Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two symbiotic pea (Pisum sativum L.) mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread formation in symbiotic root nodules were characterised with respect to dynamics of arbuscule development at 15 degrees C and 24 degrees C. Mutation of sym33 decreased mycorrhiza colonisation at both temperatures and delayed arbuscule development at 15 degrees C, whereas mutation of sym40 accelerated mycorrhiza colonisation and arbuscule senescence at 24 degrees C. The differences between the mutants and the wild-type were more pronounced at 24 degrees C, a temperature close to the optimum for pea growth. The results demonstrate that both pea genes are important in the control of arbuscular mycorrhiza development and can be considered necessary for the tripartite symbiosis in pea.     

Expression profiling of up-regulated plant and fungal genes in early and late stages of<Emphasis Type="Italic"> Medicago truncatula-Glomus mosseae</Emphasis> interactions

Suppression subtractive hybridization (SSH), expression profiling and EST sequencing identified 12 plant genes and six fungal genes that are expressed in the arbuscular mycorrhizal symbiosis between Medicago truncatula and Glomus mosseae. All the plant genes and three of the fungal genes were up-regulated in symbiotic tissues. Expression of 15 of the genes is described for the first time in mycorrhizal roots and two are novel sequences. Six M. truncatula genes were also activated during appressorium formation at the root surface, suggesting a role in this early stage of mycorrhiza establishment, whilst the other six plant genes were only induced in the late stages of mycorrhization and could be involved in the development or functioning of the symbiosis. Phosphate fertilization had no significant influence on expression of any of the plant genes. Expression profiling of G. mosseae genes indicated that two of them may be associated with appressorium development on roots and one with arbuscule formation or function. The other three fungal genes were expressed throughout the life-cycle of G. mosseae.     

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.     

Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus

In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus . A detailed expression analysis by RT-PCR revealed that SbtM1 , SbtM3 , SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter–reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

Effect of mutations in the pea genes <Emphasis Type="Italic">Sym33</Emphasis> and <Emphasis Type="Italic">Sym40</Emphasis>

Two pea (Pisum sativum L.) symbiotic mutants SGEFix(-)-1 (sym40) and SGEFix(-)-2 (sym33) with abnormalities in infection thread development and function in symbiotic root nodules have been characterised in terms of mycorrhizal colonisation of roots, shoot and root biomass accumulation and shoot and root phosphorus (P) content. The mutation in gene sym33 decreased mycorrhizal colonisation of roots (except arbuscule abundance in mycorrhizal root fragments, which increased) but did not change the effectiveness of mycorrhiza function. The mutation in sym40 did not affect either of these processes. Both mutants showed differences in plant development compared with the wild-type line SGE. The mutants had delayed flowering and pod ripening, and shoot/root biomass ratios and P accumulation also differed from those of SGE. These observations suggest that the gene mutations cause systemic changes in plant development.     

Accumulation of secondary compounds in barley and wheat roots in response to inoculation with an arbuscular mycorrhizal fungus and co-inoculation with rhizosphere bacteria

 Colonization of Hordeum vulgare L. cv. Salome (barley)and Triticum aestivum L. cv. Caprimus (wheat) roots by the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith leads to de novo synthesis of isoprenoid cyclohexenone derivatives with blumenin [9-O-(2′-O-β-glucuronosyl)-β-glucopyranoside of 6-(3-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one] as the major constituent and to transient accumulation of hydroxycinnamate amides (4-coumaroylagmatine and -putrescine). Accumulation of these compounds in mycorrhizal wheat roots started 2 weeks after sowing together with the onset of arbuscule formation and proceeded with mycorrhizal progression. Highest levels were reached in 3- to 4-week-old secondary roots (root branches of first and higher order) characterized by the formation of vesicles. In the final developmental stages, the fungus produced massive amounts of spores, enclosing the stele of older root parts (older than 5 weeks) characterized by cortical death. In these root parts, the secondary compounds were detected in trace amounts only, indicating that they were located in the cortical tissues. Some rhizosphere bacteria tested, i.e. Agrobacterium rhizogenes, Pseudomonas fluorescens, and Rhizobium leguminosarum, markedly stimulated both fungal root colonization and blumenin accumulation, thus, acting as mycorrhiza-helper bacteria (MHB). Application of blumenin itself strongly inhibited fungal colonization and arbuscule formation at early stages of mycorrhiza development. This was associated with a markedly reduced accumulation of the hydroxycinnamate amides 4-coumaroylputrescine and -agmatine. The results suggest that both the isoprenoid and the phenylpropanoid metabolism are closely linked to the developmental stage and the extent of fungal colonization. Their possible involvement in the regulation of mycorrhiza development is discussed. Accepted: 18 September 1998     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: A review of basic and applied aspects

