Root cortex development is fine-tuned by the interplay of MIGs, SCL3 and DELLAs during arbuscular mycorrhizal symbiosis.

Root development is a crucial process that determines the ability of plants to acquire nutrients, adapt to the substrate and withstand changing environmental conditions. Root plasticity is controlled by a plethora of transcriptional regulators that allow, in contrast to tissue development in animals, post-embryonic changes that give rise to new tissue and specialized cells. One of these changes is the accommodation in the cortex of hyperbranched hyphae of symbiotic arbuscular mycorrhizal (AM) fungi, called arbuscules. Arbuscule-containing cells undergo massive reprogramming to coordinate developmental changes with transport processes. Here we describe a novel negative regulator of arbuscule development, MIG3. MIG3 induces and interacts with SCL3, both of which modulate the activity of the central regulator DELLA, restraining cortical cell growth. As in a tug-of-war, MIG3-SCL3 antagonizes the function of the complex MIG1-DELLA, which promotes the cell expansion required for arbuscule development, adjusting cell size during the dynamic processes of the arbuscule life cycle. Our results in the legume plant Medicago truncatula advance the knowledge of root development in dicot plants, showing the existence of additional regulatory elements not present in Arabidopsis that fine-tune the activity of conserved central modules.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

A tandem Kunitz protease inhibitor (KPI106)-serine carboxypeptidase (SCP1) controls mycorrhiza establishment and arbuscule development in Medicago truncatula.

Plant proteases and protease inhibitors are involved in plant developmental processes including those involving interactions with microbes. Here we show that a tandem between a Kunitz protease inhibitor (KPI106) and a serine carboxypeptidase (SCP1) controls arbuscular mycorrhiza development in the root cortex of Medicago truncatula. Both proteins are only induced during mycorrhiza formation and belong to large families whose members are also mycorrhiza-specific. Furthermore, the interaction between KPI106 and SCP1 analysed using the yeast two-hybrid system is specific, indicating that each family member might have a defined counterpart. In silico docking analysis predicted a putative P1 residue in KPI106 (Lys173) that fits into the catalytic pocket of SCP1, suggesting that KPI106 might inhibit the enzyme activity by mimicking the protease substrate. In vitro mutagenesis of the Lys173 showed that this residue is important in determining the strength and specificity of the interaction. The RNA interference (RNAi) inactivation of the serine carboxypeptidase SCP1 produces aberrant mycorrhizal development with an increased number of septated hyphae and degenerate arbuscules, a phenotype also observed when overexpressing KPI106. Protease and inhibitor are both secreted as observed when expressed in Nicotiana benthamiana epidermal cells. Taken together we envisage a model in which the protease SCP1 is secreted in the apoplast where it produces a peptide signal critical for proper fungal development within the root. KPI106 also at the apoplast would modulate the spatial and/or temporal activity of SCP1 by competing with the protease substrate.

Reprogramming of plant cells by filamentous plant-colonizing microbes.

Although phylogenetically unrelated, filamentous oomycetes and fungi establish similar structures to colonize plants and they represent economically the most important microbial threat to crop production. In mutualistic interactions established by root-colonizing fungi, clear differences to pathogens can be seen, but there is mounting evidence that their infection strategies and molecular interactions have certain common features. To infect the host, fungi and oomycetes employ similar strategies to circumvent plant innate immunity. This process involves the suppression of basal defence responses which are triggered by the perception of conserved molecular patterns. To establish biotrophy, effector proteins are secreted from mutualistic and pathogenic microbes to the host tissue, where they play central roles in the modulation of host immunity and metabolic reprogramming of colonized host tissues. This review article discusses key effector mechanisms of filamentous pathogens and mutualists, how they modulate their host targets and the fundamental differences or parallels between these different interactions. The orchestration of effector actions during plant infection and the importance of their localization within host tissues are also discussed.

A versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus Glomus sp is crucial for the symbiotic relationship with plants.

