Plant lysin motif extracellular proteins are required for arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal fungi (AMF) can form a mutually beneficial symbiotic relationship with most land plants. They are known to secrete lysin motif (LysM) effectors into host root cells for successful colonization. Intriguingly, plants secrete similar types of LysM proteins; however, their role in plant-microbe interactions is unknown. Here, we show that Medicago truncatula deploys LysM extracellular (LysMe) proteins to facilitate symbiosis with AMF. Promoter analyses demonstrated that three M. truncatula LysMe genes MtLysMe1/2/3, are expressed in arbuscule-containing cells and those adjacent to intercellular hyphae. Localization studies showed that these proteins are targeted to the periarbuscular space between the periarbuscular membrane and the fungal cell wall of the branched arbuscule. M. truncatula mutants in which MtLysMe2 was knocked out via CRISPR/Cas9-targeted mutagenesis exhibited a significant reduction in AMF colonization and arbuscule formation, whereas genetically complemented transgenic plants restored wild-type level AMF colonization. In addition, knocking out the ortholog of MtLysMe2 in tomato resulted in a similar defect in AMF colonization. In vitro binding affinity precipitation assays suggested binding of MtLysMe1/2/3 with chitin and chitosan, while microscale thermophoresis (MST) assays revealed weak binding of these proteins with chitooligosaccharides. Moreover, application of purified MtLysMe proteins to root segments could suppress chitooctaose (CO8)-induced reactive oxygen species production and expression of reporter genes of the immune response without impairing chitotetraose (CO4)-triggered symbiotic responses. Taken together, our results reveal that plants, like their fungal partners, also secrete LysM proteins to facilitate symbiosis establishment.

Medicago SPX1 and SPX3 regulate phosphate homeostasis, mycorrhizal colonization, and arbuscule degradation.

To acquire sufficient mineral nutrients such as phosphate (Pi) from the soil, most plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. Attracted by plant-secreted strigolactones (SLs), the fungi colonize the roots and form highly branched hyphal structures called arbuscules inside inner cortex cells. The host plant must control the different steps of this interaction to maintain its symbiotic nature. However, how plants sense the amount of Pi obtained from the fungus, and how this determines the arbuscule lifespan, are far from understood. Here, we show that Medicago truncatula SPX-domain containing proteins SPX1 and SPX3 regulate root Pi starvation responses, in part by interacting with PHOSPHATE RESPONSE REGULATOR2, as well as fungal colonization and arbuscule degradation. SPX1 and SPX3 are induced upon Pi starvation but become more restricted to arbuscule-containing cells upon the establishment of symbiosis. This induction in arbuscule-containing cells is associated with the presence of cis-regulatory AW-boxes and transcriptional regulation by the WRINKLED1-like transcription factor WRI5a. Under Pi-limiting conditions, SPX1 and SPX3 facilitate the expression of the SL biosynthesis gene DWARF27, which could help explain the increased fungal branching in response to root exudates. Later, in arbuscule-containing cells, SPX1 and SPX3 redundantly control arbuscule degradation. Thus, SPX proteins play important roles as phosphate sensors to maintain a beneficial AM symbiosis.

Stochastic nuclear organization and host-dependent allele contribution in Rhizophagus irregularis.

