A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Fungi hijack a ubiquitous plant apoplastic endoglucanase to release a ROS scavenging ß-glucan decasaccharide to subvert immune responses.

Plant pathogenic and beneficial fungi have evolved several strategies to evade immunity and cope with host-derived hydrolytic enzymes and oxidative stress in the apoplast, the extracellular space of plant tissues. Fungal hyphae are surrounded by an inner insoluble cell wall layer and an outer soluble extracellular polysaccharide (EPS) matrix. Here, we show by proteomics and glycomics that these two layers have distinct protein and carbohydrate signatures, and hence likely have different biological functions. The barley (Hordeum vulgare) ß-1,3-endoglucanase HvBGLUII, which belongs to the widely distributed apoplastic glycoside hydrolase 17 family (GH17), releases a conserved ß-1,3;1,6-glucan decasaccharide (ß-GD) from the EPS matrices of fungi with different lifestyles and taxonomic positions. This low molecular weight ß-GD does not activate plant immunity, is resilient to further enzymatic hydrolysis by ß-1,3-endoglucanases due to the presence of three ß-1,6-linked glucose branches and can scavenge reactive oxygen species. Exogenous application of ß-GD leads to enhanced fungal colonization in barley, confirming its role in the fungal counter-defensive strategy to subvert host immunity. Our data highlight the hitherto undescribed capacity of this often-overlooked EPS matrix from plant-associated fungi to act as an outer protective barrier important for fungal accommodation within the hostile environment at the apoplastic plant-microbe interface.

Plant phosphate status drives host microbial preferences: a trade-off between fungi and bacteria.

Recent discoveries show that plant recruitment of fungi and bacteria in a non-mycorrhizal host follows different strategies dependent on phosphate availability. A new study by Morcillo et al (2019) demonstrates that volatile compounds synthesized by rhizobacteria contribute to phosphate starvation response-dependent regulation of bacterial colonization and immune system activation in Arabidopsis thaliana plants.

Transcriptomics reveal a mechanism of niche defense: two beneficial root endophytes deploy an antimicrobial GH18-CBM5 chitinase to protect their hosts.

Effector secretion is crucial for root endophytes to establish and protect their ecological niche. We used time-resolved transcriptomics to monitor effector gene expression dynamics in two closely related Sebacinales, Serendipita indica and Serendipita vermifera, during symbiosis with three plant species, competition with the phytopathogenic fungus Bipolaris sorokiniana, and cooperation with root-associated bacteria. We observed increased effector gene expression in response to biotic interactions, particularly with plants, indicating their importance in host colonization. Some effectors responded to both plants and microbes, suggesting dual roles in intermicrobial competition and plant-microbe interactions. A subset of putative antimicrobial effectors, including a GH18-CBM5 chitinase, was induced exclusively by microbes. Functional analyses of this chitinase revealed its antimicrobial and plant-protective properties. We conclude that dynamic effector gene expression underpins the ability of Sebacinales to thrive in diverse ecological niches with a single fungal chitinase contributing substantially to niche defense.

A secreted LysM effector protects fungal hyphae through chitin-dependent homodimer polymerization.

Plants trigger immune responses upon recognition of fungal cell wall chitin, followed by the release of various antimicrobials, including chitinase enzymes that hydrolyze chitin. In turn, many fungal pathogens secrete LysM effectors that prevent chitin recognition by the host through scavenging of chitin oligomers. We previously showed that intrachain LysM dimerization of the Cladosporium fulvum effector Ecp6 confers an ultrahigh-affinity binding groove that competitively sequesters chitin oligomers from host immune receptors. Additionally, particular LysM effectors are found to protect fungal hyphae against chitinase hydrolysis during host colonization. However, the molecular basis for the protection of fungal cell walls against hydrolysis remained unclear. Here, we determined a crystal structure of the single LysM domain-containing effector Mg1LysM of the wheat pathogen Zymoseptoria tritici and reveal that Mg1LysM is involved in the formation of two kinds of dimers; a chitin-dependent dimer as well as a chitin-independent homodimer. In this manner, Mg1LysM gains the capacity to form a supramolecular structure by chitin-induced oligomerization of chitin-independent Mg1LysM homodimers, a property that confers protection to fungal cell walls against host chitinases.

