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PopA is released by type III secretion from the bacterial plant pathogen Ralstonia solanacearum and triggers the hypersensitive response (HR) in tobacco. The function of PopA remains obscure, mainly because mutants lacking this protein are not altered in their ability to interact with plants. In an attempt to identify the site of PopA activity in plant cells, we generated transgenic tobacco plants expressing the popA gene under the control of an inducible promoter. Immunocytologic analysis revealed that the HR phenotype of these plants correlated with the presence of PopA at the plant plasma membrane. Membrane localization was observed irrespective of whether the protein was designed to accumulate in the cytoplasm or to be secreted by the plant cell, suggesting a general lipid-binding ability. We found that the protein had a high affinity for sterols and sphingolipids in vitro and that it required Ca2+ for both lipid binding and oligomerization. In addition, the protein was integrated into liposomes and membranes from Xenopus laevis oocytes where it formed ion-conducting pores. These characteristics suggest that PopA is part of a system that aims to attach the host cell plasma membrane and to allow molecules cross this barrier.

International audience; A root-knot nematode (RKN) parasitizing rice (Oryza sativaL.) and causing damage in Santa Catarina (SC), Rio Grande do Sul (RS) and Parana (PR) states (Brazil) was identified asMeloidogyne ottersoni(Thorne1969) Franklin1971. The species is redescribed from the Brazilian population from Meleiro (SC) and compared with the description ofM. ottersonifrom Wind Lake (Wisconsin, USA) with additional morphological, biochemical and molecular characterization. The female and male bear smaller stylets: 10-12 mu m, 14-16 mu m, respectively, when compared withM. graminicola: 12-14 mu m, 16-18 mu m, andM. oryzae: 14-16 mu m, 18-20 mu m.Meloidogyne ottersonipresents perineal patterns located on the contour of a slight protuberance. Striae are mostly continuous, never raised by transverse irregular striae, as frequently observed inM. graminicolaandM. oryzae.Meloidogyne ottersonibelongs to the RKN group 11 described by Jepson (1987); the reproduction is by meiotic parthenogenesis and the somatic chromosome number is 18. The tail of second-stage juveniles is very long and thin, and tapers to a long, narrow, irregular hyaline terminus (M. ottersoni, 20.5 mu mvs M. graminicola,17.9 mu m andM. oryzae, 22.0 mu m, respectively). The ability of the BrazilianM. ottersonipopulation to parasitize canary grass,Phalaris arundinaceaL. (type host), and barnyard grass,Echinocloa crus-galli,was confirmed. Biochemically, the esterase profile ofM. ottersonilacks any band (Est Ot0, Rm=0), which differentiates it fromM. graminicolaandM. oryzae(Est VS1, Rm=0.70 and Est O1,Rm=1.02, respectively). In Maximum Likelihood analysis of ITS, D2D3 and COXII-16S rRNA sequences, populations ofM. ottersonifrom different states of Brazil clustered together and were separated from otherMeloidogynespp., thus confirming that all four populations are very similar and conspecific.

Pachysandra terminalis (Buxaceae) was introduced from Japan to Europe in 1882. This ornamental plant is grown in northern Europe as a ground cover in shaded sites. Line patterns, more or less necrotic ringspots, and mosaic symptoms on leaves of pachysandra plants have been seen in public gardens in France (Strasbourg, Colmar, Mulhouse, and Nantes) and Germany (Freiburg im Breisgaü). Extracts of plant tissues obtained from these five sites were used for mechanical- and aphid-transmission experiments, enzyme-linked immunosorbent assays (ELISA) with antibodies directed toward a tomato strain of Alfalfa mosaic virus (AMV) (from G. Marchoux, INRA, France), and electron microscopy. All inoculations produced symptoms typical for AMV in Nicotiana tabacum cv. Xanthi-nc tobacco, Chenopodium amaranticolor, C. quinoa, Vigna unguiculata, Phaseolus vulgaris, Vicia faba, Pisum sativum, and Ocimum basilicum (2). Moreover, Medicago arborea, a new host for AMV, showed systemic mosaic on leaflets. On these and the original pachysandras, AMV was readily detected by ELISA. After isolation from three to four local lesions on Vigna unguiculata and further multiplication in tobacco, one isolate was purified. Bacilliform particles of three sizes, typical for AMV, were seen by electron microscopy. Transmissions of the strain to ELISA-negative pachysandras was achieved by mechanical inoculations (7 of 20 inoculated plants were ELISA positive) and by the aphid species Myzus persicae and Aphis craccivora (14 of 17 inoculated plants were infected). Symptoms were observed 2 months after inoculation; some of the plants remained symptomless but were AMV positive in ELISA. As early as 1970, similar symptoms were reported in pachysandra in New Jersey, and AMV was isolated from affected plants (1). However, inoculations of healthy pachysandra plants with AMV was not performed. Our results show the need for an AMV indexing protocol in the propagation of pachysandra to control its spread. References: (1) D. E. Hershman and E. H. Varney. Plant Dis. 66:1195, 1982. (2) E. M. J. Jaspars and L. Bos. 1980. Alfalfa mosaic virus. No. 229 in: Descriptions of Plant Viruses. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England.

Physostegia virginiana Benth. (false dragon head) is a perennial plant from the family Lamiaceae cultivated as an ornamental in gardens and for cut-flower production. In 2000, stunting of plants and yellow-to-brown ringspots on leaves were observed in cut-flower production in the Alpes Maritimes Department (southeast France). These symptoms greatly decreased the commercial value of the stems. The disease was attributed to Alfalfa mosaic virus (AMV) because extracts of infected plant tissues revealed typical bacilliform particles by electron microscopy, produced symptoms typical of AMV after inoculation of a range of previously described test plants (1), and reacted positively in enzyme-linked immunosorbent assays (ELISA) with antibodies raised to a tomato strain of AMV (from G. Marchoux, INRA, France). After isolation from single local lesions on Vigna unguiculata, the AMV isolate was multiplied in cv. Xanthinc tobacco, where it induced local and systemic ringspot symptoms. Infected Xanthinc plants served as sources of inocula for subsequent mechanical- and aphid (Myzus persicae)-transmission tests to healthy seedlings of P. virginiana (seeds from the botanic garden of Nancy, France; 36 plants for each inoculation procedure). Chlorotic and necrotic local lesions were observed in 25% of mechanically inoculated plants. Three months after inoculation, uninoculated leaves of all mechanically inoculated plants and 30.5% of aphid-inoculated plants tested positive for AMV based on ELISA. During the first year after inoculation, less than 10% of infected plants showed typical systemic symptoms. This proportion reached 40% during the second year. Recently, we observed similar symptoms in P. virginiana plants cultivated in public gardens in Intercourse (Pennsylvania), Toronto (Ontario, Canada) and Montreal (Quebec, Canada). Using ELISA, AMV was detected in symptomatic plants from these three additional locations. Reference: (1) L. Cardin and B. Moury. Plant Dis. 84:594, 2000.

