Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis formed between Medicago truncatula and Glomus versiforme

Many terrestrial plant species are able to form symbiotic associations with arbuscular mycorrhizal fungi. Here we have identified three cDNA clones representing genes whose expression is induced during the arbuscular mycorrhizal symbiosis formed between Medicago truncatula and an arbuscular mycorrhizal fungus, Glomus versiforme. The three clones represent M. truncatula genes and encode novel proteins: a xyloglucan endotransglycosylase-related protein, a putative arabinogalactan protein (AGP), and a putative homologue of the mammalian p110 subunit of initiation factor 3 (eIF3). These genes show little or no expression in M. truncatula roots prior to formation of the symbiosis and are significantly induced following colonization by G. versiforme. The genes are not induced in roots in response to increases in phosphate. This suggests that induction of expression during the symbiosis is due to the interaction with the fungus and is not a secondary effect of improved phosphate nutrition. In situ hybridization revealed that the putative AGP is expressed specifically in cortical cells containing arbuscules. The identification of two mycorrhiza-induced genes encoding proteins predicted to be involved in cell wall structure is consistent with previous electron microscopy data that indicated major alterations in the extracellular matrix of the cortical cells following colonization by mycorrhizal fungi.     

Expression of a xyloglucan endotransglucosylase/hydrolase gene, Mt-XTH1, from Medicago truncatula is induced systemically in mycorrhizal roots

Xyloglucan endotransglucosylase/hydrolases (XTH) are enzymes that catalyze the hydrolysis and transglycosylation of xyloglucan polymers in plant cell walls. Previously, we isolated a cDNA from mycorrhizal roots of Medicago truncatula that is predicted to encode an XTH [van Buuren, M.L., Maldonado-Mendoza, I.E., Trieu, A.T., Blaylock, L.A., Harrison, M.J., 1999. Novel genes induced during an arbuscular mycorrhizal (AM) symbiosis between M. truncatula and G. versiforme. Mol. Plant-Microb. Interact. 12, 171-181.]. Here, we identified the corresponding XTH gene, designated Mt-XTH1. The Mt-XTH1 gene contains four exons separated by three introns and resides on a 15-kb Xba1 fragment adjacent to a second XTH gene designated Mt-XTH2. Mt-XTH2 shares the same exon-intron structure as Mt-XTH1. Exons 2, 3 and 4 and introns 1 and 2 are identical to Mt-XTH1, while exon 1 and intron 3 are divergent, both in sequence and in length. Mt-XTH1 is induced following colonization of the roots by AM fungi but does not respond to changes in phosphate status. Analysis of transgenic roots expressing an Mt-XTH1 promoterColon, two colonsuidA fusion revealed that the Mt-XTH1 promoter directs expression in cells throughout the root system with significantly higher levels of activity in mycorrhizal roots. Mt-XTH1 expression is elevated not only in the regions of the roots colonized by the fungus, but also at sites distal to the infected regions. These expression patterns are consistent with activation in response to a systemic signal.     

A mycorrhiza-responsive protein in wheat roots

A small protein, designated Myk15, was found to be strongly induced in wheat ( Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane alpha-helix and may correspond to a cleavable targeting domain.     

Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations

Medicago truncatula, a diploid autogamous legume, is currently being developed as a model plant for the study of root endosymbiotic associations, including nodulation and mycorrhizal colonization. An important requirement for such a plant is the possibility of rapidly introducing and analyzing chimeric gene constructs in root tissues. For this reason, we developed and optimized a convenient protocol for Agrobacterium rhizogenes-mediated transformation of M. truncatula. This unusual protocol, which involves the inoculation of sectioned seedling radicles, results in rapid and efficient hairy root organogenesis and the subsequent development of vigorous "composite plants." In addition, we found that kanamycin can be used to select for the cotransformation of hairy roots directly with gene constructs of interest. M. truncatula composite plant hairy roots have a similar morphology to normal roots and can be nodulated successfully by their nitrogen-fixing symbiotic partner, Sinorhizobium meliloti. Furthermore, spatiotemporal expression of the Nod factor-responsive reporter pMtENOD11-gusA in hairy root epidermal tissues is indistinguishable from that observed in Agrobacterium tumefaciens-transformed lines. M. truncatula hairy root explants can be propagated in vitro, and we demonstrate that these clonal lines can be colonized by endomycorrhizal fungi such as Glomus intraradices with the formation of arbuscules within cortical cells. Our results suggest that M. truncatula hairy roots represent a particularly attractive system with which to study endosymbiotic associations in transgenically modified roots.     

