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Male and female unisexual flowers evolved from hermaphroditic ancestors, and control of flower sex is useful for plant breeding. We isolated a female-to-male sex transition mutant in melon and identified the causal gene as the carpel identity genelt;igt;CRABS CLAW (CRC)lt;/igt;. We show that the master regulator of sex determination in cucurbits, the transcription factorlt;igt;WIP1lt;/igt; whose expression orchestrates male flower development, recruits the corepressor TOPLESS to thelt;igt;CRClt;/igt; promoter to suppress its expression through histone deacetylation. Impairing TOPLESS-WIP1 physical interaction leads tolt;igt;CRClt;/igt; expression, carpel determination, and consequently the expression of the stamina inhibitor, the aminocyclopropane-1-carboxylic acid synthase 7 (lt;igt;CmACS7lt;/igt;), leading to female flower development. Our findings suggest that sex genes evolved to interfere with flower meristematic function, leading to unisexual flower development.

Male and female unisexual flowers evolved from hermaphroditic ancestors, and control of flower sex is useful for plant breeding. We isolated a female-to-male sex transition mutant in melon and identified the causal gene as the carpel identity gene CRABS CLAW (CRC) . We show that the master regulator of sex determination in cucurbits, the transcription factor WIP1 whose expression orchestrates male flower development, recruits the corepressor TOPLESS to the CRC promoter to suppress its expression through histone deacetylation. Impairing TOPLESS-WIP1 physical interaction leads to CRC expression, carpel determination, and consequently the expression of the stamina inhibitor, the aminocyclopropane-1-carboxylic acid synthase 7 ( CmACS7 ), leading to female flower development. Our findings suggest that sex genes evolved to interfere with flower meristematic function, leading to unisexual flower development.

International audience; Shapes of vegetables and fruits are the result of adaptive evolution and human selection. Modules controlling organ shape have been identified. However, little is known about signals coordinating organ development and shape. Here, we describe the characterization of a melon mutation rf1, leading to round fruit. Histological analysis of rf1 flower and fruits revealed fruit shape is determined at flower stage 8, after sex determination and before flower fertilization. Using positional cloning, we identified the causal gene as the monoecy sex determination gene CmACS7, and survey of melon germplasms showed strong association between fruit shape and sexual types. We show that CmACS7-mediated ethylene production in carpel primordia enhances cell expansion and represses cell division, leading to elongated fruit. Cell size is known to rise as a result of endoreduplication. At stage 8 and anthesis, we found no variation in ploidy levels between female and hermaphrodite flowers, ruling out endoreduplication as a factor in fruit shape determination. To pinpoint the gene networks controlling elongated versus round fruit phenotype, we analyzed the transcriptomes of laser capture microdissected carpels of wild-type and rf1 mutant. These high-resolution spatiotemporal gene expression dynamics revealed the implication of two regulatory modules. The first module implicates E2FDP transcription factors, controlling cell elongation versus cell division. The second module implicates OVATE-and TRM5-related proteins, controlling cell division patterns. Our finding highlights the dual role of ethylene in the inhibition of the stamina development and the elongation of ovary and fruit in cucurbits.

International audience; In monoecious melon (Cucumis melo), sex is determined by the differential expression of sex determination genes (SDGs) and adoption of sex-specific transcriptional programs. Histone modifications such as H3K27me3 have been previously shown to be a hallmark associated to unisexual flower development in melon; yet, no genetic approaches have been conducted for elucidating the roles of H3K27me3 writers, readers, and erasers in this process. Here we show that melon homologs to Arabidopsis LHP1, CmLHP1A and B, redundantly control several aspects of plant development, including sex expression. Cmlhp1ab double mutants displayed an overall loss and redistribution of H3K27me3, leading to a deregulation of genes involved in hormone responses, plant architecture, and flower development. Consequently, double mutants display pleiotropic phenotypes and, interestingly, a general increase of the male:female ratio. We associated this phenomenon with a general deregulation of some hormonal response genes and a local activation of male-promoting SDGs and MADS-box transcription factors. Altogether, these results reveal a novel function for CmLHP1 proteins in maintenance of monoecy and provide novel insights into the polycomb-mediated epigenomic regulation of sex lability in plants.

