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.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  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.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 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.To investigate these processes, Eitzen et al. examined the genes that were switched on when MbA cells were in contact with 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 . HoweverArabidopsis thaliana  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  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 the present study, we explored the antagonistic potential of an anamorphic Ustilaginales yeast within the leaf microbial community of Arabidopsis thaliana leaves infected with the causal agent of white rust, Albugo laibachii .In a previous study we isolated a basidiomycetous yeast from aibachii . This ye sorghum . Based obullatus . Since tMbA 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   . When thobserved . Howeverommunity . The antobserved . TogetheMbA was analyzed by Single Molecule Real-Time sequencing , 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   . The secormation . This reee1 gene . In thisdis does .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 . This resulted in complete restoration of U. maydis virulence, demonstrating that MbApep1 encodes a functional effector protein -mediated DNA transfer. In preliminary transformation assays, we expressed a cytosolic GFP reporter-gene under control of the constitutive o2tef-Promoter , 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  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 . Inoculations of A. thaliana leaves were performed as described above for A. laibachii infection assays .To study the transcriptomic response of n assays . For MbAMbA genome by using Tophat2  downregulated 1300 and upregulated 1580 genes compared to cells in axenic culture  were generated by using a poly-A enrichment and sequenced on an Illumina HiSeq4000 platform. The paired end reads were mapped to the  Tophat2 . The ana culture . Differe culture  using a  culture . In the nditions .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  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 , cell wall stress , 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  as a control. We noticed a concentration-dependent increase in relative fluorescence unit (RFU)/min for the active GH25 in molar concentrations from 2 µM to 10 µM. Whereas, for similar concentrations, mutated GH25 (GH25mut) showed no significant increase in RFU/min compared to the active version. Commercial HEWL showed a steady increase in RFU/min from 1 µM to 5.5 µM concentrations   . OverallA. thaliana and stabilize the microbial community by competing with filamentous eukaryotes   . In addiynthases .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  and U. maydis (at 28°) strains were grown in liquid YEPS light medium and maintained on potato dextrose agar plates. King’s B medium was used for culturing Syn Com bacterial members at 22°. All the strains were grown in a rotary shaker at 200 rpm. All the recipes for medium and solutions can be found in MbA grown to an optical density (600 nm) of 0.6–0.8 were centrifuged at 3500 rpm for 10 min and suspended in sterile water to reach an OD of 1.0. Next, a dilution series from 100 to 10–4 was prepared in sterile H2O. In the end, 5 μl of each dilution was spotted on CM plates supplemented with the indicated stress agents. The plates were incubated for 2 days at 22°C. Confrontation assays: at first, MbA and SynCom bacterial strains were grown to an O.D of 0.8–1. MbA cultures (10 μl) were dropped in four quadrants of a potato dextrose agar plate, previously spread with a bacterial culture. Plates were incubated for 2–4 days at 22°C.Trichoderma harzianum, # L1412, Sigma). After 20 min of incubation at room temperature, as cell wall lysis occurred, cold SCS was added to the mixture and protoplasts spun down for 10 min at 2400 rpm. They were then washed twice with SCS and resuspended with 10 ml STC . Construction of deletion mutants was performed by homologous recombination; the 5’ and 3’ flanking regions of the target genes were amplified and ligated to an antibiotic resistance cassette , was tested positive by amplification of the right border and left border areas with primers specific for the genomic locus and for the plasmid region. Additionally, two primers specific for the MbA bE and bW genes were chosen to amplify parts of the native locus. To induce filament and appressoria formation in vitro we used a Moesziomyces YEPSL culture at OD600 0.6–0.8. The cells were diluted to an OD600 of 0.2 in 2% YEPSL (for appressoria formation 100 µM 16-hydroxyhexadecanoic acid [Sigma-Aldrich] or 1% ethanol was added) and sprayed the yeast like cells on parafilm which mimics the hydrophobic plant surface. After 18 hr of incubation at 100% humidity the number of cells grown as filaments (or generating appressoria) was determined relative to the total number of total cells by using a light microscope.The complete b-locus of the solopathogenic mplified  and inseArabidopsis thaliana seeds were subjected to cold treatment for 7 days and sown on 1/2 strength Murashige Skoog (MS) medium  medium . The MS A. thaliana seedlings on MS plates were sprayed with A. laibachii as a control and GH25+ A. laibachii and Mut_GH25+A. laibachii as treatments. After 10 dpi, the seedlings were harvested, frozen in liquid nitrogen, and kept at −80°C. For DNA extraction, the frozen plant material was ground into a fine powder with mortar and pestle and treated with extraction buffer . This was followed by centrifugation after the addition of one volume phenol/chloroform/isoamylalkohol, 25:24:1 (Roth). The top aqueous layer was removed and added to one volume of isopropanol to precipitate the nucleic acids. DNA pellet obtained after centrifugation was washed with 70% EtOH and finally dissolved in 50 µl nuclease-free water. For qPCR measurements, 10 µl of GoTaq qPCR 2× Master Mix , 5 µl of DNA (~50 ng), and 1 µl of forward and reverse primer (10 µM) up to a total volume 20 µl were used. Samples were measured in triplicates in a CFX Connect real-time PCR detection system (Bio-Rad) following the protocol of A. laibachii DNA was quantified using the following oligonucleotide sequences: A. thaliana EF1-α: 5′-AAGGAGGCTGCTGAGATGAA-3′, 5′-TGGTGGTCTCGAACTTCCAG-3′; Oomycete internal transcribed spacer (ITS) 5.8 s: 5′-ACTTTCAGCAGTGGATGTCTA-3′, 5′-GATGACTCACTGAATTCTGCA-3′. Cq values obtained in case of the oomycete DNA amplification was normalized to A. thaliana DNA amplicon and then the difference between control (only Albugo) and treatment (Albugo+ GH25/Mut_GH25) was calculated by ddCq. The relative biomass of Albugo was analyzed by the formula (2−ddCq). Each data point in the graph represents three independent biological replicates.Four weeks old A. thaliana plants were fixed between two fingers and liquid latex was applied to the leaf surface by using a small brush. The latex was dried using the cold air option of a hair dryer, carefully peeled off with a thin tweezer, and immediately frozen in liquid nitrogen. Afterwards, the frozen latex pieces were grinded with liquid nitrogen and the RNA was isolated by using Trizol Reagent  according to the manufacturer’s instructions. Turbo DNA-Free Kit  was used to remove any DNA contamination in the extracted RNA. Synthesis of cDNA was performed using First Strand cDNA Synthesis Kit  according to recommended instruction starting with a concentration of 10 µg RNA. QIAprep Mini Plasmid Prep Kit  was used for isolation of plasmid DNA from bacteria after the principle of alkaline lysis. Genomic DNA was isolated using phenol–chloroform extraction protocol . MbA genome was annotated with the Augustus software tool. Secretome was investigated using SignalP4.0. Analysis of functional domains in the secreted proteins was done by Inter-Pro Scan. AntiSmash was used to predict potential secondary metabolite clusters. RNA sequencing was done at the Cologne Center for Genomics (CCG) by using a poly-A enrichment on an Illumina HiSeq4000 platform. The achieved paired end reads were mapped to the MbA and A. thaliana TAIR10 genome by using Tophat2 (MbA axenic cultures were used to generate exon and intron hints and to start a second annotation with Augustus. Heat maps were performed using the heatmap.2 function of the package gplots (version 3.0.1) in R-studio (R version 3.5.1). An analysis of variance (ANOVA) model was used for pairwise comparison of the conditions, with Tukey's HSD test to determine the significant differences among them .Sequence assembly of  Tophat2 . RNA-SeqPichia pastoris KM71H-OCH gene expression system was used to produce MBA_GH25 domain tagged with an N-terminal Polyhistidine tag (6xHis) and a C-terminal peptide containing the c-myc epitope and a 6xHis tag. The His-MspGH25 cloned into pGAPZαA vector  under the control of a constitutive promotor with an α-factor signal peptide for secretion. Expression and puriﬁcation of recombinant proteins were performed according to manufacturer’s instructions : YPD medium supplemented with 100 μg ml−1 zeocin was used for initial growth of P. pastoris strains at 28°C and 200 rpm (for liquid cultures). Production of the recombinant protein was performed in 1 L buffered  YPD medium with 2% sucrose at 28°C for 24 hr with 200 rpm shaking. Next the protein was subjected to affinity purification with a Ni-NTA-matrix according to manufacturer’s instructions . After purification, the His-MspGH25 protein was dialyzed in an exchange buffer . The purified protein was kept in 100 µl aliquots at 4°C.The Site-directed mutagenesis was performed on pGAPZα-His- MspGH25 vector according to the instructions of the QuikChange Multi Site-Directed Mutagenesis Kit  with primers targeting nucleotides of the active site of GH25.MBA from P. pastoris was quantified according to a sensitive fluorescence-based method using Molecular Probes EnzChek Lysozym-Assay-Kit . DQ lysozyme substrate (Micrococcus lysodeikticus) stock suspension (1.0 mg/ml) and 1000 units/ml HEWL stock solution were prepared according to the manufacturer's instruction. Molar concentration of the HEWL stock solution was calculated using the following website  and was found to be 11 µM. Protein concentration of MspGH25 both active and mutated version was measured in the Nanodrop 2000c spectrophotometer  according to the manufacturer’s instructions using 100 µl of sample after using 100 µl of the appropriate buffer as a blank control in glass cuvette. The molar concentrations of recombinant proteins were also calculated as above.Purified glycoside hydrolase of At the start of the reaction 50 μl of the DQ lysozyme substrate working suspension was added to each microplate well containing reaction buffer with either HEWL (in molar concentrations ranging from 0.1 to 5.5 µM) or MspGH25 (in molar concentration from 0.5 to 17.5 µM). Fluorescence intensity of each reaction was measured every 5 min to follow the kinetic of the reaction at 37°C for 60 min, using fluorescence microplate reader with fluorescein filter Tecan Inﬁnite 200 Pro plate reader . Digestion products from the DQ lysozyme substrate have an absorption maximum at ~494 nm and a fluorescence emission maximum at ~518 nm. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.Acceptance summary:Moesziomyces bullatus ex Albugo (MbA) was isolated from the biotrophic pathogen oomycete Albugo laibachii, which acts as a hub organism in Arabidopsis phyllosphere microbiome. The study promotes the establishment of Mba as a leaf epiphytic model fungus, by providing a sequenced genome, the transcriptome in response to interaction with a phyllosphere bacterial SynCom and A. laibachii and by establishing a transformation system for generation of k. o. strains by homologous recombination. The authors report that MbA antagonizes the oomycete pathogen A. laibachii, via a putatively secreted GH25 hydrolase with lysozyme activity. This important finding provides novel insights into the yet elusive molecular mechanisms involved in shaping the microbiome community on leaf surfaces through microbe-microbe interactions.Understanding functions and mechanisms of microbe-microbe interactions in plant microbiota communities is of great fundamental interest, and promising for agricultural application for example in biocontrol. This study reports on a specific molecular player mediating antagonism by a leaf-surface dwelling yeast on an oblicate biotrophic oomycete pathogen. The yeast Decision letter after peer review:[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a putative secreted hydrolase" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor, and three reviewers. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "A fungal member of the eLife in its present form.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in Moesziomyces albugensis as a genetic model as well as its characterization interesting, the reviewers detected several flaws, which prevent publication in eLife:Although the reviewers and ourself found the study original and the establishment of Moesziomyces albugensis" appears incorrect– the phylogenetic placing of "– its mode of action on Albugo is not described and– the GH25 enzyme and its function are not sufficiently characterizedMoesziomyces action on Albugo and GH25 function.We would be happy to consider an improved version of the manuscript, in which the reviewer comments have been addressed comprehensively; and which provides a better understanding of Reviewer #1:Moesziomyces albugensis as a new "model fungus" at the leaf surface, which may act as a parasite on the pathogenic oomycete of Arabidopsis Albugo laibachii. They provide a sequenced genome, the transcriptome in response to interaction with the plant, phyllosphere bacteria and the oomycete A. laibachii, a transformation system for generation of k. o. strains by homologous recombination and characterize the mating type as well as filament and appressoria forming behavior in vitro.The authors establish M. albugensis antagonizes the oomycete pathogen A. laibachii, probably via a secreted putative GH25 hydrolase. This finding and other findings, that may be expected from this research line in the future, may yield biocontrol fungi to combat agriculturally relevant oomycete infections of crop leaves.In addition, they also report some interesting biology and show that A. laibachii is very interesting, the manuscript still suffers from a few unclarities.While M.albugensis is an exciting new fungus and the identification of the effector required for parasitism of A. laibachii. This should be clarified.– From the text I did not understand whether M. albugensis lives only epiphytically or can also colonize the plant tissue (it forms appressoria on hydrophobic surfaces) or whether it is exclusively associated with – It is unclear how the bacterial SynCom was constructed and which isolates it contains.A. laibachii infection seems kind of rough and prone to subjectivity. An additional, more quantitative method should be used for quantification, for example by qPCR using an amplicon from A. laibachii DNA normalized to a plant amplicon.– The quantification of A. laibachii or whether it helps M. albugensis to parasitize A. laibachii by other means. The authors should examine the effect of isolated GH25 hydrolase on A. laibachii infection.– It is unclear whether GH25 hydrolase suppresses infection of plant leaves by A. laibachii blocked by GH25 hydrolase or M. albugensis?– At which stage of infection is Reviewer #2:Arabidopsis phyllosphere classified as Moesziomyces albugensis (Ma). The report provides phylogenetic classification, genome analysis and functional analysis of Ma inhibitory activity towards the white rust oomycete Albugo laibachii (Al). Understanding functional interaction in the plant microbiota is a field of intense research with a great fundamental interest, as well as promising applied perspectives regarding the identification of novel biocontrol agents such as potentially Ma. I found the manuscript very pleasant to read and well structured. The work appeared rigorous and conclusions are appropriately supported by the available data. A number of biocontrol yeasts have been isolated in the past, but in my opinion this study stands out by providing direct genetic evidence of the anti-rust activity of Ma, through the identification of one underlying gene (encoding a putative GH25 enzyme) and the characterization of the corresponding mutant phenotype. The Ma genome sequence and gene expression data also provide an interesting resource for further analysis of the biology of this phyllosphere fungus.The study by Eitzen et al., characterizes a yeast species isolated from 1) I would have liked to see the activity of the GH25 characterized further. I am lacking a clear hint towards GH25 molecular activity on Al and possibly other members of the phyllosphere. Does ectopic (eg in plants) expression of GH25 has inhibitory effect on Albugo? Is there any indication on which species would this enzyme act? Is it acting specifically on Albugo? What protects Ma from its own secreted hydrolase? How conserved in GH25 across fungi? Is there anything specific about its sequence (and predicted activity) as compared to other GH25s? to other glycosyde hydrolases? To what extent is GH25 activity beneficial to Ma? Could the growth of this fungus measured on Albugo-infected vs Albugo-free plants? in vitro with or without Albugo?I believe some info and discussion on these questions is important as the identification of GH25 is in my view the major novelty of this work.2) The description of statistical analyses needs attention. I particular, I could not find information on how gene expression and differentially expressed genes were determined .Reviewer #3:Albugo laibachii.The manuscript deals with the characterization of a phyllosphere yeast that is supposed to have an antagonistic effect on While the manuscript is well written, it suffers from numerous cases of overinterpretation of the results and methodological flaws, as outlined below.Moesziomyces albugensis". First, there is not even a remotely valid description of the "species", and second, it is quite obvious that after checking some of the recent literature on Moesziomyces (part of which two of the authors even co-authored) that the isolate they are investigating is clearly Moeziomycesbullatus. This needs to be changed throughout the manuscript.Abstract. It is beyond reason that the authors call their yeast isolate "Abstract. That the isolates "prevents" Albugo infections is insufficiently shown (but see later criticism on this point).Abstract. The evidence for the claim that GH25 is a major effector of the antagonism is weak, at best.Introduction. This statement is not correct. there are several studies, e.g. by the Macia-Vicente group that have shown that the local environment drives the diversity of root-associated microbiomes.Moesziomyces bullatus.Introduction. The genus Pseudozyma is obsolete. This should be termed Moesziomyces. And the species is known as well, it is Introduction. I was surprised about the statement and have the read Kurse et al., manuscript. In fact, the authors report the opposite, an infrequent sexual reproduction on grass hosts as the most probable explanation for the retainment of a sexual reproduction.Moesziomyces and that Pseudozyma aphidis is conspecific with Moesziomyces bullatus.Introduction. I really wonder why the authors cite this less comprehensive phylogeny, while two of them have shown, based on a broader sampling that in fact the species mentioned are embedded within Pseudozyma had already been