BACKGROUND: Arbuscular mycorrhizal (AM) fungi are arguably the most important symbionts of plants, offering a range of benefits to their hosts. However, the provisioning of these benefits does not appear to be uniform among AM fungal individuals, with genetic variation between fungal symbionts having a substantial impact on plant performance. Interestingly, genetic variation has also been reported within fungal individuals, which contain millions of haploid nuclei sharing a common cytoplasm. In the model AM fungus, Rhizophagus irregularis, several isolates have been reported to be dikaryotes, containing two genetically distinct types of nuclei recognized based on their mating-type (MAT) locus identity. However, their extremely coenocytic nature and lack of a known single nucleus stage has raised questions on the origin, distribution and dynamics of this genetic variation. RESULTS: Here we performed DNA and RNA sequencing at the mycelial individual, single spore and single nucleus levels to gain insight into the dynamic genetic make-up of the dikaryote-like R. irregularis C3 isolate and the effect of different host plants on its genetic variation. Our analyses reveal that parallel spore and root culture batches can have widely variable ratios of two main genotypes in C3. Additionally, numerous polymorphisms were found with frequencies that deviated significantly from the general genotype ratio, indicating a diverse population of slightly different nucleotypes. Changing host plants did not show consistent host effects on nucleotype ratio's after multiple rounds of subculturing. Instead, we found a major effect of host plant-identity on allele-specific expression in C3. CONCLUSION: Our analyses indicate a highly dynamic/variable genetic organization in different isolates of R. irregularis. Seemingly random fluctuations in nucleotype ratio's upon spore formation, recombination events, high variability of non-tandemly repeated rDNA sequences and host-dependent allele expression all add levels of variation that may contribute to the evolutionary success of these widespread symbionts.

A Homeotic Mutation Changes Legume Nodule Ontogeny into Actinorhizal-Type Ontogeny.

Some plants fix atmospheric nitrogen by hosting symbiotic diazotrophic rhizobia or Frankia bacteria in root organs known as nodules. Such nodule symbiosis occurs in 10 plant lineages in four taxonomic orders: Fabales, Fagales, Cucurbitales, and Rosales, which are collectively known as the nitrogen-fixing clade. Nodules are divided into two types based on differences in ontogeny and histology: legume-type and actinorhizal-type nodules. The evolutionary relationship between these nodule types has been a long-standing enigma for molecular and evolutionary biologists. Recent phylogenomic studies on nodulating and nonnodulating species in the nitrogen-fixing clade indicated that the nodulation trait has a shared evolutionary origin in all 10 lineages. However, this hypothesis faces a conundrum in that legume-type and actinorhizal-type nodules have been regarded as fundamentally different. Here, we analyzed the actinorhizal-type nodules formed by Parasponia andersonii (Rosales) and Alnus glutinosa (Fagales) and found that their ontogeny is more similar to that of legume-type nodules (Fabales) than generally assumed. We also show that in Medicago truncatula, a homeotic mutation in the co-transcriptional regulator gene NODULE ROOT1 (MtNOOT1) converts legume-type nodules into actinorhizal-type nodules. These experimental findings suggest that the two nodule types have a shared evolutionary origin.

High Salt Levels Reduced Dissimilarities in Root-Associated Microbiomes of Two Barley Genotypes.

Plants harbor in and at their roots bacterial microbiomes that contribute to their health and fitness. The microbiome composition is controlled by the environment and plant genotype. Previously, it was shown that the plant genotype-dependent dissimilarity of root microbiome composition of different species becomes smaller under drought stress. However, it remains unknown whether this reduced plant genotype-dependent effect is a specific response to drought stress or a more generic response to abiotic stress. To test this, we studied the effect of salt stress on two distinct barley (Hordeum vulgare L.) genotypes: the reference cultivar Golden Promise and the Algerian landrace AB. As inoculum, we used soil from salinized and degraded farmland on which barley was cultivated. Controlled laboratory experiments showed that plants inoculated with this soil displayed growth stimulation under high salt stress (200 mM) in a plant genotype-independent manner, whereas the landrace AB also showed significant growth stimulation at low salt concentrations. Subsequent analysis of the root microbiomes revealed a reduced dissimilarity of the bacterial communities of the two barley genotypes in response to high salt, especially in the endophytic compartment. High salt level did not reduce α-diversity (richness) in the endophytic compartment of both plant genotypes but was associated with an increased number of shared strains that respond positively to high salt. Among these, Pseudomonas spp. were most abundant. These findings suggest that the plant genotype-dependent microbiome composition is altered generically by abiotic stress.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Lateral root formation involving cell division in both pericycle, cortex and endodermis is a common and ancestral trait in seed plants.

