Phylogenetic, structural, and functional characterization of AMT3;1, an ammonium transporter induced by mycorrhization among model grasses.

In the arbuscular mycorrhizal (AM) symbiosis, plants satisfy part of their nitrogen (N) requirement through the AM pathway. In sorghum, the ammonium transporters (AMT) AMT3;1, and to a lesser extent AMT4, are induced in cells containing developing arbuscules. Here, we have characterized orthologs of AMT3;1 and AMT4 in four other grasses in addition to sorghum. AMT3;1 and AMT4 orthologous genes are induced in AM roots, suggesting that in the common ancestor of these five plant species, both AMT3;1 and AMT4 were already present and upregulated upon AM colonization. An artificial microRNA approach was successfully used to downregulate either AMT3;1 or AMT4 in rice. Mycorrhizal root colonization and hyphal length density of knockdown plants were not affected at that time, indicating that the manipulation did not modify the establishment of the AM symbiosis and the interaction between both partners. However, expression of the fungal phosphate transporter FmPT was significantly reduced in knockdown plants, indicating a reduction of the nutrient fluxes from the AM fungus to the plant. The AMT3;1 knockdown plants (but not the AMT4 knockdown plants) were significantly less stimulated in growth by AM fungal colonization, and uptake of both 15N and 33P from the AM fungal network was reduced. This confirms that N and phosphorus nutrition through the mycorrhizal pathway are closely linked. But most importantly, it indicates that AMT3;1 is the prime plant transporter involved in the mycorrhizal ammonium transfer and that its function during uptake of N cannot be performed by AMT4.

The family of ammonium transporters (AMT) in Sorghum bicolor: two AMT members are induced locally, but not systemically in roots colonized by arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi contribute to plant nitrogen (N) acquisition. Recent studies demonstrated the transport of N in the form of ammonium during AM symbiosis. Here, we hypothesize that induction of specific ammonium transporter (AMT) genes in Sorghum bicolor during AM colonization might play a key role in the functionality of the symbiosis. For the first time, combining a split-root experiment and microdissection technology, we were able to assess the precise expression pattern of two AM-inducible AMTs, SbAMT3;1 and SbAMT4. Immunolocalization was used to localize the protein of SbAMT3;1. The expression of SbAMT3;1 and SbAMT4 was greatly induced locally in root cells containing arbuscules and in adjacent cells. However, a split-root experiment revealed that this induction was not systemic. By contrast, a strictly AM-induced phosphate transporter (SbPt11) was expressed systemically in the split-root experiment. However, a gradient of expression was apparent. Immunolocalization analyses demonstrated that SbAMT3;1 was present only in cells containing developing arbuscules. Our results show that the SbAMT3;1 and SbAMT4 genes are expressed in root cortical cells, which makes them ready to accommodate arbuscules, a process of considerable importance in view of the short life span of arbuscules. Additionally, SbAMT3;1 might play an important role in N transfer during AM symbiosis.

Local and systemic mycorrhiza-induced protection against the ectoparasitic nematode Xiphinema index involves priming of defence gene responses in grapevine.

The ectoparasitic dagger nematode (Xiphinema index), vector of Grapevine fanleaf virus (GFLV), provokes gall formation and can cause severe damage to the root system of grapevines. Mycorrhiza formation by Glomus (syn. Rhizophagus) intraradices BEG141 reduced both gall formation on roots of the grapevine rootstock SO4 (Vitis berlandieri×V. riparia) and nematode number in the surrounding soil. Suppressive effects increased with time and were greater when the nematode was post-inoculated rather than co-inoculated with the arbuscular mycorrhizal (AM) fungus. Using a split-root system, decreased X. index development was shown in mycorrhizal and non-mycorrhizal parts of mycorrhizal root systems, indicating that both local and systemic induced bioprotection mechanisms were active against the ectoparasitic nematode. Expression analyses of ESTs (expressed sequence tags) generated in an SSH (subtractive suppressive hybridization) library, representing plant genes up-regulated during mycorrhiza-induced control of X. index, and of described grapevine defence genes showed activation of chitinase 1b, pathogenesis-related 10, glutathione S-transferase, stilbene synthase 1, 5-enolpyruvyl shikimate-3-phosphate synthase, and a heat shock proein 70-interacting protein in association with the observed local and/or systemic induced bioprotection against the nematode. Overall, the data suggest priming of grapevine defence responses by the AM fungus and transmission of a plant-mediated signal to non-mycorrhizal tissues. Grapevine gene responses during AM-induced local and systemic bioprotection against X. index point to biological processes that are related either to direct effects on the nematode or to protection against nematode-imposed stress to maintain root tissue integrity.

