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Black spot (caused by the fungal pathogen Diplocarpon rosae) is the most severe disease of roses in the outdoor landscape. Most cultivars are susceptible to this disease and its control requires repeated fungicide treatments. New rules on pesticide use encourage breeders to develop roses with a high resistance level to black spot. Growing genetically resistant rose cultivars is an alternative to chemical control of the disease. Resistance durability depends on the kind of resistance, the plant genetic background, the spatial and temporal deployment of resistances, the integration of resistances in cultural systems, and the dynamic of pathogen adaptation. To date, both dominant resistance genes and partial resistance loci have been described particularly in the genetic background of Rosa multiflora. In this study, a genotype closely related to the wild rose Rosa wichurana was examined to identify new resistance genes. Two progenies, connected by the resistant male parent, were scored for black spot resistance after natural infections in field over three and two years in three and one French locations for HW (Rosa hybrida ‘H190’ × hybrid of Rosa wichurana) and OW (Rosa chinensis ‘Old Blush’ × hybrid of Rosa wichurana) progenies, respectively. Genetic maps based on microsatellite and SNP markers were developed for HW and OW progenies, respectively. One common quantitative trait locus (QTL) was localized on linkage group 3 (LG3) of the male maps; it was revealed in the two progenies for several years and in different locations. Another QTL was identified but only in the HW progeny: it was mapped on LG5. The effectiveness of these QTLs should be confirmed against a wide range of pathogen isolates. To provide tools to study the genetic diversity of D. rosae, we sequenced two strains using Illumina paired-end sequencing technology and developed polymorphic microsatellite markers. The genome size of the two strains was estimated to 31 and 34 Mb. A set of 31 polymorphic markers was obtained. 70 monoconidial strains of D. rosae isolated from leaves of Rosa spp. with black spot symptoms were obtained from various locations in Europe and Asia. The analysis of this Eurasian pathogen collection revealed a strong genetic differentiation between pathogen populations collected from wild roses and those collected from gardens. To screen the resistance level of rose genotypes against pathogen diversity, we inoculated rooted cuttings with monoconidial strains of the pathogen under greenhouse conditions. Disease symptoms were scored 10 and 28 days post inoculation (dpi). This pathological test showed a range of resistance levels in rose against the diverse pathogen isolates collected in France and identified genotypes interesting for breeding.

Metabolic profile is a key component of fruit quality, which is a challenge to study due to great compound diversity, especially in species with high nutritional value. This study presents optimized analytical methods for metabolic profiling in the fruits of three Solanaceae species: Lycium barbarum, Lycium chinense and Solanumlycopersicum. It includes the most important chemical classes involved in nutrition and taste, i.e., carotenoids, phenolic compounds and primary compounds. Emphasis has been placed on the systematic achievement of good extraction yields, sample stability, and high response linearity using common LC-ESI-TQ-MS and GC-EI-MS apparatuses. A set of 13 carotenoids, 46 phenolic compounds and 67 primary compounds were profiled in fruit samples. Chemometrics revealed metabolic markers discriminating Lycium and Solanum fruits but also Lycium barbarum and Lycium chinense fruits and the effect of the crop environment. Typical tomato markers were found to be lycopene, carotene, glutamate and GABA, while lycibarbarphenylpropanoids and zeaxanthin esters characterized goji (Lycium spp.) fruits. Among the compounds discriminating the Lycium species, reported here for the first time to our knowledge, chlorogenic acids, asparagine and quinic acid were more abundant in Lycium chinense, whereas Lycium barbarum accumulated more lycibarbarphenylpropanoids A-B, coumaric acid, fructose and glucose.

Two environmentally stable QTLs linked to black spot disease resistance in the Rosa wichurana genetic background were detected, in different connected populations, on linkage groups 3 and 5. Co-localization between R-genes and defense response genes was revealed via meta-analysis. The widespread rose black spot disease (BSD) caused by the hemibiotrophic fungus Diplocarpon rosae Wolf. is efficiently controlled with fungicides. However, in the actual context of reducing agrochemical use, the demand for rose bushes with higher levels of resistance has increased. Qualitative resistance conferred by major genes (Rdr genes) has been widely studied but quantitative resistance to BSD requires further investigation. In this study, segregating populations connected through the BSD resistant Rosa wichurana male parent were phenotyped for disease resistance over several years and locations. A pseudo-testcross approach was used, resulting in six parental maps across three populations. A total of 45 individual QTLs with significant effect on BSD resistance were mapped on the male maps (on linkage groups (LG) B3, B4, B5 and B6), and 12 on the female maps (on LG A1, A2, A3, A4 and A5). Two major regions linked to BSD resistance were identified on LG B3 and B5 of the male maps and were integrated into a consensus map built from all three of the male maps. A meta-analysis was used to narrow down the confidence intervals of individual QTLs from three populations by generating meta-QTLs. Two 'hot spots' or meta-QTLs were found per LG, enabling reduction of the confidence interval to 10.42 cM for B3 and 11.47 cM for B5. An expert annotation of NBS-LRR encoding genes of the genome assembly of Hibrand et al. was performed and used to explore potential co-localization with R-genes. Co-localization with defense response genes was also investigated.