丛枝菌根真菌对西瓜嫁接苗生长和根系防御性酶活性的影响

在温室盆栽条件下,研究丛枝菌根(AM)真菌地表球囊霉(Glomus versiforme)对连作土壤中西瓜自根苗和嫁接苗生长、根系膜透性、丙二醛(MDA)含量和防御性酶活性的影响.结果表明： 接种AM真菌能显著增加西瓜自根苗和嫁接苗的生物量,提高根系活力,降低根系膜透性和MDA含量.接种AM真菌的自根苗地上部鲜质量、地上部干质量和根系活力分别增加了57.6%、60.0%和142.1%,而接种AM真菌的嫁接苗分别增加了26.7%、28.0%和11.0%;自根苗（C）、嫁接苗（G）、接种AM真菌自根苗（C+M）和接种AM真菌嫁接苗（G+M）的根系细胞膜透性为C>G>C+M>G+M,根系MDA含量为C>G>G+M>C+M.接种AM真菌能提高西瓜自根苗和嫁接苗根系的苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、过氧化物酶(POD)、几丁质酶和β 1,3 葡聚糖酶活性,而且接种AM真菌的西瓜自根苗和嫁接苗根系POD、PAL和β-1,3-葡聚糖酶活性的峰值比不接种的提前2周出现.接种AM真菌能激活西瓜自根苗和嫁接苗与抗逆性有关的防御性酶反应,使根系对逆境产生快速反应,从而提高其抗连作障碍的能力.     

Suppression of allene oxide cyclase in hairy roots of Medicago truncatula reduces jasmonate levels and the degree of mycorrhization with Glomus intraradices

During the symbiotic interaction between Medicago truncatula and the arbuscular mycorrhizal (AM) fungus Glomus intraradices, an endogenous increase in jasmonic acid (JA) occurs. Two full-length cDNAs coding for the JA-biosynthetic enzyme allene oxide cyclase (AOC) from M. truncatula, designated as MtAOC1 and MtAOC2, were cloned and characterized. The AOC protein was localized in plastids and found to occur constitutively in all vascular tissues of M. truncatula. In leaves and roots, MtAOCs are expressed upon JA application. Enhanced expression was also observed during mycorrhization with G. intraradices. A partial suppression of MtAOC expression was achieved in roots following transformation with Agrobacterium rhizogenes harboring the MtAOC1 cDNA in the antisense direction under control of the cauliflower mosaic virus 35S promoter. In comparison to samples transformed with 35SuidA, roots with suppressed MtAOC1 expression exhibited lower JA levels and a remarkable delay in the process of colonization with G. intraradices. Both the mycorrhization rate, quantified by fungal rRNA, and the arbuscule formation, analyzed by the expression level of the AM-specific gene MtPT4, were affected. Staining of fungal material in roots with suppressed MtAOC1 revealed a decreased number of arbuscules, but these did not exhibit an altered structure. Our results indicate a crucial role for JA in the establishment of AM symbiosis.     