done, thus, "can be renamed" is not a correct statement.Introduction. The transfer of these species into Moesziomyces bullatus or "A strain formerly classified as Pseudozyma aphidis (now Moesziomyces bullatus)".Introduction. This should read Moesziomyces albuginensis. And this also would not make phylogenetic or evolutionary sense, as the Moesziomyces bullatus strain isolated from Arabidopsis thaliana is firmly embedded within Moesziomyces bullatus and there is no evidence that is should be considered as a biologically distinct species. Also, the evidence of disease "prevention" are at most anecdotal (see later criticism).Introduction. There is no such species name as Results. This statement alone refutes the previous statement in the Introduction. If the species was isolated from and found to be closely associated with the spores of Albugo, even over years of continuous cultivation cycles, how can the authors confidently claim that the species prevents infection?Moesziomyces bullatus (as Pseudozymaaphidis) that are marked as biocontrol agent. The new strain would just be an addition to that, there is little novelty in this.Introduction. There are many strains of Results. This argumentation is impossible to follow. Just because something is isolated from a special location does not make it a new species.Pseudozyma sp." while they could show in a previous manuscript  that the species is Meosziomyces bullatus.Results. I really cannot see the reason for the authors claiming again the identification was just "Ustilago maydis is also difficult to justify, as that species is largely unrelated to Moesziomyces bullatus. Instead, other species of the genus Moesziomyces, especially additional strains of Moesziomyces bullatus should have been used.Results. This interaction does not need to be specific. The test against Moesziomyces strains and demonstrating that a lower dose of the Albugo-associated strain inhibits the infection.Results. While in general, it is interesting that the yeast showed an antogonistic effect, dose dependency should have been checked. Several species of the Ustulagoinales are known to produce biosurfactants that alter the properties of the plant cuticle. Thus, the effect observed can probably already explained by this. This could be addressed by additional experiments with a variety of Ustilago and Moesziomyces albugensis”. This lacks a lot of the usual quality control, such as genome completeness statistics, read mapping specifics, testing of the gene predictions by RNA mapping.Subsection “The genome of Moesziomyces albugensis”. Evidence should be presented that this is not the result of a mis-assembly.Subsection “The genome of Moesziomyces albugensis”. Even though "tempting", this speculation of the authors seems very far-fetched, considering that there are many unrelated groups of the Ustilaginales that cause tumors or not.Subsection “The genome of Moesziomyces albugensis”. This part is interesting, but needs to be backed up by RNASeq and metabolic profiling.Subsection “The genome of Moesziomyces albugensis”. If the authors reanalyzed that genome, they could probably see that most of the "effector losses" are the result of bad gene calls.Subsection “The genome of Moesziomyces albugensis”. I wonder why the authors do not cite or discuss their own findings on several Moesziomyces species previously characterized as non-pathogenic. Ökmen et al., had found years ago that these yeasts possess functional copies of PEP1.Subsection “The genome of Moesziomyces albugensis”. The interpretation here cannot be upheld. Given current data, it seems to be a specialty of Ustilago madis that the clusters are expanded . In addition, the "loss" or presence of the clusters is likely a simple function of phylogenetic distance. Judging from the unfortunately not well resolved phylogeny in , Kalmanozyma could simply be most unrelated to Ustilago maydis.Subsection “The genome of M. albugensis”. This is the first part of the manuscript that seems solid, even though I wonder why non infection trials on the natural host of Moesziomyces bullatus were done.Subsection “Genetic characterization of Subsection “Identification of microbe-microbe effector genes by RNA-Seq”. While this cell biology part is again rather solid, the conclusions seem much too strong. For stating that the secretion of GH25 is important, the authors would need to construct a non-secreted version under a native promotor. In addition, the metabolic profile would need to be analysed, both of the secreted metabolites and those retained in the cells. It is conceivable that the GH25 enzyme processes a non-effective precursor to generate the observed effect.As many results obtained are already discussed in the Results section, the Discussion section is sometimes repetitive. I encourage the authors to keep the Results section free from interpretation and to do this in the Discussion section. As many incorrect claims are made again in the Discussion, I do not comment on them again.Subsection “Bioinformatics and computational data analysis”. The information regarding the genome assembly and gene prediction is at best minimal. This needs to much extended, also including the necessary quality checks as indicated earlier.Figure 1. This is hardly useful to present the "phylogenetic position" of the strain. There are much more elaborate phylogenies and the authors should simply refer to them. Also, the separation into "apathogenic" and "pathogenic" strains is pointless, as they confirm what was previously known. Likely there are no apathogenic members of the Ustilaginales and the yeasts have just by chance been isolated first from the environment before the pathogenic stage was discovered.Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a secreted lysozyme" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Caroline Gutjahr as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Christian Hardtke as the Senior Editor.Thank you for submitting your article "A fungal member of the The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below only address clarity and presentation, except if you prefer yourself to add data regarding request No. 3.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:Moesziomyces bullatus ex Albugo (MbA), which represents a hub organism in the Arabidopsis phyllosphere microbiome, as a leaf epiphytic model fungus, by providing a sequenced genome, the transcriptome in response to interaction with a phyllosphere bacterial SynCom and the oomycete A. laibachii and by establishing a transformation system for generation of k. o. strains by homologous recombination.Eitzen et al., investigate a specific mechanism of antagonism by an epiphytic yeast on an obligate biotrophic oomycete pathogen. They established the yeast A. laibachii, via a putatively secreted GH25 hydrolase with lysozyme activity. This finding, and other findings that may be expected from this research line in the future, do not only add to a molecular understanding of microbial interactions on the leaf surface but may in the future yield biocontrol fungi to combat agriculturally relevant oomycete infections of crop leaves.They report that M. bullatus ex Albugo antagonizes the oomycete pathogen Essential revisions:eLife:Based on the major comments of all three reviewers and a discussion, we request to address the following points before the manuscript can be accepted by 1) The reviewers found the phylogenetic tree in Figure 1 misleading . Since a more comprehensive and correct tree is published in Kruse et al., Figure 1 should be removed from the manuscript.Albugo laibachii? This question or hypotheses should be raised and discussed in the Discussion section.2) The GH25 hydrolase exhibits lysozyme activity, which hydrolyzes β‐ linkages between the NAM and NAG saccharides. These glucans are usually found on bacterial cell walls. However, to the reviewer's knowledge, the cell wall of oomycetes is composed of β-1,3, and β-1,6 glucans. How does GH25 hydrolase inhibit the oomycete 3) The Title states that GH25 is a secreted enzyme. Unless the authors include an experiment showing secretion, statements on secretion should be toned down in the title and across the manuscript where applicable. . [Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]Reviewer #1:Moesziomyces albugensis as a new "model fungus" at the leaf surface, which may act as a parasite on the pathogenic oomycete of Arabidopsis Albugo laibachii. They provide a sequenced genome, the transcriptome in response to interaction with the plant, phyllosphere bacteria and the oomycete A. laibachii, a transformation system for generation of k. o. strains by homologous recombination and characterize the mating type as well as filament and appressoria forming behavior in vitro.The authors establish M. albugensis antagonizes the oomycete pathogen A. laibachii, probably via a secreted putative GH25 hydrolase. This finding and other findings, that may be expected from this research line in the future, may yield biocontrol fungi to combat agriculturally relevant oomycete infections of crop leaves.In addition, they also report some interesting biology and show that A. laibachii is very interesting, the manuscript still suffers from a few unclarities.While M.albugensis is an exciting new fungus and the identification of the effector required for parasitism of M. albugensis lives only epiphytically or can also colonize the plant tissue (it forms appressoria on hydrophobic surfaces) or whether it is exclusively associated with A. laibachii. This should be clarified.