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International audience; Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.

Xanthomonas oryzae pv. oryzicola is an important bacterial pathogen responsible for outbreaks of bacterial leaf streak (BLS) on rice, mostly occurring in Asia and parts of Africa. To better monitor epidemics and assess population structures, efficient tools that allow the precise identification and diagnosis of pathogenic populations are needed. In this study, we explored variable numbers of tandem repeats (VNTR) as a fast, reliable, and cost-effective molecular typing tool. Screening of three X. oryzae pv. oryzicola genome sequences (Philippine strain BLS256, Chinese strain GX01, and Malian strain MAI10) predicted 28 candidate VNTR loci. Primer pairs for polymerase chain reaction (PCR) amplification of all 28 loci were designed and applied to a panel of 20 X. oryzae pv. oryzicola strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci that are shared among Asian and African X. oryzae pv. oryzicola strains. A dendrogram constructed from 25 VNTR loci indicated that most Asian strains are clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali is related to Asian strains, pointing to a possible introduction of Asian strains to the African continent. The new VNTR-based tool described here is useful for studies of population structures and epidemiological monitoring of X. oryzae pv. oryzicola.

Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by non-ribosomal peptide synthesis (NRPS) in this genus is albicidin produced by Xanthomonas albilineans. The DNA gyrase inhibitor albicidin is not only an important virulence factor but also a possible lead structure for novel antibiotics. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. The most unexpected result of these analyses is that these two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters. Furthermore, these Xanthomonas spp. strains share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or for the secretion of peptides. In silico prediction of peptide structures according to the NRPS architecture accounts for eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas transluscens and X. oryzae pv. oryzicola. Further genome mining revealed (i) novel NRPS genes shared by Xanthomonas spp. strains GPE PC73 and XaS3 with the plant-associated bacterium Bradyrhizobium spp. strain BTAi and (ii) novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. This study revealed the significant potential of the genus Xanthomonas of producing new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of new non-ribosomally synthesized peptides in plant-bacteria interactions.

Objectives: Xanthomonas albilineans is a xylem-invading pathogen that causes leaf scald, one of the major diseases of sugarcane. Previous studies revealed that i/ two important genomic features differentiate X. albilineans from other sequenced species of Xanthomonas: genome erosion and the presence of a type III secretion system (T3SS) of the SPI-1 family (1-2), and ii/ large variability exists among strains of X. albilineans and all strains involved in outbreaks of sugarcane leaf scald disease since the late 1980s belong to the same genetic group called PFGE-B (3). In the present study, we used the genome sequence of X. albilineans strain GPE PC73 to describe all pathogenicity-related features either shared with all species of Xanthomonas or specific to X. albilineans, and to further investigate the large diversity of this species. Results Among the major virulence factors described so far in Xanthomonads, most of them are conserved in X. albilineans, except the T3SS of the Hrp family and the gum gene clusters, and Hrp T3SS effectors. The genome of X. albilineans also encodes specific pathogenicity-related factors including twelve non ribosomal peptide synthetases and five enzymes harboring a specific cellulose binding domain. Several