International audience; Meloidogyne spp., plant-parasitic nematodes present worldwide, are intensively studied because of the damage caused to a large variety of agronomically important crops. Several reports indicate that proteins from the Meloidogyne spp. dorsal gland might play an important role to allow proper establishment of a functional nematode feeding site. The precise role of these proteins in the process of feeding cell development is unknown. To gain insights into the function of these secreted M. incognita proteins, we constitutively (ectopically) expressed the nematodes dorsal gland protein 7E12 in tobacco plants. It was found that the number of galls at 8 and 16 days after nematode infection was significantly higher in transgenic plants compared to control plants. Eggs from nematodes in transgenic plants hatched faster than those in control plants. Histological analysis of nematode induced galls in transgenic plants clearly shows a different morphology. Giant feeding cells harbor more vacuoles and an increased amount of cell wall invaginations, while neighboring cells surrounding feeding cells are more numerous. These results suggest that the presence of the 7E12 protein in tobacco accelerates gall formation. This assumption is supported by our data illustrating faster gall formation and egg eclosion in transgenic plants. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Osteoporosis constitutes a major worldwide public health burden characterized by enhanced skeletal fragility. Bone metabolism is the combination of bone resorption by osteoclasts and bone formation by osteoblasts. While increase in bone resorption is considered as the main contributor of bone loss that may lead to osteoporosis, this loss is accompanied by increased bone marrow adiposity. Osteoblasts and adipocytes share the same precursor cell and an inverse relationship exists between the two lineages. Therefore, identifying signaling pathways that stimulates mesenchymal stem cells osteogenesis at the expense of adipogenesis is of major importance in order to develop new therapeutic treatments. For this purpose, we identified by transcriptomic analysis the oxytocin receptor pathway as a potential regulator of the osteoblast/adipocyte balance of human multipotent adipose-derived stem (hMADS) cells. Both oxytocin (OT) and carbetocin (Cb, a stable OT analogue) negatively modulate adipogenesis while promoting osteogenesis in both hMADS cells and human bone marrow mesenchymal stromal cells (hBMSC). Consistent with these observations ovariectomized (OVX) mice and rats, which become osteoporotic and exhibit disequilibrium of this balance, have significant decreased OT levels compared to sham-operated controls. Subcutaneous OT injection reverses bone loss in OVX mice and reduces marrow adiposity. Clinically, plasma OT levels are significantly lower in postmenopausal women developing osteoporosis than in their healthy counterparts. Taken together, these results suggest that plasma OT levels represent a novel diagnostic marker for osteoporosis and that OT administration holds promise as a potential therapy for this disease. ______________________________________________________________________________ Author contributions: C.E.: carried out most of the experiments, data analysis and interpretation, collection and assembly of data; A.B., H.B. and C.B.: analysis of bone micro-architecture and biomechanical parameters of mice, manuscript writing; L.Z., M.S. and Z.T.: microarray analysis, data analysis and interpretation; F.M.: contribution to the mice study, data analysis and interpretation; V.B., G.F.C. and L.E.: provision of patients, data analysis and interpretation; N.W.: histological analysis of mice tissues, data analysis and interpretation; E.L.: provision of study material, data analysis and interpretation; C.D.: data analysis and interpretation, manuscript writing; G.A.: conception and design, data analysis and interpretation, manuscript writing; E.A.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing.

Plant-parasitic nematodes are major agricultural pests worldwide and novel approaches to control them are sorely needed. We report the draft genome sequence of the root-knot nematode Meloidogyne incognita, a biotrophic parasite of many crops, including tomato, cotton and coffee. Most of the assembled sequence of this asexually reproducing nematode, totaling 86 Mb, exists in pairs of homologous but divergent segments. This suggests that ancient allelic regions in M. incognita are evolving toward effective haploidy, permitting new mechanisms of adaptation. The number and diversity of plant cell wall-degrading enzymes in M. incognita is unprecedented in any animal for which a genome sequence is available, and may derive from multiple horizontal gene transfers from bacterial sources. Our results provide insights into the adaptations required by metazoans to successfully parasitize immunocompetent plants, and open the way for discovering new antiparasitic strategies

International audience; Sedentary endoparasitic nematodes are root parasites that interact with their hosts in a remarkable way. These obligate biotrophic pathogens establish an intimate relationship with their host plants, inducing the redifferentiation of root cells into specialized feeding cells. The successful establishment of feeding cells is essential for nematode development. Root-knot nematodes, of the genus Meloidogyne, have evolved strategies enabling them to induce feeding cell formation in thousands of plant species, probably by manipulating fundamental elements of plant cell development. Feeding cells enlarge and are converted into multinucleate giant cells through synchronous nuclear divisions without cell division. Fully differentiated giant cells may contain more than a hundred polyploid nuclei that may have undergone extensive endoreduplication. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of the typical root gall. Giant cell formation requires extensive changes to gene expression. The induction of feeding cells remains poorly understood, but it is thought that effectors secreted by the nematode play a key role in parasitism, with potential direct effects on recipient host cells. In this review, we focus on the most recent investigations of the molecular basis of the plant–root-knot nematode interaction. Recently, microarray technology has been used to study the plant response to Meloidogyne spp. infection. Such a genome-wide expression profiling provides a global view of transcriptional changes, especially for genes involved in cell wall, transport processes and plant defense responses during giant cell formation. The identification of nematode-responsive plant genes constitutes a major step toward understanding how root-knot nematodes dramatically alter root development to induce and maintain giant cells. The characterization of nematode secretions as parasitism effectors and the development of RNAi technology should improve our understanding of the molecular events and regulatory mechanisms involved in plant parasitism. Finally, Meloidogyne genome sequences should provide further insight into plant–root-knot nematode interactions.

SummarySexual reproduction involves epigenetic reprogramming [1] comprising DNA methylation [2] and histone modifications [3–6]. In addition, dynamics of HISTONE3 (H3) variant H3.3 upon fertilization are conserved in animals, suggesting an essential role [7–9]. In contrast to H3, H3.3 marks actively transcribed regions of the genome and can be deposited in a replication-independent manner [10, 11]. Although H3 variants are conserved in plants, their dynamics during fertilization have remained unexplored. We overcame technical limitations to live imaging of the fertilization process in Arabidopsis thaliana and studied dynamics of the male-gamete-specific H3.3 [12] and the centromeric Histone Three Related 12 (HTR12) [13]. The double-fertilization process in plants produces the zygote and the embryo-nourishing endosperm [14]. We show that the zygote is characterized by replication-independent removal of paternal H3.3 and homogeneous incorporation of parental chromatin complements. In the endosperm, the paternal H3.3 is passively diluted by replication while the paternal chromatin remains segregated apart from the maternal chromatin (gonomery). Hence epigenetic regulations distinguish the two products of fertilization in plants. H3.3-replication-independent dynamics and gonomery also mark the first zygotic divisions in animal species [5, 15]. We thus propose the convergent selection of parental epigenetic imbalance involving H3 variants in sexually reproducing organisms.

