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‐β‐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.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

Linking lipid transfer with reduced arbuscule formation in tomato roots colonized by arbuscular mycorrhizal fungus under low pH stress

Arbuscules are the core structures of arbuscular mycorrhizae (AM), and arbuscule development is regulated by environmental stress, e.g., low pH. Recent studies indicate that lipid transfer from plants is essential for AM fungal colonization; however, the role of lipid transfer in arbuscule formation and the dynamics of lipid accumulation in arbuscules under low pH stress are far from well understood. In the symbiosis of tomato and Rhizophagus intraradices under contrasting pH conditions (pH 4.5 vs. pH 6.5), we investigated arbuscule formation, nutrient uptake, alkaline phosphatase activity and lipid accumulation; examined the gene expression involved in phosphate transport, lipid biosynthesis and transfer and sugar metabolism; and visualized the lipid dynamics in arbuscules. Low pH greatly inhibited arbuscule formation, in parallel with reduced phospholipid fatty acids accumulation in AM fungus and decreased P uptake. This reduction was supported by the decreased expression of plant genes encoding lipid biosynthesis and transfer. More degenerating arbuscules were observed under low pH conditions, and neutral lipid fatty acids accumulated only in degenerating arbuscules. These data reveal that, under low pH stress, reduced lipid transfer from hosts to AM fungi is responsible for the inhibited arbuscule formation.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

Two Medicago truncatula Half-ABC Transporters Are Essential for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis.     

Characterization of Mycorrhizas Formed by Glomus sp. on Roots of Hypernodulating Mutants of Lotus japonicus

Lotus japonicus hypernodulating mutants, Ljsym78-1 and Ljsym78-2, by the arbuscular mycorrhizal fungus Glomus sp. was characterized. The mutants are defective in systemic autoregulation of nodulation and nitrate inhibition, and form an excess of nodules and lateral roots. The percent root length colonized by the arbuscular mycorrhizal fungi was significantly higher for the mutant than wild-type roots. Detailed assessment of the colonization indicated that the percentage of colonization by arbuscules was increased, but that by external hyphae, internal hyphae and vesicles was decreased, in the mutant roots compared with the wild-type. The succinate dehydrogenase activity of arbuscules, external hyphae and internal hyphae showed similar trends. In addition, the majority of individual arbuscules that formed on the mutant roots had a well-developed and seemingly tough morphology. The results suggest that mutation at the Ljsym78 locus positively stimulates the growth and activity of arbuscules, but leads to reduced growth and activity of hyphae. We report the first identification of Lotus japonicus mutants that show significantly increased arbuscule formation and termed these mutants Arb⁺⁺. Received 8 August 2000/ Accepted in revised form 19 October 2000     

The PAM1 gene of petunia,required for intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi,encodes a homologue of VAPYRIN

Most terrestrial plants engage into arbuscular mycorrhizal (AM) symbiosis with fungi of the phylum Glomeromycota. The initial recognition of the fungal symbiont results in the activation of a symbiosis signalling pathway that is shared with the root nodule symbiosis (common SYM pathway). The subsequent intracellular accommodation of the fungus, and the elaboration of its characteristic feeding structures, the arbuscules, depends on a genetic programme in the plant that has recently been shown to involve the VAPYRIN gene in Medicaco truncatula. We have previously identified a mutant in Petunia hybrida, penetration and arbuscule morphogenesis 1 (pam1), that is defective in the intracellular stages of AM development. Here, we report on the cloning of PAM1, which encodes a VAPYRIN homologue. PAM1 protein localizes to the cytosol and the nucleus, with a prominent affinity to mobile spherical structures that are associated with the tonoplast, and are therefore referred to as tonospheres. In mycorrhizal roots, tonospheres were observed in the vicinity of intracellular hyphae, where they may play an essential role in the accommodation and morphogenesis of the fungal endosymbiont.     

