Vivienne Gianinazzi-Pearson

Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA‐targeted nested PCR

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon‐discriminating molecular probes were developed. The 5′ end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote‐specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon‐discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon‐discriminating primers for the second, was performed on individual trypan blue‐stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co‐existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual and mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis

Significance The arbuscular mycorrhizal symbiosis between fungi of the Glomeromycota phylum and plants involves more than two-thirds of all known plant species, including important crop species. This mutualistic symbiosis, involving one of the oldest fungal lineages, is arguably the most ecologically and agriculturally important symbiosis in terrestrial ecosystems. The Glomeromycota are unique in that their spores and coenocytic hyphae contain hundreds of nuclei in a common cytoplasm, which raises important questions about the natural selection, population genetics, and gene expression of these highly unusual organisms. Study of the genome of Rhizophagus irregularis provides insight into genes involved in obligate biotrophy and mycorrhizal symbioses and the evolution of an ancient asexual organism, and thus is of fundamental importance to the field of genome evolution. The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Cell Defense Responses Associated with Localized and Systemic Resistance to Phytophthora parasitica Induced in Tomato by an Arbuscular Mycorrhizal Fungus

The arbuscular mycorrhizal fungus Glomus mosseae is able to confer bioprotection against Phytophthora parasitica in tomato roots. Localized and induced systemic resistance (ISR) have been demonstrated to be involved in pathogen control in mycorrhizal and nonmycorrhizal roots with a split root experimental system. Decreased pathogen development in mycorrhizal and nonmycorrhizal parts of mycorrhizal root systems is associated with accumulation of phenolics and plant cell defense responses. G. mosseae-containing cortical cells in the mycorrhizal tissues are immune to the pathogen and exhibit a localized resistance response with the formation of cell wall appositions reinforced by callose adjacent to intercellular hyphae. The systemically induced resistance in nonmycorrhizal root parts is characterized by elicitation of host wall thickenings containing non-esterified pectins and PR-1a protein in reaction to intercellular pathogen hyphae, and by the formation of callose-rich encasement material around P. paras...

Medicago truncatula ENOD11: a novel RPRP-encoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells.

Leguminous plants establish endosymbiotic associations with both rhizobia (nitrogen fixation) and arbuscular mycorrhizal fungi (phosphate uptake). These associations involve controlled entry of the soil microsymbiont into the root and the coordinated differentiation of the respective partners to generate the appropriate exchange interfaces. As part of a study to evaluate analogies at the molecular level between these two plant-microbe interactions, we focused on genes from Medicago truncatula encoding putative cell wall repetitive proline-rich proteins (RPRPs) expressed during the early stages of root nodulation. Here we report that a novel RPRP-encoding gene, MtENOD11, is transcribed during preinfection and infection stages of nodulation in root and nodule tissues. By means of reverse transcription-polymerase chain reaction and a promoter-reporter gene strategy, we demonstrate that this gene is also expressed during root colonization by endomycorrhizal fungi in inner cortical cells containing recently formed arbuscules. In contrast, no activation of MtENOD11 is observed during root colonization by a nonsymbiotic, biotrophic Rhizoctonia fungal species. Analysis of transgenic Medicago spp. plants expressing pMtENOD11-gusA also revealed that this gene is transcribed in a variety of nonsymbiotic specialized cell types in the root, shoot, and developing seed, either sharing high secretion/metabolite exchange activity or subject to regulated modifications in cell shape. The potential role of early nodulins with atypical RPRP structures such as ENOD11 and ENOD12 in symbiotic and nonsymbiotic cellular contexts is discussed.

In planta histochemical staining of fungal alkaline phosphatase activity for analysis of efficient arbuscular mycorrhizal infections

A histochemical procedure was developed to visualize and estimate the proportion of arbuscular mycorrhizal infections showing fungal alkaline phosphatase activity, as compared to the total amount of fungal tissue (trypan blue staining) and of living mycelium, indicated by succinate dehydrogenase activity. In roots of Allium porrum and Platanus acerifolia, only a small proportion of living intraradical mycelium showed alkaline phosphatase activity during early infection but this increased greatly just before the mycorrhizal growth response of the host plant. Infection revealed by all three stains reached a maximum at 6 weeks after inoculation, after which the level of trypan blue stained infection remained constant but the proportion showing succinate dehydrogenase and alkaline phosphatase activity declined as the infection aged. Alkaline phosphatase activity was absent from virtually all abortive entry point hyphae formed on roots of a resistant myc−, nod− mutant of Pisum sativum, although succinate dehydrogenase activity was detected. Observations suggest that the alkaline phosphatase activity is induced by colonization of host roots and that this fungal enzyme could provide a useful marker for analyzing the symbiotic efficiency of arbuscular mycorrhizal infections.

