. It produces an enzyme known as the GH25 hydrolase, which, when purified, could reduce A. laibachii infections on plant leaves.

Bacteria, fungi and other related microorganisms cause many diseases which, like white rust, can severely affect crops. Chemical methods exist to prevent these infections but they can have many biological and ecological side effects. A solution inspired by natural interactions may be safer and more effective at managing plant diseases that affect valuable crops. Harnessing the interactions between microbes living on plants, and the GH25 enzyme, may offer better disease control.

Plants are colonized by a wide range of microorganisms. While some microbes enter the plant and establish endophytic interactions with a broad range of outcomes from beneficial to pathogenic, plant surfaces harbor a large variety of microbial organisms. Recent research has focused largely on the importance of the rhizosphere microbiota in nutrient acquisition, protection from pathogens, and boosting overall plant growth and development (Ritpitakphong et al., 2016; Walker et al., 2003; Bulgarelli et al., 2013). However, the above ground parts of the plant including the phyllosphere are colonized by diverse groups of microbes that also assist in plant protection and immunity (Busby et al., 2016; Mikiciński et al., 2016). The environment has a major impact on the microbial communities of the leaf surface, ultimately influencing their interactions with the host (Stone et al., 2018).

Scale-free network analysis was performed with the leaf microbial population of Arabidopsis thaliana (Agler et al., 2016). The majority of the interactions between kingdoms, e.g. fungi and bacteria, were found to be negative, consistent with the fact that rather the antagonistic interactions stabilize a microbial community (Coyte et al., 2015). Phyllosphere network analysis of A. thaliana identified a small number of microbes as ‘hub’ organisms, i.e. influential microbes that have severe effects on the community structure. The major hub microbe in the A. thaliana phyllosphere is the oomycete Albugo laibachii, which is a pathogenic symbiont biotrophic of Arabidopsis (Agler et al., 2016). This pathogen has been shown to significantly reduce the bacterial diversity of epiphytic and endophytic leaf habitats. Since bacteria generally comprise a large proportion of the phyllosphere microbiome (Vorholt, 2012), phylogenetic profiling of A. thaliana was also directed toward identifying a small group of bacteria that frequently colonize A. thaliana leaves. The analysis helped to develop a synthetic community of bacteria for experiments in gnotobiotic plants.

Besides bacteria and oomycetes, the microbiota of the A. thaliana leaf also comprises a broad range of fungi. Among those fungi, basidiomycete yeasts are frequently found and the most frequent ones are the epiphytic basidiomycete genus Dioszegia (Agler et al., 2016), as well as an anamorphic yeast associated with A. laibachii infection belonging to the Ustilaginales. This order includes many pathogens of important crop plants, for example corn smut and loose smut of oats, barley, and wheat are caused by Ustilago maydis, U. avenae, U. nuda, and U. tritici, respectively. Generally, the pathogenic development of smut fungi is linked with sexual recombination and plant infection is only initiated upon mating, when two haploid sporidia form a dikaryotic filament (Brefort et al., 2009). Ustilaginales Pseudozyma sp. yeasts, however, are found mostly in their anamorphic stage in nature. They tend to epiphytically colonize a wide range of habitats, where an infrequent sexual recombination might occur when they meet on a susceptible host (Kruse et al., 2017). Phylogenetic reconstruction (Kruse et al., 2017; Wang et al., 2015) showed that the smut pathogen of millet, Moesziomyces bullatus and four species of Pseudozyma, namely P. antarctica, P. aphidis, P. parantarctica, and P. rugulosa form a monophyletic group. The latter do represent anamorphic and culturable stages of M. bullatus and, hence, can be grouped to this genus. Moesziomyces strains have been reported in a number of cases to act as microbial antagonists. A strain formerly classified as Pseudozyma aphidis (now M. bullatus) inhibited Xanthomonas campestris pv. vesicatoria, X. campestris pv. campestris, Pseudomonas syringae pv. tomato, Clavibacter michiganensis, Erwinia amylovora, and Agrobacterium tumefaciens in vitro and also led to the activation of induced defense responses in tomato against the pathogen (Barda et al., 2015). It was reported that P. aphidis can parasitize the hyphae and spores of Podosphaera xanthii (Gafni et al., 2015). Pseudozyma churashimaensis was reported to induce systemic defense in pepper plants against X. axonopodis, Cucumber mosaic virus, Pepper mottle virus, Pepper mild mottle virus, and broad bean wilt virus (Lee et al., 2017).