For more than 400 million years, plants have maintained a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This evolutionary success can be traced to the role of these fungi in providing plants with mineral nutrients, particularly phosphate. In return, photosynthates are given to the fungus, which support its obligate biotrophic lifestyle. Although the mechanisms involved in phosphate transfer have been extensively studied, less is known about the reciprocal transfer of carbon. Here, we present the high-affinity Monosaccharide Transporter2 (MST2) from Glomus sp with a broad substrate spectrum that functions at several symbiotic root locations. Plant cell wall sugars can efficiently outcompete the Glc uptake capacity of MST2, suggesting they can serve as alternative carbon sources. MST2 expression closely correlates with that of the mycorrhiza-specific Phosphate Transporter4 (PT4). Furthermore, reduction of MST2 expression using host-induced gene silencing resulted in impaired mycorrhiza formation, malformed arbuscules, and reduced PT4 expression. These findings highlight the symbiotic role of MST2 and support the hypothesis that the exchange of carbon for phosphate is tightly linked. Unexpectedly, we found that the external mycelium of AM fungi is able to take up sugars in a proton-dependent manner. These results imply that the sugar uptake system operating in this symbiosis is more complex than previously anticipated.

Exploring the synergistic effects of biochar and arbuscular mycorrhizal fungi on phosphorus acquisition in tomato plants by using gene expression analyses.

Arbuscular mycorrhizal (AM) fungi are symbiotic organisms that contribute significantly to plant mineral nutrition, mainly phosphate. However, their benefits are constricted by the availability of phosphate in the soil, and thus they are recalcitrant as amendment in highly fertilized soils. Biochars are by-products of the pyrolysis of biomass in the absence of oxygen. They can improve soil properties and act as a source of nutrients for plants. However, depending on their origin, the final composition of biochars is extremely variable and thus, their efficiency unpredictable. In order to gain mechanistic insights into how the combined application of biochars and AM fungi contribute to plant phosphate nutrition and growth, we used gene expression analyses of key symbiotic marker genes. We compared for this analysis two biochars originated from very different feedstocks (chicken manure and wheat straw) on tomato plants with or without the AM fungus Rhizophagus irregularis. Our results show that the synergy between AM fungi and biochars as P biofertilizers is greatly governed by the origin of the biochar that determines the speed at which phosphate is released to the soil and absorbed by the plant. Thus, chicken manure biochar quickly impacted on plant growth by readily releasing P, but it turned out detrimental for symbiosis formation, decreasing colonization levels and expression of key symbiotic plant marker genes such as SlPT4 or SlFatM. In contrast, wheat straw biochar was inferior at improving plant growth but stimulated the establishment of the symbiosis, producing plants with the same concentration of phosphate as those with the chicken manure. Taken together, slow P releasing biochars from plant residues appears to be a more promising amendment for long terms experiments in which biofertilizers such as AM fungi are considered. Furthermore, our results indicate that implementing plant transcriptomic analyses might help to mechanistically dissect and better understand the effects of biochars on plant growth in different scenarios.

Small-secreted proteins as virulence factors in nematode-trapping fungi.

Nematode-trapping fungi (NTF), such as Arthrobotrys flagrans (Duddingtonia flagrans), are soil-borne fungi able to form adhesive trapping networks to attract and catch nematodes. In this forum piece we highlight some of their most fascinating features with a special focus on the role of small-secreted proteins in the predatory interaction.

Distinguishing friends from foes: Can smRNAs modulate plant interactions with beneficial and pathogenic organisms?

In their agro-ecological habitats, plants are constantly challenged by fungal interactions that might be pathogenic or beneficial in nature, and thus, plants need to exhibit appropriate responses to discriminate between them. Such interactions involve sophisticated molecular mechanism of signal exchange, signal transduction and regulation of gene expression. Small RNAs (smRNAs), including the microRNAs (miRNAs), form an essential layer of regulation in plant developmental processes as well as in plant adaptation to environmental stresses, being key for the outcome during plant-microbial interactions. Further, smRNAs are mobile signals that can go across kingdoms from one interacting partner to the other and hence can be used as communication as well as regulatory tools not only by the host plant but also by the colonising fungus. Here, largely with a focus on plant-fungal interactions and miRNAs, we will discuss the role of smRNAs, and how they might help plants to discriminate between friends and foes.

