Christine Arnould

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.

Effect of 2,4-Diacetylphloroglucinol on Pythium: Cellular Responses and Variation in Sensitivity Among Propagules and Species

ABSTRACT The antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) plays an important role in the suppression of plant pathogens by several strains of Pseudomonas spp. Based on the results of this study, there is variation within and among Pythium spp. to 2,4-DAPG. Also, various propagules of Pythium ultimum var. sporangiiferum, that are part of the asexual stage of the life cycle, differ considerably in their sensitivity to 2,4-DAPG. Mycelium was the most resistant structure, followed by zoosporangia, zoospore cysts, and zoospores. Additionally, we report for the first time that pH has a significant effect on the activity of 2,4-DAPG, with a higher activity at low pH. Furthermore, the level of acetylation of phloroglucinols is also a major determinant of their activity. Transmission electron microscopy studies revealed that 2,4-DAPG causes different stages of disorganization in hyphal tips of Pythium ultimum var. sporangiiferum, including alteration (proliferation, retraction, and disruption) of the plasma membrane, vacuolization, and cell content disintegration. The implications of these results for the efficacy and consistency of biological control of plant-pathogenic Pythium spp. by 2,4-DAPG-producing Pseudomonas spp. are discussed.

Hydrolytic enzyme activity of Paenibacillus sp. strain B2 and effects of the antagonistic bacterium on cell integrity of two soil-borne pathogenic fungi.

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of Sorghum bicolor and having an antagonistic activity towards soil-borne fungal pathogens, possessed extracellular cellulolytic, proteolytic, chitinolytic and pectinolytic enzyme activities. The eventual role of these lytic enzymes in cellular interactions between Paenibacillus sp. strain B2 and Phytophthora parasitica and Fusariumoxysporum was investigated by electron microscopy and molecular cytology. Electron microscopic observations showed that the presence of Paenibacillus sp. strain B2 resulted in disorganisation of cell walls and/or cell contents of P. parasitica and F. oxysporum. However, when P. parasitica was treated with commercial purified cellulase, protease, chitinase and pectinase, only protease had an inhibitory effect on mycelial growth. It is proposed that the inhibitory effect of Paenibacillus sp. strain B2 on the growth of soil-borne fungal pathogens is probably derived from more than one mechanism.

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 Ca2+ 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.

Overexpression of the Epidermis-Specific Homeodomain-Leucine Zipper IV Transcription Factor OUTER CELL LAYER1 in Maize Identifies Target Genes Involved in Lipid Metabolism and Cuticle Biosynthesis

Transcription factors of the homeodomain-leucine zipper IV (HD-ZIP IV) family play crucial roles in epidermis-related processes. To gain further insight into the molecular function of OUTER CELL LAYER1 (OCL1), 14 target genes up- or down-regulated in transgenic maize (Zea mays) plants overexpressing OCL1 were identified. The 14 genes all showed partial coexpression with OCL1 in maize organs, and several of them shared preferential expression in the epidermis with OCL1. They encoded proteins involved in lipid metabolism, defense, envelope-related functions, or cuticle biosynthesis and include ZmWBC11a (for white brown complex 11a), an ortholog of AtWBC11 involved in the transport of wax and cutin molecules. In support of the annotations, OCL1-overexpressing plants showed quantitative and qualitative changes of cuticular wax compounds in comparison with wild-type plants. An increase in C24 to C28 alcohols was correlated with the transcriptional up-regulation of ZmFAR1, coding for a fatty acyl-coenzyme A reductase. Transcriptional activation of ZmWBC11a by OCL1 was likely direct, since transactivation in transiently transformed maize kernels was abolished by a deletion of the activation domain in OCL1 or mutations in the L1 box, a cis-element bound by HD-ZIP IV transcription factors. Our data demonstrate that, in addition to AP2/EREBP and MYB-type transcription factors, members of the HD-ZIP IV family contribute to the transcriptional regulation of genes involved in cuticle biosynthesis.

