Functional diversity of arbuscular mycorrhizas extends to the expression of plant genes involved in P nutrition

This study of functional diversity considers symbiotic associations between two plant species, Medicago truncatula and Lycopersicon esculentum, and seven species of arbuscular mycorrhizal fungi (AMF). The objective was to integrate physiological analyses with molecular techniques to test whether functional diversity between AMF species is not only apparent at the level of mycorrhiza formation, plant nutrient uptake and plant growth, but also at the molecular level as observed by variation in the root expression of plant genes involved in the plant's P-starvation response. The seven species of AMF varied widely in their influence on the root expression of MtPT2 and Mt4 from M. truncatula and LePT1 and TPSI1 from L. esculentum. At one extreme was Glomus mosseae, whereby its colonization of M. truncatula resulted in the greatest reduction in MtPT2 and Mt4 gene expression and the highest level of P uptake and growth, while at the other extreme was Gigaspora rosea, whereby colonization resulted in the highest levels of MtPT2 and Mt4 gene expression and the lowest P uptake and growth. The expression of LePT1 and TPSI1 within the roots of L. esculentum was low and relatively uniform across the seven mycorrhizas, reflecting the ability of this cultivar to maintain low and constant shoot P levels despite root colonization by a broad selection of AMF. This study extends current understanding of functional diversity and shows that plants can respond differently to AMF, not only at the level of colonization, nutrient uptake and growth, but also at the level of gene expression.     

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.     

Differential regulation of a family of apyrase genes from Medicago truncatula

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.     

Grafting between model legumes demonstrates roles for roots and shoots in determining nodule type and host/rhizobia specificity

Previous grafting experiments have demonstrated that legume shoots play a critical role in symbiotic development of nitrogen-fixing root nodules by regulating nodule number. Here, reciprocal grafting experiments between the model legumes Lotus japonicus and Medicago truncatula were carried out to investigate the role of the shoot in the host-specificity of legume-rhizobia symbiosis and nodule type. Lotus japonicus is nodulated by Mesorhizobium loti and makes determinate nodules, whereas M. truncatula is nodulated by Sinorhizobium meliloti and makes indeterminate nodules. When inoculated with M. loti, L. japonicus roots grafted on M. truncatula shoots produced determinate nodules identical in appearance to those produced on L. japonicus self-grafted roots. Moreover, the hypernodulation phenotype of L. japonicus har1-1 roots grafted on wild-type M. truncatula shoots was restored to wild type when nodulated with M. loti. Thus, L. japonicus shoots appeared to be interchangeable with M. truncatula shoots in the L. japonicus root/M. loti symbiosis. However, M. truncatula roots grafted on L. japonicus shoots failed to induce nodules after inoculation with S. meliloti or a mixture of S. meliloti and M. loti. Instead, only early responses to S. meliloti such as root hair tip swelling and deformation, plus induction of the early nodulation reporter gene MtENOD11:GUS were observed. The results indicate that the L. japonicus shoot does not support normal symbiosis between the M. truncatula root and its microsymbiont S. meliloti, suggesting that an unidentified shoot-derived factor may be required for symbiotic progression in indeterminate nodules.     

The sss colonization gene of the tomato-Fusarium oxysporum f. sp. radicis-lycopersici biocontrol strain Pseudomonas fluorescens WCS365 can improve root colonization of other wild-type pseudomonas spp.bacteria

We show that the disease tomato foot and root rot caused by the pathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici can be controlled by inoculation of seeds with cells of the efficient root colonizer Pseudomonas fluorescens WCS365, indicating that strain WCS365 is a biocontrol strain. The mechanism for disease suppression most likely is induced systemic resistance. P. fluorescens strain WCS365 and P. chlororaphis strain PCL1391, which acts through the production of the antibiotic phenazine-1-carboxamide, were differentially labeled using genes encoding autofluorescent proteins. Inoculation of seeds with a 1:1 mixture of these strains showed that, at the upper part of the root, the two cell types were present as microcolonies of either one or both cell types. Microcolonies at the lower root part were predominantly of one cell type. Mixed inoculation tended to improve biocontrol in comparison with single inoculations. In contrast to what was observed previously for strain PCL1391, mutations in various colonization genes, including sss, did not consistently decrease the biocontrol ability of strain WCS365. Multiple copies of the sss colonization gene in WCS365 improved neither colonization nor biocontrol by this strain. However, introduction of the sss-containing DNA fragment into the poor colonizer P. fluorescens WCS307 and into the good colonizer P. fluorescens F113 increased the competitive tomato root tip colonization ability of the latter strains 16- to 40-fold and 8- to 16-fold, respectively. These results show that improvement of the colonization ability of wild-type Pseudomonas strains by genetic engineering is a realistic goal.     