In the present study, we explored the antagonistic potential of an anamorphic Ustilaginales yeast within the leaf microbial community of A. thaliana. We show that Moesziomyces bullatus ex Albugo on Arabidopsis (which will be referred to as MbA from further on) prevents infection by the oomycete pathogen A. laibachii and identified fungal candidate genes that were upregulated in the presence of A. laibachii, when both microbes were co-inoculated to the host plant. A knockout mutant of one of the candidates, which belongs to the glycoside hydrolase – family 25 (GH25) – was found to lose its antagonistic activity toward A. laibachii, providing mechanistic insights into fungal-oomycete antagonism within the phyllosphere microbiota. Functional characterization of GH25 will be an important step toward establishing MbA as a suitable biocontrol agent.

In a previous study we isolated a basidiomycetous yeast from Arabidopsis thaliana leaves infected with the causal agent of white rust, Albugo laibachii (Agler et al., 2016). This yeast was tightly associated with A. laibachii spore propagation. Even after years of subculturing in the lab and re-inoculation of plants with frozen stocks of A. laibachii isolate Nc14, this yeast remained highly abundant in spore isolates. Phylogenetic analyses based on fungal ITS-sequencing identified the yeast as Pseudozyma sp. Those yeasts can be found across the family of Ustilaginaceae, being closely related to pathogens of monocots like maize, barley, sugarcane, or sorghum (Zuo et al., 2019). Based on phylogenetic similarity to the pathogenic smut Moesziomyces bullatus which infects millet, several anamorphic Pseudozyma isolates were suggested to be renamed and grouped to M. bullatus (Wang et al., 2015). Since the Pseudozyma sp. that was isolated from A. laibachii spores groups into the same cluster, we classified this newly identified strain as MbA (Moesziomyces bullatus ex Albugo on Arabidopsis).

Based on the identification of MbA as having a significant effect on bacterial diversity in the Arabidopsis phyllosphere, we tested its interaction with 30 bacterial strains from 17 different species of a synthetic bacterial community (SynCom, Supplementary file 1) of Arabidopsis leaves in one-to-one plate assays. This experiment identified seven strains being inhibited by Moesziomyces, as indicated by halo formation after 7 days of co-cultivation (Figure 1—figure supplement 1). Interestingly, this inhibition was not seen when the pathogenic smut fungus U. maydis was co-cultivated with the bacteria, indicating a specific inhibition of the bacteria by MbA (Figure 1—figure supplement 1).

The primary hub microbe in the Arabidopsis phyllosphere was found to be the pathogenic oomycete A. laibachii, which was isolated in direct association with Moesziomyces (Agler et al., 2016). To test if both species interfere with each other, we deployed a gnotobiotic plate system and quantified A. laibachii infection symptoms on Arabidopsis. In control experiments, spray inoculation of only A. laibachii spores on Arabidopsis leaves led to about 33% infected leaves at 14 days post infection (dpi) (Figure 1). When the bacterial SynCom was pre-inoculated on leaves 2 days before A. laibachii spores a significant reduction of A. laibachii infection by about 50% was observed (Figure 1). However, if Moesziomyces was pre-inoculated with the bacterial SynCom, A. laibachii spore production was almost completely abolished. Similarly, the pre-inoculation of only MbA resulted in an almost complete loss of A. laibachii infection, independently of the presence of a bacterial community (Figure 1). The antagonistic effect of MbA toward A. laibachii was further confirmed using Trypan blue staining of A. laibachii infected A. thaliana leaves. A. laibachii forms long, branching filaments on Arabidopsis leaves at 15 dpi. Contrarily, in the presence of MbA, we observed mostly zoospores forming either no or very short hyphae, while further colonization of the leaf with long, branching was not observed (Figure 1—figure supplement 2). Together, our findings demonstrate that MbA holds a strong antagonistic activity toward A. laibachii, resulting in efficient biocontrol of pathogen infection. Thus, MbA is an important member of the A. thaliana phyllosphere microbial community, with a strong impact on its quantitative composition. However, despite several reports of the basidiomycete yeasts acting as antagonists, genome information of this group is rather limited. To enable a molecular understanding of how MbA acts on other members of the phyllosphere community, MbA genome information is required. We therefore sequenced the genome of MbA and established molecular tools including a protocol for stable genomic transformation to allow functional genetic approaches.

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Quantification of A. laibachii infection on A. thaliana.