The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Proteome analysis and identification of symbiosis‐related proteins from Medicago truncatula Gaertn. by two‐dimensional electrophoresis and mass spectrometry

Time‐course analysis of root protein profiles was studied by two‐dimensional gel electrophoresis and silver staining in the model plant Medicago truncatula, inoculated either with the arbuscular mycorrhizal fungus Glomus mosseae or with the nitrogen fixing bacterium Sinorhizobium meliloti. Protein modifications in relation to the development of both symbioses included down‐ and upregulations, as well as newly induced polypeptides. Matrix assisted laser desorption/ionization‐time of flight‐mass spectrometry after trypsin digestion clearly identified one polypeptide induced in nodulated roots as a M. truncatula leghemoglobin. Internal sequencing with a quadrupole time‐of‐flight mass spectrometer and database searches confirmed the induction of proteins previously described in root symbioses, and revealed the implication of other proteins. In nodulated roots, one polypeptide was identified as an elongation factor Tu from S. meliloti, while another one could not be assigned a function. In mycorrhizal roots, analyzed proteins also included a protein of unknown function, as well as a glutathione‐S‐transferase, a fucosidase, a myosin‐like protein, a serine hydroxymethyltransferase and a cytochrome‐c‐oxidase. These results emphasize the usefulness of proteome analysis in identifying molecular events occurring in plant root symbioses.

Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a locus a myc- mutant of Pisum sativum L.

Pisum sativum L. myc− mutants which fail to form arbuscular mycorrhiza have recently been identified amongst nod− mutants (Duc et al., 1989, Plant Sci. 60, 215–222). The reason for this resistance to symbiotic fungi has been investigated in the case of a ‘locus a’ mutant (P2) inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd, and Trappe. The fungal symbiont formed viable appressoria in contact with the root surface but its development was stopped at the root epidermis. Abundant material was deposited on the inner face of root cell walls adjacent to the appressoria in the P2 mutant, but not in the wild-genotype parent cultivar (Frisson) forming a symbiotic mycorrhizal infection. Fluorescence, histochemical, cytochemical and immunocytological approaches were used to characterize the paramural deposits in epidermal and hypodermal cells of the mutant. Strong fluorescence under blue light indicated the accumulation of phenolic compounds although polymers like lignin or suberin were not localized. Proteins and glycoproteins were homogeneously distributed within the paramural deposits. In the latter, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for 1,4-polysaccharide detection showed a heterogeneous composition with electron-dense points surrounded by non-reactive material, but cytological tests for cellulose and pectin gave weak responses as compared to epidermal and hypodermal walls of the wild genotype. β-1,3-Glucans indicative of callose were detected by in-situ immunolocalization in the paramural deposits below appressoria on mutant roots, but not in walls of the wild genotype. Thus, appressorium formation by G. mosseae on roots of the ‘locus a’ P. sativum mutant elicits wall modifications usually associated with activation of defence responses to pathogens. It is proposed that this locus must be involved in a key event in symbiotic infection processes in P. sativum, and the possible role of complex regulatory interactions between symbiosis and defence genes in endomycorrhiza development is discussed.

Visualization of ribosomal DNA loci in spore interphasic nuclei of glomalean fungi by fluorescence in situ hybridization

Abstract Fluorescence in situ hybridization (FISH) was applied to interphasic nuclei isolated from spores of four species of AM fungi : Scutellospora castanea, Glomus mosseae, Glomus intraradices and Gigaspora rosea. Ribosomal DNA loci were visualized using digoxigenin-labeled 25 S rDNA probes obtained by nested PCR. Several hybridization sites were detected per nucleus and an internuclear variability was observed in the number of loci. This is the first report of successful application of FISH to analyse the genomes of glomalean fungi.

Colonisation patterns of root tissues byPhytophthora nicotianae var.parasitica related to reduced disease in mycorrhizal tomato

Tomato plants pre-colonised by the arbuscular mycorrhizal fungusGlomus mosseae showed decreased root damage by the pathogenPhytophthora nicotianae var.parasitica. In analyses of the cellular bases of their bioprotective effect, a prerequisite for cytological investigations of tissue interactions betweenG. mosseae andP. nicotianae v.parasitica was to discriminate between the hyphae of the two fungi within root tissues. We report the use of antibodies as useful tools, in the absence of an appropriate stain for distinguishing hyphae ofP. nicotianae v.parasitica from those ofG. mosseae inside roots, and present observations on the colonisation patterns by the pathogenic fungus alone or during interactions in mycorrhizal roots. Infection intensity of the pathogen, estimated using an immunoenzyme labelling technique on whole root fragments, was lower in mycorrhizal roots. Immunogold labelling ofP. nicotianae v.parasitica on cross-sections of infected tomato roots showed that inter or intracellular hyphae developed mainly in the cortex, and their presence induced necrosis of host cells, the wall and contents of which showed a strong autofluorescence in reaction to the pathogen. In dual fungal infections of tomato root systems, hyphae of the symbiont and the pathogen were in most cases in different root regions, but they could also be observed in the same root tissues. The number ofP. nicotianae v.parasitica hyphae growing in the root cortex was greatly reduced in mycorrhizal root systems, and in mycorrhizal tissues infected by the pathogen, arbuscule-containing cells surrounded by intercellularP. nicotianae v.parasitica hyphae did not necrose and only a weak autofluorescence was associated with the host cells. Results are discussed in relation to possible processes involved in the phenomenon of bioprotection in arbuscular mycorrhizal plants.