Stress-resistant plants and their production

The present invention relates to plant genes involved in negative regulation of resistance to biotic and/or abiotic stress and uses thereof. More particularly, the present invention relates to plants comprising an inactivated MADS-box gene function, and having increased resistance to biotic and/or abiotic stress. The invention also relates to methods for producing modified plants having increased resistance to fungal, bacterial pathogens and/or to drought stress. In particular, the invention relates to methods for producing plants with inactivated MAD26 gene, or an ortholog thereof, and exhibiting resistance to biotic and/or abiotic stress. (Résumé d'auteur

The rice functional genomics (refuge) platform, an international hosting platform for elucidating gene function using rice as a model system

REFUGE is an international hosting platform for the elucidation of gene function using rice as a model species. It offers international scientists, notably nonrice specialists, an access to know-how, bioinformatic, biological, and molecular resources, allowing the use of rice as a model system to investigate gene function through functional genomics strategies. PhD and postdoctoral researchers, notably from the South, are the first target beneficiaries of this initiative. Visiting scientists have access to REFUGE's know-how, including bioinformatic searches, preparation of T-DNA vectors, high-throughput production of rice transformants, molecular characterization of transformants, genotyping of insertion lines from local and international collections; growth, crossing, and phenotyping in containment greenhouse; and access to cell imaging and genotyping platforms. Hosted scientists have the opportunity to carry out one to several visits from 1 to 3 months, the REFUGE staff taking care of the materials produced between two visits. The REFUGE platform provides supervision of the hosted scientists or students and covers bench fees. Visitors have to find their own travel and subsistence funds. However, REFUGE can provide assistance to scientists notably from South countries, to find an appropriate funding source for travel and subsistence. Applications should be submitted at http://www.refuge-platform.org and will be readily examined by the REFUGE scientific committee. Funded by Agropolis Fondation, http://www.agropolis-fondation.fr/, REFUGE is a collaborative venture between the joint research unit DAP -INRA, CIRAD and Montpellier University and Agronomy School- and the 'Plant genome and development' laboratory at IRD and Perpignan University. (Texte intégral

Role of LRR-RLKS in stress response and developmental processes through systematic K.O. analysis in rice

The function of a small number of Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) genes has been studied, mostly in Arabidopsis thaliana. They are involved in several important biological processes, including development, resistance/defence and adaptation to biotic and abiotic stresses. We are interested in deciphering the function of these genes in rice. In this aim, we decided to take advantage of the availability of T-DNA and Tos17 insertion mutant libraries of rice cv. Nipponbare to start a systematic screen of knock-out mutants of these genes. The rice genome contains ~320 LRRRLK genes. Our search in international collections of insertion mutant lines which are catalogued by Flanking Sequence Tag (FST) information from most of the rice insertional mutagenesis projects around the world revealed that 238 insertional mutant lines are available for the 323 genes. Phenotypical evaluation of homozygous mutants is performed under various biotic and abiotic stresses and mutant plants are observed for phenotypes at several stages of growth, from seedlings in Petri dishes to flowering and grain development stages in the greenhouse. Thus, with this project, we aim first to decipher and explore new genes involved in defence and developmental responses to stresses. Second, our analysis will be of great benefit to provide potential targets for cereal improvement, notably in the breeding of drought-tolerant and pathogen-resistant crops. This project is supported by grant #ANR-08-GENM-021 from Agence Nationale de la recherche (ANR), France. (Résumé d'auteur

RNA interference of the stress related OsMADS26 transcription factor in rice increases resistance to various pathogens and reduces plant development

Several transcription factors have been involved in rice response against biotic and abiotic stresses (Khong et al., 2008). In A. thaliana, the MADS box transcription factor AGL12 control cell division in root meristem and is involved in flowering transition (Tapia-Lopez et al., 2008). When over expressed in Catharanthus roseus cell suspension AGL12 promotes tissue like organization and alkaloid biosynthesis (Montiel et al., 2007). OsMADS26, the rice orthologous of AGL12 in A. thaliana was recently over expressed under the control of a DEX inducible promoter and described as a stress related gene Lee et al., 2008). Nevertheless its precise function remains unknown. QPCR analysis revealed that this gene is expressed both in leaves and roots and is upregulated in response to osmotic stress. In order to precise the function of OsMAD26 in stress response or in development, and in absence of insertion mutant, we had generated rice lines RNA interfered for this gene. Interfered lines were obtained with two GST tagging the 5' or the 3' end of the mRNA. Interfered lines were affected in their development (root, tillering, height, dry weight) and were delayed for their flowering time in comparison to control. Roots of interfered lines present a reduced geotropism. Interfered lines were not affected in comparison to control line in their availability to resist to drought stress. But interestingly, interfered lines present an increased resistance against Magnaeporthae grisea, Xanthomonas oryzae and the Rice Yellow Mottle Virus. This suggests that OsMADS26 is a negative regulator of biotic stress response and acts as a general activator of development. In order to identify the genes and the biological pathways regulated by OsMADS26 we have proceeded to a transcriptome comparative analysis of genes expressed in WT and RNA interfered lines. Data analysis is in progress. (Texte intégral

