Search

It has been rarely questioned as to whether the enrichment of specific bacterial taxa found in the rhizosphere of a given plant species changes with different soil types under field conditions and under similar climatic conditions. Understanding tree microbiome interactions is essential because, in contrast to annual plants, tree species require decades to grow and strongly depend on the nutritive resources of the soil. In this context, we tested using a natural toposequence the hypothesis that beech trees select specific taxa and functions in their rhizosphere based on the soil conditions and their nutritive requirements. Our 16S rRNA gene pyrosequencing analyses revealed that the soil type determines the taxa colonizing the beech rhizosphere. A rhizosphere effect was observed in each soil type, but a stronger effect was observed in the nutrient-poor soils. Although the communities varied significantly across the toposequence, we identified a core beech rhizosphere microbiome. Functionally, GeoChip analyses showed a functional redundancy across the toposequence, with genes related to nutrient cycling and to the bacterial immune system being significantly enriched in the rhizosphere. Altogether, the data suggest that, regardless of the soil conditions, trees enrich variable bacterial communities to maintain the functions necessary for their nutrition.

• The plant plasma membrane-localized NADPH oxidases, known as respiratory burst oxidase homologues (RBOHs), appear to play crucial roles in plant growth and development. They are involved in important processes, such as root hair growth, plant defence reactions and abscisic acid signalling. • Using sequence similarity searches, we identified seven putative RBOH-encoding genes in the Medicago truncatula genome. A phylogenetic reconstruction showed that Rboh gene duplications occurred in legume species. We analysed the expression of these MtRboh genes in different M. truncatula tissues: one of them, MtRbohA, was significantly up-regulated in Sinorhizobium meliloti-induced symbiotic nodules. • MtRbohA expression appeared to be restricted to the nitrogen-fixing zone of the functional nodule. Moreover, using S. meliloti bacA and nifH mutants unable to form efficient nodules, a strong link between nodule nitrogen fixation and MtRbohA up-regulation was shown. MtRbohA expression was largely enhanced under hypoxic conditions. Specific RNA interference for MtRbohA provoked a decrease in the nodule nitrogen fixation activity and the modulation of genes encoding the microsymbiont nitrogenase. • These results suggest that hypoxia, prevailing in the nodule-fixing zone, may drive the stimulation of MtRbohA expression, which would, in turn, lead to the regulation of nodule functioning

Little is known about abundances of the major bacterial taxa in agricultural soils and how they are affected by fertilization or other agricultural practices. Our aim was to determine the abundance and relative distribution of several bacterial phyla and one class, as well as the archaeal and crenarchaeal communities, and how they were affected by different fertilization regimes to examine whether specific responses of microorganisms could be identified at these high taxonomic ranks. We used real-time PCR with taxa specific primers to quantify the abundance of the Actinobacteria, Acidobacteria, Bacteriodetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Alphaproteobacteria and Crenarchaeota, as well as of the total bacteria and total archaea in soil sampled in 2002 and 2007 from a long term experimental field site subjected to six different fertilization regimes since 1956. The fertilization had affected soil pH, carbon and nitrogen, in addition to the C:N ratio. The total abundances of each taxon were affected in similar ways by the imposed treatments. By contrast, the relative abundance of certain taxa responded differently to the same fertilizer, with some taxa increasing while others decreased. Significant treatment effects were observed for the relative abundance of Alphaproteobacteria, Firmicutes, Verrucomicrobia and the Crenarchaeota and soil pH was the main driver for the observed differences. Differences between sampling years were also observed for the relative abundance of the Actinobacteria and the Bacteroidetes. Altogether, our results showed that agricultural practices can impact bacterial and archaeal phyla and classes in soil differently, which is consistent with recent work suggesting ecological coherence of members of bacterial clades at high taxonomic ranks.

International audience; Reactive Oxygen Species (ROS) are continuously produced as a result of aerobic metabolism or in response to biotic and abiotic stresses. ROS are not only toxic by-products of aerobic metabolism, but are also signalling molecules involved in several developmental processes in all organisms. Previous studies have clearly shown that an oxidative burst often takes place at the site of attempted invasion during the early stages of most plant-pathogen interactions. Moreover, a second ROS production can be observed during certain types of plant-pathogen interactions, which triggers hypersensitive cell death (HR). This second ROS wave seems absent during symbiotic interactions. This difference between these two responses is thought to play an important signalling role leading to the establishment of plant defense. In order to cope with the deleterious effects of ROS, plants are fitted with a large panel of enzymatic and non-enzymatic antioxidant mechanisms. Thus, increasing numbers of publications report the characterisation of ROS producing and scavenging systems from plants and from microorganisms during interactions. In this review, we present the current knowledge on the ROS signals and their role during plant-microorganism interactions.

Pseudomonas fluorescens C7R12 was previously shown to promote colonization of Medicago truncatula roots by Glomus mosseae BEG12. To gain more insight into the interaction between C7R12 and BEG12, the cell organization of C7R12 was characterized on adventitious roots mycorrhized or not with BEG12 and on extraradical hyphae. Bacterial cell observations were made using the immuno-fluorescence technique and confocal laser scanning microscopy. Five types of cell organization, so-called organization types (OT), were identified: small or large single cells, cells by pair and cells in microcolonies or in strings. The frequencies of each OT on the roots were expressed as the percentage of observations in which these OTs were represented. The OT frequencies on mycorrhizal and nonmycorrhizal roots differed significantly. Bacterial cells were more frequently single on mycorrhizal than on nonmycorrhizal roots, and in microcolonies and strings on nonmycorrhizal roots. Furthermore, the root area covered by bacterial cells, as assessed by image analysis, appeared to be significantly lower on mycorrhizal than on nonmycorrhizal roots. C7R12 cells were abundant on extraradical hyphae and organized both as single cells and microcolonies. Taken together, these results suggest that P. fluorescens C7R12 cells were less active and less abundant on mycorrhizal than on nonmycorrhizal roots.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; Altered flux of labile C from plant roots into soil is thought to influence growth and maintenance of microbial communities under elevated atmospheric CO2 concentrations. We studied the abundance and function of the soil microbial community at two levels of spatial resolution to assess the response of microorganisms in the rhizosphere of the whole root system and of apical root zones of Phaseolus vulgaris L. to elevated CO2 and high or low N supply. At the coarser resolution, microbial biomass C, basal respiration and phospholipid fatty acid (PLFA) patterns in the rhizosphere remained unaffected by elevated CO2, because the C flux from the whole root system into soil did not change [as shown by Haase, S., Neumann, G., Kania, A., Kuzyakov, Y., Römheld, V., Kandeler, E., 2007. Elevation of atmospheric CO2 and N-nutritional status modify nodulation, nodule carbon supply, and root exudation of Phaseolus vulgaris L. Soil Biology & Biochemistry 39, 2208–2221]. At a higher spatial resolution, more low-molecular-weight compounds were released from apical root zones under elevated CO2. Thus, at an early stage of plant growth (12 days after sowing), elevated CO2 induced an increase of enzyme activities (xylosidase, cellobiosidase and leucine-aminopeptidase) in the rhizosphere soil of apical root zones. At later stages of plant growth (21 days after sowing), however, enzyme activities (those above as well as N-acetyl-β-glucosaminidase and phosphatase) decreased under elevated CO2. The abundance of total and denitrifying bacteria in the rhizosphere soil of apical root zones was unaffected by CO2 elevation or N supply. Plant age seemed to be the main factor influencing the density of the bacterial community. In conclusion, the soil microbial community in the apical root zone responded to elevated CO2 by altered enzyme regulation (production and/or activity) and not by greater bacterial abundance.