Legume-Rhizobium nitrogen fixation is dramatically affected under drought and other environmental constraints. However, it has yet to be established as to whether such regulation of nitrogen fixation is only exerted at the whole-plant level (e.g. by a systemic nitrogen feedback mechanism) or can also occur at a local nodule level. To address this question, nodulated pea (Pisum sativum) plants were grown in a split-root system, which allowed for half of the root system to be irrigated at field capacity, while the other half was water deprived, thus provoking changes in the nodule water potential. Nitrogen fixation only declined in the water-deprived, half-root system and this result was correlated with modifications in the activities of key nodule's enzymes such as sucrose synthase and isocitrate dehydrogenase and in nodular malate content. Furthermore, the decline in nodule water potential resulted in a cell redox imbalance. The results also indicate that systemic nitrogen feedback signaling was not operating in these water-stressed plants, since nitrogen fixation activity was maintained at control values in the watered half of the split-root plants. Thus, the use of a partially draughted split-root system provides evidence that nitrogen fixation activity under drought stress is mainly controlled at the local level rather than by a systemic nitrogen signal. © 2007 American Society of Plant Biologists.

Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (β-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the over-expressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

Phytophthora parasitica is a soilborne oomycete pathogen capable of infecting a wide range of plants, including many solanaceous plants. In a first step towards large-scale gene discovery, we generated expressed sequence tags (ESTs) from a cDNA library constructed using mycelium grown in synthetic medium. A total of 3568 ESTs were assembled into 2269 contiguous sequences. Functional categorization could be performed for 65.45% of ESTs. A significant portion of the transcripts encodes proteins of common metabolic pathways. The most prominent sequences correspond to members of the elicitin family, and enzymes involved in the lipid metabolism. A number of genes potentially involved in pathogenesis were also identified, which may constitute virulence determinants.

We prepared a series of cryptogein mutants, an elicitor from Phytophthora cryptogea, with altered abilities to bind sterols and fatty acids. The induction of the early events, i.e., synthesis of active oxygen species and pH changes, in suspension tobacco cells by these mutated proteins was proportional to their ability to bind sterols but not fatty acids. Although the cryptogein-sterol complex was suggested to be a form triggering a defense reaction in tobacco, some proteins unable to bind sterols induced the synthesis of active oxygen species and pH changes. The modeling experiments showed that conformational changes after the introduction of bulky residues into the omega loop of cryptogein resemble those induced by sterol binding. These changes may be necessary for the ability to trigger the early events by elicitins. However, the ability to stimulate necrosis in suspension tobacco cells and the expression of defense proteins in tobacco plants were linked neither to the lipid binding capacity nor to the capacity to provoke the early events. On the basis of these experiments and previous results, we propose that elicitins could stimulate two signal pathways. The first one induces necroses and the expression of pathogen-related proteins, includes tyrosine protein kinases and mitogen-activated protein kinases, and depends on the overall structure and charge distribution. The second type of interaction is mediated by phospholipase C and protein kinase C. It triggers the synthesis of active oxygen species and pH changes. This interaction depends on the ability of elicitins to bind sterols.

Plant lipid transfer proteins (LTPs) are small, cysteine-rich proteins secreted into the extracellular space. They belong to the pathogenesis-related proteins (PR-14) family and are believed to be involved in several physiological processes including plant disease resistance, although their precise biological function is still unknown. Here, we show that a recombinant tobacco LTP1 is able to load fatty acids and jasmonic acid. This LTP1 binds to specific plasma membrane sites, previously characterized as elicitin receptors, and is shown to be involved in the activation of plant defense. The biological properties of this LTP1 were compared with those of LTP1-linolenic and LTP1-jasmonic acid complexes. The binding curve of the LTP1-linolenic acid complex to purified tobacco plasma membranes is comparable to the curve obtained with LTP1. In contrast, the LTP1-jasmonic acid complex shows a strongly increased interaction with the plasma membrane receptors. Treatment of tobacco plants with LTP1-jasmonic acid resulted in an enhancement of resistance toward Phytophthora parasitica. These effects were absent upon treatment with LTP1 or jasmonic acid alone. This work presents the first evidence for a biological activity of a LTP1 and points out the crucial role of protein-specific lipophilic ligand interaction in the modulation of the protein activity.

Prunus species express a more or less wide spectrum of resistance to root-knot nematodes (RKN) of the genus Meloidogyne. Among them, sources from Myrobalan plum (P. cerasifera) control all major and minor RKN species tested. In this outbreeding species, the clones P.2175 and P.2980 are heterozygous for the Ma single dominant gene and carry the alleles Ma1 and Ma3, respectively. Each allele confers a high-level resistance to the predominant RKN, M. arenaria, M. incognita and M. javanica and to the Florida isolate of an unknown Meloidogyne sp. which overcomes the resistance from peach and almond sources. The polymorphism of two coupling-phase SCAR markers tightly linked to Ma, SCAL19690 and SCAFLP2202, was evaluated within diverse diploid Prunus accessions. This material belongs to the subgenera Prunophora (Myrobalan and apricot) or Amygdalus (peach, almond and almond-peach) and includes the RKN resistance sources ‘Nemared’, ‘Alnem 1’ and ‘GF.557’. The alleles SCAL19690 and SCAFLP2202 were not present in three apricot cultivars (‘Moniqui’, ‘Luizet’ and ‘Stark Early Orange’) representative of the genetic diversity of this species and they segregated in an interspecific cross between P.2980 and apricot. These results suggest that apricot, reported as resistant to M. arenaria, M. incognita and M. javanica, and the Myrobalan plum might possess two different resistance systems. SCAL19690 and SCAFLP2202 were also absent from all tested Amygdalus material, whatever its resistance to RKN. Eight Myrobalan×Amygdalus segregating progenies including bispecific (P.2175 or P.2980×peach or almond) and trispecific (P.2175 or P.2980×almond-peach) hybrids were tested with the Florida isolate to identify individuals carrying the Ma resistance alleles. Both SCARs were then evaluated for segregation in these progenies to develop marker-assisted selection of Prunus interspecific rootstocks. SCAL19690 and SCAFLP2202 could be clearly detected and their tight linkage to Ma1 and Ma3 was confirmed. Consequently these SCARs appear to be powerful tools to screen for RKN resistance conferred by the Ma gene. They should also facilitate marker-assisted pyramiding of Ma with other resistance genes from the Amygdalus subgenus or from the botanically-related Armeniaca section.