An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves

Background: Localized introduction and transient expression of T-DNA constructs mediated by agro-infiltration of leaf tissues has been largely used in dicot plants for analyzing the transitivity and the cell-to cell movement of the RNAi signal. In cereals, however, the morphology of the leaf and particularly the structure of the leaf epidermis, prevent infiltration of a bacterial suspension in cells by simple pressure, a method otherwise successful in dicots leaves. This study aimed at establishing a rapid method for the functional analysis of rice genes based on the triggering of RNA interference (RNAi) following Agrobacterium-mediated transient transformation of leaves. Results: Using an agro-infection protocol combining a wound treatment and a surfactant, we were able to obtain in a reliable manner transient expression of a T-DNA-borne uidA gene in leaf cells of japonica and indica rice cultivars. Using this protocol to transiently inhibit gene expression in leaf cells, we introduced hairpin RNA (hpRNA) T-DNA constructs containing gene specific tags of the phytoene desaturase (OsPDS) and of the SLENDER 1 (OsSLR1) genes previously proven to trigger RNAi of target genes in stable transformants. SiRNA accumulation was observed in the agro-infected leaf area for both constructs indicating successful triggering of the silencing signal. Accumulation of secondary siRNA was observed in both stably and transiently transformed leaf tissues expressing the HpRNA OsSLR1 construct. Gene silencing signalling was investigated in monitoring the parallel time course of OsPDS-derived mRNA and siRNA accumulation in the agro-infiltrated leaf area and adjacent systemic sectors. The sensitive RT-Q-PCR method evidenced a consistent, parallel decrease of OsPDS transcripts in both the agroinfiltred and adjacent tissues, with a time lag for the latter. Conclusions: These results indicate that the method is efficient at inducing gene silencing in the agro-infected leaf area. The transfer of low amounts of siRNA, probably occurring passively through the symplastic pathway from the agro-infected area, seemed sufficient to trigger degradation of target transcripts in the adjacent tissues. This method is therefore well suited to study the cell-to-cell movement of the silencing signal in a monocot plant and further test the functionality of natural and artificial miRNA expression constructs. (Résumé d'auteur

Dissection des bases biologiques de caractères d'intérêt chez le riz : architecture et développement du système racinaire

Le riz possède un système racinaire fasciculé composé principalement de racines adventives aussi appelées racines coronaires ou nodales. Ces racines émergent d'abord des noeuds empilés à la base des tiges principales puis des ramifications aériennes. Parce que le système racinaire n'est pas directement accessible et est difficile à observer, la sélection phénotypique de variétés de riz capables d'exploiter au mieux les ressources du sol reste un défi pour les sélectionneurs. La sélection de caractères architecturaux améliorés nécessite donc l'identification des déterminants génétiques du développement racinaire. Ainsi, de nombreux travaux de recherche pour identifier et caractériser les gènes contrôlant l'architecture racinaire sont en cours chez le riz et plusieurs autres céréales. Dans cette revue, nous présentons les dernières stratégies et des résultats obtenus récemment dans l'identification de gènes et de loci à effets quantitatifs impliqués dans le développement racinaire du riz. (Résumé d'auteur

Genome-wide association mapping of leaf mass traits in a Vietnamese rice landrace panel