DNA fragments present in PFGE-B strains and absent in other strains of the pathogen were isolated by suppression subtractive hybridization (SSH). Additionally, occurrence of methylation of genomic DNA by a specific Dam methyltransferase in PFGE-B strains, but not in other strains, was experimentally demonstrated. Large inter-strain variability in X. albilineans was confirmed using multi locus sequence analysis (MLSA), clustered regularly interspaced short palindromic repeats (CRISPR) and SSH markers. Conclusions This study allowed us to identify several new candidate pathogenicity genes. In-depth functional analyses are now necessary to explore the role of these genes in the successful invasion of the sugarcane xylem vessels by X. albilineans.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight in rice, one of the most devastating diseases of rice worldwide. African X. oryzae pv. oryzae strains belong to a clear genetic group distinct from those of Asia. Three new races of the pathogen were characterized among strains from West Africa. We evaluated 107 Oryza glaberrima accessions for resistance to bacterial blight under greenhouse conditions. Six-week-old seedlings were inoculated with five different African X. oryzae pv. oryzae strains originating from the West African nations of Burkina and Mali and representing different races (A1, A2, and A3). Philippine X. oryzae pv. oryzae strain PXO86 (race 2) was also used. Most (48%) of the accessions of O. glaberrima were highly susceptible to X. oryzae pv. oryzae strains from Burkina, while 20 and 36 were resistant to X. oryzae pv. oryzae strains from Mali and the Philippines, respectively. CAPS markers and dot blot assays were used for detection of resistance genes xa5 and Xa21 from a selected set of O. glaberrima accessions. Our results suggest that the O. glaberrima germplasm contains a narrow genetic base for resistance to X. oryzae pv. oryzae. Sources of resistance identified among O. glaberrima are recommended for rice breeding programs to develop bacterial blight-resistant cultivars for West Africa.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated sequences (cas) form hypervariable loci which are widely distributed in prokaryotes. The repetitive region is characterized by short interspersed unique spacer sequences which often match to bacteriophage-derived DNA, thus providing immunity against foreign genetic elements, in particular bacteriophages. Bacteria of the genus Xanthomonas cause diseases on over 400 different host plants, including many economically important crops, such as rice, wheat, citrus, and banana plants. We screened more than 300 strains of Xanthomonas, representing ten different species (44 pathovars), for the presence of CRISPR loci. Only a few pathovars were found to possess a CRISPR locus, among them X. axonopodis pv. vasculorum, X. axonopodis pv. cassavae, X. campestris pv. raphani, X. citri pv. citri, X. oryzae pv. oryzae (Xoo), X. translucens, and X vasicola pv. musacearum. Presence/absence of CRISPR loci appeared to be conserved at the pathovar level, except for Xoo. The apparent absence of CRISPR loci from African Xoo isolates confirms previous results showing that African Xoo isolates form a phylogenetic group that is distant from the Asian Xoo group. Comparative genomics suggested that the common ancestor of all xanthomonads had two CRISPR loci which in most species/pathovars got lost during evolution. Based on DNA sequence information about the terminal spacers of 32 Asian Xoo CRISPR loci we postulate that the common ancestor of these strains had all the spacers which are nowadays still found in a few strains and that some spacers got lost during evolution in some Xoo lineages. This work represents the first proof of concept of CRISPR analysis as a molecular tool for high-resolution strain typing, phylogenetic studies, and global surveillance of a phytopathogen. (Texte intégral)