Plant NF-Y transcription factors control a wide array of biological functions enabling appropriate reproductive and developmental processes as well as adaptation to various abiotic and biotic environments. In Medicago truncatula, MtNF-YA1 was previously identified as a key determinant for nodule development and establishment of rhizobial symbiosis. Here, we highlight a new role for this protein in compatibility to Aphanomyces euteiches, a root pathogenic oomycete. The Mtnf-ya1-1 mutant plants showed better survival rate, reduced symptoms, and increased development of their root apparatus as compared to their wild-type (VVT) background A17. MtNF-YA-1 was specifically up-regulated by A. euteiches in F83005.5, a highly susceptible natural accession of M. truncatula while transcript level remained stable in A17, which is partially resistant. The role of MtNF-YA1 in F83005.5 susceptibility was further documented by reducing MtNF-YA1 expression either by overexpression of the miR169q, a microRNA targeting MtNF-YA1, or by RNAi approaches leading to a strong enhancement in the resistance of this susceptible line. Comparative analysis of the transcriptome of VVT and Mtnf-ya1-1 led to the identification of 1509 differentially expressed genes. Among those, almost 36 defense-related genes were constitutively expressed in Mtnf-ya1-1, while 20 genes linked to hormonal pathways were repressed. In summary, we revealed an unexpected dual role for this symbiotic transcription factor as a key player in the compatibility mechanisms to a pathogen.

International audience; Although it is usually admitted that arbuscular mycorrhizal (AM) fungi are key components in soil bio-functioning, little is known on the response of microbial functional diversity to AM inoculation. The aims of the present study were to determine the influence of Glomus intraradices inoculum densities on plant growth and soil microflora functional diversity in autoclaved soil or non-disinfected soil. Microbial diversity of soil treatments was assessed by measuring the patterns of in situ catabolic potential of microbial communities. The soil disinfection increased sorghum growth, but lowered catabolic evenness (4.8) compared to that recorded in the non-disinfected soil (6.5). G. intraradices inoculation induced a higher plant growth in the autoclaved soil than in the non-disinfected soil. This AM effect was positively related to inoculum density. Catabolic evenness and richness were positively correlated with the number of inoculated AM propagules in the autoclaved soil, but negatively correlated in the non-disinfected soil. In addition, after soil disinfection and AM inoculation, these microbial functionality indicators had higher values than in the autoclaved or in the non-disinfected soil without AM inoculation. These results are discussed in relation to the ecological influence of AM inoculation, with selected fungal strains and their associated microflora on native soil microbial activity

International audience; The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; One of the most serious diseases of banana is fusarium wilt, caused by Fusarium oxysporum f.sp. cubense (Foc). The objectives of this study were to isolate and identify nonpathogenic F. oxysporum strains from soils suppressive to banana wilt, and to determine the diversity of these isolates. More than 100 Fusarium strains were isolated from the rhizosphere of banana plants and identified to species level. Pathogenicity testing was carried out to confirm that these isolates were nonpathogens of banana. A PCR-based RFLP analysis of the intergenic spacer region of the ribosomal RNA operon was used to characterize the nonpathogens. The isolates were also compared with isolates of Foc from South Africa and the known biological control isolate of F. oxysporum, Fo47. The species-specific primers FOF1 and FOR1, in addition to morphological features, were used to confirm the identity of F. oxysporum isolates included in the PCR-RFLP analysis. Twelve different genotypes could be distinguished, identified by a six-letter code allocated to each isolate following digestion with the restriction enzymes HaeIII, HhaI, HinfI, MspI, RsaI and ScrfI. Eleven of these included nonpathogenic F. oxysporum isolates, and these groups could all be distinguished from the genotype that included Foc. Fo47 was included in one of the genotype groups consisting of nonpathogenic F. oxysporum isolates from South Africa.

International audience; The stimulation of rhizosphere microorganisms by exudates released from roots is important for nutrient cycling and differs between plant species. The reasons for this between-species variability are poorly understood. We studied correlations between shoot biomass, soluble and non-soluble root C concentrations and rhizosphere bacterial abundance (CFU: colony forming units) and an index of microbial activity (in vitro utilization of [U-14C]glucose by soil microorganisms). We studied Briza media and Rumex acetosella (nutrient-poor habitats), Epilobium hirsutum, Eupatorium cannabinum, Rumex obtusifolius and Urtica dioica (nutrient rich habitats) cultivated in a greenhouse for 5 weeks in a forest soil. We found significant differences among species for the bacterial abundance and microbial activity in the rhizosphere. These differences poorly reflected the nutrient richness of the common habitats for these species, possibly because the soil conditions were not optimal. Nevertheless, microbial activity was positively correlated with root soluble C concentration and shoot biomass and negatively correlated with the concentration of non-soluble C in roots. These preliminary results suggest that the carbon economy could be an important control of the between-species variability of microbial activity in the rhizosphere.

A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbioses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C35) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid membrane resulting from the absence of free or lipidA-bound C35 hopanoids or the accumulation of the C30 hopanoid diploptene. Our results also show that C35 hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage., IMPORTANCE Because bradyrhizobia provide fixed nitrogen to plants, this work has potential agronomical implications. An understanding of how hopanoids facilitate bacterial survival in soils and plant hosts may aid the engineering of more robust agronomic strains, especially relevant in regions that are becoming warmer and saline due to climate change. Moreover, this work has geobiological relevance: hopanes, molecular fossils of hopanoids, are enriched in ancient sedimentary rocks at discrete intervals in Earth history. This is the first study to uncover roles for 2Me- and C35 hopanoids in the context of an ecological niche that captures many of the stressful environmental conditions thought to be important during (2Me)-hopane deposition. Though much remains to be done to determine whether the conditions present within the plant host are shared with niches of relevance to the rock record, our findings represent an important step toward identifying conserved mechanisms whereby hopanoids contribute to fitness.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes., Author Summary Parasitic nematodes are microscopic worms that cause major diseases of plants, animals and humans. During compatible interactions, root-knot nematodes (RKN) induce the formation of galls in which redifferentiation of root cells into multinucleate and hypertrophied giant cells is essential for nematode growth and reproduction. The importance of glutathione (GSH), a major antioxidant molecule involved in plant development, in plant microbe interaction and in abiotic stress response, was analyzed during the plant-RKN interaction. Our analyses demonstrated that the gall development and functioning are characterized by an adapted GSH metabolism and that depletion of GSH content impairs nematode reproduction and modified sex ratio. This phenotype is linked to specific modifications of carbon metabolism which do not occur in uninfected roots indicating a peculiar metabolism of this neoformed organ. This first metabolomic analysis during the plant-RKN interaction highlights the regulatory role played by GSH in this pathogenic interaction and completes our vision of the role of GSH during plant-pathogen interactions. RKN sex ratio modification has previously been observed under unfavorable nematode feeding conditions suggesting that the GSH-redox system could be a general sensor of gall fitness in natural conditions.