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The genome of MbA

Genome sequence of MbA was analyzed by Single Molecule Real-Time sequencing (Pacific Biosciences, Menlo Park, CA), which lead to 69,674 mapped reads with an accuracy of 87.3% and 8596 bp sub-read length. Sequence assembly using the HGAP-pipeline (Pacific Biosciences) resulted in 31 contigs with an N₅₀ Contig Length of 705 kb. The total length of all contigs results in a predicted genome size of 18.3 Mb (Table 1). Gene prediction for the MbA genome with Augustus (Stanke et al., 2004) identified 6653 protein coding genes, of which 559 carry a secretion signal. Out of these 559, 380 are predicted to be secreted extracellularly (i.e. they do not carry membrane domains or cell-wall anchors) (Table 1). The small genome size and high number of coding genes result in a highly compact genome structure with only small intergenic regions. These are features similarly found in several pathogenic smut fungi such as U. maydis and S. reilianum (Table 1). Remarkably, both MbA and Anthracocystis flocculosa, which is another anamorphic and apathogenic yeast, show a similarly high rate of introns, while the pathogenic smut fungi have a significantly lower intron frequency (Table 1).

Table 1

	MbA	U. bromivora	S. scitamineum	S. reilianum	U. maydis	U. hordei	M. pennsylvanicum	A. flocculosa
Assembly statistics
Total contig length (Mb)	18.3		19.5	18.2	19.7	20.7	19.2	23.2
Total scaffold length (Mb)		20.5	19.6	18.4	19.8	21.15	19.2	23.3
Average base coverage	50×	154×	30×	20×	10×	25×	339×	28×
N50 contig (kb)	705.1		37.6	50.3	127.4	48.7	43.4	38.6
N50 scaffold length (kb)		877	759.2	738.5	817.8	307.7	121.7	919.9
Chromosomes	21	23		23	23	23
GC-content (%)	60.9	52.4	54.4	59.7	54	52	50.9	65.1
Coding (%)	62.8	54.4	57.8	62.6	56.3	54.3	54	66.3
Coding sequence
Percentage CDS (%)	69.5	59.8	62	65.9	61.1	57.5	56.6	54.3
Average gene size (bp)	1935	1699	1819	1858	1836	1708	1734	2097
Average gene density (gene/kb)	0.36	0.35	0.34	0.37	0.34	0.34	0.33	0.30
Protein-coding genes	6653	7233	6693	6648	6786	7113	6279	6877
Exons	11,645	11,154	10,214	9776	9783	10,907	9278	19,318
Average exon size (bp)	1091	1101	1191	1221	1230	1107	527	658
Exons/gene	1.75	1.5	1.5	1.47	1.44	1.53	1.48	2.8
tRNA genes	150	133	116	96	111	110	126	176
Noncoding sequence
Introns	9333	3921	3521	3103	2997	3161	2999	12,427
Introns/gene	1.40	0.54	0.53	0.47	0.44	0.44	0.48	1.81
Average intron length (base)	163	163	130.1	144	142	141	191.4	141
Average intergenic distance (bp)	769	1054	1114	929	1127	1186	1328	1273
Secretome
Protein with signal peptide	559		622	632	625	538	419	622
Secreted without TMD	380				467			737
– with known domain	260				264			554

1. [Brefort et al., 2014; Nadal and Gold, 2010].

To gain better insights in the genome organization of MbA, we compared its structure with the U. maydis genome, which served as a manually annotated high-quality reference genome for smut fungi (Kämper et al., 2006). Out of the 31 MbA contigs, 21 show telomeric structures and a high synteny to chromosomes of U. maydis, with three of them displaying major events of chromosomal recombination (Figure 2A). Interestingly, the Moesziomyces contig 2, on which also homologs to pathogenic loci like the U. maydis virulence cluster 2A (Kämper et al., 2006) can be found, contains parts of three different U. maydis chromosomes (Chr. 2, 5, and 20) (Figure 2—figure supplement 1). The second recombination event on contig six affects the U. maydis leaf-specific effector protein See1, which is required for tumor formation (Redkar et al., 2015). This recombination event is also found in the genome of the maize head smut S. reilianum, wherein the U. maydis chromosomes 5 and 20 recombined in the promoter region of the see1 gene (Figure 2B). In this respect it should be noted that S. reilianum, although infecting the same host, does not produce leaf tumors as U. maydis does (Schirawski et al., 2010).

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Also the third major recombination event, affecting MbA contig 8, changes the genomic context genes encoding essential virulence factors in U. maydis (stp1 and pit1/2), as well as the A mating type locus, which is important for pheromone perception and recognition of mating partners (Bölker et al., 1992). Based on the strong antibiotic activities of MbA, we mined the genome of MbA for the presence of secondary metabolite gene clusters. Using AntiSMASH, we were able to predict 13 of such clusters, of which three can be assigned to terpene synthesis, three contain nonribosomal peptide synthetases, and one cluster has a polyketide synthase as backbone genes (Figure 3—figure supplement 1). Interestingly, the secondary metabolite cluster that is involved in the production of the antimicrobial metabolite ustilagic acid in other Ustilaginomycetes is absent in MbA (Figure 3—figure supplement 1B). On the contrary, we could identify three MbA-specific metabolite clusters that could potentially be involved in the antibacterial activity of MbA (Figure 3—figure supplement 1C).