Xanthomonas oryzae pathovar oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc) cause bacterial diseases in rice: leaf blight and leaf streak, respectively. Although both the Asian and the African strains of Xoo induce similar symptoms, they are genetically different, with the African Xoo strains being more closely related to the Asian Xoc. To identify the sequences responsible for differences between African and Asian Xoo strains and their relatedness to Xoc strains, a suppression-subtractive hybridization (SSH) procedure was performed, using the African Xoo MAI1 strain as a tester and the Philippine Xoo PXO86 strain and Xoc BLS256 strain as drivers. A nonredundant set of 134 sequences from MAI1 was generated. Several DNA fragments isolated by SSH were similar to genes of unknown function, hypothetical proteins, genes related to the type III secretion system, and other pathogenicity-related genes. The specificity of various fragments was validated by Southern blot analysis. SSH sequences were compared with several xanthomonad genomes. In silico analysis revealed SSH sequences as specific to strain MAI1, revealing their potential as specific markers for further epidemiological and diagnostic studies. SSH proved to be a useful method for rapidly identifying specific genes among closely related X. oryzae strains.

La yuca (Manihot esculenta) constituye la base de la alimentación de más de 600 millones de personas en el mundo. Una de las principales limitaciones de este cultivo es la bacteriosis vascular, ocasionada por la bacteria Xanthomonas axonopodis pv. manihotis (Xam). Este artículo revisa el conocimiento actual acerca de la interacción Xanthomonas-yuca. Se presentan estudios recientes llevados a cabo sobre la diversidad y dinámica de las poblaciones de Xam empleando diferentes estrategias moleculares. Se describen los diferentes métodos desarrollados para la detección y diagnóstico de la bacteria en plantas y semillas de yuca y su contribución para reducir el impacto de la enfermedad. Se presentan los estudios encaminados a comprender los mecanismos moleculares y los genes responsables en la resistencia de la yuca a la bacteriosis vascular incluyendo los últimos avances obtenidos gracias a la aplicación de estrategias de genómica funcional. El conocimiento adquirido en los últimos años en este patosistema permitirá desarrollar mejores estrategias para el manejo de la enfermedad así como desarrollar a corto plazo variedades de yuca resistentes a la bacteriosis lo que contribuiría a resolver uno de los principales problemas de los productores pobres de yuca y le abriría un horizonte promisorio al cultivo de la yuca en el mundo. Cassava (Manihot esculenta), a starchy root crop, constitutes the source of alimentation for over 600 million people worldwide. Cassava Bacterial Blight (CBB) is caused bythe bacterium Xanthomonas axonopodis pv. manihotis (Xam). This review will focus on the current knowledge on the molecular cassava-Xam interaction. We will present the different molecular techniques developed to assess the genetic diversity and dynamics of Xam populations. We will also present different methods developed for detecting the pathogen in vegetative planting materials and true seeds and their contribution to reduce the impact of the disease. We will review different studies conducted to gain a better understanding on the molecular mechanisms and the genes involved in the cassava bacterial resistance, including the recent advances obtained using functionalgenomics. The acquired knowledge in the last years for this pathosystem will help to establish better disease control strategies and generate, in a short term, resistant cassava varieties contributing to solve one of the main problems of poor cassava farmers and this effort will open a new horizon to the cassava crop in the world.

National audience; We created a network, called ARGEBIOS (Agropolis Reverse GEnetics for BlOtic Stress), of four laboratories involved in the area of resistance to biotic stress resistance in rice to collectively analyse compromised phenotypes of T-DNA tagged lines identified by reverse genetics approach. The ARGEBIOS integrated approach aims at identifying novel signalling and effector components involved in broad-spectrum resistance to biotic stress in rice and in specific pathways leading to resistance to bacterial, fungal and viral pathogens. Candidate genes were identified from transcriptome and proteome data generated in the four laboratories and from available data in the literature. Mutant T2 rice lines in candidate genes are analysed for compromised resistance to host and non-host rice pathogens and genotyped to verify the linkage between the identified phenotype and the T-DNA insertion. So far 150 mutant lines were analysed for compromised resistance to the three rice pathogens and non-host resistance to M. grisea a d mutant phenotypes were identified in up to 20% of T-DNA screened lines. C. Brugidou's team is studying the molecular interactions between rice and rice yellow mottle virus (RYMV) to elucidate the mechanisms controlling sensitivity, tolerance and the resistance of rice to the virus. Using a combined transcriptome, proteome and bioinformatic approach, they identified deregulation of host genome expression at the beginning of viral infection process. 550 genes were identified as highly deregulated in Indica and Japonica cultivars and are involved in all functional categories. They are using the ARGEBIOS set-up to functionally validate their implication in the viral infection processes. JB Morel's team is focusing on defining the signalling components involved in durable host resistance to M. grisea strains in both Japonica and Indica subspecies. The screen of T2 lines is based upon a compatible interaction enabling to reveal both EDS (enhanced disease susceptibility) and EDR (enhanced disease resistance) typ of mutants. P. Piffanelli's team interest lies on the mechanisms leading to cross-species non-host resistance to M. grisea strains in rice. A high-throughput inoculation protocol and phenotypic analysis for compromised non-host resistance of rice Nipponbare to M.grisea strains attacking other monocots (e.g. wheat, Digitaria, barley) was set-up at CIRAD to identify signalling components involved in type I and type II cross-species resistance mechanisms. V. Verdier's team focuses on the molecular mechanisms leading to resistance to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Screening assays were performed on 30 day-old rice plants by leaf clip inoculation. Symptoms were scored by measuring lesion lengths. Most of the strains induced a susceptible reaction while strain PX0339 induced a moderate resistant reaction (MR) and was selected to screen the T-DNA mutants looking at EDS-type phenotypes. Mutant phenotypes were identified in 13% of T-DNA lines. We are now undergoing high-throughput g notyping of the identified lines following a protocol combining Southern blot and PCR analyses to confirm the linkage between T-DNA and observed phenotypes. The comparative study with fungal, bacterial and viral pathogens will enable to pinpoint shared signalling pathways likely to be potential targets to engineer durable field resistance to rice pathogens.

