Microbial Interkingdom Interactions in Roots Promote Arabidopsis Survival.

Roots of healthy plants are inhabited by soil-derived bacteria, fungi, and oomycetes that have evolved independently in distinct kingdoms of life. How these microorganisms interact and to what extent those interactions affect plant health are poorly understood. We examined root-associated microbial communities from three Arabidopsis thaliana populations and detected mostly negative correlations between bacteria and filamentous microbial eukaryotes. We established microbial culture collections for reconstitution experiments using germ-free A. thaliana. In plants inoculated with mono- or multi-kingdom synthetic microbial consortia, we observed a profound impact of the bacterial root microbiota on fungal and oomycetal community structure and diversity. We demonstrate that the bacterial microbiota is essential for plant survival and protection against root-derived filamentous eukaryotes. Deconvolution of 2,862 binary bacterial-fungal interactions ex situ, combined with community perturbation experiments in planta, indicate that biocontrol activity of bacterial root commensals is a redundant trait that maintains microbial interkingdom balance for plant health.

Proteins released into the plant apoplast by the obligate parasitic protist Albugo selectively repress phyllosphere-associated bacteria.

Biotic and abiotic interactions shape natural microbial communities. The mechanisms behind microbe-microbe interactions, particularly those protein based, are not well understood. We hypothesize that released proteins with antimicrobial activity are a powerful and highly specific toolset to shape and defend plant niches. We have studied Albugo candida, an obligate plant parasite from the protist Oomycota phylum, for its potential to modulate the growth of bacteria through release of antimicrobial proteins into the apoplast. Amplicon sequencing and network analysis of Albugo-infected and uninfected wild Arabidopsis thaliana samples revealed an abundance of negative correlations between Albugo and other phyllosphere microbes. Analysis of the apoplastic proteome of Albugo-colonized leaves combined with machine learning predictors enabled the selection of antimicrobial candidates for heterologous expression and study of their inhibitory function. We found for three candidate proteins selective antimicrobial activity against Gram-positive bacteria isolated from A. thaliana and demonstrate that these inhibited bacteria are precisely important for the stability of the community structure. We could ascribe the antibacterial activity of the candidates to intrinsically disordered regions and positively correlate it with their net charge. This is the first report of protist proteins with antimicrobial activity under apoplastic conditions that therefore are potential biocontrol tools for targeted manipulation of the microbiome.

Distinct Phyllosphere Microbiome of Wild Tomato Species in Central Peru upon Dysbiosis.

Plants are colonized by myriads of microbes across kingdoms, which affect host development, fitness, and reproduction. Hence, plant microbiomes have been explored across a broad range of host species, including model organisms, crops, and trees under controlled and natural conditions. Tomato is one of the world's most important vegetable crops; however, little is known about the microbiota of wild tomato species. To obtain insights into the tomato microbiota occurring in natural environments, we sampled epiphytic microbes from leaves of four tomato species, Solanum habrochaites, S. corneliomulleri, S. peruvianum, and S. pimpinellifolium, from two geographical locations within the Lima region of Peru over 2 consecutive years. Here, a high-throughput sequencing approach was applied to investigate microbial compositions including bacteria, fungi, and eukaryotes across tomato species and geographical locations. The phyllosphere microbiome composition varies between hosts and location. Yet, we identified persistent microbes across tomato species that form the tomato microbial core community. In addition, we phenotypically defined healthy and dysbiotic samples and performed a downstream analysis to reveal the impact on microbial community structures. To do so, we compared microbial diversities, unique OTUs, relative abundances of core taxa, and microbial hub taxa, as well as co-occurrence network characteristics in healthy and dysbiotic tomato leaves and found that dysbiosis affects the phyllosphere microbial composition in a host species-dependent manner. Yet, overall, the present data suggests an enrichment of plant-promoting microbial taxa in healthy leaves, whereas numerous microbial taxa containing plant pathogens occurred in dysbiotic leaves.Concluding, we identify the core phyllosphere microbiome of wild tomato species, and show that the overall phyllosphere microbiome can be impacted by sampling time point, geographical location, host genotype, and plant health. Future studies in these components will help understand the microbial contribution to plant health in natural systems and can be of use in cultivated tomatoes.

Microbiome profiling reveals that Pseudomonas antagonises parasitic nodule colonisation of cheater rhizobia in Lotus.

