Author response: A fungal member of the Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a GH25 lysozyme
2021-01-05

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Plants are not only challenged by pathogenic organisms but also colonized by commensal microbes. The network of interactions these microbes establish with their host and among each other is suggested to contribute to the immune responses of plants against pathogens. In wild Arabidopsis thaliana populations, the oomycete pathogen Albugo laibachii plays an influential role in structuring the leaf phyllosphere. We show that the epiphytic yeast Moesziomyces bullatus ex Albugo on Arabidopsis, a close relative of pathogenic smut fungi, is an antagonistic member of the A. thaliana phyllosphere, which reduces infection of A. thaliana by A. laibachii. Combination of transcriptomics, reverse genetics, and protein characterization identified a GH25 hydrolase with lysozyme activity as a major effector of this microbial antagonism. Our findings broaden the understanding of microbial interactions within the phyllosphere, provide insights into the evolution of epiphytic basidiomycete yeasts, and pave the way for novel biocontrol strategies. eLife digest 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. Introduction 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. Results 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. Figure 1 with 2 supplements see all Download asset Open asset Infection assay of A. laibachii on A. thaliana. Addition of a bacterial SynCom reduces the infection symptoms of A. laibachii at 14 days post infection. Those symptoms can be almost abolished by spraying MbA to the plant, independently of the presence of the bacterial community. Infections were performed in six individual replicates with 12 technical replicates. N indicates the number of infected plants that were scored for symptoms. An analysis of variance (ANOVA) model was used for pairwise comparison of the conditions, with Tukey's HSD test to determine differences among them. Different letters indicate significant differences (p-values <0.05). Figure 1—source data 1 Quantification of A. laibachii infection on A. thaliana. https://cdn.elifesciences.org/articles/65306/elife-65306-fig1-data1-v2.xlsx Download elife-65306-fig1-data1-v2.xlsx 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 Comparison of genomes and genomic features of known pathogenic and anamorphic Ustilaginomycetes. MbAU. bromivoraS. scitamineumS. reilianumU. maydisU. hordeiM. pennsylvanicumA. flocculosaAssembly statisticsTotal contig length (Mb)18.319.518.219.720.719.223.2Total scaffold length (Mb)20.519.618.419.821.1519.223.3Average base coverage50×154×30×20×10×25×339×28×N50 contig (kb)705.137.650.3127.448.743.438.6N50 scaffold length (kb)877759.2738.5817.8307.7121.7919.9Chromosomes2123232323GC-content (%)60.952.454.459.7545250.965.1Coding (%)62.854.457.862.656.354.35466.3Coding sequencePercentage CDS (%)69.559.86265.961.157.556.654.3Average gene size (bp)19351699181918581836170817342097Average gene density (gene/kb)0.360.350.340.370.340.340.330.30Protein-coding genes66537233669366486786711362796877Exons11,64511,15410,2149776978310,907927819,318Average exon size (bp)109111011191122112301107527658Exons/gene1.751.51.51.471.441.531.482.8tRNA genes15013311696111110126176Noncoding sequenceIntrons933339213521310329973161299912,427Introns/gene1.400.540.530.470.440.440.481.81Average intron length (base)163163130.1144142141191.4141Average intergenic distance (bp)769105411149291127118613281273SecretomeProtein with signal peptide559622632625538419622Secreted without TMD380467737– with known domain260264554 [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). Figure 2 with 1 supplement see all Download asset Open asset Circos comparison of MbA and U. maydis chromosome structure (A). We highlighted potential secondary metabolite clusters, secreted proteins, and gene predictions on both strands (±). (B) Homology-based comparisons identified three chromosomal recombination events, which affects the MbA contigs 2, 6, and 8. 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 MbA proteins homologous to U. maydis effector genes with known virulence function. NameHomologueQuery coverE-valueIdentity (%)U. maydis knockout phenotypeReferenceg1653UMAG_01987 (Pep1)82%3-e5660.96Complete loss of tumor formation – blocked in early stages of infectionDoehlemann et al., 2009g1828UMAG_01829 (Afu1)99%0.071.57Organ-specific effector – reduced virulence in seedling leavesSchilling et al., 2014g2626UMAG_12197 (Cce1)98%1e-4860.16Complete loss of tumor formation – blocked in early stages of infectionSeitner et al., 2018g2765UMAG_11938 (Scp2)100%1e-7393.44Reduced in virulenceKrombach et al., 2018g2910UMAG_02475 loss of tumor formation – blocked in early stages of effector – reduced virulence in seedling et al., loss of tumor formation – blocked in early stages of infectionDoehlemann et al., of virulence – reduced tumor size and et al., et al., et al., impact on tumor formation – reduced et al., A of the U. maydis genome structure is the presence