Overexpression of the Potato Monosaccharide Transporter StSWEET7a Promotes Root Colonization by Symbiotic and Pathogenic Fungi by Increasing Root Sink Strength.

Root colonization by filamentous fungi modifies sugar partitioning in plants by increasing the sink strength. As a result, a transcriptional reprogramming of sugar transporters takes place. Here we have further advanced in the characterization of the potato SWEET sugar transporters and their regulation in response to the colonization by symbiotic and pathogenic fungi. We previously showed that root colonization by the AM fungus Rhizophagus irregularis induces a major transcriptional reprogramming of the 35 potato SWEETs, with 12 genes induced and 10 repressed. In contrast, here we show that during the early colonization phase, the necrotrophic fungus Fusarium solani only induces one SWEET transporter, StSWEET7a, while represses most of the others (25). StSWEET7a was also induced during root colonization by the hemi-biotrophic fungus Fusarium oxysporum f. sp. tuberosi. StSWEET7a which belongs to the clade II of SWEET transporters localized to the plasma membrane and transports glucose, fructose and mannose. Overexpression of StSWEET7a in potato roots increased the strength of this sink as evidenced by an increase in the expression of the cell wall-bound invertase. Concomitantly, plants expressing StSWEET7a were faster colonized by R. irregularis and by F. oxysporum f. sp. tuberosi. The increase in sink strength induced by ectopic expression of StSWEET7a in roots could be abolished by shoot excision which reverted also the increased colonization levels by the symbiotic fungus. Altogether, these results suggest that AM fungi and Fusarium spp. might induce StSWEET7a to increase the sink strength and thus this gene might represent a common susceptibility target for root colonizing fungi.

Erl1, a novel era-like GTPase from Magnaporthe oryzae, is required for full root virulence and is conserved in the mutualistic symbiont Glomus intraradices.

Comparative analyses of genome sequences from several plant-infecting fungi have shown conservation and expansion of protein families with plant disease-related functions. Here, we show that this hypothesis can be extended to mutualistic symbiotic fungi. We have identified a gene encoding an Era (Escherichia coli Ras)-like GTPase in the rice blast fungus Magnaporthe oryzae and found that it is orthologous to the mature amino terminal part of the Gin1 protein from the arbuscular mycorrhizal (AM) fungus Glomus intraradices. M. oryzae Erl1 is required for full root virulence. Appressoria formation was not severely affected in Deltaerl1 strains, but invasive hyphae grew slower than in the wild type. Root browning defect of Deltaerl1 strains could be complemented by the AM gene under the control of the ERL1 promoter. Erl1 and Gin-N localized to the nucleus when carboxy-terminally labeled with green fluorescent protein (GFP). However, amino-terminal GFP-tagged versions of the proteins expressed in Aspergillus nidulans were shown to localize in the cytoplasm and to cause polarity defects. These data suggest that Erl1 and Gin-N are orthologs and might be involved in the control of hyphal growth in planta. This is the first characterization of an Era-like GTPase in filamentous fungi.

Membrane steroid-binding protein 1 induced by a diffusible fungal signal is critical for mycorrhization in Medicago truncatula.

Arbuscular mycorrhiza (AM) is a mutualistic biotrophic association that requires a complex exchange of signals between plant and fungus to allow accommodation of the mycosymbiont in the root cortex. Signal exchange happens even before physical contact, activating the plant symbiotic program. We investigated very early transcriptional responses in Medicago truncatula to inoculation with Glomus intraradices and identified four genes induced by diffusible AM fungal signals before contact. Three of them were previously shown to be mycorrhiza induced at later stages of the symbiosis, while MtMSBP1, encoding a membrane-bound steroid-binding protein, is a novel mycorrhizal marker. Expression analyses in plants defective in the symbiotic receptor kinase DMI2 allowed discrimination of two different signaling cascades involved in the perception of the diffusible signals. Thus, while some of the genes are activated in a DMI2-dependent manner, the induction of one of them encoding a proteinase inhibitor is DMI2-independent. Downregulation of MtMSBP1 by RNAi led to an aberrant mycorrhizal phenotype with thick and septated appressoria, decrease number of arbuscules and distorted arbuscule morphology. This provides genetic evidence that MtMSBP1 is critical for mycorrhiza development. We hypothesize that MtMSBP1 plays a role in sterol homeostasis in the root.