Nodule microbiota are dominated by symbiotic nitrogen-fixing rhizobia, however, other non-rhizobial bacteria also colonise this niche. Although many of these bacteria harbour plant-growth-promoting functions, it is not clear whether these less abundant nodule colonisers impact root-nodule symbiosis. We assessed the relationship between the nodule microbiome and nodulation as influenced by the soil microbiome, by using a metabarcoding approach to characterise the communities inside nodules of healthy and starved Lotus species. A machine learning algorithm and network analyses were used to identify nodule bacteria of interest, which were re-inoculated onto plants in controlled conditions to observe their potential functionality. The nodule microbiome of all tested species differed according to inoculum, but only that of Lotus burttii varied with plant health. Amplicon sequence variants representative of Pseudomonas species were the most indicative non-rhizobial signatures inside healthy L. burttii nodules and negatively correlated with Rhizobium sequences. A representative Pseudomonas isolate co-colonised nodules infected with a beneficial Mesorhizobium, but not with an ineffective Rhizobium isolate and another even reduced the number of ineffective nodules induced on Lotus japonicus. Our results show that nodule endophytes influence the overall outcome of the root-nodule symbiosis, albeit in a plant host-specific manner.

Shaping the leaf microbiota: plant-microbe-microbe interactions.

The aerial portion of a plant, namely the leaf, is inhabited by pathogenic and non-pathogenic microbes. The leaf's physical and chemical properties, combined with fluctuating and often challenging environmental factors, create surfaces that require a high degree of adaptation for microbial colonization. As a consequence, specific interactive processes have evolved to establish a plant leaf niche. Little is known about the impact of the host immune system on phyllosphere colonization by non-pathogenic microbes. These organisms can trigger plant basal defenses and benefit the host by priming for enhanced resistance to pathogens. In most disease resistance responses, microbial signals are recognized by extra- or intracellular receptors. The interactions tend to be species specific and it is unclear how they shape leaf microbial communities. In natural habitats, microbe-microbe interactions are also important for shaping leaf communities. To protect resources, plant colonizers have developed direct antagonistic or host manipulation strategies to fight competitors. Phyllosphere-colonizing microbes respond to abiotic and biotic fluctuations and are therefore an important resource for adaptive and protective traits. Understanding the complex regulatory host-microbe-microbe networks is needed to transfer current knowledge to biotechnological applications such as plant-protective probiotics.

Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors.

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts.

Microbial Hub Taxa Link Host and Abiotic Factors to Plant Microbiome Variation.

Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe-microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe-microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial "hubs," are strongly interconnected and have a severe effect on communities. By documenting these microbe-microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on "hub" microbes, which, via microbe-microbe interactions, transmit the effects to the microbial community. We analyzed two "hub" microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial "hubs" and their importance in phyllosphere microbiome structuring has crucial implications for plant-pathogen and microbe-microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via "hub" microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome "keystone" pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-associated microbiomes.

Albugo-imposed changes to tryptophan-derived antimicrobial metabolite biosynthesis may contribute to suppression of non-host resistance to Phytophthora infestans in Arabidopsis thaliana.

BACKGROUND: Plants are exposed to diverse pathogens and pests, yet most plants are resistant to most plant pathogens. Non-host resistance describes the ability of all members of a plant species to successfully prevent colonization by any given member of a pathogen species. White blister rust caused by Albugo species can overcome non-host resistance and enable secondary infection and reproduction of usually non-virulent pathogens, including the potato late blight pathogen Phytophthora infestans on Arabidopsis thaliana. However, the molecular basis of host defense suppression in this complex plant-microbe interaction is unclear. Here, we investigate specific defense mechanisms in Arabidopsis that are suppressed by Albugo infection. RESULTS: Gene expression profiling revealed that two species of Albugo upregulate genes associated with tryptophan-derived antimicrobial metabolites in Arabidopsis. Albugo laibachii-infected tissue has altered levels of these metabolites, with lower indol-3-yl methylglucosinolate and higher camalexin accumulation than uninfected tissue. We investigated the contribution of these Albugo-imposed phenotypes to suppression of non-host resistance to P. infestans. Absence of tryptophan-derived antimicrobial compounds enables P. infestans colonization of Arabidopsis, although to a lesser extent than Albugo-infected tissue. A. laibachii also suppresses a subset of genes regulated by salicylic acid; however, salicylic acid plays only a minor role in non-host resistance to P. infestans. CONCLUSIONS: Albugo sp. alter tryptophan-derived metabolites and suppress elements of the responses to salicylic acid in Arabidopsis. Albugo sp. imposed alterations in tryptophan-derived metabolites may play a role in Arabidopsis non-host resistance to P. infestans. Understanding the basis of non-host resistance to pathogens such as P. infestans could assist in development of strategies to elevate food security.