– From the text I did not understand whether M. albugensis belongs to a group of basidiomycete yeasts that has been found to lead epiphytic existence on the leaf surface. However, since the recombinant, self-compatible strain (CB1) which we generated in this study was found to form appressoria on hydrophobic surface, this hints towards the idea that in nature, the anamorphic epiphytic yeasts might still own the potential to form filamentous structure in presence of a correct mating partner. However, this remains rather speculative because the general question of how the ustilaginean yeasts actually relate to pathogenic strains remains to be solved. We added an additional clarification in subsection “Genetic characterization of MbA”.We apologize for being unclear in this point. – It is unclear how the bacterial SynCom was constructed and which isolates it contains.A. laibachii Nc14 isolate and have added those to cover more phylogenetic diversity.We apologize for not having included the Syn Com isolates list. We have added this to the supplementary now and have explained how the syncom was assembled: For this SynCom we had isolated bacteria from different sites close to Tuebingen in Germany and have sequenced partial 16S to identify taxonomy. Based on these isolates and how often we have found the bacteria on different sites , we assembled a community with maximum phylogenetic diversity. We have further isolated bacteria from spores of our unsterile The detailed information is now available in the new Supplementary file 1.– The quantification of A. laibachii infection seems kind of rough and prone to subjectivity. An additional, more quantitative method should be used for quantification, for example by qPCR using an amplicon from A. laibachii DNA normalized to a plant amplicon.A. thaliana seedling. This method of counting pustules on leaves has been intensely used for evaluating rust fungal infections. As leaves of sterile Arabidopsis plants are small, there are no more than one to two pustules per leaf. Using this range would not give enough resolution to see significant differences and therefore we decided to analyze significant more plants and leaves and focus on presence and absence. For statistical analysis, infection percentage of each seedling is subjected to analysis of variance (ANOVA) for pairwise comparison of the conditions, with Tukey’s HSD test to determine significant differences among them . We performed the experiments in six independent biological replicates with 12 technical replicates and observed significant difference between the Albugo treated seedlings and Albugo + Moeziomyces treated ones. Hence, we are convinced that the data shown in our study is robust and statistically significant.We thank this reviewer for pointing this out. Our quantification method is based on the percentage of infected leaves vs non-infected leaves in each individual Albugo biomass in A. thaliana in our new experiments where samples were treated with GH25 hydrolase and Mutant_GH25, respectively . These experiments provide direct evidence for the biological activity of the GH25 hydrolase: Albugo biomass was reduced to about 50% in GH25 treatments compared to Mutant GH25 or untreated Albugo-infections. This new data is now included in the Results section and the Materials and methods section.However, based on the suggestion of this reviewer, we performed qPCR-based relative quantification of the A. laibachii or whether it helps M. albugensis to parasitize A. laibachii by other means. The authors should examine the effect of isolated GH25 hydrolase on A. laibachii infection.– It is unclear whether GH25 hydrolase suppresses infection of plant leaves by Albugo.This is a very good question, and we think it is answered by the new data which is already mentioned in the point above: the plant infection experiments with the recombinant GH25 hydrolase show that it directly interferes with Albugo infection . Based on this finding, our next question for upcoming work will be to elaborate the molecular mechanism by which the enzyme inhibits A. laibachii blocked by GH25 hydrolase or M. albugensis?– At which stage of infection is A. thaliana leaves where colonization of Albugo is visualized by Trypan blue staining . This shows that in presence of MbA, there is little to no colonization of Albugo in the leaf surface and only short hyphae are formed. We have also provided more information in the Results.We observed a slight but not significant decrease in zoosporangial germination efficiency on treatment with the GH25 hydrolase  but found that plant colonization of Albugo is significantly reduced. This is also in line with the microscopic of Reviewer #2:Arabidopsis phyllosphere classified as Moesziomyces albugensis (Ma). The report provides phylogenetic classification, genome analysis and functional analysis of Ma inhibitory activity towards the white rust oomycete Albugo laibachii (Al). Understanding functional interaction in the plant microbiota is a field of intense research with a great fundamental interest, as well as promising applied perspectives regarding the identification of novel biocontrol agents such as potentially Ma. I found the manuscript very pleasant to read and well structured. The work appeared rigorous and conclusions are appropriately supported by the available data. A number of biocontrol yeasts have been isolated in the past, but in my opinion this study stands out by providing direct genetic evidence of the anti-rust activity of Ma, through the identification of one underlying gene (encoding a putative GH25 enzyme) and the characterization of the corresponding mutant phenotype. The Ma genome sequence and gene expression data also provide an interesting resource for further analysis of the biology of this phyllosphere fungus.The study by Eitzen et al., characterizes a yeast species isolated from 1) I would have liked to see the activity of the GH25 characterized further. I am lacking a clear hint towards GH25 molecular activity on Al and possibly other members of the phyllosphere.in Pichia pastoris, purified the proteins by Ni-NTA affinity chromatography and performed lysozyme activity assays using the recombinant proteins. This showed that the GH25 has a lysozyme activity, which is abolished by a single amino acid exchange in the predicted active site. This data is now included in the manuscript  and is described in the Results and the Materials and methods section.To address this point, we expressed the GH25 hydrolase (and an enzymatic inactive version) Does ectopic (eg in plants) expression of GH25 has inhibitory effect on Albugo?Albugo, we used the recombinant protein. As mentioned above, we did not observe a significant effect on Albugo zoospore formation . On the other hand, we found that treatment of Albugo-infected Arabidopsis with the active (but not with the inactive) GH25 significantly reduced Albugo growth . This provides direct evidence showing that the GH25 lysozyme is actually inhibiting infection of Arabidopsis by Albugo.To address the question whether the GH25 lysozyme has a direct effect on Is there any indication on which species would this enzyme act? Is it acting specifically on Albugo?Moesziomyces – Albugo interaction on Arabidopsis. However, as also suggested by reviewer 1, we believe that Moesziomyces and the GH25 could have a potential used for biocontrol in economically more relevant systems. We will therefore test its impact on Phytophthora infestans in future experiments.In this study we were focusing on the What protects Ma from its own secreted hydrolase?Moesziomyces.The most likely explanation is that the fungal cell wall does not contain a substrate of the GH25 lysozyme. We are not aware of any other protection mechanism in How conserved in GH25 across fungi? Is there anything specific about its sequence (and predicted activity) as compared to other GH25s? to other glycosyde hydrolases?g2490 is predicted has a lysozyme activity, which is essentially an enzyme that cleaves peptidoglycan in bacterial cell walls by catalyzing the hydrolysis of β‐ linkages between the NAM and NAG saccharides The active site of the GH25 hydrolase is found to contain a DXE motif which is found to be conserved between a wide range of fungi, ranging from other basidiomycetes, ascomycetes and even Chytrids . We have additionally attached the raw file for PRALINE multiple sequence analysis for conserved sites.The GH25 hydrolase encoded by gene To what extent is GH25 activity beneficial to Ma?This is an interesting question, which we cannot answer based on experimental evidence so far. What we show is that the GH25 is required and responsible for the antagonism towards Albugo. One now could hypothesize that this antagonism itself is beneficial for Moesziomyces, i.e. for interspecific competition in niche differentiation. A final answer for this question, however, will be subject of future research.Could the growth of this fungus be measured on Albugo-infected vs Albugo-free plants? in vitro with or without Albugo?Moesziomyces as a member of the leaf phyllosphere, particularly in presence of Albugo. Regarding “in-vitro” growth, this is experimentally difficult, since Albugo is an obligate biotroph which cannot be propagated in axenic culture.This is an interesting thought, which will definitely be part of future analysis on the ecological role of the GH25 for 2) The description of statistical analyses needs attention. I particular, I could not find information on how gene expression and differentially expressed genes were determined.How was gene expression level determined? How were differentially expressed genes identified? Besides, some information on RNAseq QC and bias correction is needed.Moesziomyces and Arabidopsis genome with Tophat2 and a count table was generated for Moesziomyces counts with HT-seq count. DE-genes were determined with using the "limma"-package in R on "voom"-transformed count data and sample-specific weighting to reduce variance between samples . Counts lower than 10 in average have been extracted. Differentially expressed genes were counted with a threshold of False discovery rate of 0.05 and log2FC > 0. We have the added additional