Zucchini yellow mosaic virus (ZYMV; potyviridae) represents a major pathogen of Cucurbitaceae crops. ZYMV resistance in melon PI 414723 is conditioned by a dominant allele at the Zym locus. This resistant accession restricts viral spread and does not develop mosaic symptoms, but necrosis sometimes develops in response to inoculation. In previous studies, Zym has been mapped to linkage group II of the melon genetic map. In the present study, positional cloning of the locus was undertaken, starting from the CM-AG36 SSR marker at approximately 2 cm distance. We utilized five mapping populations that share the same resistant parent, PI 414723, and analyzed a total of 1630 offspring, to construct a high-resolution genetic map of the Zym locus. Two melon BAC libraries were used for chromosome walking and for developing new markers closer to the resistance gene by BAC-end sequencing. A BAC contig was constructed, and we identified a single BAC clone, from the ZYMV susceptible genotype MR-1, that physically encompasses the resistance gene. A second clone was isolated from another susceptible genotype, WMR 29, and the two clones were fully sequenced and annotated. Additional markers derived from the sequenced region delimited the region to 17.6 kb of a sequence that harbors a NAC-like transcription factor and, depending on the genotype, either two or three R-gene homologs with a CC-NBS-LRR structure. Mapping was confirmed by saturating the map with SNP markers using a single mapping population. The same region was amplified and sequenced also in the ZYMV resistant genotype PI 414723. Because numerous polymorphic sites were noted between genotypes, we could not associate resistance with a specific DNA polymorphism; however, this study enables molecular identification of Zym and paves the way to functional studies of this important locus.

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata.

In the melon exotic accession PI 161375, the gene cmv1, confers recessive resistance to Cucumber mosaic virus (CMV) strains of subgroup II. cmv1 prevents the systemic infection by restricting the virus to the bundle sheath cells and impeding viral loading to the phloem. Here we report the fine mapping and cloning of cmv1. Screening of an F2 population reduced the cmv1 region to a 132 Kb interval that includes a Vacuolar Protein Sorting 41 gene. CmVPS41 is conserved among plants, animals and yeast and is required for post-Golgi vesicle trafficking towards the vacuole. We have validated CmVPS41 as the gene responsible for the resistance, both by generating CMV susceptible transgenic melon plants, expressing the susceptible allele in the resistant cultivar and by characterizing CmVPS41 TILLING mutants with reduced susceptibility to CMV. Finally, a core collection of 52 melon accessions allowed us to identify a single amino acid substitution (L348R) as the only polymorphism associated with the resistant phenotype. CmVPS41 is the first natural recessive resistance gene found to be involved in viral transport and its cellular function suggests that CMV might use CmVPS41 for its own transport towards the phloem., The TILLING platform is supported by the Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) and the European Research Council (ERC-SEXYPARTH). This work was supported by grants AGL2009-12698-C02-01 and AGL2012-40130-C02-01 from the Spanish Ministry of Science and Innovation, the Spanish Ministry of Econom and Competitiveness, through the “Severo Ochoa Programme for Centres of Excellence in R&D” 2016-2019 (SEV‐2015‐0533)” and the CERCA Programme/Generalitat de Catalunya.

