Transcriptomic response to Nod Factor treatments on Medicago
ABSTRACT: NGS2013-04: Transcriptomic response to Nod Factor treatments on Medicago Role of the root hair in water and nutrient uptake and the establishment of the nitrogen-fixing symbiotic interaction with rhizobia. The RNA was extracted from root hairs of Medicago: control vs treated by nod factors (2 biological replicates)
Project description:affy_ralstonia_medicago - Ralstonia solanacearum is the causal agent of the devastating bacterial wilt disease. Its infection process was studied with an in vitro inoculation procedure on intact roots of Medicago truncatula. The pathosystem involved susceptible A17 and resistant F83005.5 M truncatula lines infected with the pathogenic strain GMI1000. The mutant A17 line, Sickle, which showed a resistant phenotype was also part of the experiment. To identify host signaling pathway triggered by R. solanacearum infection with a focus on the involvment of ethylene, we used the Medicago Affymetrix array to monitore the expression profiles and the molecular process associated with initial symptoms development (12hpi) and colonization (72hpi). In order to maximize chances to observe differential gene expression, RNA samples were extracted from the root infection zone (root tips) -Three Medicago truncatula lines, A17, F83005.5 and sickle were inoculated with GMI1000 Ralstonai solanacearum strain (107 cfu/ml). RNA were extracted from root extremities (1 cm above the root tip) at time 0, 12h and 72h post inoculation. Three biological repeats were conducted normal vs disease comparison, time course, 27 arrays - Medicago
Project description:affy_ralstonia_peeters_medicago - We have identified two essential virulence determinants (GALA7, a type III secretion effector and HpaP, a chaperone-like protein) of Ralstonia solanacearum for the infection and colonisation of the host plant Medicago truncatula. The scope of this project is to identify the GALA7 and HpaP-specific transcriptome alteration. For this purpose wild type and mutant infected root material (13h and 72h postinfection) will be analysed on M. truncatula affymetrix chips. Medicago truncatula (A17 line) are grown in vitro on Farheus medium (with Nitrogen source) plantlets are inoculated with water R. solanacearum wt, gala7 and hpap mutants, and root tips are collected at 13h and 72h postinoculation. Experiment was performed 3 times independently. 4 bacteria conditions x 2 harvest times x 3 biological repeats = 24 samples Keywords: gene knock out,normal vs disease comparison,time course,treated vs untreated comparison 24 arrays - Medicago
Project description:Nitrogen assimilation in plants is a tightly regulated process that integrates developmental and environmental signals. The legume-rhizobial symbiosis results in the formation of a specialized organ called root nodule, where the rhizobia fixes atmospheric nitrogen into ammonia. Ammonia is assimilated by the plant enzyme glutamine synthetase, which is specifically inhibited by PPT. The expression of key genes related to the regulation of root nodule metabolism will likely be affected by glutamine synthetase inhibition. We used microarrays to detail the global programme of gene expression in response to Glutamine synthetase inhibition in root nodules and identified genes differentially expressed over a time course. Medicago truncatula nodulated plants (20 days post inoculation) were treated with 0.25 mM of PPT. Root nodules were harvested at 4, 8 and 24 hours after PPT application. As a control, root nodules collected just before PPT application were used (PPT 0h). Three biological replicates consisting of pools of root nodules harvested from five distinct plants were used for RNA extraction and hybridization on Affymetrix GeneChips.
Project description:Legumes interact with rhizobia, leading to the development of root nodules. Diffusible rhizobial signals were identified as Nod-LCOs. Applying Nod-LCOs on plantlet roots, we used GeneChips to detail the global programme of gene expression in response to the external application of Nod-LCOs. Overall design: To harvest tissues for transcriptome profiling, three biological replicates consisting of 10 plantlets per treatment were selected. After 6 h of incubation in the climate chamber, 10 plantlets per batch were removed from the treatment (Nod-LCOs) or control solutions and harvested. During harvest, one mm of the root tip of each plantlet was removed and discarded. The remaining 2 to 2.5 cm of the distal root region were cut off and directly frozen in liquid nitrogen.
Project description:Plants show a remarkable plasticity to adapt their root architecture to biotic and abiotic constraints of the soil environment. Although some of these modifications are fine-tuned by miRNAs, there are still shadow zones in these regulations. In the model legume Medicago truncatula, we analyzed the small RNA (smRNA) transcriptome of roots submitted to symbiotic and pathogenic interactions. Mapping on the genome and prediction of pre-miRNA hairpins allowed the identification of 416 candidates. Out of them, we found known and novel variants of 77 miRNA families, already reported in miRBase. In addition, thanks stringent criteria of miRNA prediction, 53 mtr-miRNAs were discovered, including 27 putative miRtrons. Exploring polymorphism in 26 M. truncatula ecotypes, higher polymorphism was observed in conserved rather than legume-specific miRNA genes. An average of 19 targets, mainly involved in environmental responses and signaling, was predicted per novel miRNA. In addition, taking advantage of our large number of smRNA libraries, we identified sets of miRNAs responsive to root pathogens or to symbiotic interactions and the related Nod and myc-LCO signals. 23 libraries of small RNA (smRNA) of roots submitted to symbiotic and pathogenic interactions.