Characterization of the interaction between the bacterial wilt pathogen Ralstonia solanacearum and the model legume plant Medicago truncatula

The soilborne pathogen Ralstonia solanacearum is the causal agent of bacterial wilt and attacks more than 200 plant species, including some legumes and the model legume plant Medicago truncatula. We have demonstrated that M. truncatula accessions Jemalong A17 and F83005.5 are susceptible to R. solanacearum and, by screening 28 R. solanacearum strains on the two M. truncatula lines, differential interactions were identified. R. solanacearum GMI1000 infected Jemalong A17 line, and disease symptoms were dependent upon functional hrp genes. An in vitro root inoculation method was employed to demonstrate that R. solanacearum colonized M. truncatula via the xylem and intercellular spaces. R. solanacearum multiplication was restricted by a factor greater than 1 x 10(5) in the resistant line F83005.5 compared with susceptible Jemalong A17. Genetic analysis of recombinant inbred lines from a cross between Jemalong A17 and F83005.5 revealed the presence of major quantitative trait loci for bacterial wilt resistance located on chromosome 5. The results indicate that the root pathosystem for M. truncatula will provide useful traits for molecular analyses of disease and resistance in this model plant species.     

黄花苜蓿与蒺藜苜蓿对土壤低磷胁迫适应策略的比较研究

土壤有效磷(P)含量低是限制植物生长的主要因素之一。根形态变化和根系大量分泌以柠檬酸为主的有机酸是植物适应土壤P素缺乏的重要机制。以广泛分布于我国北方的重要豆科牧草黄花苜蓿(Medicago falcata)和豆科模式植物蒺藜苜蓿(M. truncatula)为材料, 采用砂培方法, 研究了低P胁迫对其植株生长、根系形态和柠檬酸分泌的影响, 对比了两种苜蓿适应低P胁迫的不同策略。结果表明: 1)低P处理显著抑制了蒺藜苜蓿与黄花苜蓿的地上部生长, 而对地下部生长影响较小, 从而导致根冠比增加。2)低P胁迫显著降低黄花苜蓿的总根长和侧根长, 而对蒺藜苜蓿的上述根系形态指标没有显著影响。3)低P胁迫促进两种苜蓿根系的柠檬酸分泌, 无论是在正常供P还是低P胁迫条件下, 黄花苜蓿根系分泌柠檬酸量显著高于蒺藜苜蓿根系。上述结果表明, 黄花苜蓿和蒺藜苜蓿对低P胁迫的适应策略不同, 低P胁迫下, 黄花苜蓿主要通过根系大量分泌柠檬酸, 活化根际难溶态P来提高对P的吸收, 而蒺藜苜蓿维持较大的根系是其适应低P胁迫的主要策略。     

Impact of sewage sludges on Medicago truncatula symbiotic proteome

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M. truncatula symbiotic proteome.     

Mutations in DMI3 and SUNN modify the appressorium-responsive root proteome in arbuscular mycorrhiza

Modification of the Medicago truncatula root proteome during the early stage of arbuscular mycorrhizal symbiosis was investigated by comparing, using two-dimensional electrophoresis, the protein patterns obtained from non-inoculated roots and roots synchronized for Glomus intraradices appressorium formation. This approach was conducted in wild-type (J5), mycorrhiza-defective (TRV25, dmi3), and autoregulation-defective (TR122, sunn) M. truncatula genotypes. The groups of proteins that responded to appressorium formation were further compared between wild-type and mutant genotypes; few overlaps and major differences were recorded, demonstrating that mutations in DMI3 and SUNN modified the appressorium-responsive root proteome. Except for a chalcone reductase, none of the differentially displayed proteins that could be identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry previously was known as appressorium responsive. A DMI3-dependent increased accumulation of signal transduction-related proteins (dehydroascorbate reductase, cyclophilin, and actin depolymerization factor) was found to precede mycorrhiza establishment. Differences in the accumulation of proteins related to plant defense reactions, cytoskeleton rearrangements, and auxin signaling upon symbiont contact were recorded between wild-type and hypermycorrhizal genotypes, pointing to some putative pathways by which SUNN may regulate very early arbuscule formation.     

Association of highly and weakly mycorrhizal seedlings can promote the extra- and intraradical development of a common mycorrhizal network

Arbuscular mycorrhizal fungi (AMF) are key determinants of plant interactions in ecosystems. Through their effects on competition, they are regulators of the structure of communities. Conversely, the composition of plant assemblages may also influence the AMF colonization dynamics of plant species. Here, we tested under in vitro culture conditions the effects of Medicago truncatula, a highly mycorrhizal plant species, and Silene vulgaris, a weakly mycorrhizal plant species, grown single (monospecies treatments) or in combination (bispecies treatment) on the colonization dynamics of the AMF Rhizophagus irregularis MUCL 43194. The seedlings were placed in a pre-established hyphal network developing from a mature M. truncatula mycorrhizal donor plant. Extraradical mycelium (ERM) and root colonization parameters as well as root morphology were measured over a period of 12 days. An increased ERM length, total root colonization and proportion of arbuscules were noted in the bispecies treatment. Conversely, the bispecies treatment seemed to have no effect on root growth. This study also demonstrated the suitability of the in vitro culture system for studying the interactions between AMF and host plants grown as mono- and bispecies combinations.