The spectrum and inheritance of resistance to the root-knot nematode Meloidogyne hapla was studied in the diploid species Rosa multiflora and R. indica. The host suitability of seven rose rootstocks, namely five R. multiflora (K2, K1, Floradale, CE63 and CE65) and two R. indica (CE35 and Maroc) clonal accessions, was evaluated using four geographic isolates of M. hapla. Plants grown under greenhouse conditions were tested at high and durable inoculum pressure of nematodes and rated for nematode infestation five months after inoculation on a 0–5 gall index. Different host suitabilities to M. hapla were demonstrated depending on the nematode isolates: in R. multiflora, the clone K2 had a resistant (R) response to all isolates; the clone K1 ranged from intermediate (I) (isolate ‘Canada’) to resistant (other isolates), Floradale was shown to be intermediate, whereas CE63 and CE65 were moderate to good hosts (H). In R. indica, both rootstocks were good to excellent hosts for the isolate Canada but resistant to all three other isolates, thus expressing an isolate-specific resistance. A study on the genetics of resistance in R. multiflora to the M. hapla isolate Canada was then conducted using an incomplete diallel cross involving all previous clones except Floradale. A total of 120 hybrid individuals belonging to several progenies representing the cross combinations R × I, R × H, I × H and H × H were evaluated. Individuals of each progeny generally ranged into a monomodal distribution that suggests polygenic inheritance of resistance. In the family Rosaceae, the differences in the resistance genetics to the meiotic species M. hapla and to the mitotic species, M. arenaria, M. incognita and M. javanica are discussed in relation to the reproductive status of the nematodes.

A cDNA-amplification fragment length polymorphism (AFLP)-based strategy has been used to identify genes differentially expressed between two pairs of near-isogenic lines (NIL) of the root-knot nematode Meloidogyne incognita either avirulent or virulent against the tomato Mi resistance gene. Gene expression profiles from infective second-stage juveniles (J2) were compared, and 22 of the 24,025 transcript-derived fragments (TDF) generated proved to be differential, i.e., present in both avirulent NIL and absent in both virulent NIL. Fourteen of the TDF sequences did not show any significant similarity to known proteins, while eight matched reported sequences from nematodes and other invertebrates. The differential expression of nine genes was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) experiments. In situ hybridization conducted with five of the sequences showed that two were specifically expressed in the intestinal cells (HM10 and PM1), one in the subventral esophageal glands (HM1), and two in the dorsal esophageal gland of J2 (HM7 and HM12). Analysis of full-length cDNA sequences revealed the presence of a signal peptide for HM1, HM10, and HM12, indicating that the encoded proteins are putatively secreted. Since secreted products in general and esophageal gland secretions in particular are thought to be among the main M. incognita pathogenicity factors, this result suggests a possible dual role for some of the genes encoding such secretions, i.e., they could be involved in both pathogenicity and virulence or avirulence of these biotrophic parasites.

Resistance to pests and diseases is a highly desirable objective in fruit breeding but is a long-term endeavor. It requires appropriate tests for early screening and an analysis of host-parasite interactions for durability of resistance. Host resistance is considered durable when it remains effective for a long period despite an intensive exposure to the parasite. Durability has to be the key-point of any fruit breeding programme for resistance. Non-durable resistance is usually associated with a hypersensitive reaction (HR) and major gene resistance which often occur in multiple allelic series and/or complex loci. HR resistance or race-specific resistance is often rendered ineffective because the pathogen population shifts genetically to avoid the resistance gene. Resistance of a more durable nature against fungi and bacteria is often polygenic and based upon the additive or interaction effects of a few to several genes not promoting a hypersensitive reaction. The more complex the genetics of the resistance, the more difficult it is for the pathogen to evolve to a form that can overcome the resistance. Polygenic resistance is usually associated with a quantitative expression that is characterized by a continuous variation between susceptibility and resistance. To approach the number of genes involved in polygenic resistance (QTL) and the relative effect of each of these genes, they have to be mapped on the genome. Marker-assisted selection (MAS) gives the possibility to recombine QTL for resistance; major genes could be associated with QTL. The combining of resistance genes is desired to: acquire a higher level of resistance against a particular pathotype, obtain resistance against a broader range of pathotypes, and combine different mechanisms of resistance. The current situation of fruit crops - monocultivar orchards established for several years - enhances the selection pressure on the pathogen population and results in population shifts which could be dramatic. Guidelines have to be proposed to predict the evolutionary potential of pathogen populations based on analysis of their genetic structure; the most important parameters to consider are reproductive/mating systems and gene/genotype flow. Pathogens that have the greatest risk of breaking down resistance genes are those that possess a mixed reproduction system, with one sexual cycle per growing season and asexual reproduction during the epidemic phase, and a high potential for gene flow. Several examples of different strategies used by fruit breeders will be reported mainly on Coffea (coffee rust), Musa (black leaf streak disease), Prunus (root knot nematodes) and Malus (scab, mildew, fire blight). Fruit breeders can use the lessons learned from deploying resistance genes in annual crops and selecting resistance in forest trees and apply these to their strategies for fruit tree improvement

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplas-tic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

International audience; The toxic effects of wastewater samples, collected in December 1998, from upstream (U) and downstream (D) sites of the purification station of the town of Nice (South-East France on the coast of the Mediterranean Sea) were assessed undiluted and at various dilutions (75%, 50%, and 25% of collected water sample), on trout hepatocyte cultures treated for 48 or 72 h. Chemical contamination (PCBs, PAHs, Cd, Cu, Pb, and Zn) was also evaluated by chemical analysis. The water samples from the upstream site were more cytotoxic than those from the downstream site. The induction of CYP1A enzyme and metallothioneins (MTs) were selected as specific indicators of exposure to organic contaminants and metals, respectively. CYP1A-related EROD activity as well as protein expression were found to be greatly induced after 72 h exposure of the hepatocytes to the undiluted water samples (U(100%) and D(100%)), but CYP1A1 mRNA was significantly overexpressed only by samples from the upstream site. Maximal MT levels were reached after 48 h of treatment with the least concentrated water samples (U(25%) and D(25%)). Glutathione S-transferase (GST) activities were similarly increased under the same conditions. On the other hand, there was no significant glutathione peroxidase (GPx) activity response. Induction of apoptosis was analyzed by using as markers both the fragmentation of the nuclear DNA into oligonucleosomal-length fragments recognized as a "DNA ladder" and the activation of DEVD (Asp-Glu-Val-Asp)-dependent protease considered as the central mediator of programmed cell death. Significant DNA cleavage was only detectable after 72-h exposure to the most concentrated water samples from upstream sites (U(75%) and U(100%)). DEVD-dependent protease activities were significantly increased, mainly in cells exposed to U(75%) and D(25%) for 72 h. In addition, pollution-related DNA damage assessed by using the Comet assay was approximatively 1.5 times greater than that of the control level with the undiluted water samples U(100%) and D(100%), after 72-h and 48-h exposure, respectively. The present study shows that such a multibiomarker-based approach could provide complementary information, for aquatic pollution monitoring, about the early biochemical effects in cells exposed to complex chemical pollution and could be considered as early warning systems to aquatic pollution.