Studies on the model plant Arabidopsis have led to the common view that lateral roots are exclusively formed from pericycle cells and that the latter are unique in their ability to be reprogrammed into stem cells. By analysing lateral root formation in an evolutionary context, we show that lateral root primordium formation in which cortex, endodermis and pericycle are mitotically activated, is a common and ancestral trait in seed plants, whereas the exclusive involvement of pericycle evolved in the Brassicaceae. Furthermore, the endodermis can also be reprogrammed into stem cells in some species.

Plant growth-promoting rhizobacterium Pseudomonas sp. CM11 specifically induces lateral roots.

Plant growth-promoting rhizobacteria are involved in altering secondary root (SR) formation, but hitherto there has been no distinction between the different types of SRs upon induction of soil biota, and the genetic pathways involved. By using plate and soil systems, we studied the effects of the Pseudomonas strains CM11 and WCS417 on plant performance with a focus on root development. Through a combination of cellular, molecular and genetic analyses, we investigated the type of SRs induced upon CM11 and WCS417 root inoculation using genetic pathways associated with specific SR types. CM11 was shown to affect the root architecture differently from WCS417. CM11 inoculation leads to primary root arrest, whereas WCS417 reveals a longer primary root. Both CM11 and WCS417 activate the PLETHORA 3,5,7-controlled lateral root pathway, rather than the WUSCHEL-RELATED HOMEOBOX 11,12-controlled adventitious (lateral) root pathway. In addition, CM11 promotes plant growth in model and various crop species. It improves plant fitness traits, such as bigger shoots, faster bolting and higher yield in terms of seeds. Our results indicate that the root system architecture can be promoted by activation of PLETHORA 3,5,7 dependent primed lateral pre-branch sites upon inoculation with CM11, which creates great potential to gain a better understanding of root plasticity.

Plant-specific histone deacetylases are essential for early and late stages of Medicago nodule development.

Legume and rhizobium species can establish a nitrogen-fixing nodule symbiosis. Previous studies have shown that several transcription factors that play a role in (lateral) root development are also involved in nodule development. Chromatin remodeling factors, like transcription factors, are key players in regulating gene expression. However, studies have not investigated whether chromatin remodeling genes that are essential for root development are also involved in nodule development. Here, we studied the role of Medicago (Medicago truncatula) histone deacetylases (MtHDTs) in nodule development. Arabidopsis (Arabidopsis thaliana) orthologs of HDTs have been shown to play a role in root development. MtHDT expression is induced in nodule primordia and is maintained in the nodule meristem and infection zone. Conditional, nodule-specific knockdown of MtHDT expression by RNAi blocks nodule primordium development. A few nodules may still form, but their nodule meristems are smaller, and rhizobial colonization of the cells derived from the meristem is markedly reduced. Although the HDTs are expressed during nodule and root development, transcriptome analyses indicate that HDTs control the development of each organ in a different manner. During nodule development, the MtHDTs positively regulate 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 (MtHMGR1). Decreased expression of MtHMGR1 is sufficient to explain the inhibition of primordium formation.

A Remote cis-Regulatory Region Is Required for NIN Expression in the Pericycle to Initiate Nodule Primordium Formation in Medicago truncatula.

The legume-rhizobium symbiosis results in nitrogen-fixing root nodules, and their formation involves both intracellular infection initiated in the epidermis and nodule organogenesis initiated in inner root cell layers. NODULE INCEPTION (NIN) is a nodule-specific transcription factor essential for both processes. These NIN-regulated processes occur at different times and locations in the root, demonstrating a complex pattern of spatiotemporal regulation. We show that regulatory sequences sufficient for the epidermal infection process are located within a 5 kb region directly upstream of the NIN start codon in Medicago truncatula Furthermore, we identify a remote upstream cis-regulatory region required for the expression of NIN in the pericycle, and we show that this region is essential for nodule organogenesis. This region contains putative cytokinin response elements and is conserved in eight more legume species. Both the cytokinin receptor 1, which is essential for nodule primordium formation, and the B-type response regulator RR1 are expressed in the pericycle in the susceptible zone of the uninoculated root. This, together with the identification of the cytokinin-responsive elements in the NIN promoter, strongly suggests that NIN expression is initially triggered by cytokinin signaling in the pericycle to initiate nodule primordium formation.