information, in Results section.We have added additional, missing. Each file was checked for quality control of RNA-Seq reads prior to analysis with FastQC. Reads were mapped to the Reviewer #3:Albugo laibachii.The manuscript deals with the characterization of a phyllosphere yeast that is supposed to have an antagonistic effect on While the manuscript is well written, it suffers from numerous cases of overinterpretation of the results and methodological flaws, as outlined below.Moesziomyces albugensis". First, there is not even a remotely valid description of the "species", and second, it is quite obvious that after checking some of the recent literature on Moesziomyces (part of which two of the authors even co-authored) that the isolate they are investigating is clearly Moeziomyces bullatus. This needs to be changed throughout the manuscript.Abstract. It is beyond reason that the authors call their yeast isolate "Moesziomyces bullatus. Nevertheless, it needs to be specified that the characterized strain is not a pathogenic strain of Moesziomyces bullatus, which is a pathogen of millet. Despite multiple attempts, we were neither able to mate the strain with any isolate of an available collection of pathogenic Moesziomyces bullatus, nor have we been able to infect a potential host plant. We have been trying to infect millet plants also with the self-compatible CB1 strain, which is able to form appressoria. However, also this strain did not show invasive growth in any of our experiments (data not shown). Therefore, to specify the isolate that is characterized in this study, we describe it as Moesziomyces bullatus ex Albugo on Arabidopsis (MbA for simplicity) throughout the manuscript. This classifies the strain as a member of the species Moesziomyces bullatus and at the same time it reflects that it has actually been isolated from Albugo on Arabidopsis plants and not as a pathogen of millet.We accept the criticism by the reviewer. We therefore describe the characterized isolate as Abstract. That the isolates "prevents" Albugo infections is insufficiently shown (but see later criticism on this point).Albugo infection.We realized that “prevent” is not an appropriate term here and rephrased it to “reduces infection”, which underlines that we are observing a quantitative effect and not a complete prevention of Abstract. The evidence for the claim that GH25 is a major effector of the antagonism is weak, at best.We accept this criticism, which is also shared by the other reviewers and have performed a series of additional experiments which significantly strengthens this part of the study and provides direct evidence for GH25 being an effector in the observed antagonism. As described above, we produced recombinant protein for the GH25 hydrolase and showed its enzymatic activity. Most importantly, we show that enzymatically active GH25 significantly reduces infection of Arabidopsis by Albugo, which we quantified using qPCR. The additional evidence is shown in the new Figure 8 and in the new Figure 1—figure supplement 2 and Figure 7—figure supplement 2. Detailed descriptions are provided in Materials and methods section and Results of the revised manuscript.Introduction. This statement is not correct. there are several studies, e.g. by the Macia-Vicente group that have shown that the local environment drives the diversity of root-associated microbiomes.We agree and have modified the sentence accordingly.Moesziomyces bullatus.Introduction. the genus Pseudozyma is obsolete. This should be termed Moesziomyces. And the species is known as well, it is Pseudozyma to introduce the anamorphic yeasts in the manuscript and report biocontrol activity of those group yeasts which have been commonly termed “Pseudozyma” in a substantial body of literature. In accordance to the corrected phylogeny, we mentioned the transfer of the genus Pseudozyma to Moesziomyces soon after in the Introduction.We have mentioned the term Introduction. I was surprised about the statement and have the read Kurse et al., manuscript. In fact, the authors report the opposite, an infrequent sexual reproduction on grass hosts as the most probable explanation for the retainment of a sexual reproduction.We modified the sentence to avoid confusion (Introduction).Moesziomyces and that Pseudozyma aphidis is conspecific with Moesziomyces bullatus.Introduction. I really wonder why the authors cite this less comprehensive phylogeny, while two of them have shown, based on a broader sampling that in fact the species mentioned are embedded within The corrected phylogeny has now been incorporated.Introduction. The transfer of these species into Pseudozyma had already been done, thus, "can be renamed" is not a correct statement.We have modified the sentence.Moesziomyces bullatus or "A strain formerly classified as Pseudozyma aphidis (now Moesziomyces bullatus)".Introduction. This should read We have changed this as described above.Moesziomyces albuginensis. And this also would not make phylogenetic or evolutionary sense, as the Moesziomyces bullatus strain isolated from Arabidopsis thaliana is firmly embedded within Moesziomyces bullatus and there is no evidence that is should be considered as a biologically distinct species. Also, the evidence of disease "prevention" are at most anecdotal (see later criticism).Introduction. There is no such species name as We have addressed this point as described above.Results. This statement alone refutes the previous statement in the Introduction. If the species was isolated from and found to be closely associated with the spores of Albugo, even over years of continuous cultivation cycles, how can the authors confidently claim that the species prevents infection?Albugo-infected A. thaliana leaf surface. Previous study  identified this strain of Moesziomyces as being a hub microbe in the A. thaliana phyllosphere. In this study we provide direct, quantitative evidence that it antagonizes the infection of Arabidopsis by the biotrophic oomycete pathogen Albugo laibachii via the GH25 hydrolase. However, we do not want to claim that Moesziomyces sp. in general has such a function , and therefore we specified the findings to the individual isolate which we have studied in detail in this study.The fungal isolate used in this study in particular has been found associated with Introduction. There are many strains of Moesziomyces bullatus (as Pseudozyma aphidis) that are marked as biocontrol agent. The new strain would just be an addition to that, there is little novelty in this.Pseudozyma sp., which is also addressed in the introduction part of our manuscript. However, we are convinced that our study is not a mere description of an organism with biocontrol activity. An important aspect is on the multitrophic interactions in a phyllosphere microbial community, where MbA appears to be regulating the presence of several other microbes in the network. Another novelty of our approach lies in the fact that we haven’t externally introduced an organism to combat pathogenicity; since the yeast is found as a member of the microbial community in the natural environment. Most importantly, our RNAseq analysis revealed the transcriptional activation of secretory enzymes in MbA when being co-cultivated with Albugo on the plant leaf, and this approach identified the GH25 lysozyme as a key determinant for the observed microbial antagonism. To the best of our knowledge, this study shows for the first direct and functional evidence on the genetic and protein level in a fungal – oomycete antagonism on/in-planta. Thus, the novelty of this study is o on the (i) community aspect and (ii) the mechanistic evidence of the microbial antagonism, rather than in the identification of a new fungal isolate.We agree with the reviewer that there are several reports on biocontrol activity by Results. This argumentation is impossible to follow. Just because something is isolated from a special location does not make it a new species.This has been corrected as described above.Results. I really cannot see the reason for the authors claiming again the identification was just "Pseudozyma sp." while they could show in a previous manuscript  that the species is Meosziomyces bullatus.This has been corrected as described above.Ustilago maydis is also difficult to justify, as that species is largely unrelated to Moesziomyces bullatus. Instead, other species of the genus Moesziomyces, especially additional strains of Moesziomyces bullatus should have been used.Results. This interaction does not need to be specific. The test against Ustilago maydis and the MbA strain, which acts as a microbial hub in the A. thaliana phyllosphere. As suggested by this reviewer, it would be interesting to test other strains of Moesziomyces bullatus against the bacterial SynCom and Albugo to test if there is specific adaptation towards microbial interactions in different isolates of the same species. These lines will surely be followed by our future research in this context, where we want to better explore the ecology Ustilaginales yeasts in different environments.The intention of this experiment was to observe if there is any difference between the well-characterized pathogenic smut Results. While in general, it is interesting that the yeast showed an antogonistic effect, dose dependency should have been checked. Several species of the Ustulagoinales are known to produce biosurfactants that alter the properties of the plant cuticle. Thus, the effect observed can probably already explained by this. This could be addressed by additional experiments with a variety of Ustilago and Moesziomyces strains and demonstrating that a lower dose of the Albugo-associated strain inhibits the infection.A. laibachii .We agree that Ustilaginales are known to produce biosurfactants and we would not like to exclude that these secondary metabolites have significant impact on the