Medicago truncatula gene responses specific to arbuscular mycorrhiza interactions with different species and genera of Glomeromycota

Plant genes exhibiting common responses to different arbuscular mycorrhizal (AM) fungi and not induced under other biological conditions have been sought for to identify specific markers for monitoring the AM symbiosis. A subset of 14 candidate Medicago truncatula genes was identified as being potentially mycorrhiza responsive in previous cDNA microarray analyses and exclusive to cDNA libraries derived from mycorrhizal root tissues. Transcriptional activity of the selected plant genes was compared during root interactions with seven AM fungi belonging to different species of Glomus, Acaulospora, Gigaspora, or Scutellospora, and under widely different biological conditions (mycorrhiza, phosphate fertilization, pathogenic/beneficial microbe interactions, incompatible plant genotype). Ten of the M. truncatula genes were commonly induced by all the tested AM fungal species, and all were activated by at least two fungi. Most of the plant genes were transcribed uniquely in mycorrhizal roots, and several were already active at the appressorium stage of fungal development. Novel data provide evidence that common recognition responses to phylogenetically different Glomeromycota exist in plants during events that are unique to mycorrhiza interactions. They indicate that plants should possess a mycorrhiza-specific genetic program which is comodulated by a broad spectrum of AM fungi.

Symbiosis-Related Plant Genes Modulate Molecular Responses in an Arbuscular Mycorrhizal Fungus During Early Root Interactions

To gain further insight into the role of the plant genome in arbuscular mycorrhiza (AM) establishment, we investigated whether symbiosis-related plant genes affect fungal gene expression in germinating spores and at the appressoria stage of root interactions. Glomus intraradices genes were identified in expressed sequence tag libraries of mycorrhizal Medicago truncatula roots by in silico expression analyses. Transcripts of a subset of genes, with predicted functions in transcription, protein synthesis, primary or secondary metabolism, or of unknown function, were monitored in spores and germinating spores and during interactions with roots of wild-type or mycorrhiza-defective (Myc-) mutants of M. truncatula. Not all the fungal genes were active in quiescent spores but all were expressed when G. intraradices spores germinated in wild-type M. truncatula root exudates or when appressoria or arbuscules were formed in association with wild-type M. truncatula roots. Most of the fungal genes were upregulated or induced at the stage of appressorium development. Inactivation of the M. truncatula genes DMI1, DMI2/MtSYM2, or DMI3/MtSYM13 was associated with altered fungal gene expression (nonactivation or inhibition), modified appressorium structure, and plant cell wall responses, providing first evidence that cell processes modified by symbiosis-related plant genes impact on root interactions by directly modulating AM fungal activity.

Structure and composition of model cheeses influence sodium NMR mobility, kinetics of sodium release and sodium partition coefficients

The mobility and release of sodium ions were assessed in model cheeses with three different lipid/protein ratios, with or without added NaCl. The rheological properties of the cheeses were analysed using uniaxial compression tests. Microstructure was characterised by confocal laser scanning microscopy. (23)Na nuclear magnetic resonance (NMR) spectroscopy was used to study the molecular mobility of sodium ions in model cheeses through measurements of the relaxation and creation times. Greater mobility was observed in cheeses containing a lower protein content and with added NaCl. The kinetics of sodium release from the cheese to an aqueous phase was correlated with the mobility of sodium ions. The highest rates of sodium release were observed with a lower protein content and with added NaCl. The water/cheese partition coefficients of sodium increased when NaCl was added or the protein content was higher. The study highlighted the effect of model cheese characteristics on molecular and macroscopic behaviours of sodium.

Symbiosis-related pea genes modulate fungal and plant gene expression during the arbuscule stage of mycorrhiza with Glomus intraradices

The arbuscular mycorrhiza association results from a successful interaction between genomes of the plant and fungal symbiotic partners. In this study, we analyzed the effect of inactivation of late-stage symbiosis-related pea genes on symbiosis-associated fungal and plant molecular responses in order to gain insight into their role in the functional mycorrhizal association. The expression of a subset of ten fungal and eight plant genes, previously reported to be activated during mycorrhiza development, was compared in Glomus intraradices-inoculated wild-type and isogenic genotypes of pea mutated for the PsSym36, PsSym33, and PsSym40 genes where arbuscule formation is inhibited or fungal turnover modulated, respectively. Microdissection was used to corroborate arbuscule-related fungal gene expression. Molecular responses varied between pea genotypes and with fungal development. Most of the fungal genes were downregulated when arbuscule formation was defective, and several were upregulated with more rapid fungal development. Some of the plant genes were also affected by inactivation of the PsSym36, PsSym33, and PsSym40 loci, but in a more time-dependent way during root colonization by G. intraradices. Results indicate a role of the late-stage symbiosis-related pea genes not only in mycorrhiza development but also in the symbiotic functioning of arbuscule-containing cells.

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.