Progress 08/15/02 to 08/14/06
Outputs
This project was extended from 3 to 4 years at no additional cost and declined renewal because it uses a model system to investigate legume biology. The USDA no longer funds model system research through the NRI Plant-Microbe Interactions panel. We completed the project's two objectives (1) Positional cloning of the M. truncatula sunn supernodulation mutation and verification of cloning by rescuing the mutation: We were able to walk to a region containing the mutation clone the gene. In the original sunn mutant (sunn-1), a single base change was detected in a leucine-rich repeat receptor-like kinase (LRR-RLK) gene with a high degree of similarity to the Arabidopsis CLV1 gene and to LRR-RLK genes recently discovered to be involved in nodulation regulation in Lotus japonicus, pea and soybean. Three additional EMS alleles of sunn from two independent genetic screens were identified, allowing us to bypass the rescue of the phenotype. SUNN is expressed in shoots, flowers and
roots. Comparable levels of SUNN mRNA were detected in shoots and roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated CLV phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. We also identified a potential interacting gene, RLP1, which is downstream of SUNN in the genome, is 92% identical to SUNN at the nucleotide level, but lacks the kinase domain. RLP1 is expressed in the same tissue pattern as SUNN. All of this is summarized in our publication from last year. Although we are no longer funded through the USDA, we are
continuing pursuit of this research because of its high agricultural impact, having isolated a mutation in RLP1 for characterization and other supernodulation mutants. This work is currently under review at the NSF. Objective 2. Characterize the effect of the sunn mutation on gene expression at a large scale by comparison to wild type via microarray analysis: Statistical analysis of this very large data set resulted in a focus on the 24 hour post inoculation time point. The data were most reproducible at the time point, and it represents an early point in development, after the initiation of nodulation but before visible tissue differentiation . In the absence of rhizobia and 24 hours post inoculation with rhizobia, microarray analysis yielded 236 genes significantly up or down regulated in the presence of rhizobia and interestingly, 144 genes significantly up or down regulated in the absence of rhizobia. Combined with the short root phenotype, this suggests the sunn-1 mutation
affects other developmental pathways besides nodulation. Most of the differentially regulated genes are involved in signal transduction.
Impacts
Cloning of the SUNN gene is already aiding the understanding of the nodulation process. An ortholog of the gene has been extensively studied in a non-nodulating plant, Arabidopsis thaliana, and we have extended our studies to that plant as well. Combined with the microarray data which shows 144 differentially expressed genes in non-nodulating plants, this supports the theory that nodulation may not use unique genes, but rather organize genes common to all plants into unique pathways. If this is true, engineering of non-nodulating plants so that they can fix nitrogen from the air may be technically feasible.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
This project was extended from 3 to 4 years at no additional cost. We have essentially completed two objectives of the project. Objective 1. Positional cloning of the M. truncatula sunn supernodulation mutation and verification of cloning by rescuing the mutation: We were able to walk to a region containing the mutation and examine that region for candidate genes. In the original sunn mutant (sunn-1), a single base change was detected in a leucine-rich repeat receptor-like kinase (LRR-RLK) gene with a high degree of similarity to the Arabidopsis CLV1 gene and to LRR-RLK genes recently discovered to be involved in nodulation regulation in Lotus japonicus, pea and soybean. Three additional EMS alleles of sunn from two independent genetic screens were identified, allowing us to bypass the rescue of the phenotype. Although the molecular defects among alleles varied, root length was more sensitive to subtle environmental influences than to the mutated allele itself. SUNN
is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, reduced levels of SUNN mRNA were detected in roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated CLV phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. We also identified a potential interacting gene, RLP1, which is downstream of SUNN in the genome, is 92% identical to SUNN at the nucleotide level, but lacks the kinase domain. RLP1 is
expressed in the same tissue pattern as SUNN. All of this is reported in our publication. Objective 2. Characterize the effect of the sunn mutation on gene expression at a large scale by comparison to wild type via microarray analysis: Statistical analysis of this very large data set resulted in a focus on the 24 hour post inoculation time point. These data were most reproducible, and the time point represents an early point in development, after the initiation of nodulation but before visible tissue differentitation. In the absence of rhizobia and 24 hours post inoculation with rhizobia, microarray analysis yielded 236 genes significantly up or down regulated in the presence of rhizobia and interestingly, 144 genes significantly up or down regulated in the absence of rhizobia. Combined with the short root phenotype, this suggests the sunn-1 mutation affects other developmental pathways besides nodulation. Most of the differentially regulated genes are involved in signal transduction.