microbial interactions on the leaf surface. The focus and aim of this study, however, was to investigate possible functions of secreted proteins as potential microbe-microbe effectors. Thus, we decided to investigate the function of the GH25 in more detail and performed the characterization of the recombinant protein, showing that the active GH25 lysozyme is significantly inhibiting infection of Arabidopsis by Subsection “The genome of Moesziomyces albugensis”. This lacks a lot of the usual quality control, such as genome completeness statistics, read mapping specifics, testing of the gene predictions by RNA mapping.Moesziomyces and Arabidopsis genome with Tophat2 and a count table was generated for Moesziomyces counts with HT-seq count. DE-genes were determined with using the "limma"-package in R on "voom"-transformed count data and sample-specific weighting to reduce variance between samples . We have added additional information, in the Results section.We thank the reviewer for pointing out this lack of information on the RNA seq experiment. Each file was checked for quality control of RNA-Seq reads prior to analysis with FastQC. Reads were mapped to the Subsection “The genome of Moesziomyces albugensis”. Evidence should be presented that this is not the result of a mis-assembly.MbA and Moesziomyces antarctica T-34 and confirms proper assembly..We have added the new Figure 2—figure supplement 1, which shows genomic comparison of Subsection “The genome of Moesziomyces albugensis”. Even though "tempting", this speculation of the authors seems very far-fetched, considering that there are many unrelated groups of the Ustilaginales that cause tumors or not.We agree that this statement is very speculative and therefore deleted the sentence.Subsection “The genome of Moesziomyces albugensis”. This part is interesting, but needs to be backed up by RNASeq and metabolic profiling.MbA against the SynCom bacterial members on plate .Our analysis provided no evidence on upregulation of the secondary metabolite clusters in the RNA seq experiments, hence metabolic profiling was not performed further. Thus, we only speculate on them to be involved in antibacterial activity based on interaction of Subsection “The genome of Moesziomyces albugensis”. If the authors reanalyzed that genome, they could probably see that most of the "effector losses" are the result of bad gene calls.P. flocculosa genome with respect to gene loss. To avoid misunderstanding, we modified the sentence to make it clearer that we only refer to the statement by Lefebvre et al., (2013), which is apparently not in line with our observation made for MbA.We have not analyzed the Subsection “The genome of Moesziomyces albugensis”. I wonder why the authors do not cite or discuss their own findings on several Moesziomyces species previously characterized as non-pathogenic. Ökmen et al., had found years ago that these yeasts possess functional copies of PEP1.MbA”) “Moesziomyces sp., possess functional homologues of the pep1 gene, a core virulence effector of U. maydis , suggesting that such anamorphic yeasts have the potential to form infectious filamentous structures by means of sexual reproduction .”We have added the following in . In addition, the "loss" or presence of the clusters is likely a simple function of phylogenetic distance. Judging from the unfortunately not well resolved phylogeny in , Kalmanozyma could simply be most unrelated to Kalmanozyma brasiliensis.We agree and have removed the example of Subsection “Genetic characterization of M. albugensis”. This is the first part of the manuscript that seems solid, even though I wonder why non infection trials on the natural host of Moesziomyces bullatus were done.Setaria sp. and Echinochloa crus-galli for susceptibility towards transgenic self-compatible (CB1) strain of Moesziomyces sp. However, we could not observe any penetration event in the plant surface, even though the organism was seen to be forming filamentous structure. The same was true for the self-compatible CB1 strain. Thus, we cannot make any statement on a potential role of the MbA isolate as a plant pathogen in our paper. On the one hand we were not able to infect a potential host, but on the other hand we also cannot exclude that under certain conditions it could be infectious.We appreciate this suggestion by this reviewer. Actually, we tested two different cultivars of millet, Subsection “Identification of microbe-microbe effector genes by RNA-Seq”. While this cell biology part is again rather solid, the conclusions seem much too strong. For stating that the secretion of GH25 is important, the authors would need to construct a non-secreted version under a native promotor. In addition, the metabolic profile would need to be analysed, both of the secreted metabolites and those retained in the cells. It is conceivable that the GH25 enzyme processes a non-effective precursor to generate the observed effect.We agree that the statement was quite bolt in the previous version of the manuscript. However, since the experiments with the recombinant GH25 lysozyme  confirmed its role in the interaction, we decided to stay with this phrase, which is now justified in our opinion.As many results obtained are already discussed in the Results section, the Discussion section is sometimes repetitive. I encourage the authors to keep the Results section free from interpretation and to do this in the Discussion section. As many incorrect claims are made again in the Discussion, I do not comment on them again.We thank the reviewer for pointing this out. As suggested, we have omitted certain sentences in the Results section to avoid being repetitive.Subsection “Bioinformatics and computational data analysis”. The information regarding the genome assembly and gene prediction is at best minimal. This needs to much extended, also including the necessary quality checks as indicated earlier.Again, we agree with the reviewer and have extended this part in the Results section.Figure 1. This is hardly useful to present the "phylogenetic position" of the strain. There are much more elaborate phylogenies and the authors should simply refer to them. Also, the separation into "apathogenic" and "pathogenic" strains is pointless, as they confirm what was previously known. Likely there are no apathogenic members of the Ustilaginales and the yeasts have just by chance been isolated first from the environment before the pathogenic stage was discovered.We discussed this critically and, although we agree with this reviewer that there are more extensive phylogenies published, we still believe that the figure is helpful for the reader as an introduction. However, we agree with this reviewer that the term “apathogenic” is not helpful and therefore this has been changed in the revised figure legend.[Editors’ note: what follows is the authors’ response to the second round of review.]Essential revisions:Based on the major comments of all three reviewers and a discussion, we request to address the following points before the manuscript can be accepted by eLife:1) The reviewers found the phylogenetic tree in Figure 1 misleading . Since a more comprehensive and correct tree is published in Kruse et al., Figure 1 should be removed from the manuscript.As suggested, we have removed Figure 1 from the manuscript. We felt it was helpful as an introduction to the topic, but we agree that the information is not necessarily required in this manuscript.2) The GH25 hydrolase exhibits lysozyme activity, which hydrolyzes β‐ linkages between the NAM and NAG saccharides. These glucans are usually found on bacterial cell walls. However, to the reviewer's knowledge, the cell wall of oomycetes is composed of β-1,3, and β-1,6 glucans. How does GH25 hydrolase inhibit the oomycete Albugo laibachii? This question or hypotheses should be raised and discussed in the Discussion section.A. laibachii infection is surprising (we were surprised by this finding as well). The GH25 enzymes are widely conserved in the fungal kingdom and have been associated with both fungal hyperparasitism, as well as oomycete-oomycete parasitism in Phytium .We fully agree that the observed effect of the GH25 on With regard of a direct effect on the Albugo cell wall, one should mention that there is quite sparse knowledge on the actual cell wall composition and we would actually not exclude that it contains a substrate of the GH25. Although the MbA GH25 obviously has lysozyme activity, a comprehensive biochemical characterization of the fungal GH25 family is missing. Thus, it might very well be that these enzymes have unexpected/unknown substrates in the oomycete cell wall. Next, it is unknown which consequences an eventual hydrolysis of an Albugo cell wall compound might have . In the revised manuscript we now include a discussion on potential functions of GH25 to give the reader an idea on how it might act on the molecular level.A. laibachii might be an indirect one: since oomycetes can be found in association with bacteria (which is a largely unstudied/neglected topic) one could even speculate that the GH25 actually targets Albugo-associated bacteria, which might be necessary for virulence of the oomycete. While such a tempting scenario would be also in line with findings of endosymbiotic bacteria in mycorrhizal fungi (work by P. Bonfante and others), at present this is purely speculative and unless there no evidence pointing towards this direction, we’d prefer to not discuss this aspect in the present manuscript.Lastly, one cannot rule out that the effect on 3) The Title states that GH25 is a secreted enzyme. Unless the authors include an experiment showing secretion, statements on secretion should be toned down in the title and across the manuscript where applicable. .We accept this criticism and removed the word “secreted” from the Title, which now reads “…via a GH25 lysozyme”.