Fruit ripening is divided into climacteric and non-climacteric types depending on the presence or absence of a transient rise in respiration rate and the production of autocatalytic ethylene. Melon is ideal for the study of fruit ripening, as both climacteric and non-climacteric varieties exist. Two introgressions of the non-climacteric accession PI 161375, encompassed in the QTLs ETHQB3.5 and ETHQV6.3, into the non-climacteric ‘Piel de Sapo’ background are able to induce climacteric ripening independently. We report that the gene underlying ETHQV6.3 is MELO3C016540 (CmNAC-NOR), encoding a NAC (NAM, ATAF1,2, CUC2) transcription factor that is closely related to the tomato NOR (non-ripening) gene. CmNAC-NOR was functionally validated through the identification of two TILLING lines carrying non-synonymous mutations in the conserved NAC domain region. In an otherwise highly climacteric genetic background, both mutations provoked a significant delay in the onset of fruit ripening and in the biosynthesis of ethylene. The PI 161375 allele of ETHQV6.3 is similar to that of climacteric lines of the cantalupensis type and, when introgressed into the non-climacteric ‘Piel de Sapo’, partially restores its climacteric ripening capacity. CmNAC-NOR is expressed in fruit flesh of both climacteric and non-climacteric lines, suggesting that the causal mutation may not be acting at the transcriptional level. The use of a comparative genetic approach in a species with both climacteric and non-climacteric ripening is a powerful strategy to dissect the complex mechanisms regulating the onset of fruit ripening., This work was supported by the Spanish Ministry of Economy and Competitivity grant AGL2015-64625-C2-1-R, Centro de Excelencia Severo Ochoa 2016-2020, and the CERCA Programme/Generalitat de Catalunya to JGM; Spanish Ministry of Economy and Competitivity/FEDER grant AGL2015-64625-C2-2-R to AJM; EU Framework Program Horizon 2020 COST Action FA1106 Quality Fruit for networking activities to CL; European Research Council grant ERC-SEXYPARTH to AB; Chief Scientist of the Ministry of Agriculture of Israel grant no. 261-1049-13 to NK.

International audience; Fruit ripening and softening are key traits for many fleshy fruit. Since cell walls play a key role in the softening process, expansins have been investigated to control fruit over ripening and deterioration. In tomato, expression of Expansin I gene, SlExp1, during fruit ripening was associated with fruit softening. To engineer tomato plants with long shelf life, we screened for mutant plants impaired in Slexp1 function. Characterization of two induced mutations, Slexp1-6_14/211S, and Slexp1-7_Q213Stop, showed that Slexp1 loss of function leads to enhanced fruit firmness and delayed fruit ripening. Analysis of cell wall polysaccharide composition of Slexp1-7_Q213Stop mutant pointed out significant differences for uronic acid, neutral sugar and total sugar contents. Hemicelluloses chemistry analysis by endo-beta-1,4-D-glucanase hydrolysis and MALDI-TOF spectrometry revealed that xyloglucan structures were affected in the fruit pericarp of Slexp1 -7_Q213Stop mutant. Altogether, these results demonstrated that SlExp1 loss of function mutants yield firmer and late ripening fruits through modification of hemicellulose structure. These SlExp1 mutants represent good tools for breeding long shelf life tomato lines with contrasted fruit texture as well as for the understanding of the cell wall polysaccharide assembly dynamics in fleshy fruits.

PubMedID: 27171236 Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera. © 2016 Boualem et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. European Research Council Agence Nationale de la Recherche Saudi Pharmaceutical Society: ANR-10-LABX-40 ANR-11-BSV7-0024 This work was supported by BAP department in INRA, the grant Program Saclay Plant Sciences (SPS, ANR-10-LABX-40) for ABo and ABe, the Agence Nationale de la Recherche grant MELODY (ANR-11-BSV7-0024) for ABo, ABe and CD and the European Research Council grant (ERC-SEXYPARTH) for ABe. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank P. Audigier and N. Giovinazzo for plant care and V. Rittener for technical assistance. We thank M. Chatterjee for helpful discussions on the manuscript. We thank J. Fauve for providing watermelon andromonoecious accessions. This work was supported by BAP department in INRA, the grants Program Saclay Plant Sciences (SPS, ANR-10-LABX-40), ANR MELODY (ANR-11-BSV7-0024) and the European Research Council (ERC-SEXYPARTH).