A previous genome comparison of the related Ustilaginales yeast A. flocculosa with U. maydis concluded that this anamorphic strain had lost most of its effector genes, reflecting the absence of a pathogenic stage in this organism (Lefebvre et al., 2013). In contrast, MbA contains 1:1 homologs of several known effectors with a known virulence function in U. maydis (Table 2). We previously found that Moesziomyces sp. possess functional homologues of the pep1 gene, a core virulence effector of U. maydis (Sharma et al., 2019), suggesting that such anamorphic yeasts have the potential to form infectious filamentous structures by means of sexual reproduction (Kruse et al., 2017). To assess the potential virulence activity of MbA effector homologs, we expressed the homolog of the U. maydis core effector Pep1 in an U. maydis pep1 deletion strain (SG200∆01987). This resulted in complete restoration of U. maydis virulence, demonstrating that MbApep1 encodes a functional effector protein (Figure 3—figure supplement 2).

Table 2

Name	Homologue	Query cover	E-value	Identity (%)	U. maydis knockout phenotype	Reference
g1653	UMAG_01987 (Pep1)	82%	3-e56	60.96	Complete loss of tumor formation – blocked in early stages of infection	Doehlemann et al., 2009
g1828	UMAG_01829 (Afu1)	99%	0.0	71.57	Organ-specific effector – reduced virulence in seedling leaves	Schilling et al., 2014
g2626	UMAG_12197 (Cce1)	98%	1e-48	60.16	Complete loss of tumor formation – blocked in early stages of infection	Seitner et al., 2018
g2765	UMAG_11938 (Scp2)	100%	1e-73	93.44	Reduced in virulence	Krombach et al., 2018
g2910	UMAG_02475 (Stp1)	32%	3e-42	60.71	Complete loss of tumor formation – blocked in early stages of infection	Schipper, 2009
g3652	UMAG_02239 (See1)	43%	9e-11	54.90	Organ-specific effector – reduced virulence in seedling leaves	Redkar et al., 2015
g3113	UMAG_01375 (Pit2)	*	*	*	Complete loss of tumor formation – blocked in early stages of infection	Doehlemann et al., 2011
g3279	UMAG_03274 (Rsp3)	10%	5e-20	70.11	Strong attenuation of virulence – reduced tumor size and number	Ma et al., 2018
g5296	UMAG_05731 (Cmu1)	98%	3e-70	43.84	Reduced virulence	Djamei et al., 2011
g6183	UMAG_06098 (Fly1)	100%	0.0	81.85	Reduced virulence	Ökmen et al., 2018
g5835	UMAG_05302 (Tin2)	87%	8e-24	37.81	Minor impact on tumor formation – reduced anthocyanin biosynthesis	Brefort et al., 2014

A hallmark of the U. maydis genome structure is the presence of large clusters with effector genes, the expression of which is only induced during plant infection (Kämper et al., 2006). To assess the presence of potential virulence clusters in MbA, we compared all U. maydis effector gene clusters to the MbA genome, based on homology. This revealed that the 12 major effector clusters of U. maydis are present in MbA. However, while many of the clustered effector genes are duplicated in pathogenic smut fungi, MbA carries only a single copy of each effector gene. This results in the presence of ‘short’ versions of the U. maydis gene effector clusters (Figure 3—figure supplement 3). This gets particularly obvious for the biggest and most intensively studied virulence cluster of smut fungi, the effector cluster 19A (Schirawski et al., 2010; Brefort et al., 2014; Dutheil et al., 2016). In MbA, only 5 out of the 24 effector genes present in U. maydis are conserved in this cluster (Figure 3). Interestingly, some anamorphic yeasts like Kalmanozyma brasiliensis and A. flocculosa completely lost virulence clusters, while another non-pathogenic member of the Ustilaginales, Pseudozyma hubeiensis, shows an almost complete set of effectors when compared to U. maydis (Figure 3).

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Genetic characterization of MbA

To perform reverse genetics in MbA, we established a genetic transformation system based on protoplast preparation and polyethylene glycol (PEG)-mediated DNA transfer. In preliminary transformation assays, we expressed a cytosolic GFP reporter-gene under control of the constitutive o2tef-Promoter (Figure 4A). For the generation of knockout strains, a split marker approach was used to avoid ectopic integrations (Figure 4B). To allow generation of multiple knockouts, we used a selection marker-recycling system (pFLPexpC) that allows selection marker excision at each transformation round (Khrunyk et al., 2010).