Multiple reference genomes and transcriptomes for Arabidopsis thaliana.

Genetic differences between Arabidopsis thaliana accessions underlie the plant's extensive phenotypic variation, and until now these have been interpreted largely in the context of the annotated reference accession Col-0. Here we report the sequencing, assembly and annotation of the genomes of 18 natural A. thaliana accessions, and their transcriptomes. When assessed on the basis of the reference annotation, one-third of protein-coding genes are predicted to be disrupted in at least one accession. However, re-annotation of each genome revealed that alternative gene models often restore coding potential. Gene expression in seedlings differed for nearly half of expressed genes and was frequently associated with cis variants within 5 kilobases, as were intron retention alternative splicing events. Sequence and expression variation is most pronounced in genes that respond to the biotic environment. Our data further promote evolutionary and functional studies in A. thaliana, especially the MAGIC genetic reference population descended from these accessions.

Genome analyses of the sunflower pathogen Plasmopara halstedii provide insights into effector evolution in downy mildews and Phytophthora.

BACKGROUND: Downy mildews are the most speciose group of oomycetes and affect crops of great economic importance. So far, there is only a single deeply-sequenced downy mildew genome available, from Hyaloperonospora arabidopsidis. Further genomic resources for downy mildews are required to study their evolution, including pathogenicity effector proteins, such as RxLR effectors. Plasmopara halstedii is a devastating pathogen of sunflower and a potential pathosystem model to study downy mildews, as several Avr-genes and R-genes have been predicted and unlike Arabidopsis downy mildew, large quantities of almost contamination-free material can be obtained easily. RESULTS: Here a high-quality draft genome of Plasmopara halstedii is reported and analysed with respect to various aspects, including genome organisation, secondary metabolism, effector proteins and comparative genomics with other sequenced oomycetes. Interestingly, the present analyses revealed further variation of the RxLR motif, suggesting an important role of the conservation of the dEER-motif. Orthology analyses revealed the conservation of 28 RxLR-like core effectors among Phytophthora species. Only six putative RxLR-like effectors were shared by the two sequenced downy mildews, highlighting the fast and largely independent evolution of two of the three major downy mildew lineages. This is seemingly supported by phylogenomic results, in which downy mildews did not appear to be monophyletic. CONCLUSIONS: The genome resource will be useful for developing markers for monitoring the pathogen population and might provide the basis for new approaches to fight Phytophthora and downy mildew pathogens by targeting core pathogenicity effectors.

Host-microbe and microbe-microbe interactions in the evolution of obligate plant parasitism.

Research on obligate biotrophic plant parasites, which reproduce only on living hosts, has revealed a broad diversity of filamentous microbes that have independently acquired complex morphological structures, such as haustoria. Genome studies have also demonstrated a concerted loss of genes for metabolism and lytic enzymes, and gain of diversity of genes coding for effectors involved in host defense suppression. So far, these traits converge in all known obligate biotrophic parasites, but unexpected genome plasticity remains. This plasticity is manifested as transposable element (TE)-driven increases in genome size, observed to be associated with the diversification of virulence genes under selection pressure. Genome expansion could result from the governing of the pathogen response to ecological selection pressures, such as host or nutrient availability, or to microbial interactions, such as competition, hyperparasitism and beneficial cooperations. Expansion is balanced by alternating sexual and asexual cycles, as well as selfing and outcrossing, which operate to control transposon activity in populations. In turn, the prevalence of these balancing mechanisms seems to be correlated with external biotic factors, suggesting a complex, interconnected evolutionary network in host-pathogen-microbe interactions. Therefore, the next phase of obligate biotrophic pathogen research will need to uncover how this network, including multitrophic interactions, shapes the evolution and diversity of pathogens.

A novel structural effector from rust fungi is capable of fibril formation.

It has been reported that filament-forming surface proteins such as hydrophobins are important virulence determinants in fungi and are secreted during pathogenesis. Such proteins have not yet been identified in obligate biotrophic pathogens such as rust fungi. Rust transferred protein 1 (RTP1p), a rust protein that is transferred into the host cytoplasm, accumulates around the haustorial complex. To investigate RTP1p structure and function, we used immunocytological, biochemical and computational approaches. We found that RTP1p accumulates in protuberances of the extra-haustorial matrix, a compartment that surrounds the haustorium and is separated from the plant cytoplasm by a modified host plasma membrane. Our analyses show that RTP1p is capable of forming filamentous structures in vitro and in vivo. We present evidence that filament formation is due to ß-aggregation similar to what has been observed for amyloid-like proteins. Our findings reveal that RTP1p is a member of a new class of structural effectors. We hypothesize that RTP1p is transferred into the host to stabilize the host cell and protect the haustorium from degradation in later stages of the interaction. Thus, we provide evidence for transfer of an amyloid-like protein into the host cell, which has potential for the development of new resistance mechanisms against rust fungi.

