The phosphate starvation response regulator PHR2 antagonizes arbuscule maintenance in Medicago.

Phosphate starvation response (PHR) transcription factors play essential roles in regulating phosphate uptake in plants through binding to the P1BS cis-element in the promoter of phosphate starvation response genes. Recently, PHRs were also shown to positively regulate arbuscular mycorrhizal colonization in rice and lotus by controlling the expression of many symbiotic genes. However, their role in arbuscule development has remained unclear. In Medicago, we previously showed that arbuscule degradation is controlled by two SPX proteins that are highly expressed in arbuscule-containing cells. Since SPX proteins bind to PHRs and repress their activity in a phosphate-dependent manner, we investigated whether arbuscule maintenance is also regulated by PHR. Here, we show that PHR2 is a major regulator of the phosphate starvation response in Medicago. Knockout of phr2 showed reduced phosphate starvation response, symbiotic gene expression, and fungal colonization levels. However, the arbuscules that formed showed less degradation, suggesting a negative role for PHR2 in arbuscule maintenance. This was supported by the observation that overexpression of PHR2 led to enhanced degradation of arbuscules. Although many arbuscule-induced genes contain P1BS elements in their promoters, we found that the P1BS cis-elements in the promoter of the symbiotic phosphate transporter PT4 are not required for arbuscule-containing cell expression. Since both PHR2 and SPX1/3 negatively affect arbuscule maintenance, our results indicate that they control arbuscule maintenance partly via different mechanisms. While PHR2 potentiates symbiotic gene expression and colonization, its activity in arbuscule-containing cells needs to be tightly controlled to maintain a successful symbiosis in Medicago.

Discriminating symbiosis and immunity signals by receptor competition in rice.

Plants encounter various microbes in nature and must respond appropriately to symbiotic or pathogenic ones. In rice, the receptor-like kinase OsCERK1 is involved in recognizing both symbiotic and immune signals. However, how these opposing signals are discerned via OsCERK1 remains unknown. Here, we found that receptor competition enables the discrimination of symbiosis and immunity signals in rice. On the one hand, the symbiotic receptor OsMYR1 and its short-length chitooligosaccharide ligand inhibit complex formation between OsCERK1 and OsCEBiP and suppress OsCERK1 phosphorylating the downstream substrate OsGEF1, which reduces the sensitivity of rice to microbe-associated molecular patterns. Indeed, OsMYR1 overexpression lines are more susceptible to the fungal pathogen Magnaporthe oryzae, whereas Osmyr1 mutants show higher resistance. On the other hand, OsCEBiP can bind OsCERK1 and thus block OsMYR1-OsCERK1 heteromer formation. Consistently, the Oscebip mutant displayed a higher rate of mycorrhizal colonization at early stages of infection. Our results indicate that OsMYR1 and OsCEBiP receptors compete for OsCERK1 to determine the outcome of symbiosis and immunity signals.

A simple and versatile fluorochrome-based procedure for imaging of lipids in arbuscule-containing cells.

The arbuscular mycorrhizal (AM) symbiosis is characterized by the reciprocal exchange of nutrients. AM fungi are oleaginous microorganisms that obtain essential fatty acids from host plants. A lipid biosynthesis and delivery pathway has been proposed to operate in inner root cortex cells hosting arbuscules, a cell type challenging to access microscopically. Despite the central role lipids play in the association, lipid distribution patterns during arbuscule development are currently unknown. We developed a simple co-staining method employing fluorophore-conjugated Wheat Germ Agglutinin (WGA) and a lipophilic blue fluorochrome, Ac-201, for the simultaneous imaging of arbuscules and lipids distributed within arbuscule-containing cells in high resolution. We observed lipid distribution patterns in wild-type root infection zones in a variety of plant species. In addition, we applied this methodology to mutants of the Lotus japonicus GRAS transcription factor RAM1 and the Oryza sativa half-size ABC transporter STR1, both proposed to be impaired in the symbiotic lipid biosynthesis-delivery pathway. We found that lipids accumulated in cortical cells hosting stunted arbuscules in Ljram1 and Osstr1, and observed lipids in the arbuscule body of Osstr1, suggesting that in the corresponding plant species, RAM1 and STR1 may not be essential for symbiotic lipid biosynthesis and transfer from arbuscule-containing cells, respectively. The versatility of this methodology has the potential to help elucidate key questions on the complex lipid dynamics fostering AM symbioses.

Arbuscular-Mycorrhizal Symbiosis in Medicago Regulated by the Transcription Factor MtbHLHm1;1 and the Ammonium Facilitator Protein MtAMF1;3.