See relatedEditorialSeecover image Spirometry has been established as an essential test for diagnosing and monitoring respiratory disease, particularly asthma and COPD, as well as in occupational health surveillance. In Australia and New Zealand, there is currently no pathway for spirometry operators in community‐based healthcare settings to demonstrate spirometry competence. The Australia and New Zealand Society of Respiratory Science (ANZSRS) has identified a need for developing a pathway for operators working in community‐based practices in Australia and New Zealand to demonstrate spirometry competence and certification. Spirometry certification provides evidence to patients, clients, employers and organizations that an individual has participated in an assessment process that qualifies them to perform spirometry to current international spirometry standards set out by the American Thoracic Society and the European Respiratory Society (ATS/ERS). This document describes a competence assessment pathway that incorporates a portfolio and practical assessment. The completion of this pathway and the award of certification confer an individual is competent to perform spirometry for 3 years, after which re‐certification is required. The adoption of this competency assessment and certification process by specialist organizations, and the commitment of operators performing spirometry to undergo this process, will enhance spirometry quality and practice in community‐based healthcare settings. ANZSRSAustralian and New Zealand Society of Respiratory ScienceARTPAssociation for Respiratory Technology and PhysiologyATS/ERSAmerican Thoracic Society/European Respiratory SocietyCBSCCommunity‐Based Spirometry CompetencyCOPDchronic obstructive pulmonary diseaseQIPQuality Innovation PerformanceIntroductionThe ANZSRS Community‐Based Spirometry Competency working groupThe value of certificationExisting models of competence assessmentPathway for certification in Australia and New Zealand Stage 1: Spirometry training and assessment Stage 2: Work experience and skill development Stage 3: Portfolio assessment Stage 4: Practical assessmentApplying for certification Initial application Re‐certification Resources and supporting documents Key performance indicatorsDiscussionConclusionReferencesIn 2017, the Australian and New Zealand Society of Respiratory Science (ANZSRS) published a position statement on the minimum requirements for spirometry training courses.The current paper is a supplement to the 2017 position statement and presents a process for assessing competency and awarding certification to trained operators performing diagnostic spirometry and occupational health screening spirometry in community‐based health settings. These may include, but are not limited to, the following: community and primary healthcare services, general practice, private practice specialist services, occupational health services, non‐tertiary hospitals, research facilities, pathology services, outreach services, allied health services and pharmacies.Spirometry is performed in community‐based health services across the world. Despite its wide use, the quality of the results obtained in this setting does not always meet international practice standards.The ANZSRS board recognized the need to improve the standard of spirometry in community‐based healthcare settings. Members of the special interest group, the ANZSRS Community‐Based Spirometry Competency (CBSC) working group, were selected by the board of the ANZSRS through an Expression of Interest process. All members of the CBSC working group recognized the importance of improving spirometry competence in community‐based healthcare settings and had extensive experience in developing, coordinating and facilitating spirometry training programmes in Australia and New Zealand. Several members were authors of the ANZSRS Spirometry Training position paper.To address the needs of spirometry operators, physicians and the community, the CBSC working group has developed a model for assessing competence of spirometry operators in community‐based settings. The model has been derived following a review of current international and national processes for competence assessment in spirometry and other disciplines. The assessment tools presented in Supplementary Information include tools developed and used by the authors in various spirometry training programmes that align with the 2017 ANZSRS Spirometry Training position paper.The aim of this position paper is to provide a model for assessing competence in spirometry. It also provides potential assessment tools for organizations that have the capability to implement and administer spirometry competency assessments in the future.The working group agreed that the model for assessing competence would result in operator certification, which is dependent on the demonstration of spirometry competencies. For certification to be awarded, the candidate would demonstrate the application of spirometry knowledge and skills independent of the training environment. At the time of preparing this paper, there were no legal requirements for operators to be certified to perform spirometry in Australia and New Zealand.Certification is a method used to provide confidence that an individual possesses the skills and knowledge to perform their job to current practice standards.Staphylococcus aureus (MRSA) infection when compared to staff who are not certified for the task.Education and training set a minimum standard of practice, whereas certification recognizes an individual's knowledge and expertise in a field. For example, in Californian hospitals where leaders are certified in infection prevention and control, there are lower rates rate of methicillin‐resistant Limited models for assessing spirometry competency in community‐based services exist. Many of the elements of these programmes were reviewed by the working group for broad principles that could be adopted. In the UK, the Association for Respiratory Technology and Physiology (ARTP) certifies operators for 3 years at one of the three different levels: performing, performing and interpreting or only interpreting spirometry tests.There is a sparsity of literature regarding the uptake of formal competency recognition and its impact on spirometry quality. The National Health Service (NHS) in the UK, in collaboration with the ARTP have introduced a national health register for spirometry performance and interpretation.The responsibility for awarding spirometry certification in recognition of competence is the role of specialist organizations, referred to as the ‘certifiers’ in the paper. The certifier will have the resources to receive applications for certification, receive uploaded assessment evidence and outcomes, maintain a database of certified operators and qualified assessors, field enquiries, provide support to operators and assessors and send reminders for re‐certification.The proposed model for certifying operators is outlined in Figure The model is flexible and fair in that evidence can be collected through a variety of pathways. Assessment tools, used by the authors in existing spirometry training programmes, have been reviewed by the working party for use in this pathway. Although these tools have not been formally evaluated, the working party is confident that they are valid and reliable, by virtue of their current use to assess participants completing established, and in some instances QIP accredited, spirometry training programmes in Australia and New Zealand. Extensive discussion by working group members has resulted in the development of assessment tool templates recommended for use by the certifier to adapt as required. The assessment tools; information for certifiers, candidates and assessors; and further information about the competency framework are provided in Supplementary Information.Candidates for certification will be assessed against specified spirometry competency elements and performance criteria by independent assessors as outlined in Candidates are advised to complete a spirometry training programme that includes the mandatory components as outlined in the ANZSRS spirometry training position statement.et al.Workplace experience builds the candidate's confidence and experience in performing quality spirometry. It is recommended that candidates undergo a period of work‐based practice after completion of a spirometry training programme before applying for certification. Candidates will be advised to maintain a logbook of spirometry tests performed in the workplace which would be validated by a workplace supervisor or mentor and provide evidence of ongoing spirometry practice. The supervisor need not be experienced in spirometry but should be able to confirm the number of spirometry tests performed by the candidate. There is no evidence to support a defined minimum number of tests that operators must perform to attain and maintain competence, although a positive correlation between acceptability and frequency of tests performed by an operator has been shown in one study by Bellia Candidates will be required to provide a portfolio of a minimum of 10 client tests performed in the workplace, including an analysis of test quality, interpretation and quality control measures consistent with the current ATS/ERS standards and guidelines  and a logbook of the number of spirometry tests performed in the workplace.Portfolio assessment completion (if part of a recently completed spirometry training) or portfolio of test results (if not completed as part of spirometry training).Practical assessment completion.The candidate will apply for certification with the certifier. Detailed instructions and description of the evidence required by the certifier will be made available to the candidate. The evidence required to recognize spirometry competence will include:The evidence will be submitted to the certifier by the candidate. If the evidence provided is sufficient to recognize the candidate as competent to perform spirometry to current standards,Spirometry operators will need to be re‐certified every three years. The evidence required to show maintenance of competence may include a spirometry portfolio, record of frequency of practice, spirometry refresher training certificate, spirometry auditing or other