International audience

How flowers separate males and females Most flowering plant families have bisexual flowers with both male and female function. However, most members of the Cucurbiticeae family, which includes melons, cucumbers, and gourds, have unisexual flowers. To understand this difference in sex expression, Boualem et al. identified a cucumber gene expressed in the female flowers. Mutations in this gene were associated with solely male flowers. By integrating this finding into a sex determination model, the authors explain how unisexual flowers can coexist in the same plant. Science, this issue p. 688

Background: [br/] Cucumber (Cucumis sativus) belongs to the Cucurbitaceae family that includes more than 800 species. The cucumber genome has been recently sequenced and annotated. Transcriptomics and genome sequencing of many plant genomes are providing information on candidate genes potentially related to agronomically important traits. To accelerate functional characterization of these genes in cucumber we have generated an EMS mutant population that can be used as a TILLinG platform for reverse genetics. [br/] Principal Findings: [br/] A population of 3,331 M2 mutant seed families was generated using two EMS concentrations (0.5% and 0.75%). Genomic DNA was extracted from M2 families and eight-fold pooled for mutation detection by ENDO1 nuclease. To assess the quality of the mutant collection, we screened for induced mutations in five genes and identified 26 mutations. The average mutation rate was calculated as 1/1147 Kb giving rise to approximately 320 mutations per genome. We focused our characterization on three missense mutations, G33C, S238F and S249F identified in the CsACS2 sex determination gene. Protein modeling and crystallography studies predicted that mutation at G(33) may affect the protein function, whereas mutations at S-238 and S-249 may not impair the protein function. As predicted, detailed phenotypic evaluation showed that the S238F and the S249F mutant lines had no sexual phenotype. In contrast, plants homozygous for the G33C mutation showed a complete sexual transition from monoecy to andromonoecy. This result demonstrates that TILLinG is a valuable tool for functional validation of gene function in crops recalcitrant to transgenic transformation. [br/] Conclusions: [br/] We have developed a cucumber mutant population that can be used as an efficient reverse genetics tool. The cucumber TILLinG collection as well as the previously described melon TILLinG collection will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in cucurbits in general.

International audience; BACKGROUND: Ordered collections of mutants serve as invaluable tools in biological research. TILLING (Targeting Induced Local Lesions IN Genomes) provides an efficient method to discover, in mutagenized populations, the possible phenotypes controlled by gene sequences whose function is unknown. This method can replace transgenic techniques for the functional validation of cloned genes, especially in the case of transformation-recalcitrant plants such as cucumber. RESULTS: We report the development of a TILLING cucumber population, generated by EMS mutagenesis in the Poinsett76 genetic background. The population was evaluated by screening for morphological mutations, and a range of developmental, pigmentation and spontaneous lesion mutants were observed. Suitability for detecting single nucleotide polymorphism in selected genes has been tested by screening a sample of amplicons, with detection rate of 1 SNP in ~1 Mbp. CONCLUSION: The population described in this Research Note represents a useful asset in cucumber research, to be exploited for forward genetic screens and functional genomics purposes.

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: The availability of genetic and genomic resources for melon has increased significantly, but functional genomics resources are still limited for this crop. TILLING is a powerful reverse genetics approach that can be utilized to generate novel mutations in candidate genes. A TILLING resource is available for cantalupensis melons, but not for inodorus melons, the other main commercial group. [Results]: A new ethyl methanesulfonate-mutagenized (EMS) melon population was generated for the first time in an andromonoecious non-climacteric inodorus Piel de Sapo genetic background. Diverse mutant phenotypes in seedlings, vines and fruits were observed, some of which were of possible commercial interest. The population was first screened for mutations in three target genes involved in disease resistance and fruit quality (Cm-PDS, Cm-eIF4E and Cm-eIFI(iso)4E). The same genes were also tilled in the available monoecious and climacteric cantalupensis EMS melon population. The overall mutation density in this first Piel de Sapo TILLING platform was estimated to be 1 mutation/1.5 Mb by screening four additional genes (Cm-ACO1, Cm-NOR, Cm-DET1 and Cm-DHS). Thirty-three point mutations were found for the seven gene targets, six of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was demonstrated for a loss-of-function mutation in the Phytoene desaturase gene, which is involved in carotenoid biosynthesis. [Conclusions]: The TILLING approach was successful at providing new mutations in the genetic background of Piel de Sapo in most of the analyzed genes, even in genes for which natural variation is extremely low. This new resource will facilitate reverse genetics studies in non-climacteric melons, contributing materially to future genomic and breeding studies., This work was supported by projects GEN2003-20237-C06-02/03, funded by the Ministerio de Educación y Ciencia (Spain), and GEN2006-27773-C2-1/2, funded by the ERA-PG programme.