Root systems of most land plants are colonised by arbuscular mycorrhiza fungi. The symbiosis supports nutrient acquisition strategies predominantly associated with plant access to inorganic phosphate. The nutrient acquisition is enhanced through an extensive network of external fungal hyphae that extends out into the soil, together with the development of fungal structures forming specialised interfaces with root cortical cells. Orthologs of the bHLHm1;1 transcription factor, previously described in soybean nodules (GmbHLHm1) and linked to the ammonium facilitator protein GmAMF1;3, have been identified in Medicago (Medicago truncatula) roots colonised by AM fungi. Expression studies indicate that transcripts of both genes are also present in arbuscular containing root cortical cells and that the MtbHLHm1;1 shows affinity to the promoter of MtAMF1;3. Both genes are induced by AM colonisation. Loss of Mtbhlhm1;1 expression disrupts AM arbuscule abundance and the expression of the ammonium transporter MtAMF1;3. Disruption of Mtamf1;3 expression reduces both AM colonisation and arbuscule development. The respective activities of MtbHLHm1;1 and MtAMF1;3 highlight the conservation of putative ammonium regulators supporting both the rhizobial and AM fungal symbiosis in legumes.

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.

OsADK1, a novel kinase regulating arbuscular mycorrhizal symbiosis in rice.

Arbuscular mycorrhizal (AM) symbiosis relies on the formation of arbuscules for efficient nutrient exchange between plants and AM fungi. In this study, we identified a novel kinase gene in rice named OsADK1 (Arbuscule Development Kinase 1) that is required for arbuscule development. By obtaining OsADK1pro::GUS transgenic rice plants and also generating Osadk1 mutants via CRISPR/Cas9 technique, OsADK1 was revealed to be specifically induced in the arbusculated cortical cells and mutations in OsADK1 resulted in an extremely low colonisation rate (c. 3%) of rice roots by AM fungus Rhizophagus irregularis. In the mutant roots, the very few observed arbuscules nearly all arrested at an early 'trunk-forming' phase without forming any branches. Increasing the inoculum strength of AM fungus or cocultivation with a wild-type nurse plant did not result in the rescue of the arbuscule phenotype. Transcriptome sequencing of both nursed and un-nursed Osadk1 mutants then revealed that the mutation of OsADK1 could greatly affect the AM symbiotic programme, including many key transcription factors such as RAM1 and WRI5. OsADK1 therefore represents a new rice kinase that is required for arbuscule branching. Its identification opens a new window to explore the elaborate signal transduction pathway that determines arbuscule development during plant-fungus symbiosis.

A SPX domain-containing phosphate transporter from Rhizophagus irregularis handles phosphate homeostasis at symbiotic interface of arbuscular mycorrhizas.

Reciprocal symbiosis of > 70% of terrestrial vascular plants with arbuscular mycorrhizal (AM) fungi provides the fungi with fatty acids and sugars. In return, AM fungi facilitate plant phosphate (Pi) uptake from soil. However, how AM fungi handle Pi transport and homeostasis at the symbiotic interface of AM symbiosis is poorly understood. Here, we identify an SPX (SYG1/Pho81/XPR1) domain-containing phosphate transporter, RiPT7 from Rhizophagus irregularis. To characterize the RiPT7 transporter, we combined subcellular localization and heterologous expression studies in yeasts with reverse genetics approaches during the in planta phase. The results show that RiPT7 is conserved across fungal species and expressed in the intraradical mycelia. It is expressed in the arbuscules, intraradical hyphae and vesicles, independently of Pi availability. The plasma membrane-localized RiPT7 facilitates bidirectional Pi transport, depending on Pi gradient across the plasma membrane, whereas the SPX domain of RiPT7 inhibits Pi transport activity and mediates the vacuolar targeting of RiPT7 in yeast in response to Pi starvation. Importantly, RiPT7 silencing hampers arbuscule development of R. irregularis and symbiotic Pi delivery under medium- to low-Pi conditions. Collectively, our findings reveal a role for RiPT7 in fine-tuning of Pi homeostasis across the fungal membrane to maintain the AM development.

A phosphate starvation response-regulated receptor-like kinase, OsADK1, is required for mycorrhizal symbiosis and phosphate starvation responses.