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We decided to apply the transformation system to study the MbA mating type loci in more detail. Although phylogenetically closely related to U. hordei, which has a bi-polar mating system, MbA owns a tetrapolar mating system whereby both mating type loci are physically not linked. This situation is similar to the mating type structure in the pathogenic smut U. maydis (Figure 4A). The a-locus, which encodes a pheromone receptor system that is required for sensing and fusion of compatible cells, is located on contig 6. The b-locus can be found on contig 1. This multiallelic mating locus contains two genes (b-East and b-West), which code for a pair of homeodomain transcription factors. Upon mating of compatible cells, pathogenic and sexual development are triggered by a heterodimeric bE/bW complex (Brefort et al., 2009). Since the MbA genome is completely equipped with mating type genes, we first deployed a screen for potential mating partners. To this end, we screened wild M. bullatus isolates to find a suitable mating partner, but we could not observe any mating event (Figure 5—figure supplement 1). To test if MbA is able to undergo pathogenic differentiation in the absence of mating, we generated a self-compatible strain (CB1) that carries compatible b-mating alleles: to construct the CB1 strain, we used compatible alleles of the b-East and b-West genes of the barley smut U. hordei, a pathogen that is the phylogenetically most closely related to MbA and amenable to reverse genetics. The native MbA locus was replaced by the compatible U. hordei b-East and b-West gene alleles via homologous recombination (Figure 5B).

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Appressoria quantification of MbA.

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Incubation of the MbA CB1 on charcoal plates led to the formation of aerial hyphae with the characteristic fluffy phenotype of filamentous strains like the self-compatible, solopathogenic U. maydis SG200 strain (Figure 5C). A second established method to induce filament formation in smuts is on hydrophobic parafilm (Mendoza-Mendoza et al., 2009). Quantification after 18 hr incubation of MbA CB1 on parafilm resulted in the formation of filaments comparable to those of the U. maydis SG200 strain (Figure 5D). While about 17% of MbA wild-type cells showed filaments, the CB1 strain with compatible b-genes showed 38% filamentous growth.

Formation of appressoria is a hallmark of pathogenic development in smut fungi (Mendoza-Mendoza et al., 2009). While the switch from yeast-like growth to filamentous development is the first step in the pathogenic development of smut fungi, host penetration is accompanied by the formation of a terminal swelling of infectious hyphae, termed ‘appressoria’. Induction of appressoria formation in vitro can be induced by adding 100 µM of the cutin monomer 16-hydroxyhexadecanoic acid (HDD) to the fungal cells prior to cell spraying onto a hydrophobic surface (Mendoza-Mendoza et al., 2009). In the absence of HDD, only about 8% of the U. maydis SG200 cells and 14% of the MbA cells formed appressoria on parafilm 24 hr after spraying (Figure 5E). Addition of 100 µM HDD resulted in a significant induction of appressoria in both U. maydis and MbA, demonstrating that MbA does hold the genetic repertoire to form infection structures in vitro. Together, the analysis of the recombinant CB1 strain indicates that MbA can sense pathogenesis-related surface cues and produce penetration structures to a similar level as that seen for the pathogenic model organism U. maydis.

Identification of microbe–microbe effector genes by RNA-Seq

To study the transcriptomic response of MbA to different biotic interactions, RNA sequencing was performed. The MbA transcriptome was profiled in five different conditions (Figure 6A; cells in axenic culture versus cells on-planta, on-planta + SynCom, on-planta + A. laibachii, on-planta + SynCom + A. laibachii). Inoculations of A. thaliana leaves were performed as described above for A. laibachii infection assays (Figure 6A). For MbA RNA preparation, the epiphytic microbes were peeled from the plant tissue by using liquid latex (see Materials and methods section for details).

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Gene expression data of candidate effector genes.

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The libraries of the 15 samples (five conditions in three biological replicates each) were generated by using a poly-A enrichment and sequenced on an Illumina HiSeq4000 platform. The paired end reads were mapped to the MbA genome by using Tophat2 (Kim et al., 2013). The analysis revealed that MbA cells on A. thaliana leaves (on-planta) downregulated 1300 and upregulated 1580 genes compared to cells in axenic culture (Figure 6B, Supplementary file 2). Differentially expressed genes were determined with the ‘limma’-package in R on ‘voom’ (Figure 6—figure supplement 1) using a False discovery rate threshold of 0.05 and log2FC > 0. A gene ontology (GO) terms analysis revealed that, among the downregulated genes, 50% were associated with primary metabolism (Figure 6—figure supplement 2). In the two conditions in which A. laibachii was present, we observed upregulation of 801 genes. Among these genes, 411 genes were specific to co-incubation of MbA with A. laibachii and SynCom while 174 were specific to incubation with A. laibachii only. A set of 216 genes was shared in both conditions (Figure 6B).

