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.

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.

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.

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.

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.

Symbiotic Fungi Control Plant Root Cortex Development through the Novel GRAS Transcription Factor MIG1.

In an approaching scenario of soil nutrient depletion, root association with soil microorganisms can be key for plant health and sustainability [1-3]. Symbiotic arbuscular mycorrhizal (AM) fungi are major players in helping plants growing under nutrient starvation conditions. They provide plants with minerals like phosphate and, furthermore, act as modulators of plant growth altering the root developmental program [4, 5]. However, the precise mechanisms involved in this latter process are not well understood. Here, we show that AM fungi are able to modulate root cortex development in Medicago truncatula by activating a novel GRAS-domain transcription factor, MIG1, that determines the size of cortical root cells. MIG1 expression peaks in arbuscule-containing cells, suggesting a role in cell remodeling during fungal accommodation. Roots ectopically expressing MIG1 become thicker due to an increase in the number and width of cortical cells. This phenotype is fully counteracted by gibberellin (GA) and phenocopied with a GA biosynthesis inhibitor or by expression of a dominant DELLA (Δ18DELLA1) protein. MIG1 downregulation leads to malformed arbuscules, a phenotype rescued by Δ18DELLA1, suggesting that MIG1 intersects with the GA signaling to control cell morphogenesis through DELLA1. DELLA1 was shown to be a central node controlling arbuscule branching [6-8]. Now we provide evidence that, together with MIG1, DELLA1 is responsible for radial cortical cell expansion during arbuscule development. Our data point toward DELLA proteins being not only longitudinal root growth repressors [9] but also positive regulators of cortical radial cell expansion, extending the knowledge of how DELLAs control root growth.

Arbuscular mycorrhiza Symbiosis Induces a Major Transcriptional Reprogramming of the Potato SWEET Sugar Transporter Family.

Biotrophic microbes feeding on plants must obtain carbon from their hosts without killing the cells. The symbiotic Arbuscular mycorrhizal (AM) fungi colonizing plant roots do so by inducing major transcriptional changes in the host that ultimately also reprogram the whole carbon partitioning of the plant. AM fungi obtain carbohydrates from the root cortex apoplast, in particular from the periarbuscular space that surrounds arbuscules. However, the mechanisms by which cortical cells export sugars into the apoplast for fungal nutrition are unknown. Recently a novel type of sugar transporter, the SWEET, able to perform not only uptake but also efflux from cells was identified. Plant SWEETs have been shown to be involved in the feeding of pathogenic microbes and are, therefore, good candidates to play a similar role in symbiotic associations. Here we have carried out the first phylogenetic and expression analyses of the potato SWEET family and investigated its role during mycorrhiza symbiosis. The potato genome contains 35 SWEETs that cluster into the same four clades defined in Arabidopsis. Colonization of potato roots by the AM fungus Rhizophagus irregularis imposes major transcriptional rewiring of the SWEET family involving, only in roots, changes in 22 of the 35 members. None of the SWEETs showed mycorrhiza-exclusive induction and most of the 12 induced genes belong to the putative hexose transporters of clade I and II, while only two are putative sucrose transporters from clade III. In contrast, most of the repressed transcripts (10) corresponded to clade III SWEETs. Promoter-reporter assays for three of the induced genes, each from one cluster, showed re-localization of expression to arbuscule-containing cells, supporting a role for SWEETs in the supply of sugars at biotrophic interfaces. The complex transcriptional regulation of SWEETs in roots in response to AM fungal colonization supports a model in which symplastic sucrose in cortical cells could be cleaved in the cytoplasm by sucrose synthases or cytoplasmic invertases and effluxed as glucose, but also directly exported as sucrose and then converted into glucose and fructose by cell wall-bound invertases. Precise biochemical, physiological and molecular analyses are now required to profile the role of each potato SWEET in the arbuscular mycorrhizal symbiosis.

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.

Breaking down walls to live in harmony.

Some of the proteins and enzymes that allow bacteria to enter living fungal cells and cause rice seedling blight have been identified.

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.

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.