of large clusters with effector genes, the of which is only induced 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 This revealed that the 12 major effector clusters of U. maydis are present in However, while many of the effector genes are in pathogenic smut fungi, MbA only a of each effector This results in the presence of of the U. maydis gene effector clusters (Figure 3—figure supplement This for the and most virulence cluster of smut fungi, the effector cluster (Schirawski et al., et al., 2014; et al., 2016). In MbA, only 5 of the effector genes present in U. maydis are in this cluster (Figure Interestingly, some anamorphic yeasts like and A. flocculosa completely lost virulence clusters, while another member of the Pseudozyma an almost complete of effectors when compared to U. maydis (Figure Figure with supplements see all Download asset Open asset of the virulence cluster in pathogenic smut fungi and anamorphic smut yeasts indicate genes with to each related gene are indicated in and effector genes are shown in encoding proteins without a secretion signal are shown in white (Brefort et al., characterization of MbA To reverse in MbA, we established a genetic transformation system based on and In transformation we expressed a control of the (Figure For the of knockout a was used to (Figure To allow of we used a system that at each transformation et al., 2010). Figure Download asset Open asset transformation of that could be by with and into the genome (B) of the that was used to deletion homologous We to the transformation system to study the MbA mating type loci in more closely related to U. which has a mating MbA a mating system both mating type loci are not This is to the mating type structure in the pathogenic smut U. maydis (Figure The which encodes a pheromone system that is required for and of is on contig The can be found on contig This mating contains two genes and which for a of mating of pathogenic and sexual development are by a (Brefort et al., 2009). Since the MbA genome is completely with mating type genes, we deployed a for potential mating To this we wild M. bullatus isolates to a suitable mating but we could not mating event (Figure supplement 1). To test if MbA is able to pathogenic in the absence of mating, we a strain that to the we used of the and genes of the smut U. a pathogen that is the most closely related to MbA and to reverse The MbA was by the U. and gene homologous recombination (Figure Figure 5 with 1 supplement see all Download asset Open asset The MbA strain MbA mating type genes, the ones of U. can be found on two different chromosomes to the mating type system of U. (B) To a strain the genes of U. were at the MbA the MbA strain strain a on and filamentous U. maydis haploid strain and strain were used as and respectively. and of and formation in strain was in three For this cells for filament formation and cells for formation were analyzed and indicate on a surface, filament and formation in strain were significantly different test for – when compared to MbA wild type and to the of the U. maydis strain U. maydis haploid strain was used as control (Kämper et al., 2006). 20 Figure data 1 of Download of the MbA on led to the formation of hyphae with the of filamentous strains like the U. maydis strain (Figure A second established to induce filament formation in is on et al., 2009). Quantification after of MbA on resulted in the formation of filaments to those of the U. maydis strain (Figure While about of MbA cells showed the strain with showed filamentous of is a of pathogenic development in smut fungi et al., 2009). While the from growth to filamentous development is the step in the pathogenic development of smut fungi, host is by the formation of a of infectious hyphae, of formation in vitro can be induced by of the acid to the fungal cells to spraying a surface et al., 2009). In the absence of only about of the U. maydis cells and of the MbA cells on after spraying (Figure Addition of resulted in a significant of in both U. maydis and MbA, demonstrating that MbA does the genetic to form infection structures in Together, the analysis of the strain indicates that MbA can surface and produce structures to a as that seen for the pathogenic model organism U. of effector genes by To study the response of MbA to different sequencing was The MbA was in different (Figure cells in cells SynCom, A. laibachii, SynCom A. of A. thaliana leaves were performed as above for A. laibachii infection (Figure For MbA the epiphytic microbes were from the plant by using Materials and methods for Figure with 2 supplements see all Download asset Open asset analysis of used for the analysis in (B) MbA genes after spraying of haploid cells the A. thaliana leaf A total number of genes were upregulated in response to A. laibachii in the presence and absence of bacterial the genes, were upregulated in both file 2). of the A. MbA genes that are predicted to secreted these genes, were as candidate effector genes, based on their and prediction to for extracellularly Figure data 1 Gene data of candidate effector Download