An STE12 gene identified in the mycorrhizal fungus Glomus intraradices restores infectivity of a hemibiotrophic plant pathogen.

Mechanisms of root penetration by arbuscular mycorrhizal (AM) fungi are unknown and investigations are hampered by the lack of transformation systems for these unculturable obligate biotrophs. Early steps of host infection by hemibiotrophic fungal phytopathogens, sharing common features with those of AM fungal colonization, depend on the transcription factor STE12. Using degenerated primers and rapid amplification of cDNA ends, we isolated the full-length cDNA of an STE12-like gene, GintSTE, from Glomus intraradices and profiled GintSTE expression by real-time and in situ RT-PCR. GintSTE activity and function were investigated by heterologous complementation of a yeast ste12Delta mutant and a Colletotrichum lindemuthianum clste12Delta mutant. * Sequence data indicate that GintSTE is similar to STE12 from hemibiotrophic plant pathogens, especially Colletotrichum spp. Introduction of GintSTE into a noninvasive mutant of C. lindemuthianum restored fungal infectivity of plant tissues. GintSTE expression was specifically localized in extraradicular fungal structures and was up-regulated when G. intraradices penetrated roots of wild-type Medicago truncatula as compared with an incompatible mutant. Results suggest a possible role for GintSTE in early steps of root penetration by AM fungi, and that pathogenic and symbiotic fungi may share common regulatory mechanisms for invasion of plant tissues.

Preferential colonization of Solanum tuberosum L. roots by the fungus Glomus intraradices in arable soil of a potato farming area.

The symbiosis between plant roots and arbuscular mycorrhizal (AM) fungi has been shown to affect both the diversity and productivity of agricultural communities. In this study, we characterized the AM fungal communities of Solanum tuberosum L. (potato) roots and of the bulk soil in two nearby areas of northern Italy, in order to verify if land use practices had selected any particular AM fungus with specificity to potato plants. The AM fungal large-subunit (LSU) rRNA genes were subjected to nested PCR, cloning, sequencing, and phylogenetic analyses. One hundred eighty-three LSU rRNA sequences were analyzed, and eight monophyletic ribotypes, belonging to Glomus groups A and B, were identified. AM fungal communities differed between bulk soil and potato roots, as one AM fungal ribotype, corresponding to Glomus intraradices, was much more frequent in potato roots than in soils (accounting for more than 90% of sequences from potato samples and less than 10% of sequences from soil samples). A semiquantitative heminested PCR with specific primers was used to confirm and quantify the AM fungal abundance observed by cloning. Overall results concerning the biodiversity of AM fungal communities in roots and in bulk soils from the two studied areas suggested that potato roots were preferentially colonized by one AM fungal species, G. intraradices.

Differential Signaling and Sugar Exchanges in Response to Avirulent Pathogen- and Symbiont-Derived Molecules in Tobacco Cells.

Plants interact with microbes whose ultimate aim is to exploit plant carbohydrates for their reproduction. Plant-microbe interactions (PMIs) are classified according to the nature of their trophic exchanges: while mutualistic microbes trade nutrients with plants, pathogens unilaterally divert carbohydrates. The early responses following microbe recognition and the subsequent control of plant sugar distribution are still poorly understood. To further decipher PMI functionality, we used tobacco cells treated with microbial molecules mimicking pathogenic or mutualistic PMIs, namely cryptogein, a defense elicitor, and chitotetrasaccharide (CO4), which is secreted by mycorrhizal fungi. CO4 was perceived by tobacco cells and triggered widespread transient signaling components such as a sharp cytosolic Ca2+ elevation, NtrbohD-dependent H2O2 production, and MAP kinase activation. These CO4-induced events differed from those induced by cryptogein, i.e., sustained events leading to cell death. Furthermore, cryptogein treatment inhibited glucose and sucrose uptake but not fructose uptake, and promoted the expression of NtSUT and NtSWEET sugar transporters, whereas CO4 had no effect on sugar uptake and only a slight effect on NtSWEET2B expression. Our results suggest that microbial molecules induce different signaling responses that reflect microbial lifestyle and the subsequent outcome of the interaction.