International audience; Late blight, caused by the pathogen Phytophthora spp. (Oomycete), is one of the most damaging diseases of Solanaceae. The deployment of resistant varieties is the most effective, economical and environmentally safe control of Phytophthora. Contrary to monogenic resistances that are easily overcome by the pathogen, polygenic resistances generally confer a durable control of disease severity. However, their molecular basis remains unknown since no resistance QTl to Phytophthora has ever been characterized. Phy-P5, the major effect Phytophthora capsici resistance QTl in pepper, has been mapped on the chromosome P5. It was detected in 3 pepper accessions and displayed a broad-spectrum resistance to several P. capsici isolates. We airn to determine the molecular basis of Phy-PS by isolating the responsible gene deploying a positional cloninq strategy.

Supplementary Informations : 15 p. ; Sex determination in plants leads to the development of unisexual flowers from an originally bisexual floral meristem. This mechanism results in the enhancement of outcrossing and promotes genetic variability, the consequences of which are advantageous to the evolution of a species. In melon, sexual forms are controlled by identity of the alleles at the andromonoecious (a) and gynoecious (g) loci. We previously showed that the a gene encodes an ethylene biosynthesis enzyme, mACS-7, that represses stamen development in female flowers. Here we show that the transition from male to female flowers in gynoecious lines results from epigenetic changes in the promoter of a transcription factor, CmWIP1. This natural and heritable epigenetic change resulted from the insertion of a transposon, which is required for initiation and maintenance of the spreading of DNA methylation to the CmWIP1 promoter. Expression of CmWIP1 leads to carpel abortion, resulting in the development of unisexual male flowers. Moreover, we show that CmWIP1 indirectly represses the expression of the andromonoecious gene, CmACS-7, to allow tamen development. Together our data indicate a model in which CmACS-7 and CmWIP1 interact to control the development of male, female and hermaphrodite flowers in melon.

La présente invention a pour objet une combinaison de deux éléments génétiques pour le contrôle du développement du type floral d'une plante dicotylédone, ladite combinaison comprenant, respectivement : un premier élément génétique de contrôle (A/a) présent dans une plante dicotylédone, sous la forme d'un allèle dominant (A), et d'un allèle récessif (a); et un second élément génétique de contrôle (G/g) présent dans une plante dicotylédone, sous la forme d'un allèle dominant (G), et d'un allèle récessif (g), étant entendu qu'au moins le second élément génétique de contrôle a été artificiellement introduit dans ladite plante dicotylédone. La combinaison ci-dessus permet de contrôler, et/ou de modifier le sexe des fleurs de plantes dicotylédones.

BackgroundAcidity is an essential component of the organoleptic quality of fleshy fruits. However, in these fruits, the physiological and molecular mechanisms that control fruit acidity remain unclear. In peach theDlocus controls fruit acidity; low-acidity is determined by the dominant allele. Using a peach progeny of 208 F2trees, theDlocus was mapped to the proximal end of linkage group 5 and co-localized with major QTLs involved in the control of fruit pH, titratable acidity and organic acid concentration and small QTLs for sugar concentration. To investigate the molecular basis of fruit acidity in peach we initiated the map-based cloning of theDlocus.ResultsIn order to generate a high-resolution linkage map in the vicinity of theDlocus, 1,024 AFLP primer combinations were screened using DNA of bulked acid and low-acid segregants. We also screened a segregating population of 1,718 individuals for chromosomal recombination events linked to theDlocus and identified 308 individuals with recombination events close toD. Using these recombinant individuals we delimited theDlocus to a genetic interval of 0.4 cM. We also constructed a peach BAC library of 52,000 clones with a mean insert size of 90 kb. The screening of the BAC library with markers tightly linked toDlocus indicated that 1 cM corresponds to 250 kb at the vicinity of theDlocus.ConclusionIn the present work we presented the first high-resolution genetic map ofDlocus in peach. We also constructed a peach BAC library of approximately 15× genome equivalent. This fine genetic and physical characterization of theDlocus is the first step towards the isolation of the gene(s) underlying fruit acidity in peach.