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics.

BACKGROUND: Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10-20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5' ends of mRNA or use of RNA ligations. RESULTS: We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3' end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing. CONCLUSIONS: EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.

Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana.

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires "effectors" to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the "CHXCs", by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterin-requiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata.

Cell specialization in cyanobacterial biofilm development revealed by expression of a cell-surface and extracellular matrix protein.

Cyanobacterial biofilms are ubiquitous and play important roles in diverse environments, yet, understanding of the processes underlying the development of these aggregates is just emerging. Here we report cell specialization in formation of Synechococcus elongatus PCC 7942 biofilms-a hitherto unknown characteristic of cyanobacterial social behavior. We show that only a quarter of the cell population expresses at high levels the four-gene ebfG-operon that is required for biofilm formation. Almost all cells, however, are assembled in the biofilm. Detailed characterization of EbfG4 encoded by this operon revealed cell-surface localization as well as its presence in the biofilm matrix. Moreover, EbfG1-3 were shown to form amyloid structures such as fibrils and are thus likely to contribute to the matrix structure. These data suggest a beneficial 'division of labor' during biofilm formation where only some of the cells allocate resources to produce matrix proteins-'public goods' that support robust biofilm development by the majority of the cells. In addition, previous studies revealed the operation of a self-suppression mechanism that depends on an extracellular inhibitor, which supresses transcription of the ebfG-operon. Here we revealed inhibitor activity at an early growth stage and its gradual accumulation along the exponential growth phase in correlation with cell density. Data, however, do not support a threshold-like phenomenon known for quorum-sensing in heterotrophs. Together, data presented here demonstrate cell specialization and imply density-dependent regulation thereby providing deep insights into cyanobacterial communal behavior.

The Leaf Microbiome of Arabidopsis Displays Reproducible Dynamics and Patterns throughout the Growing Season.

Leaves are primarily responsible for the plant's photosynthetic activity. Thus, changes in the leaf microbiota, which includes deleterious and beneficial microbes, can have far-reaching effects on plant fitness and productivity. Identifying the processes and microorganisms that drive these changes over a plant's lifetime is, therefore, crucial. In this study, we analyzed the temporal dynamics in the leaf microbiome of Arabidopsis thaliana, integrating changes in both composition and microbe-microbe interactions via the study of microbial networks. Field-grown Arabidopsis were used to monitor leaf bacterial, fungal and oomycete communities throughout the plant's natural growing season (extending from November to March) over three consecutive years. Our results revealed the existence of conserved temporal patterns, with microbial communities and networks going through a stabilization phase of decreased diversity and variability at the beginning of the plant's growing season. Despite a high turnover in these communities, we identified 19 "core" taxa persisting on Arabidopsis leaves across time and plant generations. With the hypothesis these microbes could be playing key roles in the structuring of leaf microbial communities, we conducted a time-informed microbial network analysis which showed core taxa are not necessarily highly connected network "hubs," and "hubs" alternate with time. Our study shows that leaf microbial communities exhibit reproducible dynamics and patterns, suggesting the potential of using our understanding of temporal trajectories in microbial community composition to design experiments aimed at driving these communities toward desired states. IMPORTANCE Utilizing plant microbiota to promote plant growth and plant health is key to more environmentally friendly agriculture. A major bottleneck in the engineering of plant-beneficial microbial communities is the low persistence of applied microbes under filed conditions, especially considering plant leaves. Indeed, although many leaf-associated microorganisms have the potential to promote plant growth and protect plants from pathogens, few of them are able to survive and thrive over time. In our study, we could show that leaf microbial communities are very variable at the beginning of the plant growing season but become more and more similar and less variable as the season progresses. We further identify a cohort of 19 "core" microbes, systematically present on plant leaves that would make these microbes exceptional candidates for future agricultural applications.

Predicting Lifestyle from Positive Selection Data and Genome Properties in Oomycetes.