The haploid ascomycete Diplocarpon rosae is the causal agent of black spot disease on roses, a widespread and devastating disease in the outdoor landscape. In this study, we established a Eurasian collection of 77 monoconidial strains of D. rosae: 50 strains collected on cultivated roses in Europe and Asia, and 27 strains on wild roses in Kazakhstan. To provide tools to describe its biology and to study its genetic diversity, we sequenced two strains of D. rosae using Illumina paired-end technology. The genome sizes of these two strains were estimated at 31.1 and 35.2 Mb, which are two times smaller than the genome size of the unique strain previously published. A BUSCO analysis confirmed a genome duplication of the strain previously sequenced and partial gene duplication of strains analysed in this study. Using the two genome sequences, 27 polymorphic microsatellite markers were identified. Polymorphism analysis of the 77 strains revealed a strong genetic differentiation between strains from cultivated and wild roses, and a lower diversity within the fungal population from cultivated roses compared to the population from wild roses. Pathogenicity of 10 strains was evaluated on 9 rose cultivars inoculated in the greenhouse. Disease scoring allowed the classification of strains into three groups and the characterization of resistance of rose cultivars. Good correlation observed between resistance scoring in greenhouse conditions and in the field indicates that pathogenicity assays in controlled conditions could be very useful in the near future to rapidly characterize the resistance of new rose varieties to black spot disease., This work was supported by the CASDAR no. C-2014-06 from the French Ministry of Agriculture, Agrifood and Forestry. The authors thank Markus Linde for kindly supplying German infected leaves, Célia Calmels for microsatellite analysis, Sandrine Pierre for the contribution to assays on whole plants, Phenotic and Experimental unit Horticole for plant management in the greenhouse and in the field, respectively, Thibault Leroy for reads assembly of the Kazakh strain, and Biogenouest for read assembly of the German strain. All microsatellite data were obtained on the ANAN platform (M. Bahut). This work was financially supported by SFR 4207 Quasav.

ERIST Angers, Centre Pays de la Loire, DOCANG-2019/74; International audience

Slow growing pathogenic mycobacteria utilize host-derived lipids and accumulate large amounts of triacylglycerol (TAG) in the form of intracytoplasmic lipid inclusions (ILI), serving as a source of carbon and energy during prolonged infection. Mycobacterium abscessus is an emerging and rapidly growing species capable to induce severe and chronic pulmonary infections. However, whether M. abscessus, like Mycobacterium tuberculosis, possesses the machinery to acquire and store host lipids, remains unaddressed. Herein, we aimed at deciphering the contribution of the seven putative M. abscessus TAG synthases (Tgs) in TAG synthesis/accumulation thanks to a combination of genetic and biochemical techniques and a well-defined foamy macrophage (FM) model along with electron microscopy. Targeted gene deletion and functional complementation studies identified the MAB_3551c product, Tgs1, as the major Tgs involved in TAG production. Tgs1 exhibits a preference for long acyl-CoA substrates and site-directed mutagenesis demonstrated that His144 and Gln145 are essential for enzymatic activity. Importantly, in the lipid-rich intracellular context of FM, M. abscessus formed large ILI in a Tgs1-dependent manner. This supports the ability of M. abscessus to assimilate host lipids and the crucial role of Tgs1 in intramycobacterial TAG production, which may represent important mechanisms for long-term storage of a rich energy supply. This article is protected by copyright. All rights reserved.

In mycobacteria, MmpL proteins represent key components that participate in the biosynthesis of the complex cell envelope. Whole genome analysis of a spontaneous rough morphotype variant of Mycobacterium abscessus subsp. bolletii identified a conserved tyrosine that is crucial for the function of MmpL family proteins. Isogenic smooth (S) and rough (R) variants differed by a single mutation linked to a Y842H substitution in MmpL4a. This mutation caused a deficiency in glycopeptidolipid production/transport in the R variant and a gain in the capacity to produce cords in vitro. In zebrafish, increased virulence of the M. bolletii R variant over the parental S strain was found, involving massive production of serpentine cords, abscess formation and rapid larval death. Importantly, this finding allowed us to demonstrate an essential role of Tyr842 in several different MmpL proteins, including Mycobacterium tuberculosis MmpL3. Structural homology models of MmpL4a and MmpL3 identified two additional critical residues located in the transmembrane regions TM10 and TM4 that are facing each other. We propose that these central residues are part of the proton-motive force that supplies the energy for substrate transport. Hence, we provide important insights into mechanistic/structural aspects of MmpL proteins as lipid transporters and virulence determinants in mycobacteria.

International audience; Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNF in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

International audience; Within tuberculous granulomas, a subpopulation of Mycobacterium tuberculosis resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with M. tuberculosis-containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with M. bovis BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a lipY deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI+3) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI+3 profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI+3 profiles in the ΔlipY mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.

Rose is the world’s most important ornamental plant with economic, cultural and symbolic value. Roses are cultivated worldwide and sold as garden roses, cut flowers and potted plants. Rose has a complex genome with high heterozygosity and various ploidy levels. Our objectives were (i) to develop the first high-quality reference genome sequence for the genusRosaby sequencing a doubled haploid, combining long and short read sequencing, and anchoring to a high-density genetic map and (ii) to study the genome structure and the genetic basis of major ornamental traits.We produced a haploid rose line fromR. chinensis‘Old Blush’ and generated the first rose genome sequence at the pseudo-molecule scale (512 Mbp with N50 of 3.4 Mb and L75 of 97). The sequence was validated using high-density diploid and tetraploid genetic maps. We delineated hallmark chromosomal features including the pericentromeric regions through annotation of TE families and positioned centromeric repeats using FISH. Genetic diversity was analysed by resequencing eightRosaspecies. Combining genetic and genomic approaches, we identified potential genetic regulators of key ornamental traits, including prickle density and number of flower petals. A roseAPETALA2homologue is proposed to be the major regulator of petals number in rose. This reference sequence is an important resource for studying polyploidisation, meiosis and developmental processes as we demonstrated for flower and prickle development. This reference sequence will also accelerate breeding through the development of molecular markers linked to traits, the identification of the genes underlying them and the exploitation of synteny acrossRosaceae.