Andromonoecy is a widespread sexual system in angiosperms characterized by plants carrying both male and bisexual flowers. In melon, this sexual form is controlled by the identity of the alleles at the andromonoecious ( a ) locus. Cloning of the a gene reveals that andromonoecy results from a mutation in the active site of 1-aminocyclopropane-1-carboxylic acid synthase. Expression of the active enzyme inhibits the development of the male organs and is not required for carpel development. A causal single-nucleotide polymorphism associated with andromonoecy was identified, which suggests that the a allele has been under recent positive selection and may be linked to the evolution of this sexual system.

International audience

International audience

Highly active antiretroviral therapy (HAART) is associated with a dramatic clinical benefit to HIV-infected patients through significant plasma viremia reduction and CD4(+) T cells increase. In previous reports, HIV-specific CD4(+) and/or CD8(+) T cell responses have been studied separately during HAART; therefore the relationship between these two virus-specific populations is currently not well understood. In this study, both HIV-specific CD4(+) and CD8(+) T cell responses were investigated using a large panel of well-defined T cell epitope peptides in 24 HIV-1-infected patients undergoing HAART, with undetectable viral load and CD4(+) T cell count/= 350/mm(3). One-third of the patients had CD4(+) T cells able to proliferate when exposed to HIV-1 protein fragments but only two patients displayed polyclonal responses. In addition the majority (78%) of HAART-treated patients displayed no or monospecific CD8(+) T cell responses and the phenotypic analysis of these HIV-specific CD8(+) T cells demonstrated the absence of terminally differentiated effectors. In conclusion, the experimental approach used in this study shows that CD4(+) T cell responses may persist during HAART but are not associated with strong CD8(+) T cell responses

Although the availability of genetic and genomic resources for Cucurbita pepo has increased significantly, functional genomic resources are still limited for this crop. In this direction, we have developed a high throughput reverse genetic tool: the first TILLING (Targeting Induced Local Lesions IN Genomes) resource for this species. Additionally, we have used this resource to demonstrate that the previous EMS mutant population we developed has the highest mutation density compared with other cucurbits mutant populations. The overall mutation density in this first C. pepo TILLING platform was estimated to be 1/133 Kb by screening five additional genes. In total, 58 mutations confirmed by sequencing were identified in the five targeted genes, thirteen of which were predicted to have an impact on the function of the protein. The genotype/phenotype correlation was studied in a peroxidase gene, revealing that the phenotype of seedling homozygous for one of the isolated mutant alleles was albino. These results indicate that the TILLING approach in this species was successful at providing new mutations and can address the major challenge of linking sequence information to biological function and also the identification of novel variation for crop breeding., Financial support was provided by the Spanish Project INIA (Instituto Nacional de Investigacion y Tecnologia Agraria y Almentaria) RTA2011-00044C02-01, the ANR MELODY (ANR-11-BSV7-0024), the European Research Council (ERCSEXYPARTH), FEDER, and FSE funds. NVD has been awarded a grant by the Andalusian Institute of Agronomy Research IFAPA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Although the mouse spleen dendritic cell (DC) is perhaps the most intensively studied DC type, little has been published concerning its human equivalent. In this report, rare event flow cytometry and in situ immunofluorescence were used to study the surface phenotype and distribution of HLA-DR(+) CD3(-)14(-)16(-)19(-) human spleen DC. Spleens from organ donors with different clinical histories were used. Most (81% +/- 9%; n = 14) spleen DCs expressed high levels of the integrin CD11c. CD11c(+) DCs were distributed in 3 distinct regions-the peri-arteriolar T-cell zones, the B-cell zones, and the marginal zone, where they formed a ring of cells surrounding the white pulp, just inside a ring of CD14(+) red pulp macrophages, apparently more regularly organized than the previously described marginating DC population in the mouse spleen. The T-cell zones contained CD86(+) DCs, among which a subpopulation expressed CD83. These mature/activated CD86(+) DCs represented a minority (12% +/- 8%) of total spleen DCs in most organ donors: most spleen DCs are immature. In 3 of 18 (17%) donors, however, most (54%-81%) of spleen DCs were CD86(+), suggesting that in vivo DC activation had occurred. In one donor, a radical shift in DC distribution from the marginal zone to the T-cell zones was also observed. This activation of spleen DCs in vivo was reminiscent of the effects of experimental microbial product injection in mice, and it seemed to correlate with bacterial infection or multiple trauma. (Blood. 2001;97:3470-3477)