International audience; # The establishment and development of plant–microorganism interactions involve impressive transcriptomic reprogramming of target plant genes. The symbiont (Sinorhizobium meliloti) and the root knot-nematode pathogen (Meloidogyne incognita) induce the formation of new root organs, the nodule and the gall, respectively. # • Using laser-assisted microdissection, we specifically monitored, at the cell level, Medicago gene expression in nodule zone II cells, which are preparing to receive rhizobia, and in gall giant and surrounding cells, which play an essential role in nematode feeding and constitute the typical root swollen structure, respectively. # • We revealed an important reprogramming of hormone pathways and C1 metabolism in both interactions, which may play key roles in nodule and gall neoformation, rhizobia endocytosis and nematode feeding. Common functions targeted by rhizobia and nematodes were mainly down-regulated, whereas the specificity of the interaction appeared to involve up-regulated genes. # • Our transcriptomic results provide powerful datasets to unravel the mechanisms involved in the accommodation of rhizobia and root-knot nematodes. Moreover, they raise the question of host specificity and the evolution of plant infection mechanisms by a symbiont and a pathogen

National audience; Le développement d’une agriculture durable passe par le développement, ou l’amélioration, des critères utilisés pour évaluer a priori la durabilité d’un gène de résistance ainsi que de ceux utilisés pour définir des stratégies de déploiement des gènes de résistance dans l’agro-écosystème. Face à ces enjeux, notre ambition est de proposer des méthodes innovantes, tant expérimentales que basées sur la modélisation, de gestion durable des résistances. Pour ce faire, nous travaillerons sur le pathosystème piment - virus Y de la pomme de terre (PVY) selon 4 axes : (1) identifier les facteurs génétiques (et leur polymorphisme) chez la plante qui sont ciblés par le virus lors du contournement des résistances, (2) caractériser l’effet des mutations dans les facteurs de résistance de type eIF4E et dans le facteur de virulence viral en terme de durabilité, (3) modéliser et valider la dynamique démo- génétique d’une population virale à l’échelle d’une plante hôte et (4) définir des stratégies d’allocation spatio-temporelle d’un ensemble de gènes de résistance à l’échelle d’un agro-écosystème. Ainsi au cours de ce projet nous mettrons tout d’abord au point une méthodologie innovante d’évaluation de la durabilité potentielle de gènes de résistance indépendamment de l’effet du fond génétique de la plante. Par ailleurs les données expérimentales produites permettront de valider un modèle décrivant de l’échelle du gène à celle de l’agro-écosystème, la dynamique démo-génétique des populations virales. Ce modèle constituera un outil fiable d’expérimentation numérique pour la recherche d’organisation spatio-temporelle des cultures protégeant la durabilité des résistances. Enfin, de nouvelles connaissances fondamentales sur les interactions plantes-virus seront générées : exploration du rôle des isoformes d’eIF4E dans le contournement des allèles pvr2 de résistance par le PVY, étude par pyroséquençage de la dynamique intra-hôte de plusieurs variants viraux.

International audience; Since their introduction in south-eastern France around 1999, new, ‘emerging’ (EM) strains of watermelon mosaic virus (WMV) coexist with the ‘classic’ (CL) strains present for more than 40 years. This situation constitutes a unique opportunity to estimate the frequency of recombinants appearing in the few years following introduction of new strains of a plant RNA virus. Molecular analyses performed on more than 1000 isolates from epidemiological surveys (2004–2008) and from experimental plots (2009–2010), and targeting only recombinants that became predominant in at least one plant, revealed at least seven independent CL/EM or EM/EM recombination events. The frequency of recombinants involving at least one EM parent in the natural populations tested was on the order of 1 %. No new recombinant was detected for more than 1 year, and none but one in more than one location. In tests comparing host range and aphid transmissibility, the new recombinants did not display a better fitness than their ‘parental’ isolates. No recombinant was detected from artificial mixed infections of CL and EM isolates of various hosts after testing more than 1500 subcultures obtained after single-aphid transmission. These results constitute one of the first estimations of the frequency of recombinants in natural conditions for a plant RNA virus. This suggests that although viable recombinants of WMV are not rare, and although recombination may potentially lead to new highly damaging strains, the new recombinants observed so far had a lower fitness than the parental strains and did not emerge durably in the populations.

Motivation: Identification of conserved motifs in biological sequences is crucial to unveil common shared functions. Many tools exist for motif identification, including some that allow degenerate positions with multiple possible nucleotides or amino acids. Most efficient methods available today search conserved motifs in a set of sequences, but do not check for their specificity regarding to a set of negative sequences. Results: We present a tool to identify degenerate motifs, based on a given classification of amino acids according to their physico-chemical properties. It returns the top K motifs that are most frequent in a positive set of sequences involved in a biological process of interest, and absent from a negative set. Thus, our method discovers discriminative motifs in biological sequences that may be used to identify new sequences involved in the same process. We used this tool to identify candidate effector proteins secreted into plant tissues by the root knot nematode Meloidogyne incognita. Our tool identified a series of motifs specifically present in a positive set of known effectors while totally absent from a negative set of evolutionarily conserved housekeeping proteins. Scanning the proteome of M.incognita, we detected 2579 proteins that contain these specific motifs and can be considered as new putative effectors. Availability and Implementation: The motif discovery tool and the proteins used in the experiments are available at http://dtai.cs.kuleuven.be/ml/systems/merci. Contact: celine.vens@cs.kuleuven.be Supplementary Information: Supplementary data are available at Bioinformatics online.

International audience; Inconspicuous basidiomycetes from the order Sebacinales are known to be involved in a puzzling variety of mutualistic plant-fungal symbioses (mycorrhizae), which presumably involve transport of mineral nutrients. Recently a few members of this fungal order not fitting this definition and commonly referred to as ‘endophytes’ have raised considerable interest by their ability to enhance plant growth and to increase resistance of their host plants against abiotic stress factors and fungal pathogens. Using DNA-based detection and electron microscopy, we show that Sebacinales are not only extremely versatile in their mycorrhizal associations, but are also almost universally present as symptomless endophytes. They occurred in field specimens of bryophytes, pteridophytes and all families of herbaceous angiosperms we investigated, including liverworts, wheat, maize, and the non-mycorrhizal model plant Arabidopsis thaliana. They were present in all habitats we studied on four continents. We even detected these fungi in herbarium specimens originating from pioneering field trips to North Africa in the 1830s/40s. No geographical or host patterns were detected. Our data suggest that the multitude of mycorrhizal interactions in Sebacinales may have arisen from an ancestral endophytic habit by specialization. Considering their proven beneficial influence on plant growth and their ubiquity, endophytic Sebacinales may be a previously unrecognized universal hidden force in plant ecosystems.

International audience; The main effects of antagonistic rhizobacteria on plant pathogenic fungi are antibiosis, fungistasis or an indirect constraint through the induction of a plant defence response. To explore different biocontrol mechanisms, an in vitro confrontation assay was conducted with the rhizobacterium Pseudomonas fluorescens Pf29Arp as a biocontrol agent of the fungus Gaeumannomyces graminis var. tritici (Ggt) on wheat roots. In parallel with the assessment of disease extension, together with the bacterial and fungal root colonization rates, the transcript levels of candidate fungal pathogenicity and plant-induced genes were monitored during the 10-day infection process. The bacterial inoculation of wheat roots with the Pf29Arp strain reduced the development of Ggt-induced disease expressed as attack frequency and necrosis length. The growth rates of Ggt and Pf29Arp, monitored through quantitative polymerase chain reaction of DNA amounts with a part of the Ggt 18S rDNA gene and a specific Pf29Arp strain detection probe, respectively, increased throughout the interactions. Bacterial antagonism and colonization had no significant effect on root colonization by Ggt. The expression of fungal and plant genes was quantified in planta by quantitative reverse transcription-polymerase chain reaction during the interactions thanks to the design of specific primers and an innovative universal reference system. During the early stages of the tripartite interaction, several of the fungal genes assayed were down-regulated by Pf29Arp, including two laccases, a β-1,3-exoglucanase and a mitogen-activated protein kinase. The plant host glutathione-S-transferase gene was induced by Ggt alone and up-regulated by Pf29Arp bacteria in interaction with the pathogen. We conclude that Pf29Arp antagonism acts through the alteration of fungal pathogenesis and probably through the activation of host defences

L'article original est publié par The American Society of Plant Biologists; International audience; Cytosolic/nuclear molecular chaperones of the heat shock protein families HSP90 and HSC70 are conserved and essential proteins in eukaryotes. These proteins have essentially been implicated in the innate immunity and abiotic stress tolerance in higher plants. Here, we demonstrate that both chaperones are recruited in Arabidopsis (Arabidopsis thaliana) for stomatal closure induced by several environmental signals. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are compromised in the dark-, CO2-, flagellin 22 peptide-, and abscisic acid (ABA)-induced stomatal closure. HSC70-1 and HSP90 proteins are needed to establish basal expression levels of several ABA-responsive genes, suggesting that these chaperones might also be involved in ABA signaling events. Plants overexpressing HSC70-1 or with reduced HSP90.2 activity are hypersensitive to ABA in seed germination assays, suggesting that several chaperone complexes with distinct substrates might tune tissue-specific responses to ABA and the other biotic and abiotic stimuli studied. This study demonstrates that the HSC70/HSP90 machinery is important for stomatal closure and serves essential functions in plants to integrate signals from their biotic and abiotic environments.