evidence deemed appropriate by the certifier.Examples of resources for use by the certifier, the candidate and assessors are found in The ATS and ERS provide clear guidelines and recommendations for grading spirometry test quality.The working group encountered several obstacles during the development of the certification model. First, there is limited evidence to recommend a model for spirometry competency assessment and certification in Australia and New Zealand. To this end, the proposed pathway is a result of extensive discussions and consensus by the working group, who have recognized expertise in spirometry practice, training and assessment in Australia and New Zealand. Second, the proposed model is not yet supported by a specialist organization, that is the certifier, so solutions to implementation problems, arising during working group discussions, were purely hypothetical. Consequently, the working group felt that a flexible pathway may better align with potential certifiers' existing resources and infrastructure. Third, resources used in established spirometry training programmes were collated to produce the assessment tools, and the working party deemed the developed tools were sufficiently robust to assess competence. However, a formal evaluation of the tools will provide confidence to all stakeholders that the certification model is able to deliver its intended objectives. Lastly, although all spirometry operators will have access to the certification pathway, it is a voluntary process not anticipated to be mandated in the near future.This document outlines a model for certification of operators performing spirometry in community‐based practices in Australia and New Zealand. The model allows operators to demonstrate skills and knowledge previously gained through training and workplace practice. Certification will provide confidence to employers, patients and clients that an operator is competent to perform spirometry to current international practice standards. Currently, few programmes exist against which to benchmark the proposed pathway to achieving spirometry competence, and this is a limitation. Future work will include developing key performance indicators against which to measure the success of the pathway to certification and to inform future improvements.The authors  are facilitators and assessors in spirometry training programmes that provide a service for fees or through government funding. Income generated from fees covers the costs incurred in training and assessing course participants and related to that training only. The authors or the organizations they work for have the potential to earn income as assessors on a fee for service basis should the certification process be established.AppendixS1. Definition of terms and abbreviations.Appendix S2. Overview of certification framework.Appendix S3. Standards for performing spirometry.Appendix S4. Information for candidates.Appendix S5. Information for assessors.Appendix S6. Assessment requirements and performance evidence.Appendix S7. Spirometry competency elements and performance criteria.Appendix S8. Competency elements: portfolio and practical assessments.Appendix S9. Spirometry portfolio resources.Appendix S10. Spirometry practical resources.Appendix S11. References.Click here for additional data file.Visual Abstract Spirometry Certification: A pathway to improving practice in community‐based healthcare settings.Click here for additional data file.


Greater clarity on the nature of clinical utility is desirable. Of itself it may not bring about greater use of tests, including WGS (whole-genome sequencing), not least because clinical utility studies when performed may not confirm predicted changes in patient outcome. The notion that single “pivotal” clinical utility studies will achieve uptake needs to be questioned and that the evidence base for tests is likely to rely on patchworks of imperfect evidence embraced. It builds on existing frameworks, particularly that proposed by Fryback and Thornbury2 and is particularly useful in offering WGS-specific examples of clinical utility studies. The emphasis on comparison is welcome, and is an aspect of test evaluation which has been strangely lacking3. Clear demonstration of how a new test will lead to improvement relative to existing test strategies, must be the foundation of any decisions on whether a test is recommended for wide use, irrespective of whether the impact falls under the category of analytical or clinical validity or clinical utility.Evaluation of tests is difficult. I have seen just how difficult in the work I have done contributing to decision making on NICE’s Diagnostic Assessment Committee and the National Screening Committee in the UK over the last decade. “Clinical utility of genomic sequencing: a measurement toolkit”Clinical utility” is a misnomer; its intent is to reflect how a new test will impact on patient outcome and judgement on what constitutes this must be contributed to widely.The current perspective is wholly clinical. Utility in particular will vary with perspective, and account must be taken of this. Patients and carers have legitimate views as do policymakers and funders and the toolkit should reflect these and other stakeholder views as much as the medical community. “The toolkit largely focuses on what should be measured. Equal emphasis needs to be devoted to how, with more space devoted to study design and the relative strengths and weaknesses of different methods of data collection. The toolkit would benefit from greater scrutiny of its examples commenting on the role of chance, bias and confounding on the interpretation of the results. Accumulation of data in a way which does not maximise its validity wastes everyone’s time.5.The authors acknowledge their lack of health economic expertise, so advice on capturing the cost-effectiveness of WGS should be enhanced, particularly because achieving diagnosis more efficiently is often central to the WGS case. There is an absence of any mention of modelling which has a critical role in evidence linkage, both for the purposes of establishing effectiveness and cost-effectivenessEvaluators are likely to be bewildered by the range of outcomes they are being asked to evidence, so the advice ideally needs to assist with prioritisation or strategies to help build a portfolio of evidence which will convince funders and society that introduction of WGS does more good than harm (an issue which is barely mentioned in the current toolkit). As for test evaluation, generally, emphasising the key claims central to any value proposition and pre-eminent threats to safety should be the first concerns.6. This is particularly true for the task of summarising evidence from the disparate aspects clinical utility. However, to truly create a convincing portfolio of evidence, the ambition should be to incorporate key evidence relating to analytical and clinical validity too.Finally, advice is needed on the synthesis of evidence as much as its initial collection. The toolkit cites meta-analyses of evidence, which illustrate that improvements to secondary research can be madeAlthough the checklist does represent a useful contribution, it is one which needs to be built on further:7. So other issues need to be examined to see whether there are other important rate-limiting steps to the up-take of WGS. Funding, in my view, is the major constraint. While research funders have been quick to back investment in the technology they have been cautious in supporting its evaluation, which, as the toolkit highlights, is complex requiring dedicated and experienced evaluation teams. Related to this, we also have to consider whether geneticists advocating the technologies really believe they should have to go beyond analytical validity. As a colleague recently put it when we were discussing a potential trial to demonstrate clinical utility, “Why should we have to do more than other tests?” And we should acknowledge that there is some truth in this sentiment as historically new tests have had to do little more than demonstrate their laboratory performance. We also need to consider that clinical utility studies, when performed, are not always favourable, and that however persuasive the rationale, the hoped-for benefits are not realised in practice. The toolkit contains at least one example where the benefits were less than that might have been hoped for when they were examined rigorously8.Even if the clinical utility toolkit is optimised, will this prove sufficient in getting genetic tests into practice? This seems unlikely as ideas on the evidence requirements for tests in general, and genetic tests in particular have been available for some timeA last word of caution is that decisions on tests will probably always rely on patchworks of imperfect evidence. The notion, which underlies many evaluation frameworks that a “pivotal” clinical utility study will at some stage firmly establish the effectiveness of a test needs to be questioned. Making more of what we already have by better collating and synthesising evidence portfolios may be as fruitful as holding out for the mirage of the perfect study which will single-handedly tip the balance of getting a new WGS into practice. Targeted augmentation of a body of evidence to demonstrate specific missing aspects of a value proposition may be more valuable than pursuing a clinical utility study for its own sake.Further information on research design is available in the Reporting Summary


There is mounting interest in promoting - and evaluating efforts that improve healthy aging in age-friendly communities. Additionally, there is increasing recognition that multi-sectoral engagement beyond the aging network is needed to maximize impact and sustainability. Within the context of collective impact, this paper reviews a framework that explicates public health activities in collaboration across a range of stakeholders in age-friendly communities. Metrics demonstrating evidence of five categorical roles, processes and outcomes will be presented including: 1) Connecting and Convening; 2) Coordinating; 3) Collecting and Disseminating Data; 4) Communicating; and 5) Complementing and Supplementing. Examples that illustrate evidence vis-à-vis the components and phases of collective impact will be presented.