As evidenced in parasitism, host and niche shifts are a source of genomic and phenotypic diversification. Exemplary is a reduction in the core metabolism as parasites adapt to a particular host, while the accessory genome often maintains a high degree of diversification. However, selective pressures acting on the genome of organisms that have undergone recent lifestyle or host changes have not been fully investigated. Here, we developed a comparative genomics approach to study underlying adaptive trends in oomycetes, a eukaryotic phylum with a wide and diverse range of economically important plant and animal parasitic lifestyles. Our analysis reveals converging evolution on biological processes for oomycetes that have similar lifestyles. Moreover, we find that certain functions, in particular carbohydrate metabolism, transport, and signaling, are important for host and environmental adaptation in oomycetes. Given the high correlation between lifestyle and genome properties in our oomycete dataset, together with the known convergent evolution of fungal and oomycete genomes, we developed a model that predicts plant pathogenic lifestyles with high accuracy based on functional annotations. These insights into how selective pressures correlate with lifestyle may be crucial to better understand host/lifestyle shifts and their impact on the genome.

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

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.

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.

Amyloid Proteins in Plant-Associated Microbial Communities.

Amyloids have proven to be a widespread phenomenon rather than an exception. Many proteins presenting the hallmarks of this characteristic beta sheet-rich folding have been described to date. Particularly common are functional amyloids that play an important role in the promotion of survival and pathogenicity in prokaryotes. Here, we describe important developments in amyloid protein research that relate to microbe-microbe and microbe-host interactions in the plant microbiome. Starting with biofilms, which are a broad strategy for bacterial persistence that is extremely important for plant colonization. Microbes rely on amyloid-based mechanisms to adhere and create a protective coating that shelters them from external stresses and promotes cooperation. Another strategy generally carried out by amyloids is the formation of hydrophobic surface layers. Known as hydrophobins, these proteins coat the aerial hyphae and spores of plant pathogenic fungi, as well as certain bacterial biofilms. They contribute to plant virulence through promoting dissemination and infectivity. Furthermore, antimicrobial activity is an interesting outcome of the amyloid structure that has potential application in medicine and agriculture. There are many known antimicrobial amyloids released by animals and plants; however, those produced by bacteria or fungi remain still largely unknown. Finally, we discuss amyloid proteins with a more indirect mode of action in their host interactions. These include virulence-promoting harpins, signaling transduction that functions through amyloid templating, and root nodule bacteria proteins that promote plant-microbe symbiosis. In summary, amyloids are an interesting paradigm for their many functional mechanisms linked to bacterial survival in plant-associated microbial communities.

Obtaining deeper insights into microbiome diversity using a simple method to block host and nontargets in amplicon sequencing.

Profiling diverse microbiomes is revolutionizing our understanding of biological mechanisms and ecologically relevant problems, including metaorganism (host + microbiome) assembly, functions and adaptation. Amplicon sequencing of multiple conserved, phylogenetically informative loci has therefore become an instrumental tool for many researchers. Investigations in many systems are hindered, however, since essential sequencing depth can be lost by amplification of nontarget DNA from hosts or overabundant microorganisms. Here, we introduce "blocking oligos", a low-cost and flexible method using standard oligonucleotides to block amplification of diverse nontargets and software to aid their design. We apply them primarily in leaves, where exceptional challenges with host amplification prevail. A. thaliana-specific blocking oligos applied in eight different target loci reduce undesirable host amplification by up to 90%. To expand applicability, we designed universal 16S and 18S rRNA gene plant blocking oligos for targets that are conserved in diverse plant species and demonstrate that they efficiently block five plant species from five orders spanning monocots and dicots (Bromus erectus, Plantago lanceolata, Lotus corniculatus, Amaranth sp., Arabidopsis thaliana). These can increase alpha diversity discovery without biasing beta diversity patterns and do not compromise microbial load information inherent to plant-derived 16S rRNA gene amplicon sequencing data. Finally, we designed and tested blocking oligos to avoid amplification of 18S rRNA genes of a sporulating oomycete pathogen, demonstrating their effectiveness in applications well beyond plants. Using these tools, we generated a survey of the A. thaliana leaf microbiome based on eight loci targeting bacterial, fungal, oomycete and other eukaryotic microorganisms and discuss complementarity of commonly used amplicon sequencing regions for describing leaf microbiota. This approach has potential to make questions in a variety of study systems more tractable by making amplicon sequencing more targeted, leading to deeper, systems-based insights into microbial discovery. For fast and easy design for blocking oligos for any nontarget DNA in other study systems, we developed a publicly available R package.