Root-specific camalexin biosynthesis controls the plant growth-promoting effects of multiple bacterial strains.

Plants in their natural ecosystems interact with numerous microorganisms, but how they influence their microbiota is still elusive. We observed that sulfatase activity in soil, which can be used as a measure of rhizosphere microbial activity, is differently affected by Arabidopsis accessions. Following a genome-wide association analysis of the variation in sulfatase activity we identified a candidate gene encoding an uncharacterized cytochrome P450, CYP71A27 Loss of this gene resulted in 2 different and independent microbiota-specific phenotypes: A lower sulfatase activity in the rhizosphere and a loss of plant growth-promoting effect by Pseudomonas sp. CH267. On the other hand, tolerance to leaf pathogens was not affected, which agreed with prevalent expression of CYP71A27 in the root vasculature. The phenotypes of cyp71A27 mutant were similar to those of cyp71A12 and cyp71A13, known mutants in synthesis of camalexin, a sulfur-containing indolic defense compound. Indeed, the cyp71A27 mutant accumulated less camalexin in the roots upon elicitation with silver nitrate or flagellin. Importantly, addition of camalexin complemented both the sulfatase activity and the loss of plant growth promotion by Pseudomonas sp. CH267. Two alleles of CYP71A27 were identified among Arabidopsis accessions, differing by a substitution of Glu373 by Gln, which correlated with the ability to induce camalexin synthesis and to gain fresh weight in response to Pseudomonas sp. CH267. Thus, CYP71A27 is an additional component in the camalexin synthesis pathway, contributing specifically to the control of plant microbe interactions in the root.

Plant species-specific recognition of long and short ß-1,3-linked glucans is mediated by different receptor systems.

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe-derived or modified-self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying ß-glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different ß-glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) ß-1,3-glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long ß-1,3-glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short ß-1,3-glucans. Hydrolysis of the ß-1,6 side-branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long-chain ß-glucans. Moreover, in contrast to the recognition of short ß-1,3-glucans in A. thaliana, perception of long ß-1,3-glucans in N. benthamiana and rice is independent of CERK1, indicating that ß-glucan recognition may be mediated by multiple ß-glucan receptor systems.

Focus on How Plants Engage With Beneficial Microorganisms While at the Same Time Restricting Pathogens.

Plants live in a world filled with microbes, and spend their lives engaged in the delicate dance of nurturing beneficial interactions while simultaneously reducing disease-causing interactions. How do plants engage with beneficial microorganisms while at the same time restricting pathogens? was recently selected in a crowd-sourced effort as the top, unanswered question in the field of molecular plant-microbe interactions. Elaborating on this question and setting the stage for this focus issue, the Top10 review by Thoms, Liang and Haney examines the way multiple inputs are integrated to initiate programs of immunity or mutualistic symbiosis, and how this shapes the microbiome. This comprehensive review describes the current landscape of the field, focusing on the plant-microbe-soil continuum, but providing ideas for extending these concepts to leaves, where much of the research on immunity has centered. Other papers in this issue examine the simultaneous interaction of plants with beneficial and pathogenic microorganisms, as well as many diverse relationships with beneficial microbes that can improve plant health by increasing access to nutrients or by decreasing disease.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Unraveling the sugar code: the role of microbial extracellular glycans in plant-microbe interactions.