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls the development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.     

The Jasmonic Acid Signalling Pathway Restricts the Development of the Arbuscular Mycorrhizal Association in Tomato

The role of the jasmonate signalling pathway in modulating the establishment of the arbuscular mycorrhiza (AM) symbiosis between tomato plants and Glomus intraradices fungus was studied. The consequences of AM formation due to the blockage of the jasmonate signalling pathway were studied in experiments with plant mutants impaired in JA perception. The tomato jai-1 mutant (jasmonic acid insensitive 1) failed to regulate colonization and was more susceptible to fungal infection, showing accelerated colonization. The frequency and the intensity of fungal colonization were greatly increased in the jai-1 insensitive mutant plants. In parallel, the systemic effects on mycorrhization due to the activation of the jasmonate signalling pathway by foliar application of MeJA were evaluated and histochemical and molecular parameters of mycorrhizal intensity and efficiency were measured. Histochemical determination of fungal infectivity and fungal alkaline phosphatase activity reveal that the systemic application of MeJA was effective in reducing mycorrhization and mainly affected fungal phosphate metabolism and arbuscule formation, analyzed by the expression of GiALP and the AM-specific gene LePT4, respectively. The results of the present study clearly show that JA participates in the susceptibility of tomato to infection by arbuscular mycorrhizal fungi, and it seems that arbuscular colonization in tomato is tightly controlled by the jasmonate signalling pathway.     

An Indigenous Drought-Tolerant Strain of Glomus intraradices Associated with a Native Bacterium Improves Water Transport and Root Development in Retama sphaerocarpa

The effects of interactions between Bacillus thuringiensis, a drought-adapted bacterium, and two isolates of Glomus intraradices, an arbuscular mycorrhizal (AM) fungus, on Retama sphaerocarpa, a drought-adapted legume, were investigated. The fungal isolates were an indigenous drought-tolerant and a nonindigenous drought-sensitive isolate. Shoot length and root growth, symbiotic parameters, water transport (in terms of percent relative plant water uptake), and volumetric soil moisture and soil enzymatic activities in response to microbial inoculations were evaluated. Retama plants colonized by G. intraradices plus Bacillus possessed similar shoot length after 30 days from sowing compared with noninoculated Retama plants after 150 days. Inoculation with drought-adapted bacterium increased root growth by 201%, but maximum root development was obtained by co-inoculation of B. thuringiensis and the indigenous G. intraradices. Nodules were formed only in plants colonized by autochthonous AM fungi. Relative water uptake was higher in inoculated than in noninoculated Retama plants, and these inoculants depleted soil water content concomitantly. G. intraradices-colonized Retama reached similar shoot length irrespective of the fungal origin, but there were strong differences in relative water uptake by plants colonized by each one of the fungi. Indigenous G. intraradices-colonized roots (evaluated as functional alkaline phosphatase staining) showed the highest intensity and arbuscule richness when associated with B. thuringiensis. The interactive microbial effects on Retama plants were more relevant when indigenous microorganisms were involved. Co-inoculation of autochthonous microorganisms reduced by 42% the water required to produce 1 mg of shoot biomass. This is the first evidence of the effectiveness of rhizosphere bacterium, singly or associated with AM fungus, in increasing plant water uptake, which represents a positive microbial effect on plants grown under drought environments.     