NIN is essential for development of symbiosomes, suppression of defence and premature senescence in Medicago truncatula nodules.

NIN (NODULE INCEPTION) is a transcription factor that plays a key role during root nodule initiation. However, its role in later nodule developmental stages is unclear. Both NIN mRNA and protein accumulated at the highest level in the proximal part of the infection zone in Medicago truncatula nodules. Two nin weak allele mutants, nin-13/16, form a rather normal nodule infection zone, whereas a fixation zone is not formed. Instead, a zone with defence responses and premature senescence occurred and symbiosome development gets arrested. Mutations in nin-13/16 resulted in a truncated NIN lacking the conserved PB1 domain. However, this did not cause the nodule phenotype as nin mutants expressing NINΔPB1 formed wild-type-like nodule. The phenotype is likely to be caused by reduced NIN mRNA levels in the cytoplasm. Transcriptome analyses of nin-16 nodules showed that expression levels of defence/senescence-related genes are markedly increased, whereas the levels of defence suppressing genes are reduced. Although defence/senescence seems well suppressed in the infection zone, the transcriptome is already markedly changed in the proximal part of infection zone. In addition to its function in infection and nodule organogenesis, NIN also plays a major role at the transition from infection to fixation zone in establishing a functional symbiosis.

A nuclear-targeted effector of Rhizophagus irregularis interferes with histone 2B mono-ubiquitination to promote arbuscular mycorrhisation.

Arguably, symbiotic arbuscular mycorrhizal (AM) fungi have the broadest host range of all fungi, being able to intracellularly colonise root cells in the vast majority of all land plants. This raises the question how AM fungi effectively deal with the immune systems of such a widely diverse range of plants. Here, we studied the role of a nuclear-localisation signal-containing effector from Rhizophagus irregularis, called Nuclear Localised Effector1 (RiNLE1), that is highly and specifically expressed in arbuscules. We showed that RiNLE1 is able to translocate to the host nucleus where it interacts with the plant core nucleosome protein histone 2B (H2B). RiNLE1 is able to impair the mono-ubiquitination of H2B, which results in the suppression of defence-related gene expression and enhanced colonisation levels. This study highlights a novel mechanism by which AM fungi can effectively control plant epigenetic modifications through direct interaction with a core nucleosome component. Homologues of RiNLE1 are found in a range of fungi that establish intimate interactions with plants, suggesting that this type of effector may be more widely recruited to manipulate host defence responses.

Duplication of Symbiotic Lysin Motif Receptors Predates the Evolution of Nitrogen-Fixing Nodule Symbiosis.

Rhizobium nitrogen-fixing nodule symbiosis occurs in two taxonomic lineages: legumes (Fabaceae) and the genus Parasponia (Cannabaceae). Both symbioses are initiated upon the perception of rhizobium-secreted lipochitooligosaccharides (LCOs), called Nod factors. Studies in the model legumes Lotus japonicus and Medicago truncatula showed that rhizobium LCOs are perceived by a heteromeric receptor complex of distinct Lys motif (LysM)-type transmembrane receptors named NOD FACTOR RECEPTOR1 (LjNFR1) and LjNFR5 (L. japonicus) and LYSM DOMAIN CONTAINING RECEPTOR KINASE3 (MtLYK3)-NOD FACTOR PERCEPTION (MtNFP; M. truncatula). Recent phylogenomic comparative analyses indicated that the nodulation traits of legumes, Parasponia spp., as well as so-called actinorhizal plants that establish a symbiosis with diazotrophic Frankia spp. bacteria share an evolutionary origin about 110 million years ago. However, the evolutionary trajectory of LysM-type LCO receptors remains elusive. By conducting phylogenetic analysis, transcomplementation studies, and CRISPR-Cas9 mutagenesis in Parasponia andersonii, we obtained insight into the origin of LCO receptors essential for nodulation. We identified four LysM-type receptors controlling nodulation in P. andersonii: PanLYK1, PanLYK3, PanNFP1, and PanNFP2 These genes evolved from ancient duplication events predating and coinciding with the origin of nodulation. Phylogenetic and functional analyses associated the occurrence of a functional NFP2-orthologous receptor to LCO-driven nodulation. Legumes and Parasponia spp. use orthologous LysM-type receptors to perceive rhizobium LCOs, suggesting a shared evolutionary origin of LCO-driven nodulation. Furthermore, we found that both PanLYK1 and PanLYK3 are essential for intracellular arbuscule formation of mutualistic endomycorrhizal fungi. PanLYK3 also acts as a chitin oligomer receptor essential for innate immune signaling, demonstrating functional analogy to CHITIN ELECITOR RECEPTOR KINASE-type receptors.