Most land plants associate with arbuscular mycorrhizal (AM) fungi to secure mineral nutrient acquisition, especially that of phosphorus. A phosphate starvation response (PHR)-centered network regulates AM symbiosis. Here, we identified 520 direct target genes for the rice transcription factor OsPHR1/2/3 during AM symbiosis using transcriptome deep sequencing and DNA affinity purification sequencing. These genes were involved in strigolactone biosynthesis, transcriptional reprogramming, and bidirectional nutrient exchange. Moreover, we identified the receptor-like kinase, Arbuscule Development Kinase 1 (OsADK1), as a new target of OsPHR1/2/3. Electrophoretic mobility shift assays and transactivation assays showed that OsPHR2 can bind directly to the P1BS elements within the OsADK1 promoter to activate its transcription. OsADK1 appeared to be required for mycorrhizal colonization and arbuscule development. In addition, hydroponic experiments suggested that OsADK1 may be involved in plant Pi starvation responses. Our findings validate a role for OsPHR1/2/3 as master regulators of mycorrhizal-related genes involved in various stages of symbiosis, and uncover a new RLK involved in AM symbiosis and plant Pi starvation responses.

DLK2 regulates arbuscule hyphal branching during arbuscular mycorrhizal symbiosis.

D14 and KAI2 receptors enable plants to distinguish between strigolactones (SLs) and karrikins (KARs), respectively, in order to trigger appropriate environmental and developmental responses. Both receptors are related to the regulation of arbuscular mycorrhiza (AM) formation and are members of the RsbQ-like family of α,ß-hydrolases. DLK2 proteins, whose function remains unknown, constitute a third clade from the RsbQ-like protein family. We investigated whether the tomato SlDLK2 is a new regulatory component in the AM symbiosis. Genetic approaches were conducted to analyze SlDLK2 expression and to understand SlDLK2 function in AM symbiosis. We show that SlDLK2 expression in roots is AM-dependent and is associated with cells containing arbuscules. SlDLK2 ectopic expression arrests arbuscule branching and downregulates AM-responsive genes, even in the absence of symbiosis; while the opposite effect was observed upon SlDLK2 silencing. Moreover, SlDLK2 overexpression in Medicago truncatula roots showed the same altered phenotype observed in tomato roots. Interestingly, SlDLK2 interacts with DELLA, a protein that regulates arbuscule formation/degradation in AM roots. We propose that SlDLK2 is a new component of the complex plant-mediated mechanism regulating the life cycle of arbuscules in AM symbiosis.

A purple acid phosphatase, GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean.

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter-ß-glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule-containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild-type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.

Understanding dynamics of Rhizophagus irregularis ontogenesis in axenically developed coculture through basic and advanced microscopic techniques.

Detailed information on structural changes that occur during ontogenesis of Rhizophagus irregularis in axenically developed coculture is limited. Our study aims to investigate the series of events that occur during mycorrhizal ontogenesis under axenic condition through basic and advanced microscopic techniques followed by comparison among these to identify the suitable technique for rapid and detailed analysis of mycorrhizal structures. Three stages were identified in mycorrhizal ontogenesis from initiation (preinfection stage of hyphae; its branching, infection and appressoria formation; epidermal opening; and hyphal entry), progression (arbuscular development; hyphal coils and vesicles) to maturity (extraradical spores). Scanning electron microscopy was found to be an efficient tool for studying spatial three-dimensional progression. Adding to the advantages of advanced microscopy, potential of autofluorescence to explore the stages of symbiosis nondestructively was also established. We also report imaging of ultrathin sections by bright field microscopy to provide finer details at subcellular interface. Owing to the merits of nondestructive sampling, ease of sample preparation, autofluorescence (no dye required), no use of toxic chemicals, rapid analysis and in depth characterization confocal laser scanning microscopy was identified as the most preferred technique. The method thus developed can be used for detailed structural inquisition of mycorrhizal symbiosis both in in planta and in an in vitro system.

Salicylic acid improves arbuscular mycorrhizal symbiosis, and chickpea growth and yield by modulating carbohydrate metabolism under salt stress.

Salt stress is a major abiotic stress restricting plant growth and reproductive yield. Salicylic acid (SA) and arbuscular mycorrhizal (AM) symbioses play key roles in eliminating adverse effects of salt stress by modulating ion homeostasis and carbohydrate metabolism in crop plants. Sugars synthesized via carbohydrate metabolism act as osmotic adjustors and signaling molecules in activation of various defense responses against salt stress. The present study investigated the role of SA (0.5 mM) seed priming in establishment of AM symbiosis with Rhizoglomus intraradices and the impact on growth, ion-homeostasis, nutrient uptake, and sugar metabolism in Cicer arietinum L. (chickpea) genotypes under salt stress. Salinity had a negative correlation with plant growth and AM symbiosis in both genotypes with more negative effects in relatively salt-sensitive genotype than tolerant. SA enhanced the percent root colonization by significantly increasing the number of arbuscules and vesicles under salt stress. AM symbiosis was more effective in improving root biomass, root to shoot ratio, and nutrient acquisition than SA, while SA was more effective in maintaining ion equilibrium and modulating carbohydrate metabolism and reproductive yield when compared with AM inoculation. SA priming directed the utilization of total soluble sugars (TSS) towards reproductive attributes more efficiently than did AM inoculation by activating TSS metabolic consumption. In AM plants, TSS concentrations were more directed towards sink demand by the fungus itself rather than developing reproductive structures. SA priming further increased sugar export to roots of AM plants, thus favored AM symbiosis. Hence, SA seed priming-induced improvement in AM symbiosis can be a promising strategy in achieving sustainable production of chickpea genotypes under salt stress.