To defend against microbial invaders but also to establish symbiotic programs, plants need to detect the presence of microbes through the perception of molecular signatures characteristic of a whole class of microbes. Among these molecular signatures, extracellular glycans represent a structurally complex and diverse group of biomolecules that has a pivotal role in the molecular dialog between plants and microbes. Secreted glycans and glycoconjugates such as symbiotic lipochitooligosaccharides or immunosuppressive cyclic ß-glucans act as microbial messengers that prepare the ground for host colonization. On the other hand, microbial cell surface glycans are important indicators of microbial presence. They are conserved structures normally exposed and thus accessible for plant hydrolytic enzymes and cell surface receptor proteins. While the immunogenic potential of bacterial cell surface glycoconjugates such as lipopolysaccharides and peptidoglycan has been intensively studied in the past years, perception of cell surface glycans from filamentous microbes such as fungi or oomycetes is still largely unexplored. To date, only few studies have focused on the role of fungal-derived cell surface glycans other than chitin, highlighting a knowledge gap that needs to be addressed. The objective of this review is to give an overview on the biological functions and perception of microbial extracellular glycans, primarily focusing on their recognition and their contribution to plant-microbe interactions.

Serendipita indica E5'NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization.

Extracellular adenosine 5'-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant-fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography-tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5'NT, an enzymatically active ecto-5'-nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5'NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5'NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus-root interactions and that fungal-derived enzymes can modify apoplastic metabolites to promote fungal accommodation.

A secreted fungal histidine- and alanine-rich protein regulates metal ion homeostasis and oxidative stress.

Like pathogens, beneficial endophytic fungi secrete effector proteins to promote plant colonization, for example, through perturbation of host immunity. The genome of the root endophyte Serendipita indica encodes a novel family of highly similar, small alanine- and histidine-rich proteins, whose functions remain unknown. Members of this protein family carry an N-terminal signal peptide and a conserved C-terminal DELD motif. Here we report on the functional characterization of the plant-responsive DELD family protein Dld1 using a combination of structural, biochemical, biophysical and cytological analyses. The crystal structure of Dld1 shows an unusual, monomeric histidine zipper consisting of two antiparallel coiled-coil helices. Similar to other histidine-rich proteins, Dld1 displays varying affinity to different transition metal ions and undergoes metal ion- and pH-dependent unfolding. Transient expression of mCherry-tagged Dld1 in barley leaf and root tissue suggests that Dld1 localizes to the plant cell wall and accumulates at cell wall appositions during fungal penetration. Moreover, recombinant Dld1 enhances barley root colonization by S. indica, and inhibits H2 O2 -mediated radical polymerization of 3,3'-diaminobenzidine. Our data suggest that Dld1 has the potential to enhance micronutrient accessibility for the fungus and to interfere with oxidative stress and reactive oxygen species homeostasis to facilitate host colonization.

Root-associated fungal microbiota of nonmycorrhizal Arabis alpina and its contribution to plant phosphorus nutrition.

Most land plants live in association with arbuscular mycorrhizal (AM) fungi and rely on this symbiosis to scavenge phosphorus (P) from soil. The ability to establish this partnership has been lost in some plant lineages like the Brassicaceae, which raises the question of what alternative nutrition strategies such plants have to grow in P-impoverished soils. To understand the contribution of plant-microbiota interactions, we studied the root-associated fungal microbiome of Arabis alpina (Brassicaceae) with the hypothesis that some of its components can promote plant P acquisition. Using amplicon sequencing of the fungal internal transcribed spacer 2, we studied the root and rhizosphere fungal communities of A. alpina growing under natural and controlled conditions including low-P soils and identified a set of 15 fungal taxa consistently detected in its roots. This cohort included a Helotiales taxon exhibiting high abundance in roots of wild A. alpina growing in an extremely P-limited soil. Consequently, we isolated and subsequently reintroduced a specimen from this taxon into its native P-poor soil in which it improved plant growth and P uptake. The fungus exhibited mycorrhiza-like traits including colonization of the root endosphere and P transfer to the plant. Genome analysis revealed a link between its endophytic lifestyle and the expansion of its repertoire of carbohydrate-active enzymes. We report the discovery of a plant-fungus interaction facilitating the growth of a nonmycorrhizal plant under native P-limited conditions, thus uncovering a previously underestimated role of root fungal microbiota in P cycling.

FGB1 and WSC3 are in planta-induced ß-glucan-binding fungal lectins with different functions.