Long-Term Warming and Nitrogen Addition Have Contrasting Effects on Ecosystem Carbon Exchange in a Desert Steppe.

Desert steppe, a unique ecotone between steppe and desert in Eurasia, is considered highly vulnerable to global change. However, the long-term impact of warming and nitrogen deposition on plant biomass production and ecosystem carbon exchange in a desert steppe remains unknown. A 12-year field experiment was conducted in a Stipa breviflora desert steppe in northern China. A split-design was used, with warming simulated by infrared radiators as the primary factor and N addition as the secondary factor. Our long-term experiment shows that warming did not change net ecosystem exchange (NEE) or total aboveground biomass (TAB) due to contrasting effects on C4 (23.4% increase) and C3 (11.4% decrease) plant biomass. However, nitrogen addition increased TAB by 9.3% and NEE by 26.0% by increasing soil available N content. Thus, the studied desert steppe did not switch from a carbon sink to a carbon source in response to global change and positively responded to nitrogen deposition. Our study indicates that the desert steppe may be resilient to long-term warming by regulating plant species with contrasting photosynthetic types and that nitrogen deposition could increase plant growth and carbon sequestration, providing negative feedback on climate change.

Comparative genomics of the nonlegume Parasponia reveals insights into evolution of nitrogen-fixing rhizobium symbioses.

Nodules harboring nitrogen-fixing rhizobia are a well-known trait of legumes, but nodules also occur in other plant lineages, with rhizobia or the actinomycete Frankia as microsymbiont. It is generally assumed that nodulation evolved independently multiple times. However, molecular-genetic support for this hypothesis is lacking, as the genetic changes underlying nodule evolution remain elusive. We conducted genetic and comparative genomics studies by using Parasponia species (Cannabaceae), the only nonlegumes that can establish nitrogen-fixing nodules with rhizobium. Intergeneric crosses between Parasponia andersonii and its nonnodulating relative Trema tomentosa demonstrated that nodule organogenesis, but not intracellular infection, is a dominant genetic trait. Comparative transcriptomics of P. andersonii and the legume Medicago truncatula revealed utilization of at least 290 orthologous symbiosis genes in nodules. Among these are key genes that, in legumes, are essential for nodulation, including NODULE INCEPTION (NIN) and RHIZOBIUM-DIRECTED POLAR GROWTH (RPG). Comparative analysis of genomes from three Parasponia species and related nonnodulating plant species show evidence of parallel loss in nonnodulating species of putative orthologs of NIN, RPG, and NOD FACTOR PERCEPTION Parallel loss of these symbiosis genes indicates that these nonnodulating lineages lost the potential to nodulate. Taken together, our results challenge the view that nodulation evolved in parallel and raises the possibility that nodulation originated ∼100 Mya in a common ancestor of all nodulating plant species, but was subsequently lost in many descendant lineages. This will have profound implications for translational approaches aimed at engineering nitrogen-fixing nodules in crop plants.

GeneNoteBook, a collaborative notebook for comparative genomics.