Detection of Arbuscular Mycorrhizal Fungal Gene Expression by In Situ Hybridization.

The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.

Host-Induced Gene Silencing of Arbuscular Mycorrhizal Fungal Genes via Agrobacterium rhizogenes-Mediated Root Transformation in Medicago truncatula.

Host-induced gene silencing (HIGS) is a methodology that allows the downregulation of genes in organisms living in close association with a host and that are not amenable or recalcitrant to genetic modifications. This method has been particularly used for oomycetes and for filamentous fungi interacting with plants, including the fungi of the arbuscular mycorrhizal symbiosis. Here, we present a protocol developed in our laboratory to downregulate genes from the obligate symbiont Rhizophagus irregularis in symbiosis with Medicago truncatula plants.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

Allorecognition upon Fungal Cell-Cell Contact Determines Social Cooperation and Impacts the Acquisition of Multicellularity.

Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.

RiCRN1, a Crinkler Effector From the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis, Functions in Arbuscule Development.

Arbuscular mycorrhizal (AM) symbiosis is one of the most prominent and beneficial plant-microbe interactions that facilitates mineral nutrition and confers tolerance to biotic and abiotic stresses. AM fungi colonize the root cortex and develop specialized structures called arbuscules where the nutrient exchange takes place. Arbuscule development is a highly controlled and coordinated process requiring the involvement of many plant proteins recruited at that interface. In contrast, much less is known about the fungal proteins involved in this process. Here, we have identified an AM fungal effector that participates in this developmental step of the symbiosis. RiCRN1 is a crinkler (CRN) effector that belongs to a subfamily of secreted CRN proteins from R. irregularis. CRNs have been so far only functionally characterized in pathogenic microbes and shown to participate in processes controlling plant cell death and immunity. RiCRN1 accumulates during symbiosis establishment parallel to MtPT4, the gene coding for an arbuscule-specific phosphate transporter. Expression in Nicotiana benthamiana leaves and in Medicago truncatula roots suggest that RiCRN1 is not involved in cell death processes. RiCRN1 dimerizes and localizes to nuclear bodies, suggesting that, similar to other CRNs, it functions in the plant nucleus. Downregulation of RiCRN1 using host-induced gene silencing led to an impairment of the symbiosis in M. truncatula and to a reduction of MtPT4, while ectopic expression of RiCRN1, surprisingly, led to a drastic reduction in arbuscule size that correlated with a decrease not only in MtPT4 but also in MtBCP1, a marker for initial stages of arbuscule development. Altogether, our results suggest that a tightly regulated expression in time and space of RiCRN1 is critical for symbiosis progression and for the proper initiation of arbuscule development.

Plant signals and fungal perception during arbuscular mycorrhiza establishment.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that need their plant hosts to complete their life cycle. In the absence of the plant, germlings arrest growth after a few days and retract most of their cytoplasm back into the multinuclear spores. The spores can germinate again during more favorable conditions. How AM fungi recognize compatible host roots and activate their symbiotic program is not yet understood. However, research in this field in the last years has shed light into this topic. We, and others, have approached some of these aspects by studying changes in fungal gene expression observed at early stages of development, before and at the plant recognition stage in an attempt to identify genes and proteins featuring as key regulators in the switch between the asymbiotic and symbiotic style of life. The molecular bases of this recognition process are now starting to be understood and point to common signaling pathways shared with other microbe-plant associations and to arbuscular mycorrhiza specific signaling pathways.