A petunia mutant affected in intracellular accommodation and morphogenesis of arbuscular mycorrhizal fungi

The regulation of the arbuscular mycorrhizal (AM) symbiosis is largely under the control of a genetic programme of the plant host. This programme includes a common symbiosis signalling pathway that is shared with the root nodule symbiosis. Whereas this common pathway has been investigated in detail, little is known about the mycorrhiza-specific regulatory steps upstream and downstream of the common pathway. To get further insight in the regulation of the AM symbiosis, a transposon-mutagenized population of Petunia hybrida was screened for mutants with defects in AM development. Here, we describe a petunia mutant, penetration and arbuscule morphogenesis1 (pam1), which is characterized by a strong decrease in colonization by three different AM fungi. Penetrating hyphae are frequently aborted in epidermal cells. Occasionally the fungus can progress to the cortex, but fails to develop arbuscules. The resulting hyphal colonization of the cortex in mutant plants does not support symbiotic acquisition of phosphate and copper by the plant. Expression analysis of three petunia orthologues of the common SYM genes LjPOLLUX, LjSYMRK and MtDMI3 indicates that pam1 is not mutated in these genes. We conclude that the PAM1 gene may play a specific role in intracellular accommodation and morphogenesis of the fungal endosymbiont.     

Interactions between Lotus japonicus genotypes and arbuscular mycorrhizal fungi

Abstract

Interactions between three genotypes (Ljsym 71-1, Ljsym 71-2 and Ljsym 72) of Lotus japoicus and one isolate from each of four species of arbuscular mycorrhizal fungi (Glomus sp. R-10, Glomus intraradices, Glomus etunicatum, and Gigaspora margarita) were investigated and compared with the wild-type ‘Gifu’ B-129. All the three genotypes showed no or defective internal colonization after inoculation with these AM fungi. In Ljsym72 mutant, the AM fungi produced deformed appressoria on the root surface, but failed to form any internal structures (internal hyphae, arbuscules and vesicles) except only in Glomus intraradices. The Ljsym71-1 and Ljsym71-2 mutants had more deformed appressoria and occasionally formed internal hyphae, arbuscules and vesicles, depending on AM fungi used. Wild-type ‘Gifu’ (nod⁺myc⁺) plants had typical colonization. The colonization of mutants by several fungi varied and provides a basis for studying recognition and compatibility between plants and mycorrhizal fungal species. These mutants also will be useful in studies of the genetics of the symbiosis between plant species and AM fungi.     

Isolation of two different phenotypes of mycorrhizal mutants in the model legume plant Lotus japonicus after EMS-treatment

Lotus japonicus has been proposed as a model plant for the molecular genetic study of plant-microbe interaction including Mesorhizobium loti and arbuscular mycorrhizal (AM) fungi. Non-mycorrhizal mutants of Lotus japonicus were screened from a collection of 12 mutants showing non-nodulating (Nod-), ineffectively nodulating (Fix-) and hypernodulating (Nod++) phenotypes with monogenic recessive inheritance induced by EMS (ethylmethane sulfonate) mutagenesis. Three mycorrhizal mutant lines showing highly reduced arbuscular mycorrhizal colonization were obtained. All of them were derived from Nod- phenotypes. In Ljsym72, the root colonization by Glomus sp. R-10 is characterized by poor development of the external mycelium, formation of extremely branched appressoria, and the blocking of hyphal penetration at the root epidermis. Neither arbuscules nor vesicles were formed in Ljsym72 roots. Fungal recognition on the root surface was strongly affected by the mutation in the LjSym72 gene. Unique characteristics in mutant lines Ljsym71-1 and Ljsym71-2 were the overproduction of deformed appressoria and arrested hyphal penetration of the exodermis. Small amounts of internal colonization including degenerated arbuscule formation occurred infrequently in these types of mutants. Not only fungal development on the root surface but also that in the root exodermis and cortex was affected by the mutation in LjSym71 gene. These mutants represent a key advance in molecular research on the AM symbiosis.     

A set of fluorescent protein‐based markers expressed from constitutive and arbuscular mycorrhiza‐inducible promoters to label organelles,membranes and cytoskeletal elements in Medicago truncatula