丛枝菌根真菌对西瓜嫁接苗生长和根系防御性酶活性的影响

在温室盆栽条件下,研究丛枝菌根(AM)真菌地表球囊霉(Glomus versiforme)对连作土壤中西瓜自根苗和嫁接苗生长、根系膜透性、丙二醛(MDA)含量和防御性酶活性的影响.结果表明： 接种AM真菌能显著增加西瓜自根苗和嫁接苗的生物量,提高根系活力,降低根系膜透性和MDA含量.接种AM真菌的自根苗地上部鲜质量、地上部干质量和根系活力分别增加了57.6%、60.0%和142.1%,而接种AM真菌的嫁接苗分别增加了26.7%、28.0%和11.0%;自根苗（C）、嫁接苗（G）、接种AM真菌自根苗（C+M）和接种AM真菌嫁接苗（G+M）的根系细胞膜透性为C>G>C+M>G+M,根系MDA含量为C>G>G+M>C+M.接种AM真菌能提高西瓜自根苗和嫁接苗根系的苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、过氧化物酶(POD)、几丁质酶和β 1,3 葡聚糖酶活性,而且接种AM真菌的西瓜自根苗和嫁接苗根系POD、PAL和β-1,3-葡聚糖酶活性的峰值比不接种的提前2周出现.接种AM真菌能激活西瓜自根苗和嫁接苗与抗逆性有关的防御性酶反应,使根系对逆境产生快速反应,从而提高其抗连作障碍的能力.     

A phosphate transporter from Medicago truncatula involved in the acquisition of phosphate released by arbuscular mycorrhizal fungi

Many plants have the capacity to obtain phosphate via a symbiotic association with arbuscular mycorrhizal (AM) fungi. In AM associations, the fungi release phosphate from differentiated hyphae called arbuscules, that develop within the cortical cells, and the plant transports the phosphate across a symbiotic membrane, called the periarbuscular membrane, into the cortical cell. In Medicago truncatula, a model legume used widely for studies of root symbioses, it is apparent that the phosphate transporters known to operate at the root-soil interface do not participate in symbiotic phosphate transport. EST database searches with short sequence motifs shared by known phosphate transporters enabled the identification of a novel phosphate transporter from M. truncatula, MtPT4. MtPT4 is significantly different from the plant root phosphate transporters cloned to date. Complementation of yeast phosphate transport mutants indicated that MtPT4 functions as a phosphate transporter, and estimates of the K(m) suggest a relatively low affinity for phosphate. MtPT4 is expressed only in mycorrhizal roots, and the MtPT4 promoter directs expression exclusively in cells containing arbuscules. MtPT4 is located in the membrane fraction of mycorrhizal roots, and immunolocalization revealed that MtPT4 colocalizes with the arbuscules, consistent with a location on the periarbuscular membrane. The transport properties and spatial expression patterns of MtPT4 are consistent with a role in the acquisition of phosphate released by the fungus in the AM symbiosis.     

Silencing of the Rac1 GTPase MtROP9 in Medicago truncatula stimulates early mycorrhizal and oomycete root colonizations but negatively affects rhizobial infection

RAC/ROP proteins (ρ-related GTPases of plants) are plant-specific small G proteins that function as molecular switches within elementary signal transduction pathways, including the regulation of reactive oxygen species (ROS) generation during early microbial infection via the activation of NADPH oxidase homologs of plants termed RBOH (for respiratory burst oxidase homolog). We investigated the role of Medicago truncatula Jemalong A17 small GTPase MtROP9, orthologous to Medicago sativa Rac1, via an RNA interference silencing approach. Composite M. truncatula plants (MtROP9i) whose roots have been transformed by Agrobacterium rhizogenes carrying the RNA interference vector were generated and infected with the symbiotic arbuscular mycorrhiza fungus Glomus intraradices and the rhizobial bacterium Sinorhizobium meliloti as well as with the pathogenic oomycete Aphanomyces euteiches. MtROP9i transgenic lines showed a clear growth-reduced phenotype and revealed neither ROS generation nor MtROP9 and MtRBOH gene expression after microbial infection. Coincidently, antioxidative compounds were not induced in infected MtROP9i roots, as documented by differential proteomics (two-dimensional differential gel electrophoresis). Furthermore, MtROP9 knockdown clearly promoted mycorrhizal and A. euteiches early hyphal root colonization, while rhizobial infection was clearly impaired. Infected MtROP9i roots showed, in part, extremely swollen noninfected root hairs and reduced numbers of deformed nodules. S. meliloti nodulation factor treatments of MtROP9i led to deformed root hairs showing progressed swelling of its upper regions or even of the entire root hair and spontaneous constrictions but reduced branching effects occurring only at swollen root hairs. These results suggest a key role of Rac1 GTPase MtROP9 in ROS-mediated early infection signaling.     