A symbiosis-dedicated SYNTAXIN OF PLANTS 13II isoform controls the formation of a stable host-microbe interface in symbiosis.

Arbuscular mycorrhizal (AM) fungi and rhizobium bacteria are accommodated in specialized membrane compartments that form a host-microbe interface. To better understand how these interfaces are made, we studied the regulation of exocytosis during interface formation. We used a phylogenetic approach to identify target soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (t-SNAREs) that are dedicated to symbiosis and used cell-specific expression analysis together with protein localization to identify t-SNAREs that are present on the host-microbe interface in Medicago truncatula. We investigated the role of these t-SNAREs during the formation of a host-microbe interface. We showed that multiple syntaxins are present on the peri-arbuscular membrane. From these, we identified SYNTAXIN OF PLANTS 13II (SYP13II) as a t-SNARE that is essential for the formation of a stable symbiotic interface in both AM and rhizobium symbiosis. In most dicot plants, the SYP13II transcript is alternatively spliced, resulting in two isoforms, SYP13IIα and SYP13IIß. These splice-forms differentially mark functional and degrading arbuscule branches. Our results show that vesicle traffic to the symbiotic interface is specialized and required for its maintenance. Alternative splicing of SYP13II allows plants to replace a t-SNARE involved in traffic to the plasma membrane with a t-SNARE that is more stringent in its localization to functional arbuscules.

Earliest colonization events of Rhizophagus irregularis in rice roots occur preferentially in previously uncolonized cells.

Arbuscular mycorrhizal (AM) fungi form a symbiotic association with several plant species. An arbuscule, a finely branched structure of AM fungi, is formed in root cells and plays essential roles in resource exchange. Because arbuscules are ephemeral, host cells containing collapsed arbuscules can be recolonized, and a wide region of roots can be continuously colonized by AM fungi, suggesting that repetitive recolonization in root cells is required for continuous mycorrhization. However, recolonization frequency has not been quantified because of the lack of appropriate markers for visualization of the cellular processes after arbuscule collapse; therefore, the nature of the colonization sequence remains uncertain. Here we observed that a green fluorescent protein (GFP)-tagged secretory carrier membrane protein (SCAMP) of rice was expressed even in cells with collapsed arbuscules, allowing live imaging coupled with GFP-SCAMP to evaluate the colonization and recolonization sequences. The average lifetime of intact arbuscules was 1-2 d. Cells with collapsed arbuscules were rarely recolonized and formed a new arbuscule during the observation period of 5 d, whereas de novo colonization occurred even in close proximity to cells containing collapsed arbuscules and contributed to the expansion of the colonized region. Colonization spread into an uncolonized region of roots but sparsely into a previously colonized region having no metabolically active arbuscule but several intercellular hyphae. Therefore, we propose that a previously colonized region tends to be intolerant to new colonization in rice roots. Our observations highlight the overlooked negative impact of the degeneration stage of arbuscules in the colonization sequence.

Nitrate regulates rhizobial and mycorrhizal symbiosis in common bean (Phaseolus vulgaris L.).

Nitrogen-limited conditions are considered to be a prerequisite for legume-rhizobial symbiosis, but the effects of nitrate-rich conditions on symbiotic status remain poorly understood. We addressed this issue by examining rhizobial (Rhizobim tropici) and arbusclar mycorrhizal (Glomus intraradices) symbiosis in Phaseolus vulgaris L. cv. Negro Jamapa under nitrate pre-incubation and continuous nitrate conditions. Our results indicate that nitrate pre-incubation, independent of the concentration, did not affect nodule development. However, the continuous supply of nitrate at high concentrations impaired nodule maturation and nodule numbers. Low nitrate conditions, in addition to positively regulating nodule number, biomass, and nitrogenase activity, also extended the span of nitrogen-fixing activity. By contrast, for arbuscular mycorrhizae, continuous 10 and 50 mmol/L nitrate increased the percent root length colonization, concomitantly reduced arbuscule size, and enhanced ammonia transport without affecting phosphate transport. Therefore, in this manuscript, we have proposed the importance of nitrate as a positive regulator in promoting both rhizobial and mycorrhizal symbiosis in the common bean.