In the presence of A. laibachii, mainly metabolism- and translation-dependent genes were upregulated, which might indicate that MbA can access a new nutrient source in the presence of A. laibachii (Figure 6—figure supplement 2). Among all A. laibachii-induced MbA genes, 18 genes encode proteins carrying a secretion signal peptide and having no predicted transmembrane domain (Figure 6C). After excluding proteins being predicted to be located in intracellular organelles, nine candidate genes remained as potential microbe–microbe-dependent effectors, i.e. MbA genes that are induced by A. laibachii, show no or low expression in axenic culture, and encode for putative secreted proteins (Figure 6C). Interestingly, four of these genes encode putative glycoside hydrolases. Furthermore, two genes encode putative peptidases, one gene likely encodes an alkaline phosphatase and two encode uncharacterized proteins (Figure 6C).

To directly test the eventual antagonistic function of those genes toward A. laibachii, we selected the two predicted glycoside hydrolases-encoding genes g5 and g2490 (GH43 and GH25) and the gene encoding the uncharacterized protein g5755 for gene deletion in MbA. The respective mutant strains were tested in stress assays to assess, whether the gene deletions resulted in general growth defects. Wild-type and mutant MbA strains were exposed to different stress conditions including osmotic stress (sorbitol, NaCl), cell wall stress (calcofluor, Congo-red), and oxidative stress (H₂O₂). Overall, in none of the tested conditions we observed a growth defect of the deletion mutants in comparison to wild-type MbA (Figure 7—figure supplement 1). To test an eventual impact of the deleted genes in the antagonism of the two microbes, the MbA deletion strains were each pre-inoculated on A. thaliana leaves prior to A. laibachii infection. Deletion of g5 resulted in a significant but yet marginal increase of A. laibachii disease symptoms, while deletion of g5755 had no effect on A. laibachii. We therefore considered these two genes being not important for the antagonism of MbA toward A. laibachii. Strikingly, the MbA Δg2490 strain almost completely lost its biocontrol activity toward A. laibachii. This phenotype was reproduced by two independent g2490 deletion strains (Figure 7A). To check if this dramatic loss of microbial antagonism is specific to the deletion of g2490, in-locus genetic complementation of strain Δg2490_1 was performed via homologous recombination. The resulting strain MbA Δg2490/compl regained the ability to suppress A. laibachii infection, confirming that the observed phenotype specifically resulted from the deletion of the g2490 gene (Figure 7B). Together, these results demonstrate that the biocontrol of the pathogenic oomycete A. laibachii by the basidiomycete yeast MbA is determined by the secretion of a previously uncharacterized GH25 enzyme, which is transcriptionally activated specifically when both microbes are co-colonizing the A. thaliana leaf surface.

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Infection data of A. laibachii on A. thaliana.

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Healthy plants in natural habitats are extensively colonized by microbes, therefore it has been hypothesized that the immune system and the microbiota may instruct each other beyond the simple co-evolutionary arms race between plants and pathogens (Vannier et al., 2019). Community members as individuals or in a community context have been reported to confer extended immune functions to their plant host. Root endophytic bacteria for example were found to protect A. thaliana and stabilize the microbial community by competing with filamentous eukaryotes (Durán et al., 2018). A large inhibitory interaction network was found in the leaf microbiome of A. thaliana and genome mining was used to identify over 1000 predicted natural product biosynthetic gene clusters (BGCs) (Helfrich et al., 2018). In addition, the bacterium Brevibacillus sp. leaf 182 isolate was found to inhibit half of the 200 strains isolated from A. thaliana phyllosphere. Further analysis revealed that Brevibacillus sp. leaf 182 produces a trans-acyltransferase polyketide synthase-derived antibiotic, macrobrevin along with other putative polyketide synthases (Helfrich et al., 2018).