The International Rice Functional Genomics Consortium (IRFGC), has been working together to collaborate on setting up a functional genomics platform to systematically study rice gene functions. International efforts since the last few years have produced rice mutant resources that are a powerful functional genomics tool to identify the function of the sequenced rice genes. Within a Generation Challenge Program project the consortium works together towards the identification of genes that can contribute to a phenotype of resistance/tolerance to abiotic/biotic stresses. A list of over five hundred candidate rice Stress Associated Genes (SAGs) was collated based on genes revealed by microarray analysis induced under various stresses, as well as known functions from other plants. In a reverse genetic strategy, we are using sequence-indexed knockout mutant resources developed by the rice international community to identify insertions in the target stress associated genes, from among a collection of over sixty thousand insertion sites in the rice genome (http://orygenesdb.cirad.fr/). These include Tos17 transposons, heterologous Ac-Ds transposons and T-DNA inserts that are identified by available flanking sequence tags (FSTs) that are positioned on the complete genome sequence. In addition an available TILLING population is used to supplement the mutant coverage, and also provide non-transgenic stocks for field testing. To resolve gene redundancy, gain-of-function overexpression lines are being generated by transformation of appropriate constructs, and available activation tag populations will also be accessed for overexpression mutants. To facilitate novel gene discovery a forward genetics mutant screen is being carried out with insertion tagged genotypes that have been sequence indexed, thus aiding their further analysis. The selected collection of mutant genotypes is being phenotyped for drought and disease stress parameters in greenhouse and field-based screens. Genotypes with altered stress tolerance phenotypes will be tested for other abiotic/biotic stresses. Whole genome microarray analysis will be conducted to identify the downstream genes and characterize the stress response mechanism involved. The genes with validated function for stress resistance in rice are then a source for comparative genetics studies in other crop plants.

Cassava bacterial blight, caused by Xanthomonas axonopodis pv. manihotis (Xam), is a widespread disease that affects cassava (Manihot esculenta Crantz). Studies on the pathogen population structure, pathogen diagnosis, identification and expression of plant genes involved in resistance have been carried out. Different molecular techniques were developed to assess the genetic diversity among the Xampopulations. Characterization of Xam population dynamics over time had enable us to determine the different factors that are associated with resistance breakdown and those that influence the genetic structure or virulence phenotypes of the pathogen's population. Methods for detecting the pathogen in vegetative planting materials and true seeds were developed and contributed to reduce the impact of the disease. To better understand the genetics of resistance a quantitative trait loci (QTLs) approach was developed. Using a PCR-based strategy with degenerate primers we isolated two resistance gene candidates in cassava. We also characterized a region of a chromosome rich in R-gene like sequence. In this review we also report the main results obtained by transcript profiling methodologies, cDNA-AFLP and ESTs developed by the authors to characterize the genes involved in disease resistance. All together these techniques allowed the identification of molecular markers either associated to CBB resistance or that may represent putative genes involved in disease resistance. This article reviews current knowledge on the molecular cassava-Xam interactions.

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.

International audience; Cassava suffers from bacterial blight attack in all growing regions. Control by resistance is unstable due to high genotype–environment interactions. Identifying genes for resistance to African strains of Xanthomonas axonopodis pv. manihotis can support breeding efforts. Five F1 cassava genotypes deriving from the male parent ‘CM2177-2’ and the female parent ‘TMS30572’ were used to produce 111 individuals by backcrossing to the female parent. In all, 16 genotypes among the mapping population were resistant to stem inoculation by four strains of X. axonopodis pv. manihotis from different locations in Africa, and 19 groups with differential reactions to the four strains were identified, suggesting that the strains represent different pathotypes. Four genotypes were resistant to leaf inoculation, and three were resistant to both stem and leaf inoculations. Genotypes with susceptible, moderately resistant, and resistant reactions after leaf and stem inoculation partly differed in their reactions on leaves and stems. Based on the genetic map of cassava, single-marker regression analysis of disease severity after stem-puncture inoculation was performed. Eleven markers were identified, explaining between 16 and 33.3% of phenotypic variance of area under disease progress curve. Five markers on three and one linkage groups from the female- and malederived framework of family CM8820, respectively, seem to be weakly associated with resistance to four strains of X. axonopodis pv. manihotis. Based on the segregation of alleles from the female of family CM8873, one marker was significantly associated with resistance to two X. axonopodis pv. manihotis strains, GSPB2506 and GSPB2511, whereas five markers were not linked to any linkage group. The quantitative trait loci (QTL) mapping results also suggest that the four African strains belong to four different pathotypes. The identified pathotypes should be useful for screening for resistance, and the QTL and markers will support breeding for resistance.