Lipids Mediate Arbuscule Development and Senescence in Tomato Roots Colonized by Arbuscular Mycorrhizae Fungus under Drought Stress.

Arbuscular mycorrhizae (AM) symbiosis can enhance plant resistance to drought stress (DS). This study aimed to investigate the DS effects on lipids at different stages of symbiosis and to link lipid profiles to arbuscule dynamics in tomato roots colonized by AM fungi. DS increased mycorrhizal colonization and arbuscule abundance at an early stage but decreased them at a later stage, delayed arbuscule development, and accelerated arbuscule senescence at a later stage. DS decreased the contents of phospholipids (PLs) and saturated neutral lipids (NLs) at the early stage but increased the contents of saturated PLs and unsaturated NLs at the late stage. Specifically, DS inhibited AM-specific PL contents but increased AM-specific NL contents, which was supported by the expression of RAM2, STR/STR2. These data indicate the negative effect of DS on AM symbiosis and arbuscule dynamics with the effect size depending on the symbiosis stage, which highlights the importance of the symbiosis stage under abiotic stress.

Enhancing arbuscular mycorrhiza symbiosis effectiveness through the involvement of the tomato GRAS transcription factor SCL3/SlGRAS18.

Arbuscular mycorrhizal (AM) fungi improve plant growth, nutrition, fitness and stress tolerance while AM fungi obtain carbohydrates and lipids from the host. This whole process of mutual benefit requires substantial alterations in the structural and functional aspects of the host root cells. These modifications ultimately culminate in the formation of arbuscules, which are specialized intraradical and highly branched fungal structures. Arbuscule-containing cells undergo massive reprogramming to hosting arbuscule and members of the GRAS transcription factor family have been characterized as AM inducible genes which play a pivotal role in these process. Here, we show a functional analysis for the GRAS transcription factor SCL3/SlGRAS18 in tomato. SlGRAS18 interacts with SlDELLA, a central regulator of AM formation. Silencing of SlGRAS18 positively impacts arbuscule development and the improvement in symbiotic status, favouring flowering and therefore progress in the formation and development of fruits in SlGRAS18 silenced plants which parallel to a discernible pattern of mineral nutrient redistribution in leaves. Our results advance the knowledge of GRAS transcription factors involved in the formation and establishment of AM symbiosis and provide experimental evidence for how specific genetic alterations can lead to more effective AM symbiosis.

TETRASPANIN 8-1 from Phaseolus vulgaris plays a key role during mutualistic interactions.

Arbuscular mycorrhizal (AM) fungi and rhizobia form two of the most important plant-microbe associations for the assimilation of phosphorus (P) and nitrogen (N). Symbiont-derived signals are able to coordinate the infection process by triggering multiple responses in the plant root, such as calcium influxes and oscillations, increased reactive oxygen species (ROS), cytoskeletal rearrangements and altered gene expression. An examination was made of the role of tetraspanins, which are transmembrane proteins that self-organize into tetraspanin web regions, where they recruit specific proteins into platforms required for signal transduction, membrane fusion, cell trafficking, and ROS generation. In plant cells, tetraspanins are scaffolding proteins associated with root radial patterning, biotic and abiotic stress responses, cell fate determination, plasmodesmata and hormonal regulation. Some plant tetraspanins, such as Arabidopsis thaliana TETRASPANIN 8 and TETRASPANIN 9 (AtTET8 and AtTET9) are associated with exosomes during inter-kingdom communication. In this study, a homolog of AtTET8, PvTET8-1, in common bean (Phaseolus vulgaris L. var. Negro Jamapa) was examined in roots during interactions with Rhizobium tropici and Rhizophagus irregularis. The promoter of PvTET8-1 contained several cis-acting regulatory DNA elements potentially related to mutualistic interactions, and PvTET8-1 was transcriptionally activated during AM fungal and rhizobial associations. Silencing it decreased the size and number of nodules, nitrogen fixation, and mycorrhizal arbuscule formation, whereas overexpressing it increased the size and number of nodules, and mycorrhizal arbuscule formation but decreased nitrogen fixation. PvTET8-1 appears to be an important element in both of these mutualistic interactions, perhaps through its interaction with NADPH oxidase and the generation of ROS during the infection processes.