Impact of a pesticide cocktail (fenhexamid, folpel, deltamethrin) on the abundance of Glomeromycota in two agricultural soils.

Pesticide contamination of the environment can result from agricultural practices. Persistence of pesticide residues is a threat to the soil biota including plant roots and beneficial microorganisms, which support an important number of soil ecosystem services. Arbuscular mycorrhizal fungi (AMF) are key symbiotic microorganisms contributing to plant nutrition. In the present study, we assessed whether AMF could indicate eventual side effects of pesticides when directly applied to field soils. We evaluated the ecotoxicological impact of a cocktail of three commonly used agricultural pesticides (fenhexamid, folpel, deltamethrin) on the abundance and composition of the AMF community in vineyard (Montagne de Saint-Emilion) and arable (Martincourt) soils subjected to different agricultural practices. The dissipation of applied pesticides was monitored by multiresidual analyses to determine the scenario of exposure of the AMF community. Diversity analysis before application of the pesticide cocktail showed that the AMF communities of vineyard soils, subjected to mechanical weeding or grass cover, and of the arable soil subjected to intensive agriculture, were dominated by Glomerales. Ribotypes specific to each soil and to each agricultural practice in the same soil were found, with the highest abundance and diversity of AMF being observed in the vineyard soil with a grass-cover. The abundance of the global AMF community (Glomeromycota) and of three taxa of AMF (Funneliformis mosseae, Claroideoglomus etunicatum/C. claroideum) was evaluated after pesticide application. The abundance of Glomeromycota decreased in both soils after pesticide application while the abundance of Claroideoglomus and F. mosseae decreased only in the arable soil. These results show that higher doses of pesticide exposure did not affect the global abundance, but altered the composition, of the AMF community. Resilience of the AMF community composition was observed only in the vineyard soil, where F. mosseae was the most tolerant taxon to pesticide exposure.

Exploring root symbiotic programs in the model legume Medicago truncatula using EST analysis.

We report on a large-scale expressed sequence tag (EST) sequencing and analysis program aimed at characterizing the sets of genes expressed in roots of the model legume Medicago truncatula during interactions with either of two microsymbionts, the nitrogen-fixing bacterium Sinorhizobium meliloti or the arbuscular mycorrhizal fungus Glomus intraradices. We have designed specific tools for in silico analysis of EST data, in relation to chimeric cDNA detection, EST clustering, encoded protein prediction, and detection of differential expression. Our 21 473 5'- and 3'-ESTs could be grouped into 6359 EST clusters, corresponding to distinct virtual genes, along with 52 498 other M.truncatula ESTs available in the dbEST (NCBI) database that were recruited in the process. These clusters were manually annotated, using a specifically developed annotation interface. Analysis of EST cluster distribution in various M.truncatula cDNA libraries, supported by a refined R test to evaluate statistical significance and by 'electronic northern' representation, enabled us to identify a large number of novel genes predicted to be up- or down-regulated during either symbiotic root interaction. These in silico analyses provide a first global view of the genetic programs for root symbioses in M.truncatula. A searchable database has been built and can be accessed through a public interface.

Sugar exchanges in arbuscular mycorrhiza: RiMST5 and RiMST6, two novel Rhizophagus irregularis monosaccharide transporters, are involved in both sugar uptake from the soil and from the plant partner.

Arbuscular mycorrhizal (AM) fungi are associated with about 80% of land plants. AM fungi provide inorganic nutrients to plants and in return up to 20% of the plant-fixed CO2 is transferred to the fungal symbionts. Since AM fungi are obligate biotrophs, unraveling how sugars are provided to the fungus partner is a key for understanding the functioning of the symbiosis. In this study, we identified two new monosaccharide transporters from Rhizophagus irregularis (RiMST5 and RiMST6) that we characterized as functional high affinity monosaccharide transporters. RiMST6 was characterized as a glucose specific, high affinity H(+) co-transporter. We provide experimental support for a primary role of both RiMST5 and RiMST6 in sugar uptake directly from the soil. The expression patterns of RiMSTs in response to partial light deprivation and to interaction with different host plants were investigated. Expression of genes coding for RiMSTs was transiently enhanced after 48 h of shading and was unambiguously dependent on the host plant species. These results cast doubt on the 'fair trade' principle under carbon-limiting conditions. Therefore, in light of these findings, the possible mechanisms involved in the modulation between mutualism and parasitism in plant-AM fungus interactions are discussed.

