Project description:Casuarina glauca belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in C. glauca. Symbiosis between C. glauca and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition.
Project description:Alnus glutinosa belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in A. glutinosa. Symbiosis between A. glutinosa and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition.
Project description:Casuarina glauca belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in C. glauca. Symbiosis between C. glauca and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition. Microarrays were designed by Imaxio (Clermont Ferrand, France ; http://www.imaxio.com/index.php) which has been accredited by Agilent Technologies (Palo Alto, CA, USA; http://www.home.agilent.com/agilent/home.jspx) as a certified service provider for microarray technologies. Based on 14327 annotated unigenes for C. glauca, 60mers probes were designed using eArray software (1 probe per unigene) and custom 8 x 15K Oligo Microarrays were manufactured by Agilent.
Project description:Alnus glutinosa belongs to a family of angiosperms called actinorhizal plants because they can develop nitrogen-fixing nodules in association with the soil bacteria Frankia. The aim of this transcriptomic study was to get a global view of the plant symbiotic genetic program and to identify new key plant genes that control nodulation during symbiosis in A. glutinosa. Symbiosis between A. glutinosa and Frankia was obtained after inoculation of young plant with a concentrated culture of the bacteria. Inoculation was performed in a medium depleted in nitrogen which favors the induction of nitrogen fixing symbiosis. For this study we considered two stages of symbiosis: - an early stage where inoculated roots were harvested 7 days after inoculation with the bacteria and compared to two controls (non-inoculated roots grown with or without nitrogen and harvested at the same time) - a late stage where nodules (nitrogen-fixing specific organs) were harvested 21 days after inoculation and compared to non-inoculated roots harvested on the day of inoculation (which is our reference time 0d). Three biological replicates were used for each condition. Microarrays were designed by Imaxio (Clermont Ferrand, France ; http://www.imaxio.com/index.php) which has been accredited by Agilent Technologies (Palo Alto, CA, USA; http://www.home.agilent.com/agilent/home.jspx) as a certified service provider for microarray technologies. Based on 14327 annotated unigenes for A. glutinosa, 60mers probes were designed using eArray software (1 probe per unigene) and custom 8 x 15K Oligo Microarrays were manufactured by Agilent
Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high.
Project description:Background: Frankia sp. strains are actinobacteria that form N2-fixing root nodules on angiosperms. Several reference genome sequences are available enabling transcriptome studies in Frankia sp. Genomes from Frankia sp. strains differ markedly in size, a consequence proposed to be associated with a high number of indigenous transposases, more than 200 of which are found in Frankia sp. strain CcI3 used in this study. Because Frankia exhibits a high degree of cell heterogeneity as a consequence of its mycelial growth pattern, its transcriptome is likely to be quite sensitive to culture age. This study focuses on the behavior of the Frankia sp. strain CcI3 transcriptome as a function of nitrogen source and culture age. Results: To study global transcription in Frankia sp. CcI3 grown under different conditions, complete transcriptomes were determined using high throughput RNA deep sequencing. Samples varied by time (five days vs. three days) and by culture conditions (NH4+ added vs. N2 fixing). Assembly of millions of reads revealed more diversity of gene expression between five-day and three-day old cultures than between three day old cultures differing in nitrogen sources. Heat map analysis organized genes into groups that were expressed or repressed under the various conditions compared to median expression values. Twenty-one SNPs common to all three transcriptome samples were detected indicating culture heterogeneity in this slow-growing organism. Significantly higher expression of transposase ORFs was found in the five-day and N2-fixing cultures, suggesting that N starvation and culture aging provide conditions for on-going genome modification. Transposases have previously been proposed to participate in the creating the large number of gene duplication or deletion in host strains. Subsequent RT-qPCR experiments confirmed predicted elevated transposase expression levels indicated by the mRNA-seq data. Conclusions: The overall pattern of gene expression in aging cultures of CcI3 suggests significant cell heterogeneity even during normal growth on ammonia. The detection of abundant transcription of nif (nitrogen fixation) genes likely reflects the presence of anaerobic, N-depleted microsites in the growing mycelium of the culture, and the presence of significantly elevated transposase transcription during starvation indicates the continuing evolution of the Frankia sp. strain CcI3 genome, even in culture, especially under stressed conditions. These studies also sound a cautionary note when comparing the transcriptomes of Frankia grown in root nodules, where cell heterogeneity would be expected to be quite high. Detection of gene expression variance among Frankia HfpCci3 (Cci3) cells grown in ammonium chloride for three days, five days and HfpCci3 cells grown in nitrogen fixing conditions for three days using mRNA-seq