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; International audience; Background: Fruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening. Methodology/Principal Findings: To engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect. Conclusions/Significance: We constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.

National audience

Three amino acid loop extension homeodomain transcription factors (TALE HD TFs) act as life cycle regulators in green algae and land plants. In mosses these regulators are required for the deployment of the sporophyte developmental program. We demonstrate that mutations in either of two TALE HD TF genes, OUROBOROS or SAMSARA, in the brown alga Ectocarpus result in conversion of the sporophyte generation into a gametophyte. The OUROBOROS and SAMSARA proteins heterodimerise in a similar manner to TALE HD TF life cycle regulators in the green lineage. These observations demonstrate that TALE-HD-TF-based life cycle regulation systems have an extremely ancient origin, and that these systems have been independently recruited to regulate sporophyte developmental programs in at least two different complex multicellular eukaryotic supergroups, Archaeplastida and Chromalveolata., eLife digest Brown algae and land plants are two groups of multicellular organisms that have been evolving independently for over a billion years. Their last common ancestor is thought to have existed as a single cell; then, complex multicellular organisms would have appeared separately in each lineage. Comparing brown algae and land plants therefore helps us understand the rules that guide how multicellular organisms evolve from single-celled ancestors. During their life cycles, both brown algae and land plants alternate between two multicellular forms: the gametophyte and the sporophyte. The gametophyte develops sexually active reproductive cells, which, when they merge, create the sporophyte. In turn, spores produced by the sporophyte give rise to the gametophyte. Specific developmental programs are deployed at precise points in the life cycle to make either a sporophyte or a gametophyte. Two proteins known as TALE HD transcription factors help to control the life cycle of single-celled algae related to land plants. Similar proteins are also required for the sporophyte to develop at the right time in land plants known as mosses. This suggests that, when multicellular organisms emerged in this lineage, life cycle TALE HD transcription factors were recruited to orchestrate the development of the sporophyte. However, it was not clear whether TALE HD transcription factors play equivalent roles in other groups, such as brown algae. To address this question, Arun, Coelho et al. examined two mutants of the brown alga Ectocarpus, which produce gametophytes when the non-mutated alga would have made sporophytes. Genetic analyses revealed that these mutated brown algae carried changes in two genes that encode TALE HD transcription factors, indicating that these proteins also regulate the formation of sporophytes in brown algae. Taken together, the results suggest that TALE HD transcription factors were originally tasked with controlling life cycles, and then have been independently harnessed in both land plants and brown algae to govern the formation of sporophytes. This means that, regardless of lineage, the same fundamental forces may be shaping the evolutionary paths that lead to multicellular organisms. Proteins similar to TALE HD transcription factors also regulate life cycles in other groups such as fungi and social amoebae, which indicates that their role is very ancient. It now remains to be explored whether such proteins control life cycles and developmental programs in other multicellular organisms, such as animals.