Project description:The involvement of ROS in the legume – Rhizobium symbiotic interaction has been highlighted (Santos et al., 2001; Rubio et al., 2004). This interaction is characterized by the formation of a new organ on the root, the nodule and by the penetration, in parallel, of the bacteria into the root tissue via an infection thread (IT) (Parniske and Downie, 2003; Gage, 2004). H2O2 production has been shown in ITs during the Medicago – Sinorhizobium meliloti interaction (Santos et al., 2001). Moreover, S. meliloti mutants impaired in H2O2 detoxification mechanism possess in planta symbiotic phenotypes. As H2O2 production is known to orchestrate plant gene expression, our goal is focused on identifying the H2O2 regulated genes in the symbiotic process. We focus our analysis in the M. truncatula transcriptome as we are characterising the S. meliloti H2O2 transcriptome. -M. truncatula seedlings were grown in axenic condition on modified Fahraeus medium during seven days. Then they were transferred on new plates supplemented either with dimethyl sulfoxide (DMSO, mock treatement) or diphenylene iodonium (DPI, 10 µM). Twenty four hours later, they were inoculated either with water (mock inoculation; DMSO-H2O and DPI-H2O) or wild type S. meliloti 2011 strain (DMSO-INOC and DPI-INOC). Roots without apexes were harvested 48 hours after inoculation. After RNA extraction (Trizol), quality of the treatment was verified by RT-PCR (ENOD11: inoculation efficiency; GSHS1: DNA contamination; MTC27: constitutive) Keywords: treated vs untreated comparison 8 arrays - Medicago
Project description:Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the alphaproteobacteria. Here, we provide evidence that in broad host range rhizobia the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations bridging gaps in signalling cascades and providing a competitive advantage. Overall design: To study the influence of flavonids as well as the complete absence of quorum sensing signals on gene expression in Sinorhizobium fredii NGR234, we analyzed in total 12 samples: Thereby we compared the NGR234 wild type to NGR234 wild type treated with 1 µM apigenin as a flavoniod. Furthermore, we compared the gene expression in NGR234 quorum sensing double mutant strain to the NGR234 double mutant supplemented with 1 µM apigenin.
Project description:affy_pathogen_medicago - In compatible interaction between plants and biotrophic microorganisms, neoformation of organs occurs to ensure an efficient relationship between both partners. During the interaction between Medicago truncatula and Sinorhizobium meliloti, bacteria induce the development of root nodule with a permanent meristem, and chronically infect plant cells from zone II before differentiating into atmospheric nitrogen fixing bacteroids. M. truncatula is also plant host for root-knot nematodes, such as Meloidogyne incognita. During this compatible pathogenic interaction, root-knot nematodes induce redifferenciation of root cells from the vascular cylinder into specialized feeding cells called “giant cells”. Hyperplasia and hypertrophy of the surrounding cells lead to the formation of typical root galls. This phenomenon invokes host pathways in common with those necessary for nitrogen-nodule formation, suggesting that nematode and rhizobia exploit a relative common strategy of plant cell infection at the cellular and molecular levels. In order to highlight key genes involved in gall and nodule developments, parallel laser microdissection of giant cells from galls and cells from zone II of nodules, followed by transcriptomic analysis, were performed. The RNA pools were extracted from these cells, amplified and used for transcriptomic studies with M. truncatula Affymetrix DNA chips. Keywords: organ comparison Overall design: 12 arrays - Medicago infected with M. incognita or S. meliloti.
Project description:A comparative assessment between both technologies, RNASeq and microarrays to detect differential expression in Arabidopsis transcriptome. The sequencing approach use High-throughput sequencing on different Solexa technologies (GAII,HiSeq2000 multiplex or not) Wild type samples were analyzed from 2 tissus (flower buds and leaves) which have a very contrasted transcriptomic profile (i.e very high number of genes differentially expressed). The RNA was extracted from 2 tissus Flower Buds and Leaves from Arabidopsis. The associated GEO series with array part is GSE45345
Project description:12plex_medicago_2013-08 - r108 in symbiosis with rhizobia wt or rhizobia mutant for baca. - Two experiments to compare the transcriptomic response of medicago plants: Agar medium versus Phytagel medium (exp1) and rhizobium WT versus BacA (exp2). - Medicago truncatula ecotype R108 was inoculated with the symbiotic rhizobium Sinorhizobium meliloti strain Sm1021 and with its derivative mutant delta bacA. Nodules were collected 13 days after inoculation, and RNA were prepared for transcriptome analysis, there were three biological independant experiements. Overall design: rhizobium WT versus BacA. 3 dye-swap - gene knock out