International audience; Scab is an important disease in pear orchards caused by an ascomycetous fungus, Venturia pirina. Young leaves, stems and fruits at all stages of their development are susceptible. Breeding has been undertaken to obtain new hybrids with a good level of resistance to V. pirina. In this context, a better understanding of the host-parasite relationship is necessary. A microscopic study of susceptibility and resistance symptoms on leaves and stems was undertaken on a susceptible cultivar ('Angelys') and a resistant cultivar ('Navara'). Cultivars were grafted onto the rootstock 'Kirchensaller', grown in greenhouse conditions and inoculated with a mixture of local strains of V. pirina. The samples were prepared for confocal micoscopy, scanning electron microscopy and histological studies. On 'Angelys' and 'Navara', the early stages of host-parasite relationships observed by confocal microscopy are similar. A subcuticular primary stroma develops slowly on the susceptible host, 'Angelys'. Often hyphae of the fungus progress deeply between epidermal cells in leaves and stems. The leaf and stem tissue cells show no important changes. Sporulation occurs on leaves 12 to 14 days and on stems 18 to 21 days after inoculation. On the resistant host 'Navara', conidia germinate and form an appressorium, but only a reduced primary subcuticular stroma is observed on the leaf. The cultivar 'Navara's' resistance reaction is of the hypersensitive type. Microscopic analysis shows collapse and necrosis of the epidermal cells; the underlying parenchyma cells undergo important changes. The susceptibility and the resistance symptoms observed on pear leaves with V. pirina are very similar to the symptoms observed with Venturia inaqualis on apple trees but severe symptoms are observed on pear stems with destruction of superficial tissues and deep changes in the cortical parenchyma. The symptom of resistance on stems appears later and is very different from those observed on leaves.

International audience; Expression profiling of two paralogous arbuscular mycorrhizal (AM)-specific blue copper-binding gene (MtBcp1a and MtBcp1b) isoforms was performed by real-time quantitative polymerase chain reaction in wild-type Medicago truncatula Jemalong 5 (J5) during the mycorrhizal development with Glomus intraradices for up to 7 weeks. Time-course analysis in J5 showed that expression of both MtBcp1 genes increased continuously and correlated strongly with the colonization intensity and arbuscule content. MtPT4, selected as a reference gene of the functional plant-fungus association, showed a weaker correlation to mycorrhizal development. In a second experiment, a range of mycorrhizal mutants of the wild-type J5 was assessed. Strictly AM-penetration-defective TRV25-C and TRV25-D (dmi3, Mtsym13), hypomycorrhizal TR25 and TR89 (dmi2, Mtsym2) mutants, and a hypermycorrhizal mutant TRV17 (sunn, Mtsym12) were compared with J5 3 and 7 weeks after inoculation. No MtBcp1 transcripts were detected in the mutants blocked at the appressoria stage. Conversely, TR25, TR89, and J5 showed a gradual increase of the expression of both MtBcp1 genes in 3- and 7-week-old plants, similar to the increase in colonization intensity and arbuscule abundance. The strong correlation between the expression level of AM-specific blue copper-binding protein-encoding genes and AM colonization may imply a basic role in symbiotic functioning for these genes, which may serve as new molecular markers of arbuscule development in M. truncatula.

International audience; Plants encounter throughout their life all kinds of microorganisms, such as bacteria, fungi, or oomycetes, with either friendly or unfriendly intentions. During evolution, plants have developed a wide range of defense mechanisms against attackers. In return, adapted microbes have developed strategies to overcome the plant lines of defense, some of these microbes engaging in mutualistic or parasitic endosymbioses. By sensing microbe presence and activating signaling cascades, the plasma membrane through its dynamics plays a crucial role in the ongoing molecular dialogue between plants and microbes. This review describes the contribution of endocytosis to different aspects of plant–microbe interactions, microbe recognition and development of a basal immune response, and colonization of plant cells by endosymbionts. The putative endocytic routes for the entry of microbe molecules or microbes themselves are explored with a special emphasis on clathrin-mediated endocytosis. Finally, we evaluate recent findings that suggest a link between the compartmentalization of plant plasma membrane into microdomains and endocytosis.

13 pages; International audience; Microbial communities are of major importance in the decomposition of soil organic matter. However, the identities and dynamics of the populations involved are still poorly documented. We investigated, in an 11-month field experiment, how the initial biochemical quality of crop residues could lead to specific decomposition patterns, linking biochemical changes undergone by the crop residues to the respiration, biomass, and genetic structure of the soil microbial communities. Wheat, alfalfa, and rape residues were incorporated into the 0-15 cm layer of the soil of field plots by tilling. Biochemical changes in the residues occurring during degradation were assessed by near-infrared spectroscopy. Qualitative modifications in the genetic structure of the bacterial communities were determined by bacterial-automated ribosomal intergenic spacer analysis. Bacterial diversity in the three crop residues at early and late stages of decomposition process was further analyzed from a molecular inventory of the 16S rDNA. The decomposition of plant residues in croplands was shown to involve specific biochemical characteristics and microbial community dynamics which were clearly related to the quality of the organic inputs. Decay stage and seasonal shifts occurred by replacement of copiotrophic bacterial groups such as proteobacteria successful on younger residues with those successful on more extensively decayed material such as Actinobacteria. However, relative abundance of proteobacteria depended greatly on the composition of the residues, with a gradient observed from alfalfa to wheat, suggesting that this bacterial group may represent a good indicator of crop residues degradability and modifications during the decomposition process.

International audience; The three closely related Arabidopsis basic leucine zipper (bZIP) transcription factors TGA2, TGA5 and TGA6 are required for the establishment of the salicylic acid (SA)-dependent plant defense response systemic acquired resistance, which is effective against biotrophic pathogens. Here we show that the same transcription factors are essential for the activation of jasmonic acid (JA)- and ethylene (ET)-dependent defense mechanisms that counteract necrotrophic pathogens: the tga256 triple mutant is impaired in JA/ET-induced PDF1.2 and b-CHI expression, which correlates with a higher susceptibility against the necrotroph Botrytis cinerea. JA/ET induction of the trans-activators ERF1 and ORA59, which act upstream of PDF1.2, was slightly increased (ERF1)or unaffected (ORA59). PDF1.2 expression can be restored in the tga256 mutant by increased expression of ORA59, as observed in the tga256 jin1 quadruple mutant, which lacks the transcription factor JIN1/AtMYC2 that functions as a negative regulator of the JA/ET-dependent anti-fungal defense program. Whereas JA/ET-induced PDF1.2 expression is strongly suppressed by SA in wild-type plants, no negative effect of SA on PDF1.2 expression was observed in the tga256 jin1 quadruple mutant. These results imply that the antagonistic effects of TGA factors and JIN1/AtMYC2 on the JA/ET pathway are necessary to evoke the SA-mediated suppression of JA/ET-induced defense responses.