GintAMT3 - a Low-Affinity Ammonium Transporter of the Arbuscular Mycorrhizal Rhizophagus irregularis.

Nutrient acquisition and transfer are essential steps in the arbuscular mycorrhizal (AM) symbiosis, which is formed by the majority of land plants. Mineral nutrients are taken up by AM fungi from the soil and transferred to the plant partner. Within the cortical plant root cells the fungal hyphae form tree-like structures (arbuscules) where the nutrients are released to the plant-fungal interface, i.e., to the periarbuscular space, before being taken up by the plant. In exchange, the AM fungi receive carbohydrates from the plant host. Besides the well-studied uptake of phosphorus (P), the uptake and transfer of nitrogen (N) plays a crucial role in this mutualistic interaction. In the AM fungus Rhizophagus irregularis (formerly called Glomus intraradices), two ammonium transporters (AMT) were previously described, namely GintAMT1 and GintAMT2. Here, we report the identification and characterization of a newly identified R. irregularis AMT, GintAMT3. Phylogenetic analyses revealed high sequence similarity to previously identified AM fungal AMTs and a clear separation from other fungal AMTs. Topological analysis indicated GintAMT3 to be a membrane bound pore forming protein, and GFP tagging showed it to be highly expressed in the intraradical mycelium of a fully established AM symbiosis. Expression of GintAMT3 in yeast successfully complemented the yeast AMT triple deletion mutant (MATa ura3 mep1Δ mep2Δ::LEU2 mep3Δ::KanMX2). GintAMT3 is characterized as a low affinity transport system with an apparent Km of 1.8 mM and a V max of 240 nmol(-1) min(-1) 10(8) cells(-1), which is regulated by substrate concentration and carbon supply.

Fungal elicitation of signal transduction-related plant genes precedes mycorrhiza establishment and requires the dmi3 gene in Medicago truncatula.

Suppressive subtractive hybridization and expressed sequence tag sequencing identified 29 plant genes which are upregulated during the appressorium stage of mycorrhiza establishment between Medicago truncatula J5 (Myc+) and Glomus mosseae. Eleven genes coding plant proteins with predicted functions in signal transduction, transcription, and translation were investigated in more detail for their relation to early events of symbiotic interactions. Expression profiling showed that the genes are activated not only from the appressorium stage up to the fully established symbiosis in the Myc+ genotype of M. truncatula, but also when the symbionts are not in direct cell contact, suggesting that diffusible fungal molecules (Myc factors) play a, role in the induction of a signal-transduction pathway. Transcript accumulation in roots of a mycorrhiza-defective Myc- dmi3 mutant of M. truncatula is not modified by appressorium formation or diffusible fungal molecules, indicating that the signal transduction pathway is required for a successful G. mosseae-M. truncatula interaction leading to symbiosis development. The symbiotic nodulating bacterium Sinorhizobium meliloti does not activate the 11 genes, which supposes early discrimination by plant roots between the microbial symbionts.

Repetitive DNA sequences include retrotransposons in genomes of the Glomeromycota.

Twenty-five repetitive elements are first described in the genomes of the arbuscular mycorrhizal (AM) fungi Gigaspora margarita, Gig. rosea and Glomus mosseae. Nineteen repetitive DNA sequences isolated by genomic library screening and four by self-priming PCR had no homology to known DNA sequences, except for two Gig. margarita sequences and one Gig. rosea sequence which showed amino acid similarity to retrotransposons. Part of the Gig. rosea sequence was also similar to a DNA transposon. Two other retrotransposon sequences were isolated using PCR targeting of reverse transcriptase and ribonuclease H domains. Evidence is provided for three gypsy-like LTR retrotransposon and two non-LTR retrotransposon sequences in the AM fungal genomes. Four contain stop codons indicating that they cannot be active. Expression of three retrotransposons was not detected in germinating spores or intraradical hyphae of Gig. margarita. Southern blot analyses indicated that these three sequences are dispersed in the genome and that two are methylated. Sequence analysis of different GmarLTR1 copies showed they have undergone mutations by transitions, which may have been induced by cytosine methylation. Transposable elements may have played a major role in shaping genome structure and size during evolution of the Glomeromycota.