SUMMARY: Analysis and comparison of genomic and transcriptomic datasets have become standard procedures in biological research. However, for non-model organisms no efficient tools exist to visually work with multiple genomes and their metadata, and to annotate such data in a collaborative way. Here we present GeneNoteBook: a web based collaborative notebook for comparative genomics. GeneNoteBook allows experimental and computational researchers to query, browse, visualize and curate bioinformatic analysis results for multiple genomes. GeneNoteBook is particularly suitable for the analysis of non-model organisms, as it allows for comparing newly sequenced genomes to those of model organisms. AVAILABILITY AND IMPLEMENTATION: GeneNoteBook is implemented as a node.js web application and depends on MongoDB and NCBI BLAST. Source code is available at https://github.com/genenotebook/genenotebook. Additionally, GeneNoteBook can be installed through Bioconda and as a Docker image. Full installation instructions and online documentation are available at https://genenotebook.github.io. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mutant analysis in the nonlegume Parasponia andersonii identifies NIN and NF-YA1 transcription factors as a core genetic network in nitrogen-fixing nodule symbioses.

●Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume-rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. ●It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume-rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. ●Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. ●This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis.

A lysin motif effector subverts chitin-triggered immunity to facilitate arbuscular mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) fungi greatly improve mineral uptake by host plants in nutrient-depleted soil and can intracellularly colonize root cortex cells in the vast majority of higher plants. However, AM fungi possess common fungal cell wall components such as chitin that can be recognized by plant chitin receptors to trigger immune responses, raising the question as to how AM fungi effectively evade chitin-triggered immune responses during symbiosis. In this study, we characterize a secreted lysin motif (LysM) effector identified from the model AM fungal species Rhizophagus irregularis, called RiSLM. RiSLM is one of the highest expressed effector proteins in intraradical mycelium during the symbiosis. In vitro binding assays show that RiSLM binds chitin-oligosaccharides and can protect fungal cell walls from chitinases. Moreover, RiSLM efficiently interferes with chitin-triggered immune responses, such as defence gene induction and reactive oxygen species production in Medicago truncatula. Although RiSLM also binds to symbiotic (lipo)chitooligosaccharides it does not interfere significantly with symbiotic signalling in Medicago. Host-induced gene silencing of RiSLM greatly reduces fungal colonization levels. Taken together, our results reveal a key role for AM fungal LysM effectors to subvert chitin-triggered immunity in symbiosis, pointing to a common role for LysM effectors in both symbiotic and pathogenic fungi.

Plant-Specific Histone Deacetylases HDT1/2 Regulate GIBBERELLIN 2-OXIDASE2 Expression to Control Arabidopsis Root Meristem Cell Number.

Root growth is modulated by environmental factors and depends on cell production in the root meristem (RM). New cells in the meristem are generated by stem cells and transit-amplifying cells, which together determine RM cell number. Transcription factors and chromatin-remodeling factors have been implicated in regulating the switch from stem cells to transit-amplifying cells. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific histone deacetylases, HDT1 and HDT2, regulate a second switch from transit-amplifying cells to expanding cells. Knockdown of HDT1/2 (hdt1,2i) results in an earlier switch and causes a reduced RM cell number. Our data show that HDT1/2 negatively regulate the acetylation level of the C19-GIBBERELLIN 2-OXIDASE2 (GA2ox2) locus and repress the expression of GA2ox2 in the RM and elongation zone. Overexpression of GA2ox2 in the RM phenocopies the hdt1,2i phenotype. Conversely, knockout of GA2ox2 partially rescues the root growth defect of hdt1,2i These results suggest that by repressing the expression of GA2ox2, HDT1/2 likely fine-tune gibberellin metabolism and they are crucial for regulating the switch from cell division to expansion to determine RM cell number. We propose that HDT1/2 function as part of a mechanism that modulates root growth in response to environmental factors.

Microsymbiont discrimination mediated by a host-secreted peptide in Medicago truncatula.

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.