Leaf traits are often strongly correlated with yield, which poses a major challenge in rice breeding. In the present study, using a panel of Vietnamese rice landraces genotyped with 21,623 single-nucleotide polymorphism markers, a genome-wide association study (GWAS) was conducted for several leaf traits during the vegetative stage. Vietnamese landraces are often poorly represented in panels used for GWAS, even though they are adapted to contrasting agrosystems and can contain original, valuable genetic determinants. A panel of 180 rice varieties was grown in pots for four weeks with three replicates under nethouse conditions. Different leaf traits were measured on the second fully expanded leaf of the main tiller, which often plays a major role in determining the photosynthetic capacity of the plant. The leaf fresh weight, turgid weight and dry weight were measured; then, from these measurements, the relative tissue weight and leaf dry matter percentage were computed. The leaf dry matter percentage can be considered a proxy for the photosynthetic efficiency per unit leaf area, which contributes to yield. By a GWAS, thirteen QTLs associated with these leaf traits were identified. Eleven QTLs were identified for fresh weight, eleven for turgid weight, one for dry weight, one for relative tissue weight and one for leaf dry matter percentage. Eleven QTLs presented associations with several traits, suggesting that these traits share common genetic determinants, while one QTL was specific to leaf dry matter percentage and one QTL was specific to relative tissue weight. Interestingly, some of these QTLs colocalize with leaf- or yield-related QTLs previously identified using other material. Several genes within these QTLs with a known function in leaf development or physiology are reviewed

Genome-wide association mapping for root traits in a panel of rice accessions from Vietnam

Background: Despite recent sequencing efforts, local genetic resources remain underexploited, even though they carry alleles that can bring agronomic benefits. Taking advantage of the recent genotyping with 22,000 single-nucleotide polymorphism markers of a core collection of 180 Vietnamese rice varieties originating from provinces from North to South Vietnam and from different agrosystems characterized by contrasted water regimes, we have performed a genome-wide association study for different root parameters. Roots contribute to water stress avoidance and are a still underexploited target for breeding purpose due to the difficulty to observe them. Results: The panel of 180 rice varieties was phenotyped under greenhouse conditions for several root traits in an experimental design with 3 replicates. The phenotyping system consisted of long plastic bags that were filled with sand and supplemented with fertilizer. Root length, root mass in different layers, root thickness, and the number of crown roots, as well as several derived root parameters and shoot traits, were recorded. The results were submitted to association mapping using a mixed model involving structure and kinship to enable the identification of significant associations. The analyses were conducted successively on the whole panel and on its indica (115 accessions) and japonica (64 accessions) subcomponents. The two associations with the highest significance were for root thickness on chromosome 2 and for crown root number on chromosome 11. No common associations were detected between the indica and japonica subpanels, probably because of the polymorphism repartition between the subspecies. Based on orthology with Arabidopsis, the possible candidate genes underlying the quantitative trait loci are reviewed. Conclusions: Some of the major quantitative trait loci we detected through this genome-wide association study contain promising candidate genes encoding regulatory elements of known key regulators of root formation and development

Genome-wide association study of a panel of vietnamese rice landraces reveals new QTLs for tolerance to water deficit during the vegetative phase

Background: Drought tolerance is a major challenge in breeding rice for unfavorable environments. In this study, we used a panel of 180 Vietnamese rice landraces genotyped with 21,623 single-nucleotide polymorphism markers to perform a genome-wide association study (GWAS) for different drought response and recovery traits during the vegetative stage. These landraces originate from different geographical locations and are adapted to different agrosystems characterized by contrasted water regimes. Vietnamese landraces are often underrepresented in international panels used for GWAS, but they can contain original genetic determinants related to drought resistance. Results: The panel of 180 rice varieties was phenotyped under greenhouse conditions for several drought-related traits in an experimental design with 3 replicates. Plants were grown in pots for 4 weeks and drought-stressed by stopping irrigation for an additional 4 weeks. Drought sensitivity scores and leaf relative water content were measured throughout the drought stress. The recovery capacity was measured 2 weeks after plant rewatering. Several QTLs associated with these drought tolerance traits were identified by GWAS using a mixed model with control of structure and kinship. The number of detected QTLs consisted of 14 for leaf relative water content, 9 for slope of relative water content, 12 for drought sensitivity score, 3 for recovery ability and 1 for relative crop growth rate. This set of 39 QTLs actually corresponded to a total of 17 different QTLs because 9 were simultaneously associated with two or more traits, which indicates that these common loci may have pleiotropic effects on drought-related traits. No QTL was found in association with the same traits in both the indica and japonica subpanels. The possible candidate genes underlying the quantitative trait loci are reviewed. Conclusions: Some of the identified QTLs contain promising candidate genes with a function related to drought tolerance by osmotic stress adjustment