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod⁻, Myc⁺). Received: 3 March 1997 / Accepted: 12 May 1997     

Fungal genes related to calcium homeostasis and signalling are upregulated in symbiotic arbuscular mycorrhiza interactions

Fluctuations in intracellular calcium levels generate signalling events and regulate different cellular processes. Whilst the implication of Ca²⁺ in plant responses during arbuscular mycorrhiza (AM) interactions is well documented, nothing is known about the regulation or role of this secondary messenger in the fungal symbiont. The spatio-temporal expression pattern of putatively Ca²⁺-related genes of Glomus intraradices BEG141 encoding five proteins involved in membrane transport and one nuclear protein kinase, was investigated during the AM symbiosis. Expression profiles related to successful colonization of host roots were observed in interactions of G. intraradices with roots of wild-type Medicago truncatula (line J5) compared to the mycorrhiza-defective mutant dmi3/Mtsym13. Symbiotic fungal activity was monitored using stearoyl-CoA desaturase and phosphate transporter genes. Laser microdissection based-mapping of fungal gene expression in mycorrhizal root tissues indicated that the Ca²⁺-related genes were differentially upregulated in arbuscules and/or in intercellular hyphae. The spatio-temporal variations in gene expression suggest that the encoded proteins may have different functions in fungal development or function during symbiosis development. Full-length cDNA obtained for two genes with interesting expression profiles confirmed a close similarity with an endoplasmic reticulum P-type ATPase and a Vcx1-like vacuolar Ca²⁺ ion transporter functionally characterized in other fungi and involved in the regulation of cell calcium pools. Possible mechanisms are discussed in which Ca²⁺-related proteins G. intraradices BEG141 may play a role in mobilization and perception of the intracellular messenger by the AM fungus during symbiotic interactions with host roots.     

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.     

Regulatory genes of garden pea (Pisum sativum L.) controlling the development of nitrogen-fixing nodules and arbuscular mycorrhiza: a review of basic and applied aspects

The review sums up the long experience of the authors and other researchers in studying the genetic system of garden pea (Pisum sativum L.), which controls sthe development of nitrogen-fixing symbiosis and arbuscular mycorrhiza. A justified phenotypic classification of pea mutants is presented. Progress in identifying and cloning symbiotic genes is adequately reflected. The feasibility of using double inoculation as a means of increasing the plant productivity is demonstrated, in which the potential of a tripartite symbiotic system (pea plants-root nodule bacteria-arbuscular mycorrhiza) is mobilized.     

The Lotus japonicus acyl‐acyl carrier protein thioesterase FatM is required for mycorrhiza formation and lipid accumulation of Rhizophagus irregularis

Arbuscular mycorrhiza (AM) fungi establish symbiotic interactions with plants, providing the host plant with minerals, i.e. phosphate, in exchange for organic carbon. Arbuscular mycorrhiza fungi of the order Glomerales produce vesicles which store lipids as an energy and carbon source. Acyl‐acyl carrier protein (ACP) thioesterases (Fat) are essential components of the plant plastid‐localized fatty acid synthase and determine the chain length of de novo synthesized fatty acids. In addition to the ubiquitous FatA and FatB thioesterases, AM‐competent plants contain an additional, AM‐specific, FatM gene. Here, we characterize FatM from Lotus japonicus by phenotypically analyzing fatm mutant lines and by studying the biochemical function of the recombinant FatM protein. Reduced shoot phosphate content in fatm indicates compromised symbiotic phosphate uptake due to reduced arbuscule branching, and the fungus shows reduced lipid accumulation accompanied by the occurrence of smaller and less frequent vesicles. Lipid profiling reveals a decrease in mycorrhiza‐specific phospholipid forms, AM fungal signature fatty acids (e.g. 16:1ω5, 18:1ω7 and 20:3) and storage lipids. Recombinant FatM shows preference for palmitoyl (16:0)‐ACP, indicating that large amounts of 16:0 fatty acid are exported from the plastids of arbuscule‐containing cells. Stable isotope labeling with [¹³C₂]acetate showed reduced incorporation into mycorrhiza‐specific fatty acids in the fatm mutant. Therefore, colonized cells reprogram plastidial de novo fatty acid synthesis towards the production of extra amounts of 16:0, which is in agreement with previous results that fatty acid‐containing lipids are transported from the plant to the fungus.