DNA polymorphism and variation in virulence of #Xanthomonas axonopodis$ pv. #manihotis$ (Xam), the causal agent of cassava bacterial blight, were studied within a pathogen population from Venezuela. Collections were made in several fields at different sites within an edaphoclimatic zone where cassava is a major crop. DNA polymorphism was assessed by RFLP analysis, using an Xam plasmidic DNA sequence (ptbB) as a probe to determine the relatedness of 91 Venezuelan isolates. A high degree of polymorphism existed among the isolates, whether collected from the same or different fields. Based on a multiple correspondence analysis, the Xam population was distributed into eight clusters and no correlation was observed between genetic diversity and geographic origin. One set of haplotype strains representing the range of variability detected in Venezuela was further characterized by another RFLP analysis using two repetitive genomic probes (pBS6 and pBS8) to establish the usefulness of these probes and their complementarity with the ptbB probe. Variation for virulence was observed in the Xam Venezuelan collection by inoculating a set of cassava cultivars with 28 isolates of the pathogen, each representig a haplotype. Understanding the genetic and pathogenic variation in the pathogen population is useful for designing cassava bacterial blight management strategies. (Résumé d'auteur)

Le manioc est une culture importante en Colombie puisqu'elle couvre environ 196 000 ha. L'un des facteurs limitants de la production est une maladie causée par une bactérie : #Xanthomonas axonopodis$ pv. #manihotis$ (Xam). La distribution géographique de la maladie et son incidence sont précisées dans les différentes zones écologiques de la Colombie. Les souches de cette bactérie collectées dans trois zones ont été caractérisées selon une approche moléculaire, leur agressivité étant évaluée sur la plante. Vingt-six groupes de souches de Xam ont pu être définis et leur répartition dans les 3 zones écologiques analysée. Dans une zone de haute altitude, la variabilité de l'agent pathogène est très faible. L'impact de cette étude pour l'amélioraiton variétale vis-à-vis de la résistance à la bactériose est discuté. Les perspectives d'étude sur les gènes de résistance sont également abordées. (Résumé d'auteur)

Les travaux sur les ennemis des plantes cultivees s'organisent le plus souvent selon une approche binomiale qui prend en compte, par exemple, une plante et un parasite ou une plante et un ravageur. Dans le cas du manioc en Afrique, pour des ennemis peu nombreux et qui offrent une grande diversite de traits biologiques et de relations avec la plante, des travaux ont ete conduits depuis pres d'un quart de siecle par les chercheurs de l'Orstom en Cote d'Ivoire, au Congo et au Togo et sont actuellement poursuivies au Benin et en France. Il s'agit de la mosaique africaine du manioc et de son insecte vecteur #Bemisia tabaci$, de la bacteriose vasculaire dont l'agent est #Xanthomonas campestris$ pv #manihotis$, de la cochenille du manioc #Phenacoccus manihoti$, et de l'acarien vert, #Mononychellus progresivus$. Les couples manioc/maladie ou manioc/ravageur ont tout d'abord ete envisages sans considerer le systeme parasitaire dans son ensemble en depit de convergences evidentes dues a un environnement commun, a des analogies d'effet sur la plante hote et a des interactions probables entre les differents ennemis de la meme culture. Nous avons ici elabore une approche biocenotique qui permet d'integrer differentes actions de recherche, orientee vers l'analyse du fonctionnement des systemes plante-parasites et plante-ravageurs et qui permet de formuler et d'evaluer les options de gestion integree de cette biocenose parasitaire. La comparaison "transversale" des modeles plantes-parasites-ravageurs aborde successivement les agents nuisibles (maladies et ravageurs) et leur variabilite, leurs cycles biologiques et les facteurs environnementaux qui les conditionnent, les relations avec la plante (variabilite genetique, physiologie et conduite de la culture), l'incidence des entomophages et entomopathogenes sur les phytophages et les perspectives d'integration offertes par la modelisation. (Resume d'auteur)

International audience