International audience; Leptosphaeria maculans, the cause of blackleg or phoma stem canker of oilseed rape and canola (Brassica napus) is becoming a model system for the study of genetic relationships between host and pathogen. Knowledge of this relationship has progressed from the discovery of differential reactions among canola varieties and pathogen isolates to the elucidation of specific resistance genes in the host and the corresponding avirulence genes in the pathogen. The use of a race-specific resistance strategy to manage this disease has been of great value in Canada, Australia and Europe; however, due to the pathogen's propensity to change, new pathotypes or races have been observed to arise rapidly. This paper reviews these developments and discusses applications of this knowledge to applied management of this disease in B. napus; Leptosphaeria maculans, l'agent causal de la jambe noire ou de la nécrose du collet (phoma) chez le colza et le canola (Brassica napus), est en voie de devenir un système modèle pour l'étude des relations génétiques entre l'hôte et l'agent pathogène. Les connaissances associées à ces relation ont progressé, et ce, de la découverte d'interactions différentielles entre les variétés de canola et les isolats de L. maculans jusqu'à l'élucidation des gènes spécifiques de la résistance chez l'hôte et des gènes d'avirulence correspondants chez l'agent pathogène. L'utilisation d'une stratégie faisant appel à la résistance spécifique pour gérer cette maladie a été des plus efficaces au Canada, en Australie et en Europe. Toutefois, à cause de la tendance naturelle de L. maculans à s'adapter nous avons observé l'apparition rapide de nouveaux pathotypes ou de nouvelles races. Cet article passe en revue ces progrés et discute des applications de ces connaissances à la gestion de la maladie chez B. napus

Plant diseases caused by pathogenic microorganisms remain a major limitation in many crop production systems. Nonetheless, constitutive and inducible defense mechanisms render most plants inaccessible to pathogens, making disease an exception rather than a common outcome of plant-microbe interactions. Defense mechanisms and associated pathogen resistance were thus of key interest to many plant pathologists, and many of the molecular mechanisms underlying resistance have been elucidated over the last few decades. In recent years, the analysis of physiological and molecular determinants accounting for successful infection and eventual disease has become a topic of prime scientific interest. The hunt is now on for pathogen effectors subverting the host cell and for the plant compatibility functions manipulated by these effectors. An understanding of the molecular mechanisms underlying successful infection should make it possible to develop new crop protection strategies based on interference with compatibility to prevent disease. This review is addressing plant susceptibility and highlights a number of host processes that have been shown to be induced or subverted to facilitate infection. In particular, we focus on those processes that appear to be manipulated by filamentous fungal and oomycete pathogens.

/ Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as ‘‘guards’’. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity.Abstract Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as ‘‘guards’’. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity., Author Summary / Plant and animal bacterial pathogens have evolved to produce virulence factors, called type III effectors, which are injected into host cells to suppress host defences and provide an environment beneficial for pathogen growth. Type III effectors from pathogenic bacteria display enzymatic activities, often mimicking an endogenous eukaryotic activity, to target host signalling pathways. Elucidation of strategies used by pathogens to manipulate host protein activities is a subject of fundamental interest in pathology. PopP2 is a YopJ-like effector from the soil borne root pathogen Ralstonia solanacearum. Here, in addition to demonstrating PopP2 ability to stabilize the expression of its cognate Arabidopsis RRS1-R resistance protein and physically interact with it, we investigated the enzymatic activity of PopP2. Bacterial YopJ-like effectors are predicted to act as acetyl-transferases on host components. However, only two YopJ-like proteins from animal pathogens have been shown to be active acetyltransferases. We show that PopP2 displays autoacetyltransferase activity targeting a lysine residue wellconserved among YopJ-like family members. This lysine is a critical residue since its mutation prevents autoacetylation of PopP2 and abolishes its recognition by the host. This study provides new clues on the multiple properties displayed by bacterial type III effectors that may be used to target defense-related host components.

No abstract, L'objectif de cette thèse est de contribuer à faire progresser les connaissances sur les interactions bénéfiques entre les plantes et les microorganismes en évaluant la contribution des systèmes de sécrétion de type III (SST3). Une synthèse des connaissances disponibles relatives aux SST3 chez les Pseudomonas non pathogènes, saprotrophes ou mutualistes, présentée chapitre I, montre que les SST3 ne sont pas cantonnés aux interactions parasites ou pathogènes avec les plantes. Dans l’étude expérimentale présentée chapitre II, nous avons utilisé différents génotypes de Medicago truncatula Gaertn. cv. Jemalong capables (Myc+) ou non (Myc-) d’établir une symbiose mycorhizienne. Ce travail nous a permis de montrer que les Pseudomonas spp. fluorescents possédant un SST3 (SST3+) sont préférentiellement associés aux racines mycorhizées des génotypes Myc+ de M. truncatula (J5 et TRV48) plutôt qu’aux racines du mutant Myc- (TRV25) et au sol nu. Ainsi, la plante seule n’est pas à l’origine de la présence accrue des Pseudomonas SST3+. La colonisation de la racine par les champignons mycorhizogènes à arbuscules (CMA), le développement du mycélium intraradiculaire et/ou la formation associée d’arbuscules, sont également déterminants. Dans l’étude présentée chapitre III, nous avons comparé les effets de la souche modèle promotrice de mycorhization (MHB) P. fluorescens C7R12 (SST3+) et de son mutant C7SM7 (SST3-), sur la mycorhization et la croissance de M. truncatula dans un sol non stérile. Ce travail a permis de montrer que le SST3 de C7R12 contribue à l’effet MHB de la bactérie. La promotion de la colonisation de la racine de M. truncatula par les CMA indigènes induite par le SST3 de C7R12 s’est traduite par une amélioration de la croissance de la plante. En revanche, l’inactivation du SST3 chez C7SM7 a eu un impact délétère sur la colonisation de la racine de M. truncatula par les CMA du sol étudié et sur la croissance de la plante. L’observation d’effets quantitatifs opposés entre C7R12 et C7SM7, nous a conduits à nous interroger sur l’existence d’un effet différentiel de l’inoculation de ces bactéries sur la structure et la diversité des communautés des microorganismes associés. Dans une étude présentée chapitre IV, le suivi dynamique en parallèle de la structure des communautés totales bactériennes (B-RISA) et fongiques (F-RISA) et de la colonisation de la racine par les CMA a été réalisée. Aucun effet de l’inoculation n’a été observé sur la structure des communautés fongiques de la rhizosphère ou des racines. En revanche, la structure des communautés bactériennes a varié selon que les plantes aient été inoculées ou non et selon la souche inoculée. Néanmoins, ces différences ont été observées plusieurs semaines après les effets de l’inoculation de C7R12 ou de C7SM7 sur la colonisation de la racine par les CMA. Ce décalage dans le temps, suggère que les différences observées dans la structure des communautés bactériennes pourraient être une conséquence plutôt qu’une cause des variations observées sur la mycorhization de M. truncatula. Nos résultats n’ont pas permis de mettre en évidence d’effets de l’inoculation sur la diversité des populations des bactéries fixatrices d’azote présentes dans les nodosités de M. truncatula. L’analyse des séquences de la grande sous-unité de l’ADN ribosomique (LSU rDNA) amplifiées à partir d’ADN extrait des racines, a montré pour les plantes inoculées et non inoculées, que les populations de CMA étaient majoritairement apparentées à Glomus intraradices. Un groupe d’isolats spécifiquement associé aux racines inoculées avec C7R12 et apparenté à G. claroideum a été décrit. Le groupe spécifique pourrait être associé à l’amélioration de la mycorhization observée dans les racines inoculées avec C7R12. Néanmoins, compte tenu de sa faible représentation numérique (8%), il semble probable que l’inoculation de C7R12 ait aussi un effet quantitatif sur la colonisation de la racine de M. truncatula par les CMA. etc