Activity of Trichogramma, assessed by trapping with host-eggs, was observed as early as April and continued until October in the vineyards and their surrounding biotopes. Trichogramma showed the greatest activity in April-May, July and September. Parasitized eggs were found before the main laying period of the first generation of the grapevine moths Lobesia botrana Den. and Schiff. and Eupoecilia ambiguella Hb. Four native species, Trichogramma cacoeciae Marchal, Trichogramma daumalae Dugast and Voegele, Trichogramma evanescens Westwood and Trichogramma principium Sugonjaev and Sorokina were found.

Elicitins secreted by phytopathogenic Phytophthora spp. are proteinaceous elicitors of plant defense mechanisms and were demonstrated to load, carry, and transfer sterols between membranes. The link between elicitor and sterol-loading properties was assessed with the use of site-directed mutagenesis of the 47 and 87 cryptogein tyrosine residues, postulated to be involved in sterol binding. Mutated cryptogeins were tested for their ability to load sterols, bind to plasma membrane putative receptors, and trigger biological responses. For each mutated elicitin, the chemical characterization of the corresponding complexes with stigmasterol (1:1 stoichiometry) demonstrated their full functionality. However, these proteins were strongly altered in their sterol-loading efficiency, specific binding to high-affinity sites, and activities on tobacco cells. Ligand replacement experiments strongly suggest that the formation of a sterol-elicitin complex is a requisite step before elicitins fasten to specific binding sites. This was confirmed with the use of two sterol-preloaded elicitins. Both more rapidly displaced labeled cryptogein from its specific binding sites than the unloaded proteins. Moreover, the binding kinetics of elicitins are related to their biological effects, which constitutes the first evidence that binding sites could be the biological receptors. The first event involved in elicitin-mediated cell responses is proposed to be the protein loading with a sterol molecule.

Two major forms of glutathione S-transferase are known in Drosophila melanogaster: GST D and GST 2. In the present paper we report the existence of a third major form of glutathione S-transferase in Drosophila simulans. Induction with phenobarbital revealed a different regulation of GST between these species. Despite the fact that these two species are closely related, there was a difference in the expression profile of the enzyme implicated in the detoxification system, suggesting variations in capacity to suit their environment.

International audience; Phytophthora spp. secrete proteins called elicitins in vitro that can specifically induce hypersensitive response and systemic acquired resistance in tobacco. In Phytophthora parasitica, the causal agent of black shank, most isolates virulent on tobacco are unable to produce elicitins in vitro. Recently, however, a few elicitin-producing P. parasitica strains virulent on tobacco have been isolated. We investigated the potential diversity of elicitin genes in P. parasitica isolates belonging to different genotypes and with various virulence levels toward tobacco as well as elicitin expression pattern in vitro and in planta. Although elicitins are encoded by a multigene family, parAl is the main elicitin gene expressed. This gene is highly conserved among isolates, regardless of the elicitin production and virulence levels toward tobacco. Moreover, we show that elicitin-producing P. parasitica isolates virulent on tobacco down regulate parAl expression during compatible interactions, whichever host plant is tested. Conversely, one elicitin-producing P. parasitica isolate that is pathogenic on tomato and avirulent on tobacco still expresses parAl in the compatible interaction. Therefore, some P. parasitica isolates may evade tobacco recognition by down regulating parA1 in planta. The in planta down regulation of parA1 may constitute a suitable mechanism for P. parasitica to infect tobacco without deleterious consequences for the pathogen.

The patch exploitation strategy of females of the insect parasitoid Trichogramma brassicae was studied on patches containing different proportions of hosts that were previously attacked by conspecific females. On average, T. brassicae females spent more time on patches of higher quality, and all patches were reduced to the same level of profitability before being left. This appeared to be in accordance to the optimal predictions of the Charnov Marginal Value Theorem. The proximate leaving mechanisms involved were analyzed by means of a Cox proportional hazards model. Each oviposition in a healthy host appeared to have an incremental influence on the patch residence time, whereas each rejection of a healthy host or of a host that was previously attacked by the same female (i.e., self-superparasitism) had a decremental effect. These patch leaving mechanisms did not change according to the quality of the patch the females were exploiting. A Monte Carlo simulation was developed around the results of the Cox regression model. The results suggest that this set of patch leaving rules seems to provide the females with a sufficient way to reach the predictions of the Charnov model. Among the different mechanisms involved, the incremental effect associated with each oviposition in a healthy host appeared to play the most important role. The relationship between the proximate mechanistic rules adopted by the females and the ultimate prediction of the Charnov model is discussed. Key words: patch leaving rules, Marginal Value Theorem, Cox regression model, parasitoids, Trichogramma, Monte Carlo simulation. [Behav Ecol 11:577–586 (2000)]

The root-knot nematodes Meloidogyne chitwoodi and M. fallax are potato parasites that have recently been designated as quarantine pests in Europe. The related and more widely distributed species M. hapla may be found associated with them in field conditions. Because of their morphological resemblance, specific diagnosis of these three species is a difficult task. Therefore, simple and reliable diagnostic tools are urgently needed, for both routine identification of these nematodes and implementation of efficient management strategies and/or phytosanitary regulations. Here is presented an overview of the research recently conducted at INRA Antibes (FR) in order to develop molecular tools for the characterization of these nematodes, based on satellite DNA sequences isolated from their genome.

A homozygous flightless strain, obtained by selection from a laboratory population of Harmonia axyridis Pallas (Coleoptera, Coccinellidae), has been reared under controlled conditions since 1982 after sampling in China. The adults of this flightless strain have normal elytra and wings but drop almost vertically when they attempt to fly. The origin of this mutation, whether laboratory or natural, is discussed. As the mutation does not affect the fitness of the beetles, this flightless strain can be used in biological control. Releasing flightless adults rather than larvae should provide the potential for more continuous control of aphids by both larvae and adults.