In this study, we describe the role of the basidiomycete yeast MbA, which we previously co-isolated with the oomycete pathogen A. laibachii and now characterized as an antagonistic driver in the A. thaliana phyllosphere. A. laibachii inhibits in vitro growth of seven members of a bacterial leaf SynCom and, most strikingly, strongly suppresses disease progression and reproduction of the pathogenic oomycete A. laibachii on A. thaliana. MbA is a member of the Ustilaginales, which had previously been classified into the group of pathogenic smut fungi of the Moesziomyces bullatus species (Kruse et al., 2017). Our genome analysis identified the anamorphic yeasts M. rugulosus, M. aphidis, and M. antarcticus, which had previously been classified as ‘Pseudozyma sp.’, as the closest relatives of MbA. Anamorphic Ustilaginales yeasts are long known and have been used for biotechnological applications and also biocontrol (Boekhout, 2011). Mannosylerythritol lipids produced by M. antarcticus are known to act as biosurfactants and are of great interest for pharmaceutical applications (Kitamoto et al., 1990; Morita et al., 2007). Glycolipids like flocculosin produced by A. flocculosa or ustilagic acid characterized in the smut fungus U. maydis have antifungal activity. Those compounds destabilize the membrane of different fungi and thus serve as biocontrol agents against powdery mildews or gray mold (Cheng et al., 2003; Mimee et al., 2005; Teichmann et al., 2007).

We identified 13 potential secondary metabolite gene clusters in MbA, including non-ribosomal peptide synthases and polyketide synthetase. Interaction among microbes within the same habitat is believed to have given rise to a variety of secondary metabolites (Schroeckh et al., 2009; Rutledge and Challis, 2015). The presence of Streptomyces rapamycinicus was shown to activate an otherwise silent polyketide synthase gene cluster, fgnA, in Aspergillus fumigatus. The resultant compound proved to be a potent fungal metabolite that inhibited the germination of S. rapamycinicus spores (Stroe et al., 2020). Therefore, secondary metabolite gene clusters and their corresponding products may confer a competitive advantage to fungi over the bacteria that reside in the same environment.

What is still under investigation is the relation of anamorphic yeasts with the related pathogenic smuts. Many smut fungi including the model species U. maydis are dimorphic organisms. In their saprophytic phase they grow as haploid non-pathogenic yeast cells. Only on appropriate host surfaces, haploid cells switch to filamentous growth and expression of pathogenicity-related genes is only activated upon mating in the filamentous dikaryon. A prime prerequisite for pathogenic development is therefore the ability of mating (Bölker, 2001; Nadal and Gold, 2010). Our genome analysis identified a tetrapolar mating system with a complete set of mating genes in MbA. Looking more closely on the phylogeny of different mating genes it appears that all sequenced Moesziomyces strains have the same pheromone receptor type (Figure 7—figure supplement 3). Together with our unsuccessful mating assays, this suggests that all sequenced strains of this species have the same mating type and, therefore, are unable to mate. Mating type bias after spore germination was reported for Ustilago bromivora, which leads to a haplo-lethal allele linked to the MAT-2 locus (Rabe et al., 2016). In this case, an intratetrad mating event rescues pathogenicity in nature as the second mating partner is not viable after spore germination. Together with the observation that anamorphic Moesziomyces yeasts are ubiquitous in nature, one could hypothesize that these fungi are highly competitive in their haploid form and antagonism might have led to the selection of one viable mating type. This eventually adapted to the epiphytic life style. At the same time, one should not dismiss the possibility that in the presence of a viable mating partner and suitable host surface, the yeasts can undergo potential sexual reproduction and thereby revitalizing the gene pool from time to time.

Transcriptome analysis showed that epiphytic growth of MbA on A. thaliana leads to massive transcriptional changes particularly in primary metabolism, which might reflect adaptation to the nutritional situation on the plant surface. Moreover, MbA showed specific transcriptional responses to a bacterial community, as well as to A. laibachii when being co-inoculated on plant leaves. The presence of A. laibachii resulted in the induction of primary metabolism and biosynthesis pathways, which might reflect enhanced growth of MbA in the presence of A. laibachii.