In the root endophyte Serendipita indica, several lectin-like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in ß-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long-chain ß1-3-glucan but has no affinity for shorter ß1-3- or ß1-6-linked glucose oligomers. Comparative analysis with the previously identified ß-glucan-binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require ß1-6-linked glucose for efficient binding to branched ß1-3-glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two ß-glucan-binding lectins. Our results highlight the importance of the ß-glucan cell wall component in plant-fungus interactions and the potential of ß-glucan-binding lectins as specific detection tools for fungi in vivo.

The inconspicuous gatekeeper: endophytic Serendipita vermifera acts as extended plant protection barrier in the rhizosphere.

In nature, beneficial and pathogenic fungi often simultaneously colonise plants. Despite substantial efforts to understand the composition of natural plant-microbe communities, the mechanisms driving such multipartite interactions remain largely unknown. Here we address how the interaction between the beneficial root endophyte Serendipita vermifera and the pathogen Bipolaris sorokiniana affects fungal behaviour and determines barley host responses using a gnotobiotic soil-based split-root system. Fungal confrontation in soil resulted in induction of B. sorokiniana genes involved in secondary metabolism and a significant repression of genes encoding putative effectors. In S. vermifera, genes encoding hydrolytic enzymes were strongly induced. This antagonistic response was not activated during the tripartite interaction in barley roots. Instead, we observed a specific induction of S. vermifera genes involved in detoxification and redox homeostasis. Pathogen infection but not endophyte colonisation resulted in substantial host transcriptional reprogramming and activation of defence. In the presence of S. vermifera, pathogen infection and disease symptoms were significantly reduced despite no marked alterations of the plant transcriptional response. The activation of stress response genes and concomitant repression of putative effector gene expression in B. sorokiniana during confrontation with the endophyte suggest a reduction of the pathogen's virulence potential before host plant infection.

Nutrient exchange in arbuscular mycorrhizal symbiosis from a thermodynamic point of view.

To obtain insights into the dynamics of nutrient exchange in arbuscular mycorrhizal (AM) symbiosis, we modelled mathematically the two-membrane system at the plant-fungus interface and simulated its dynamics. In computational cell biology experiments, the full range of nutrient transport pathways was tested for their ability to exchange phosphorus (P)/carbon (C)/nitrogen (N) sources. As a result, we obtained a thermodynamically justified, independent and comprehensive model of the dynamics of the nutrient exchange at the plant-fungus contact zone. The predicted optimal transporter network coincides with the transporter set independently confirmed in wet-laboratory experiments previously, indicating that all essential transporter types have been discovered. The thermodynamic analyses suggest that phosphate is released from the fungus via proton-coupled phosphate transporters rather than anion channels. Optimal transport pathways, such as cation channels or proton-coupled symporters, shuttle nutrients together with a positive charge across the membranes. Only in exceptional cases does electroneutral transport via diffusion facilitators appear to be plausible. The thermodynamic models presented here can be generalized and adapted to other forms of mycorrhiza and open the door for future studies combining wet-laboratory experiments with computational simulations to obtain a deeper understanding of the investigated phenomena.

ß-glucan: Crucial component of the fungal cell wall and elusive MAMP in plants.

Plant innate immunity relies in first place on the detection of invading microbes. Thus, plants evolved receptors to sense unique molecules of the microbe, the so called microbe-associated molecular patterns or MAMPs. The best studied fungal MAMP is chitin, an important structural building block of the fungal cell wall. Over the past years several plant receptors for chitin have been characterized as well as different strategies adopted by fungi to evade chitin recognition. Despite its strong activity as an elicitor of plant defense chitin represents only a small percentage of the cell wall of most fungi compared to other complex sugars. ß-glucan, the most abundant fungal cell wall polysaccharide, also serves as a MAMP, but the mechanisms of ß-glucan perception and signaling in plants are largely unknown. In contrast to that the ß-glucan recognition and signaling machineries are well characterized in mammals. The C-type lectin receptor Dectin-1 is a key component of these machineries. In this review we describe valuable knowledge about the existence of at least one ß-glucan receptor in plants and about the hindrances in ß-glucan research. Additionally we discuss possible future perspectives of glucan research and the possibility to transfer the gathered knowledge from mammalian systems to plants.