Summary - Description of four new species and four populations of Rotylenchus (Nematoda: Hoplolaimidae). Proposal a tabular key - Four new species from France are described and figured: Rotylenchus ascalpi sp. n., characterised by an annulated, rounded and set off lip region; R. corsicus sp. n., characterised by a rounded, annulated lip region; R. echelimae sp. n., characterised by a truncated, smooth lip region; R. provincialis sp. n., characterised by a conoïd rounded, annulated lip region and an unannulated tail terminus. The measurements, figures and main morphological differences in relation to the original descriptions of four other populations are given: Rotylenchus capensis , R. eximius, R. magnus jaeni and R. magnus magnus. An emended diagnosis of the genus, a list of valid species and species inquirendae vel dubiae and a tabular key to species are proposed. Quatre nouvelles espèces de Rotylenchus sont décrites et figurées: Rotylenchus ascalpi sp. n., caractérisé par une région céphalique arrondie, comportant de cinq à sept anneaux; R. corsicus sp. n. caractérisé par une région céphalique arrondie, annelée; R. echelimae sp. n., caractérisé par une région céphalique tronconique non annelée; R. provincialis sp. n., caractérisé par une région céphalique annelée, conoïde-arrondie et par l'extrémité caudale lisse. Pour des populations de quatre autres espèces, Rotylenchus capensis, R. eximius, R. magnus jaeni, R. magnus magnus, les mensurations, les figures et les différences morphologiques majeures par rapport aux descriptions originales sont données. Une diagnose amendée du genre, la liste des espèces valides et des species inquirendae vel dubiae ainsi qu'une clé tabulaire pour les espèces sont proposées.

Simple laboratory tests were developed to evaluate the potential efficiency of Trichogramma cacoeciae Marchal for biological control against Lobesia botrana Denis & Schiffermuller, a major pest in vineyards. From different host egg densities in the patches, discovery and exploitation rates were studied for eggs of L. botrana and for eggs of the reference the factitious host, Ephestia kuehniella Zeller. The effect of the wasp age, the contact time between parasitoid and host eggs, and the presence of kairomones (scales) on these rates also was tested. Mortality was induced by the presence of the parasitoid in L. botrana eggs. The two rates were differently modified according to host species, host density, presence of kairomones, and by wasp age. Discovery rates were density-dependent according to host density and the other factors taken into account, except for younger Trichogramma with L. botrana eggs. Exploitation rate was always inversely density-dependent according to host density. Behavioral and physiological aspects of these relationships and interest for biological control of these tests were discussed.

Aphis gossypii is an aphid species that is found throughout the world and is extremely polyphagous. It is considered a major pest of cotton and cucurbit species. In Europe, A. gossypii is assumed to reproduce exclusively by apomictic parthenogenesis. The present study investigates the genetic diversity of A. gossypii in a microgeographic, fragmented habitat consisting of eight glasshouses of cucurbit crops. This analysis, which was based on the results from seven microsatellite loci, has confirmed that A. gossypii populations in southern France are primarily asexual, as only 12 nonrecombinant genotypic classes (clones) were identified from 694 aphids. Moreover, a high proportion of the aphids (87%) had one of three common genotypes. No significant correlation was found between genotypic class and host plant species. Within a glasshouse population of A. gossypii, a significant reduction in clonal diversity was observed as the spring/summer season progressed. The final predominance of a clone could result from interclonal competition. At the microgeographic level (i.e. glasshouses within a 500-m radius), significant genetic subdivision was detected and could be attributed to founder effects and the limitation of gene flow imposed by the enclosed nature of the glasshouse structure. Finally, the three common clones of A. gossypii detected in 1996 reappeared in spring 1997 following the winter extinction, together with rare clones that had not previously been seen. The probability that A. gossypii overwinters within refuges at a microgeographic scale from which populations are renewed each spring is discussed.

L'un des moyens d'ameliorer la qualite et la rentabilite des cultures consiste a accroitre la reistance des plantes aux agents pathogenes. Pour ce faire, deux voies principales: on cherche a activer la signalisation cellulaire responsable de la resistance naturelle des especes aux maladies; ou l'on ≪ vaccine ≫ les plantes par transgenese.

BACKGROUNDThe increasing use of macrolides especially in the treatment ofHelicobacter pylori infection has led to an increase in resistant strains. The resistance of H pylori to macrolides, especially clarithromycin, is one of the major causes of eradication failure. In H pylori, clarithromycin resistance is due to point mutations localised in domain V of 23S rRNA.AIMTo develop a molecular technique based on amplification of a relevant fragment of the 23S rRNA and colorimetric hybridisation in liquid phase to detect directly in biopsy specimens the type of mutation associated with resistance of H pylori to clarithromycin.METHODSGastric biopsy samples from 61 patients were submitted to this test. The results were compared with standard methods (determination of minimal inhibition concentration, polymerase chain reaction/restriction fragment length polymorphism, and/or DNA sequencing) in order to evaluate the test and to define the cut off values, specificity, and sensitivity.RESULTSThe 14 biopsy samples in which H pylori was not detected did not give a positive result in any assay, and the 14 samples harbouring strains susceptible to clarithromycin gave a positive result with the wild type probe as expected. The 33 biopsy specimens containing resistant strains always gave a positive signal with one of the probes detecting resistant organisms, but in eight cases they also reacted with the wild type probe, indicating that a mixture of resistant and susceptible organisms was present.CONCLUSIONThe importance of this new assay is that it allows the detection of multiple genotypes corresponding to either heterogeneous genotypes or mixed infections. Moreover, it allows in a single step not only the detection of H pylori but also the determination of its susceptibility to clarithromycin directly in biopsy specimens without the need for culture.

For the first time, mariner elements were found in insect parasitic nematodes. Full-length elements were isolated from the rhabditid Heterorhabditis bacteriophora. They were 1279 bp long, flanked by two 30-bp inverted repeats, and were able to encode a putative 358-amino acid transposase. These elements were present in about 30 copies in the H. bacteriophora genome, but their distribution among closely related Heterorhabditis and Steinernema genera was patchy. DNA and encoded peptide sequences of H. bacteriophora mariners showed greater similarity to the mariner of the coleopteran Carpelimus sp. than to the mariners of the rhabditid Caenorhabditis elegans. The possibility of horizontal transfer was investigated by examination of a host for Heterorhabditis nematodes, a beetle of the Phyllophaga sp. Mariner elements were found in this insect, but they were not very similar to the H. bacteriophora elements. Finally, the H. bacteriophora mariners formed a group with those of invertebrates, suggesting vertical transmission from a common ancestor.