A set of MbA genes encoding secreted hydrolases was induced by A. laibachii and one of these genes which encodes a putative GH25 hydrolase with similarity to Chalaropsis type lysozymes appeared to be essential for the biocontrol of A. laibachii. Initially discovered in the fungi Chalaropsis sp., this group of proteins is largely present in bacteria as well as phages, for example the germination-specific muramidase from Clostridium perfringens S40 (Chen et al., 1997). The bacterial muramidase, cellosyl from Streptomyces coelicolor (Rau et al., 2001), also belongs to the Chalaropsis type of lysozyme. These proteins are proposed to cleave the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) in the bacterial peptidoglycan. Specifically, the β-1,4 N,6-O-diacetylmuramidase activity allows the Chalaropsis type lysozyme to degrade the cell wall of Staphylococcus aureus, in contrast to the commercially available HEWL (Rau et al., 2001). Despite differences in structure and molecular weight from HEWL, the GH25 of MbA has lysozyme activity against the gram positive bacterium Micrococcus lysodeikticus in a fluorogenic assay. This highlights the overall biochemical functionality of the recombinant glycoside hydrolase. The glycoside Hydrolase 25 family is predicted to have an active site motif DXE that is highly conserved across the fungal kingdom (Figure 7—figure supplement 4). The structure of glycoside hydrolase family 25 from Aspergillus fumigates was characterized and the presence of N-terminal signal peptide was considered to indicate an extracellular secretion of the protein with possible antimicrobial properties (Korczynska et al., 2010). The role of the secreted hydrolase in the fungal kingdom is not completely explored yet. The presence of such hydrolases has in many cases been hypothesized to be associated with hyperparasitism of fungi parasitizing fungi (Hyde et al., 2019) or oomycetes parasitizing oomycetes (Horner et al., 2012). Our results might therefore indicate a cross kingdom hyperparasitism event between a fungus and an oomycete. Previous work on microbial communities has indicated that negative interactions stabilize microbial communities. Hyperparasitism is such a negative interaction with a strong eco-evolutionary effect on pathogen–host interactions and therefore on community stability (Parratt and Laine, 2016). MbA might therefore regulate A. laibachii infection and reduce disease severity. The qPCR evaluation of oomycete biomass strongly points toward the idea that A. laibachii is a direct target of antagonism for MBA. Since we observed reduced formation of A. laibachii in the presence of MbA, we also tested if the GH25 lysozyme would suppress zoospore germination. However, we could not detect a significant reduction of A. laibachii zoosporangia germination upon treatment with active GH25 lysozyme (Figure 7—figure supplement 5), suggesting that the GH25 lysozyme interferes with A. laibachii at a later stage of infection. As A. laibachii has been shown to reduce microbial diversity (Agler et al., 2016), MbA might increase diversity through hyperparasitism of A. laibachii. At the same time this increased diversity might have caused the need for more secondary metabolites to evolve in the MbA genome to defend against niche competitors. Through its close association with A. laibachii, MbA could be a key regulator of the A. thaliana microbial diversity and therefore relevant for plant health beyond the regulation of A. laibachii infection.

In conclusion, the secreted hydrolase we identified as a main factor of A. laibachii inhibition has great potential to act as antimicrobial agent. The isolated compound is not only valuable per se in an ecological context. It can further lay the grounds for exploring other microbial bioactive compounds that mediate inter-species and inter-kingdom crosstalk. A main goal of our future studies will be to understand on the mechanistic level, how the GH-25 suppresses A. laibachii, and at which developmental step the oomycete infection is blocked. It will be particularly interesting to elucidate if and how the GH25 enzyme activity directly interferes with the A. laibachii cell wall. While the canonical lysozyme substrate is found in bacterial cell walls, a detailed biochemical characterization of substrate specificity will be required to pinpoint potential target sites in the oomycete cell wall. Also, to the best of our knowledge there is no detailed information on the A. laibachii cell wall structure and composition. One could speculate if GH25 activity directly affects cell wall integrity of A. laibachii, or if a modification of the cell wall structure interferes with pathogenic development, e.g. by interfering with cellular differentiation, blocking signal perception, or by triggering a host defense response.

Since the GH-25 enzyme is well conserved among Ustilaginales including pathogenic species, it will also be tempting to elucidate whether the species-specific antagonism identified here is broadly conserved among Ustilaginales fungi and oomycetes. We further will investigate potential responses by the host plant and how this impacts A. laibachii growth upon MbA colonization. Functional investigation of these interactions can provide meaningful insights as to why certain yeasts prefer to colonize specific environments. At the same time, it will be worth exploring how the basidiomycete yeasts influence the bacterial major colonizers of the phyllosphere.

Genome information and RNA sequencing have been submitted to NCBI Genbank and are available under the following links: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148670.

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Author details

1. Katharina Eitzen

   1. Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Center for Molecular Biosciences, Cologne, Germany
   2. Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   Conceptualization, Investigation, Visualization, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

2. Priyamedha Sengupta

   Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Center for Molecular Biosciences, Cologne, Germany

   Contribution

   Investigation, Visualization, Methodology, Writing - review and editing

   Competing interests

   No competing interests declared

3. Samuel Kroll

   Max Planck Institute for Plant Breeding Research, Cologne, Germany

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Eric Kemen

   1. Max Planck Institute for Plant Breeding Research, Cologne, Germany
   2. Department of Microbial Interactions, IMIT/ZMBP, University of Tübingen, Tübingen, Germany

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   eric.kemen@uni-tuebingen.de

   Competing interests

   No competing interests declared

5. Gunther Doehlemann

   Institute for Plant Sciences and Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne, Center for Molecular Biosciences, Cologne, Germany

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   g.doehlemann@uni-koeln.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7353-8456

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