A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment

The majority of vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. These symbioses, termed arbuscular mycorrhizas, are mutually beneficial, and the fungus delivers phosphate to the plant while receiving carbon. In these symbioses, phosphate uptake by the arbuscular mycorrhizal fungus is the first step in the process of phosphate transport to the plant. Previously, we cloned a phosphate transporter gene involved in this process. Here, we analyze the expression and regulation of a phosphate transporter gene (GiPT) in the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices during mycorrhizal association with carrot or Medicago truncatula roots. These analyses reveal that GiPT expression is regulated in response to phosphate concentrations in the environment surrounding the extra-radical hyphae and modulated by the overall phosphate status of the mycorrhiza. Phosphate concentrations, typical of those found in the soil solution, result in expression of GiPT. These data imply that G. intraradices can perceive phosphate levels in the external environment but also suggest the presence of an internal phosphate sensing mechanism.     

Expression of symbiotic and nonsymbiotic globin genes responding to microsymbionts on Lotus japonicus

Leguminous plants have both symbiotic and nonsymbiotic hemoglobin (sym- and nonsym-Hb) genes. Three symbiotic (LjLb1, 2, 3) and one nonsymbiotic (LjNSG1) Hb genes were isolated from a genomic library of Lotus japonicus MG20 Miyakojima. Two motif sequences (AAAGAT and CTCTT) critical for nodule specific expression were conserved on the 5'-upstream sequence of LjLb1, 2 and 3. The 5'-upstream region of LjNSG1 contained the sequence consensus for nonsym-Hb. RT-PCR with specific primer sets for each LjLb gene showed that all the sym-Hb genes (LjLb1, 2, 3) were expressed specifically and strongly in root nodules. The expression of LjLb1, 2 and 3 could not be detected in root, leaf or stem of a mature plant, whereas low level expression was detected in seedlings by RT-PCR. This suggests that sym-Hbs may have another unknown function besides being oxygen transporter for the microsymbiont in symbiotic nitrogen fixation. The expression of LjNSG1, examined with RT-PCR, was detected at low level in root, leaf and stem. The expression of LjNSG1 was enhanced in root nodules, whereas it was repressed in roots colonized by mycorrhizal fungi Glomus sp. R10. The repression of the nonsym-Hb gene was also observed in the roots of Medicago sativa colonized by Glomus sp. R10.     

Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula

Plant genes induced during early root colonization of Medicago truncatula Gaertn. J5 by a growth-promoting strain of Pseudomonas fluorescens (C7R12) have been identified by suppressive subtractive hybridization. Ten M. truncatula genes, coding proteins associated with a putative signal transduction pathway, showed an early and transient activation during initial interactions between M. truncatula and P. fluorescens, up to 8 d after root inoculation. Gene expression was not significantly enhanced, except for one gene, in P. fluorescens-inoculated roots of a Myc(-)Nod(-) genotype (TRV25) of M. truncatula mutated for the DMI3 (syn. MtSYM13) gene. This gene codes a Ca(2+) and calmodulin-dependent protein kinase, indicating a possible role of calcium in the cellular interactions between M. truncatula and P. fluorescens. When expression of the 10 plant genes was compared in early stages of root colonization by mycorrhizal and rhizobial microsymbionts, Glomus mosseae activated all 10 genes, whereas Sinorhizobium meliloti only activated one and inhibited four others. None of the genes responded to inoculation by either microsymbiont in roots of the TRV25 mutant. The similar response of the M. truncatula genes to P. fluorescens and G. mosseae points to common molecular pathways in the perception of the microbial signals by plant roots.