We analysed Drosophila melanogaster cytochrome P450s (P450) through the measurements of four enzymatic activities: ethoxycoumarin- O -deethylase, ethoxyresorufin- O -deethylase, lauric acid hydroxylation, and testosterone hydroxylation. We did these measurements in two Drosophila strains: one is susceptible to insecticides (Canton S ) and the other is resistant to insecticides by enhanced P450 activities (RDDT R ). In addition, we also treated the flies with eight chemicals ( β -naphtoflavone, benzo- α -pyrene, 3-methylcholanthrene, phenobarbital, aminopyrine, rifampicin, prochloraz, and clofibrate) known to induce genes from the families CYP1 , CYP2 , CYP3 , CYP4 , and CYP6 . Metabolisation of all the substrates by P450 from flies microsomes was observed. The chemicals had different effects on these activities, ranging from induction to inhibition. The effects of these chemicals varied with the strains as most of them were ineffective on the RDDT R strain. The results showed that P450-dependent activities are numerous in Drosophila . Regulation features of these activities are complex. The availability of mutant strains as RDDT R should allow fundamental studies of P450 in insects.

National audience

Reorganization of the microtubule network is important for the fast isodiametric expansion of giant-feeding cells induced by root-knot nematodes. The efficiency of microtubule reorganization depends on the nucleation of new microtubules, their elongation rate and activity of microtubule severing factors. New microtubules in plants are nucleated by cytoplasmic or microtubule-bound γ-tubulin ring complexes. Here we investigate the requirement of γ-tubulin complexes for giant feeding cells development using the interaction between Arabidopsis and Meloidogyne spp. as a model system. Immunocytochemical analyses demonstrate that γ-tubulin localizes to both cortical cytoplasm and mitotic microtubule arrays of the giant cells where it can associate with microtubules. The transcripts of two Arabidopsis γ-tubulin (TUBG1 and TUBG2) and two γ-tubulin complex proteins genes (GCP3 and GCP4) are upregulated in galls. Electron microscopy demonstrates association of GCP3 and γ-tubulin as part of a complex in the cytoplasm of giant cells. Knockout of either or both γ-tubulin genes results in the gene dose-dependent alteration of the morphology of feeding site and failure of nematode life cycle completion. We conclude that the γ-tubulin complex is essential for the control of microtubular network remodelling in the course of initiation and development of giant-feeding cells, and for the successful reproduction of nematodes in their plant hosts., Author Summary Among plant pathogens, sedentary endoparasitic nematodes are one of the most damaging pests in global agriculture. Nematodes are greatly resistant due to a broad physiological variability, consequently difficult to fight against. The use of pesticides is highly pollutant to the environment and therefore new strategies must be envisaged. As nematodes induce fragmentation and long-term rearrangements of the plant cytoskeleton during infection, manipulation of cytoskeleton components necessary for parasitism could be used as targets for the development of resistant plants provoking the awareness of biotechnology companies and crop breeders in developing new strategies for the control of pathogen infection. Here we report the first stable γ-tubulin-GFP expressing plant line and provide compelling evidence for the physical interaction between components of the γTuRC, γ-tubulin and γ-tubulin-complex protein 3 (GCP3) as part of free cytoplasmic and microtubules associated complexes. We show here that γTuRC is an essential component of the microtubule nucleation machinery during giant cell development. The reduction of γ-tubulin and GCP4 levels compromises γTuRC functioning and affects microtubule nucleation in giant-feeding cells, delaying their development and affecting nematode reproduction. We conclude that upregulation of microtubule nucleation induced by γTuRC is essential for the nematode parasitism and this process can be targeted in order to protect plants against nematode infection.

National audience

Hepatocytes and keratinocytes are among the most widely used cells in pharmaco-toxicology, but a limitation of these models is the provision of human tissues on a regular basis. The suitability of HepG2, HaCaT and HESV cell lines as an acceptable substitute for primary cultures was examined. In these cell types, the effects of 3-methylcholanthrene (3-MC) were analysed on CYP1A1 gene expression, a crucial CYP subfamily in the activation of chemical carcinogens. Ethoxyresorufin O -deethylase (EROD) activity was never detected in HESV cells, but in other cell types it was stimulated in a concentration-dependent manner (maximal induction, 1–2.5 μ m ). Above this peak induction the effect fell rapidly. Northern blot analysis of CYP1A1 mRNA agreed with the trends obtained for EROD values. However, the decrease of the EROD activity observed at the highest 3-MC concentrations was not correlated with CYP1A1 mRNA reduction. This study also demonstrated that 3-MC is capable of significantly inducing CYP1A1 in HaCaT cells (17-fold over control), as in human hepatocytes (six- to 18-fold) and HepG2 (fourfold). Therefore, in contrast to SV40-immortalized keratinocytes (HESV), spontaneously immortalized keratinocytes (HaCaT) may constitute a valuable tool for studying epidermal CYP1A1 gene regulation by xenobiotics.

Malaria is a crucial problem in public health care, affecting 200 million people annually, two million of whom die. Chloroquine resistance has become widespread and alternative agents including quinoline derivatives, pyrimethamine and qinghaosu derivatives are used now in malaria treatment. These compounds were comparatively studied for their cytotoxicity and CYP induction capability in rat hepatocyte primary cultures. Chloroquine, mefloquine, amodiaquine and arteflene had IC(50)s of approximately 40 mum, whereas quinidine, primaquine, quinine and pyrimethamine were less toxic (IC(50)s of approx. 300 mum). CYP induction was also tested by using PROD, ECOD and EROD activities as markers. Primaquine, pyrimethamine, quinine, mefloquine and chloroquine provoked an approximately 1.5-fold induction of PROD activity over control. Concerning ECOD activity, amodiaquine and pyrimethamine led to a approximately 5.1- and 2.5-fold increases, respectively, whereas a 12-fold induction was obtained with primaquine. EROD activity was only induced by primaquine (approx. eightfold). This induction was dose dependent and correlated to a rise in CYP1A1 mRNA level. Finally, induction by primaquine appeared to be mediated via the Ah receptor, as indicated by its suppression by 8-MP, a compound interfering with the binding of activated AhR to DNA. These findings may have pharmacotoxicological implications and should be considered in the design of therapeutic protocols.

We developed and evaluated two different methods to improve the detection of the most prevalent virus of rose in Europe, Prunus necrotic ring-spot virus (PNRSV). Immunocapture-reverse transcription-polymerase chain reaction was estimated to be about 100 times more sensitive than double-antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and showed an equivalent specificity. Based on the observation that PNRSV multiplies actively in young growing tissues (axillary shoots and cuttings), an in vitro culture method allowing rapid (about 15 days) and homogeneous development of dormant axillary buds with high virus titers was standardized. ELISA tests of these young shoots showed, in some cases, a 104 to 105 increase in sensitivity in comparison to adjacent leaf tissues from the rose mother plants. Between 21 and 98% (depending on the season) more samples were identified as positive by using ELISA on samples from shoot tips grown in vitro rather than on leaves collected directly from the PNRSV-infected mother plants. This simple method of growing shoot tips in vitro improved the confidence in the detection of PNRSV and eliminated problems in sampling appropriate tissues.