International audience; Using their 1 amino cyclopropane-1-carboxylic acid (ACC) deaminase activity, many rhizobacteria can divert ACC from the ethylene biosynthesis pathway in plant roots. To investigate the role of this microbial activity in plant responses to plant growth-promoting rhizobacteria (PGPR), we analyzed the effects of acdS knock-out and wild-type PGPR strains on two phenotypic responses to inoculation –root hair elongation and root system architecture– in Arabidopsis thaliana. Our work shows that rhizobacterial AcdS activity has a negative effect on root hair elongation, as expected from the reduction of ethylene production rate in root cells, while it has no impact on root system architecture. This suggests that PGPR triggered root hair elongation is independent of ethylene biosynthesis or signaling pathway. In addition, it does indicate that AcdS activity alters local regulatory processes, but not systemic regulations such as those that control root architecture. Our work also indicates that root hair elongation induced by PGPR inoculation is probably an auxin-independent mechanism. These findings were unexpected since genetic screens for abnormal root hair development mutants led to the isolation of ethylene and auxin mutants. Our work hence shows that studying the interaction between a PGPR and the model plant Arabidopsis is a useful system to uncover new pathways involved in plant plasticity.

Stomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H2O2 production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA and elicitors share common signaling events, it led us to question whether stomatal movements and H2O2 production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine–Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production.

National audience; Mécanisme évolutif bien connu chez les plantes, l’hybridation interspécifique a été relativement peu étudiée dans certains groupes taxonomiques, comme les champignons. Chez les champignons phytopathogènes, l’hybridation entre une espèce indigène et une espèce exotique peut pourtant conduire à l’émergence de nouvelles maladies des plantes.

International audience; Bacterial strains from mycorrhizal roots (three belonging to Comamonadaceae and one to Oxalobacteraceae) and from non-mycorrhizal roots (two belonging to Comamonadaceae) of Medicago truncatula and two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) were tested for their effect on the in vitro saprophytic growth of Glomus mosseae BEG12 and on its colonization of M. truncatula roots. Only the Oxalobacteraceae strain, isolated from barrel medic mycorrhizal roots, and the reference strain P. fluorescens C7R12 promoted both the saprophytic growth and root colonization of G. mosseae BEG12, indicating that they acted as mycorrhiza helper bacteria. Greatest effects were achieved by P. fluorescens C7R12 and its influence on the saprophytic growth of G. mosseae was compared to that on Gigaspora rosea BEG9 to determine if the bacterial stimulation was fungal specific. This fungal specificity, together with plant specificity, was finally evaluated by comparing bacterial effects on arbuscular mycorrhizal symbiosis when each of the fungal species was inoculated to two different plant species (M. truncatula and Lycopersicon esculentum). The results obtained showed that promotion of saprophytic growth by P. fluorescens C7R12 was expressed in vitro towards G. mosseae but not towards G. rosea. Bacterial promotion of mycorhization was also expressed towards G. mosseae, but not G. rosea, in roots of M. truncatula and L. esculentum. Taken together, results indicated that enhancement of arbuscular mycorrhiza development was only induced by a limited number of bacteria, promotion by the most efficient bacterial strain being fungal and not plant specific.

International audience; Chemical inputs used in red fruit production can be detrimental to the environment and pesticides are not always efficient in controlling plant diseases. In addition, it is suspected that conventional agricultural practices have a negative impact on antioxidant activities in red fruits. Therefore new cultural approaches are required in order to produce red fruits of high quality. This involves an integrated programme of plant breeding and development of sustainable agricultural practices. Antioxidant activity was compared in leaves and berries of six raspberry cultivars for the selection of varieties for a breeding programme. This showed that cultivars that had the highest antioxidant activity in leaves had the lowest activity in berries. Biotisation is a new complementary biotechnological approach, which consists of inoculating young plants with beneficial microorganisms such as bacteria and mycorrhizal fungi for improving plant growth and tolerance to biotic and abiotic stresses. Biotisation at the step of acclimatisation of in vitro produced plantlets was shown to be efficient in increasing plant growth and water content in plant leaves grown under low nutrient conditions. A PCR-based diagnostic test was used to check the presence of inoculated mycorrhizal fungi and the absence of P. fragariae var. rubi in plant roots.

The rhizosphere – the soil compartment influenced by the root, including the root itself – is the most-active microbial habitat in soils. Indeed, the release by plant roots of a significant part of their photosynthates promotes microbial abundance and activities in the rhizosphere. This investment made by plants is paid back by microbial functions, which contribute to plant nutrition and protection against soil-borne diseases. Indeed, rhizosphere microorganisms play a major role in plant growth and health and, …

International audience; Nitric oxide (NO) is involved in diverse physiological processes in plants, including growth, development, response to pathogens, and interactions with beneficial microorganisms. In this work, a dedicated microarray representing the widest database available of NO-related transcripts in plants has been produced with 999 genes identified by a cDNA amplified fragment length polymorphism analysis as modulated in Medicago truncatula roots treated with two NO donors. The microarray then was used to monitor the expression of NO-responsive genes in M. truncatula during the incompatible interaction with the foliar pathogen Colletotrichum trifolii race 1 and during the symbiotic interaction with Sinorhizobium meliloti 1021. A wide modulation of NO-related genes has been detected during the hypersensitive reaction or during nodule formation and is discussed with special emphasis on the physiological relevance of these genes in the context of the two biotic interactions. This work clearly shows that NO-responsive genes behave differently depending on the plant organ and on the type of interaction, strengthening the need to consider regulatory networks, including different signaling molecules.

Parmi les variétés d’ignames Dioscorea alata cultivées aux Antilles françaises, certaines sont tolérantes et d’autres sensibles à l’anthracnose, maladie fongique causée par Colletotrichum gloeosporioides. Nous avons montré que : — Le champignon réalise son installation en procédant par pénétration directe dans la cuticule et ce, sur les deux faces des feuilles de l’igname, qu’elle soit sensible ou tolérante. À la différence des variétés tolérantes, sur lesquelles l’extériorisation de Colletotrichum gloeosporioides est plus lente, les variétés sensibles laissent apparaître les premiers symptômes au bout de 72-75 heures avec une relative rapidité d’extension et de sporulation du champignon. — Celui-ci, à partir de foyers infectieux, sur l’igname, peut se disperser aériennement à la faveur de la pluie et du vent. — Il peut être hébergé par différentes espèces végétales spontanées, dont des Fabacées, Convolvulacées, Loganiacées et Poacées., The cultivated varieties of Dioscorea alata in F.W.I. are resistant or sensitive to Colletotrichum gloeosporioides. This fungus infest all varieties throught the cuticule of both faces of leaves. Spores grow and appressorium appear after 8 or 9 hours under optimal température (21 to 28°C) and relative humidity (40 to 80%) conditions. Further symptoms appear quite quickly on sensitive varieties (72 or 75 hours) wheras resistant plants have light indications. The sporulating fungus can expand from infested leaves by wind rain or via indigenous plants from the Fabaceae, Convolvulaceae, Logoniaceae and Poaceae.