Medicago truncatula is widely used for analyses of arbuscular mycorrhizal (AM) symbiosis and nodulation. To complement the genetic and genomic resources that exist for this species, we generated fluorescent protein fusions that label the nucleus, endoplasmic reticulum, Golgi apparatus, trans‐Golgi network, plasma membrane, apoplast, late endosome/multivesicular bodies (MVB), transitory late endosome/ tonoplast, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin, microtubules, periarbuscular membrane (PAM) and periarbuscular apoplastic space (PAS) and expressed them from the constitutive AtUBQ10 promoter and the AM symbiosis‐specific MtBCP1 promoter. All marker constructs showed the expected expression patterns and sub‐cellular locations in M. truncatula root cells. As a demonstration of their utility, we used several markers to investigate AM symbiosis where root cells undergo major cellular alterations to accommodate their fungal endosymbiont. We demonstrate that changes in the position and size of the nuclei occur prior to hyphal entry into the cortical cells and do not require DELLA signaling. Changes in the cytoskeleton, tonoplast and plastids also occur in the colonized cells and in contrast to previous studies, we show that stromulated plastids are abundant in cells with developing and mature arbuscules, while lens‐shaped plastids occur in cells with degenerating arbuscules. Arbuscule development and secretion of the PAM creates a periarbuscular apoplastic compartment which has been assumed to be continuous with apoplast of the cell. However, fluorescent markers secreted to the periarbuscular apoplast challenge this assumption. This marker resource will facilitate cell biology studies of AM symbiosis, as well as other aspects of legume biology.     

A Medicago truncatula mutant hyper-responsive to mycorrhiza and defective for nodulation

One key strategy for the identification of plant genes required for mycorrhizal development is the use of plant mutants affected in mycorrhizal colonisation. In this paper, we report a new Medicago truncatula mutant defective for nodulation but hypermycorrhizal for symbiosis development and response. This mutant, called B9, presents a poor shoot and, especially, root development with short laterals. Inoculation with Glomus intraradices results in significantly higher root colonisation of the mutant than the wild-type genotype A17 (+20% for total root length, +16% for arbuscule frequency in the colonised part of the root, +39% for arbuscule frequency in the total root system). Mycorrhizal effects on shoot and root biomass of B9 plants are about twofold greater than in the wild-type genotype. The B9 mutant of M. truncatula is characterised by considerably higher root concentrations of the phytoestrogen coumestrol and by the novel synthesis of the coumestrol conjugate malonyl glycoside, absent from roots of wild-type plants. In conclusion, this is the first time that a hypermycorrhizal plant mutant affected negatively for nodulation (Myc⁺⁺, Nod ^−/+ phenotype) is reported. This mutant represents a new tool for the study of plant genes differentially regulating mycorrhiza and nodulation symbioses, in particular, those related to autoregulation mechanisms.     

Polygalacturonase activity and location in arbuscular mycorrhizal roots of Allium porrum L.

Polygalacturonase activity and location were analysed in leek roots (Allium porrum L.) colonized by Glomus versiforme (Karst.) Berch, an arbuscular mycorrhizal (AM) fungus. Polygalacturonase activity in mycorrhizal roots did not differ quantitatively from that found in nonmycorrhizal roots on all of the four harvesting dates. Fractionation of mycorrhizal root extracts by ion-exchange chromatography showed that expression of polygalacturonase was specific to the mutualistic association. Immunofluorescence and immunogold experiments were carried out to locate the polygalacturonase in mycorrhizal roots using a polyclonal antibody raised against a Fusarium moniliforme endopolygalacturonase. Immunolabelling was observed all over the arbuscules (intracellular fungal structures) but particularly at the interface between the arbuscule and the plant membrane. Since pectins are located in this area, we suggest that polygalacturonase produced during the symbiosis could play a role in plant pectin degradation.     

Nitrate induction of root hair density is mediated by TGA1/TGA4 and CPC transcription factors in Arabidopsis thaliana

Root hairs are specialized cells that are important for nutrient uptake. It is well established that nutrients such as phosphate have a great influence on root hair development in many plant species. Here we investigated the role of nitrate on root hair development at a physiological and molecular level. We showed that nitrate increases root hair density in Arabidopsis thaliana. We found that two different root hair defective mutants have significantly less nitrate than wild‐type plants, suggesting that in A. thaliana root hairs have an important role in the capacity to acquire nitrate. Nitrate reductase‐null mutants exhibited nitrate‐dependent root hair phenotypes comparable with wild‐type plants, indicating that nitrate is the signal that leads to increased formation of root hairs. We examined the role of two key regulators of root hair cell fate, CPC and WER, in response to nitrate treatments. Phenotypic analyses of these mutants showed that CPC is essential for nitrate‐induced responses of root hair development. Moreover, we showed that NRT1.1 and TGA1/TGA4 are required for pathways that induce root hair development by suppression of longitudinal elongation of trichoblast cells in response to nitrate treatments. Our results prompted a model where nitrate signaling via TGA1/TGA4 directly regulates the CPC root hair cell fate specification gene to increase formation of root hairs in A. thaliana.