Sebacinales - one thousand and one interactions with land plants.

20 I 21 II 21 III 23 IV 29 V 33 VI 35 36 36 References 36 SUMMARY: Root endophytism and mycorrhizal associations are complex derived traits in fungi that shape plant physiology. Sebacinales (Agaricomycetes, Basidiomycota) display highly diverse interactions with plants. Although early-diverging Sebacinales lineages are root endophytes and/or have saprotrophic abilities, several more derived clades harbour obligate biotrophs forming mycorrhizal associations. Sebacinales thus display transitions from saprotrophy to endophytism and to mycorrhizal nutrition within one fungal order. This review discusses the genomic traits possibly associated with these transitions. We also show how molecular ecology revealed the hyperdiversity of Sebacinales and their evolutionary diversification into two sister families: Sebacinaceae encompasses mainly ectomycorrhizal and early-diverging saprotrophic species; the second family includes endophytes and lineages that repeatedly evolved ericoid, orchid and ectomycorrhizal abilities. We propose the name Serendipitaceae for this family and, within it, we transfer to the genus Serendipita the endophytic cultivable species Piriformospora indica and P. williamsii. Such cultivable Serendipitaceae species provide excellent models for root endophytism, especially because of available genomes, genetic tractability, and broad host plant range including important crop plants and the model plant Arabidopsis thaliana. We review insights gained with endophytic Serendipitaceae species into the molecular mechanisms of endophytism and of beneficial effects on host plants, including enhanced resistance to abiotic and pathogen stress.

Convergent evolution of filamentous microbes towards evasion of glycan-triggered immunity.

896 I. 896 II. 896 III. 897 IV. 898 V. 899 VI. 899 900 References 900 SUMMARY: All filamentous microbes produce and release a wide range of glycans, which are essential determinants of microbe-microbe and microbe-host interactions. Major cell wall constituents, such as chitin and ß-glucans, are elicitors of host immune responses. The widespread capacity for glycan perception in plants has driven the evolution of various strategies that help filamentous microbes to evade detection. Common strategies include structural and chemical modifications of cell wall components as well as the secretion of effector proteins that suppress chitin- and ß-glucan-triggered immune responses. Thus, the necessity to avoid glycan-triggered immunity represents a driving force in the convergent evolution of filamentous microbes towards its suppression.

Host-related metabolic cues affect colonization strategies of a root endophyte.

The mechanisms underpinning broad compatibility in root symbiosis are largely unexplored. The generalist root endophyte Piriformospora indica establishes long-lasting interactions with morphologically and biochemically different hosts, stimulating their growth, alleviating salt stress, and inducing local and systemic resistance to pathogens. Cytological studies and global investigations of fungal transcriptional responses to colonization of barley and Arabidopsis at different symbiotic stages identified host-dependent colonization strategies and host-specifically induced effector candidates. Here, we show that in Arabidopsis, P. indica establishes and maintains biotrophic nutrition within living epidermal cells, whereas in barley the symbiont undergoes a nutritional switch to saprotrophy that is associated with the production of secondary thinner hyphae in dead cortex cells. Consistent with a diversified trophic behavior and with the occurrence of nitrogen deficiency at the onset of saprotrophy in barley, fungal genes encoding hydrolytic enzymes and nutrient transporters were highly induced in this host but not in Arabidopsis. Silencing of the high-affinity ammonium transporter PiAMT1 gene, whose transcripts are accumulating during nitrogen starvation and in barley, resulted in enhanced colonization of this host, whereas it had no effect on the colonization of Arabidopsis. Increased levels of free amino acids and reduced enzymatic activity for the cell-death marker VPE (vacuolar-processing enzyme) in colonized barley roots coincided with an extended biotrophic lifestyle of P. indica upon silencing of PiAMT1. This suggests that PiAmt1 functions as a nitrogen sensor mediating the signal that triggers the in planta activation of the saprotrophic program. Thus, host-related metabolic cues affect the expression of P. indica's alternative lifestyles.