A highly manageable hydroponic system based on the nutrient film technique was designed to provide accurate regulation of the physicochemical and microbiological environment of roots to investigate the rhizoplane microbial dynamics of rose plants. Bacterial communities adhering to roots were subjected to pH and active free chlorine regulation, to determine up to what point they were established. PCR-SSCP fingerprinting showed that pH was a potentially strong stabilisation factor for root microbial communities. Active free chlorine had no quantitative effect when applied at a concentration of 0.15 mg l-1, but did have a selective effect on well established bacterial communities, which were resistant to this strong physicochemical perturbation. Thus, for the control of soilborne diseases, the established community should be investigated, to determine its prior resistance to inoculation or to manage its physicochemical environment, increasing its robustness and excluding biological perturbation., Un système pratique de culture hydroponique a été conçu à l'aide de films nutritifs pour permettre une régulation précise des paramètres physico-chimiques de racines de rosiers afin d'étudier l'évolution dynamique de la communauté microbienne racinaire. Les racines ont été soumises à des pH et des concentrations de chlore libre actif variés afin de déterminer les valeurs limites d'altération des communautés bactériennes adhérentes. Les empreintes moléculaires obtenues par PCR-SSCP ont montré que le pH est un facteur de stabilisation fort pour les communautés microbiennes racinaires. Le chlore actif libre n'a pas d'effet quantitatif sur la communauté microbienne lorsqu'il est appliqué à une concentration de 0,15 mg l-1, mais il a un effet sélectif sur les communautés bactériennes bien établies, et favorise les populations résistantes à cette forte perturbation physico-chimique. Ainsi, pour le contrôle des maladies issues du sol, les communautés microbiennes racinaires devraient être étudiées afin de déterminer leur résistance aux conditions environnementales appliquées pour augmenter leur robustesse et réduire les perturbations biologiques.

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; The colonization process of tomato roots inoculated separately or/and simultaneously by a pathogenic Fusarium oxysporum f. sp. lycopersici strain Fol8 and the protective F. oxysporum strain Fo47, genetically tagged with the red and green fluorescent protein genes, respectively, was studied in a hydroponic culture. Plants were coinoculated with Fol8 and Fo47 at two conidial concentration ratios of 1/1 and 1/100, in which biological control was not effective or effective, respectively. First observation of fungi on root was possible 48 h after inoculation at a high inoculum level and 5 days post inoculation at the lower concentration of inoculum. The pattern of root colonization was similar for both strains with the initial development of hyphal network on the upper part of taproot, followed by the growth of hyphae towards the elongation zone, lateral roots and root apices. Finally, the whole elongation zone and root apex were invaded by both strains but no specific infection sites were observed. When coinoculated, both strains could grow very closely or even at the same spot on the root surface. At the nonprotective ratio, Fol8 was the successful colonizer, but application of Fo47 at a concentration 100 times >Fol8 delayed vessel colonization by the pathogen.

The cultivated varieties of Dioscorea alata in F.W.I. are resistant or sensitive to Colletotrichum gloeosporioides. This fungus infest all varieties throught the cuticule of both faces of leaves. Spores grow and appressorium appear after 8 or 9 hours under optimal température (21 to 28°C) and relative humidity (40 to 80%) conditions. Further symptoms appear quite quickly on sensitive varieties (72 or 75 hours) wheras resistant plants have light indications. The sporulating fungus can expand from infested leaves by wind rain or via indigenous plants from the Fabaceae, Convolvulaceae, Logoniaceae and Poaceae.; Parmi les variétés d’ignames Dioscorea alata cultivées aux Antilles françaises, certaines sont tolérantes et d’autres sensibles à l’anthracnose, maladie fongique causée par Colletotrichum gloeosporioides. Nous avons montré que : — Le champignon réalise son installation en procédant par pénétration directe dans la cuticule et ce, sur les deux faces des feuilles de l’igname, qu’elle soit sensible ou tolérante. À la différence des variétés tolérantes, sur lesquelles l’extériorisation de Colletotrichum gloeosporioides est plus lente, les variétés sensibles laissent apparaître les premiers symptômes au bout de 72-75 heures avec une relative rapidité d’extension et de sporulation du champignon. — Celui-ci, à partir de foyers infectieux, sur l’igname, peut se disperser aériennement à la faveur de la pluie et du vent. — Il peut être hébergé par différentes espèces végétales spontanées, dont des Fabacées, Convolvulacées, Loganiacées et Poacées.

National audience; The rhizosphere is the volume of soil which is influenced by plant root activity. Plant roots release 20% of the photosynthates into the rhizosphere. This process sustains the development of an active free living microflora in the rhizosphere compared to the bulk soil. Plants establish also symbiotic associations with soil microorganisms. Arbuscular mycorrhizas (AM) are mutualistic symbioses between soil fungi and plant roots. AM contribute to plant adaptation to low fertility environments. AM are established by 80% of terrestrial plant families including major crop plants. AM are also ancient symbiosis (400 million years). AM are known to be non-specific associations. However, the plant community is affected by the diversity of AM fungi, and reciprocally we have recently shown that the genetic diversity of AM fungi differ according to the plant species. These observations are consistent by the long joint evolution of plants and AM fungi. The evolution of plants and AM fungi is expected not to have occurred independently from the rhizosphere microflora leading to the emergence of bacteria adapted to mycorrhizal roots, these bacteria being able to influence AM establishment. To test this hypothesis, the genetic structures of bacterial communities associated with Medicago truncatula J5 (Myc+/Nod+) and its symbiosis-defective mutants TRV48 (Myc+/Nod-) and TRV25 (Myc-/Nod-) were compared as follows. DNA was extracted directly from the rhizosphere. Bacterial DNA polymorphism was assessed by an A-RISA (Automated-Ribosomal Intergenic Spacer Analysis) DNA fingerprint. The obtained fingerprints represented the genetic structure of bacterial communities. The genetic structures of bacterial communities were compared by analyzing A-RISA fingerprints with a Principal Component Analysis (PCA). Molecular markers explaining the differences in the genetic structures of the communities associated with M. truncatula genotypes were cloned and sequenced and the corresponding bacterial groups were identified. The cloned markers were further used to design a DNA probe. The probe was used to screen bacterial isolates from mycorrhizal and non-mycorrhizal roots and identify those harboring the sequences of cloned markers. Diversity of isolates showing a positive hybridization signal with the DNA probe was characterized by BOX-PCR. The genetic structure of bacterial communities associated with mycorrhizal and non-mycorrhizal genotypes differed significantly. Phylogenetic analysis of the molecular markers explaining these differences indicated that they belong to Comamonadaceae, Oxalobacteriaceae, and Rubrivivax sub-group. The isolates harboring sequences highly similar to that of the cloned markers were clustered in 25 groups on the basis of their BOX-PCR fingerprints, indicating a high diversity of these isolates. The distribution of the isolates in the different BOX-PCR groups differed significantly upon their origin (mycorrhizal/non-mycorrhizal roots). The effects of isolates representative of the genetic diversity described here above are currently being evaluated to test the value of our initial hypothesis.