International audience; * • The oomycete Phytophthora parasitica is a soilborne pathogen infecting numerous plants. The infection process includes an initial biotrophic stage, followed by a necrotrophic stage. The aim here was to identify genes that are involved in the late stages of infection. * • Using the host tomato and a transformed strain of P. parasitica expressing the green fluorescent protein (GFP), the various infection steps from recognition of the host to the colonization of plant tissues were studied. This late stage was selected to generate 4000 ESTs (expressed sequence tags), among which approx. 80% were from the pathogen. * • Comparison with an EST data set created previously from in vitro growth of P. parasitica allowed the identification of several genes, the expression of which might be regulated during late stages of infection. * • Changes in gene expression of several candidate genes predicted from in silico analysis were validated by quantitative RT–PCR experiments. These results give insights into the molecular bases of the necrotrophic stage of an oomycete pathogen.

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

The aim of the present study was to isolate clones of genes which are likely to be involved in wax deposition on barley leaves. Of particular interest were those genes which encode proteins that take part in the synthesis and further modification of very long chain fatty acids (VLCFAs), the precursors of waxes. Previously, it had been shown that wax deposition commences within a spatially well-defined developmental zone along the growing barley leaf (Richardson et al. in Planta 222:472–483, 2005). In the present study, a barley microarray approach was used to screen for candidate contig-sequences (www.barleybase.org) that are expressed particularly in those leaf zones where wax deposition occurs and which are expressed specifically within the epidermis, the site of wax synthesis. Candidate contigs were used to screen an established in-house cDNA library of barley. Six full-length coding sequences clones were isolated. Based on sequence homologies, three clones were related to Arabidopsis CER6/CUT1, and these clones were termed HvCUT1;1, HvCUT1;2 and HvCUT1;3. A fourth clone, which was related to Arabidopsis Fiddlehead (FDH), was termed HvFDH1;1. These clones are likely to be involved in synthesis of VLCFAs. A fifth and sixth clone were related to Arabidopsis CER1, and were termed HvCER1;1 and HvCER1;2. These clones are likely to be involved in the decarbonylation pathway of VLCFAs. Semi-quantitative RT-PCR confirmed microarray expression data. In addition, expression analyses at 10-mm resolution along the blade suggest that HvCUT1;1 (and possibly HvCUT1;2) and HvCER1;1 are involved in commencement of wax deposition during barley leaf epidermal cell development.

Special Feature; International audience

Diplôme : Dr. d'Universite; Root-knot nematodes, genus Meloidogyne, are obligate biotrophic parasites. During a compatible interaction, the second stage juveniles (J2) penetrate into the root, migrate intercellularly and become sedentary to initiate the differentiation of a feeding site formed by 5 to 7 giant cells in the vascular cylinder. Secreted proteins play a key role during the plant/nematode interaction. A typical gallis formed around the feeding site. Stylet secretions are produced in oesophageal glands and injected in the root during parasitism.The first objective of this work was the identification of biological functions involved in the adaptation of the parasite to its host environment and in the modulation the host responses. We initiated a differential approach and a candidate gene approach and identified proteins of unknown function, proteases, a putative secreted protein, a glutathione S-transferase (GST) and a peroxiredoxine (PRX). The GST was localized in the subventral oesophageal glands of infective J2 and detected in M. incognita stylet secretions purified from infective J2. The PRX was localised in the excretory pore of infective J2 and in the hypodermis of sedentary stages. The role of these two proteins in parasitism is discussed. Root-knot nematodes have been so far refractory to transformation. Recent works demonstrated the efficiency of RNAi for the silencing of parasitism genes on J2. This strategy is limited by the recovery of targeted transcripts a few days after J2 treatment. In order to ensure a long-lasting silencing we developed a new and original tool, presented in a last chapter.; Les nématodes à galle du genre Meloidogyne sont des vers microscopiques biotrophes, endoparasites obligatoires des racines. Lors de l’interaction compatible, la larve de second stade (L2) pénètre dans la racine, migre de façon intercellulaire, se sédentarise et initie la formation d’un site nourricier formé de 5 à 7 cellules géantes dans la zone du cylindre vasculaire. Les protéines des sécrétions des nématodes jouent un rôle majeur lors de l’interaction plante/nématode. Les sécrétions du stylet sont produites dans les glandes oesophagiennes et injectées dans la racine au cours du parasitisme. L’objectif de ce travail était d’abord d’identifier les fonctions biologiques impliquées dans l’adaptationdu parasite à l’environnement de la plante et dans la modulation des défenses de la plante. Par une approche différentielle et une approche gène candidat, des protéines de fonction inconnue, des protéases, une protéine potentiellement sécrétée, une glutathion S-transférase (GST) et une péroxirédoxine (PRX) ont été identifiées. La GST, localisée dans les glandes oesophagiennes, est présente dans les sécrétions du stylet de L2 infestantes de M. incognita. La PRX est localisée au niveau du pore excréteur des L2 et de l’hypoderme des stades sédentaires. Le rôle de ces deux protéines dans le processus parasitaire est discuté. L’analyse fonctionnelle de gènes du nématode est limitée par l’absence d’outils de transformation. L’efficacité transitoire de l’ARN interférence pour inhiber des gènes du parasitisme dans les L2 a été démontrée. Afin de garantir un « silencing » durable, la mise au point d’un nouvel outil sera développée dans une dernière partie.

International audience

Selection in plant parasites for virulence on resistant hosts and the resulting effects on parasite fitness may be considered as a driving force in host-parasite coevolution. In the present study, we tested the hypothesis that a fitness cost may be associated with nematode virulence, using the interaction between the parthenogenetic species Meloidogyne incognita and tomato as a model system. The reproductive parameters of near-isogenic lines of the nematode, selected for avirulence or virulence against the tomato Mi resistance gene, were analysed and combined into a reproductive index that was taken as a measure of fitness. The lower fitness of the virulent lines on the susceptible tomato cultivar showed for the first time that a measurable fitness cost is associated with unnecessary virulence in the nematode. Although parthenogenesis should theoretically lead to little genetic variability, such cost may impose a direct constraint on the coevolution between the plant and the nematode populations, and suggests an adaptive significance of trade-offs between selected characters and fitness-related traits. These results indicate that, although plant resistance can be broken, it might prove durable in some conditions if the virulent nematodes are counterselected in susceptible plants, which could have important consequences for the management of resistant cultivars in the field.

International audience

International audience