National audience; Most aerobic organisms have developed an active strategy for iron uptake. In dicotyledon plants, this strategy involves (i) the excretion of protons, (ii) the reduction of Fe(III) by reductases, and (iii) plasmalemma transport of Fe(II) by iron transporters. In microorganisms, this strategy is based on the synthesis of siderophores and ferri-siderophore membrane receptors, in iron stress conditions. Pyoverdines are the major class of siderophores synthesized by fluorescent pseudomonads. Taking into account the strong iron competition in the rhizosphere and the high affinity of pyoverdines for Fe(III), these molecules are expected to interfere with the iron nutrition of plants, as they do with rhizospheric microbes. The impact of Fe-pyoverdine on iron nutrition of Arabidopsis thaliana was compared to that of Fe-EDTA. Iron chelated to pyoverdine was incorporated in a more efficient way than when chelated to EDTA leading to an increased plant growth and chlorophyll content. Over accumulation of iron in an over-expressor ferritin transgene of A. thaliana was enhanced upon supplementation with Fe-pyoverdine compared to Fe-EDTA. A transgene knockout iron transporter IRT1 of A. thaliana showed a significant lower iron and chlorophyll content when supplemented with Fe-EDTA than the wild-type, but not when supplemented with Fe-pyoverdine. This observation indicates that in contrast with iron chelated to EDTA, iron from pyoverdine was incorporated through a different transporter than IRT1. Incorporation of iron from Fe-pyoverdine was consistent with the presence of pyoverdine in planta as shown by ELISA and by tracing 15N of 15N-pyoverdine. Altogether, these data demonstrate the acquisition of iron from Fe-pyoverdine by A. thaliana and indicate that this acquisition could be related to an as yet undescribed pathway. Researches are underway to identify this pathway and to localise pyoverdine in planta.

ummary The induction of resistance to disease during plant development is widespread in the plant kingdom. Resistance appears at different stages of host development, varies with plant age or tissue maturity, may be specific or broad-spectrum and is driven by diverse mechanisms, depending on plant–pathogen interactions. Studies of these forms of resistance may help us to evaluate more exhaustively the plethora of levels of regulation during development, the variability of the defense potential of developing hosts and may have practical applications, making it possible to reduce pesticide applications. Here, we review the various types of developmental resistance in plants and current knowledge of the molecular and cellular processes involved in their expression. We discuss the implications of these studies, which provide new knowledge from the molecular to the agrosystem level. Contents Summary 405 I. Introduction 405 II. The many forms of developmental resistance 406 III. Molecular mechanisms of developmental resistance 410 IV. Relationships between defense and development in plants 412 V. Concluding remarks 413 Acknowledgements 413 References 413

International audience; In order to investigate potential links existing between Gaeumannomyces graminis var. tritici (Ggt) population structure and disease development during polyetic take-all epidemics in sequences of Ggt host cereals, seven epidemics in fields with different cropping histories were monitored during the seasons 2001/2002 (two fields), 2002/2003 (two fields) and 2003/2004 (three fields). Take-all incidence and severity were measured at stem elongation and Ggt populations were characterized. The 73 isolates collected in the two fields in 2001/2002 were distributed into two multilocus genotypes, G1 and G2 according to amplified fragment length polymorphism analysis. A monolocus molecular marker amplified by F-12 random amplification polymorphism DNA primer sizing between 1.9 and 2.0 kb that gave strictly the same distinction between the two multilocus genotypes was further applied to measure G1/G2 frequencies among Ggt populations in all fields (266 isolates). The ratios of G1 to G2 differed between fields with different cropping histories. A linear relationship between G2 frequency among Ggt populations and disease severity at stem elongation was measured during the three cropping seasons. When take-all decline was observed, G2 frequencies were low in first wheat crops, highest in short-term sequences and intermediate in longer sequences of consecutive crops of Ggt host cereals. This pattern could be the result of population selection by environmental conditions, in particular by microbial antagonism during the parasitic phase of the fungus. In order to better understand take-all epidemic dynamics, the distinction between these two genotypes could be a basis to develop models that link approaches of quantitative epidemiology and advances in population genetics of Ggt

Foreword; International audience

National audience; Arbuscular mycorrhizae (AM) are ancient symbiosis (400 millions years) between Glomeromycetes and roots of 80% of terrestrial plant families. AM are known to be non-specific associations. However, the plant community is affected by the diversity of AM fungi (van der Heijden et al., 1998), and reciprocally we have recently shown that the genetic diversity of AM fungi differ according to the plant species. These observations are consistent by the long joint evolution of plants and AM fungi. This evolution is expected to not have occurred independently from the rhizosphere microflora. To test this hypothesis, we have compared the genetic structure of bacterial communities associated with Medicago truncatula J5 (Myc+/Nod+) and its symbiosis-defective mutants TRV48 (Myc+/Nod-) and TRV25 (Myc-/Nod-). DNA was extracted from the corresponding rhizospheres and its polymorphism was assessed by A-RISA DNA fingerprint. Based on Principal Component Analysis of the corresponding fingerprints, markers explaining the differences of structure between the communities associated with M. truncatula genotypes were sequenced and identified. These markers were further used to design probes used to screen isolates from mycorrhized roots that belonged to the corresponding microbial groups. The diversity of these isolates was characterized by BOX-PCR. The genetic structure of bacterial communities associated with mycorrhized and non-mycorrhized plants differed significantly. Phylogenetic analysis of the molecular markers explaining these differences indicated that they belong to Comamonadaceae, Oxalobacteriaceae, and Rubrivivax sub-group. The isolates harboring sequences highly homologous to that of markers affiliated to these microbial groups were clustered in 25 groups on the basis of their BOX-PCR fingerprints, indicating a high diversity of these isolates. The distribution of the isolates in the different BOX-PCR groups differed significantly upon their origin (mycorrhized/non-mycorrhized roots). The effects of isolates representative of the genetic diversity described here above is currently being evaluated to test the value of our initial hypothesis.

International audience; In the context of increasing international concern for food and environmental quality, use of Plant Growth-Promoting Rhizobacteria (PGPR) for reducing chemical inputs in agriculture is a potentially important issue. PGPR are root-colonizing bacteria that exert beneficial effects on plant growth and development, but they can be also employed in the control of plant pathogens, for enhancing the efficiency of fertilizers, and for degrading xenobiotic compounds. This book provides an update by renowned international experts on the most recent advances in the ecology of these important bacteria, the application of innovative methodologies for their study, their interaction with the host plant, and their potential application in agriculture.

Summary Potential differences between Bt-maize (MEB307 expressing the insecticidal Cry1Ab protein) and a near-isogenic non-Bt variety (Monumental) in their influence on the garden snail (Helix aspersa), soil microarthropods (Collembola, Actinedida, Acaridida, Gamasida and Oribatida) and mycorrhizal fungi were studied. Growing snails were caged in microcosms allowing the development of Bt or non-Bt-maize (Zea mays L.) on a sandy loam soil. After 3 months exposure, survival and growth of snails were similar in both treatments. Cry1Ab protein was detected in the Bt-maize leaves (22–42.2 μg Bt protein g−1 dry wt), in the snail tissues (0.04–0.11 μg Bt-protein g−1 dry wt) and in their faeces (0.034–5 μg Bt-protein g−1 dry wt). Total soil microarthropod abundance and diversity were similar between control (non-Bt-maize) and the genetically modified (GM) Bt-maize microcosms. The mycorrhizal colonization of roots did not differ between Bt and non-Bt-maize (frequency of mycorrhizal roots was 88.7% and 83.3% respectively). The mycorrhizal infectivity of soils, expressed as MI50 (minimum soil dry weight required to colonize 50% of plants) was measured using red clover. MI50 was similar for soils where Bt or non-Bt-maize was cultivated for 4 months. The detection of Cry1Ab protein in the viscera and faeces of H. aspersa exposed to Bt-maize indicates that snails contribute to the transfer of the Bt-protein from plant to soil or snail predators. This may constitute an alternative route of exposure for Bt-protein in soil, but this was without a negative influence on mycorrhizal fungi or microarthropods. Results showed that Bt-maize was not toxic for the selected non-target species exposed for 3 or 4 months. The microcosms and analyses used in this study represent new methods for assessing effects of chronic exposure to GM plants of several diverse, yet ecologically and temporally associated species. As the soil organisms we studied can also be used in standardized ecotoxicological tests (XP X31-205-2 for mycorrhizal fungi, ISO 11267 for Collembola and ISO 15952 for snails), microcosm exposures represent a way to link laboratory and field methods for the ecotoxicological evaluation of GM plants.