We are verifying expression patterns with Real Time PCR and this work is being prepared for publication.
Impacts
Cloning of the SUNN gene is already aiding the understanding of the nodulation process. An ortholog of the gene has been extensively studied in a non-nodulating plant, Arabidopsis thaliana, and we have extended our studies to that plant as well. Combined with the microarray data which shows 144 differentially expressed genes in non-nodulating plants, this supports the theory that nodulation may not use unique genes, but rather organize genes common to all plants into unique pathways. If this is true, engineering of non-nodulating plants so that they can fix nitrogen from the air may be technically feasible.
Publications
- Schnabel, E., Journet, E.P., de Carvalho-Niebel, F. Duc, G., and Frugoli, J. (2005) The Medicago truncatula SUNN gene encoding a CLV1-like leucine-rich repeat receptor kinase regulates both nodule number and root length, Plant Molecular Biology 58:809-822.
- Frugoli, J. (2005) Medicago truncatula as a model plant in Plant Molecular Biology, in Handbook of New Technologies for Genetic Improvement of Legumes, P.B. Kirti, ed., to be published by the Haworth Press, New York December 2005.
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Progress 01/01/04 to 12/31/04
Outputs
This project is 2.5 years into its 3 year duration. We have completed objective 1 and made significant progress on objective two. Objective 1. Positional cloning of the M. truncatula sunn supernodulation mutation and verification of cloning by rescuing the mutation: We were able to walk to a region containing the mutation and examine that region for candidate genes. In the original sunn mutant (sunn-1), a single base change was detected in a leucine-rich repeat receptor-like kinase (LRR-RLK) gene with a high degree of similarity to the Arabidopsis CLV1 gene and to LRR-RLK genes recently discovered to be involved in nodulation regulation in Lotus japonicus, pea and soybean. Three additional EMS alleles of sunn from two independent genetic screens were identified, allowing us to bypass the rescue of the phenotype. Although the molecular defects among alleles varied, root length was more sensitive to subtle environmental influences than to the mutated allele itself. SUNN
is expressed in shoots, flowers and roots. Although previously reported grafting experiments showed that the presence of the mutated SUNN gene in roots does not confer an obvious phenotype, comparable levels of SUNN mRNA were detected in shoots and roots. SUNN and the previously identified genes HAR1 (Lotus japonicus) and NARK (Glycine max) are orthologs based on gene sequence and synteny between flanking sequences. Comparison of related LRR receptor kinases determined that all nodulation autoregulation genes identified to date are the closest legume relatives of AtCLV1 by sequence, yet sunn, har and nark mutants do not display the fasciated CLV phenotype. The M. truncatula region is syntenic with duplicated regions of Arabidopsis chromosomes 2 and 4, none of which harbor CLV1 or any other LRR receptor kinase genes. Objective 2. Characterize the effect of the sunn mutation on gene expression at a large scale by comparison to wild type via microarray analysis: All of the microarray
hybridizations have been completed, allowing the expression level of thousands of genes to be compared over five time points in the nodulation process (0, 24, 48, 72 and 120 hours post inoculation) in both wild type and sunn mutants. Statistical analysis of this very large data set in ongoing. As might be expected given the higher nodule number in sunn mutants, genes involved in protein biosynthesis, transcription elongation, nodulation and transport are elevated in inoculated sunn plants versus wild type. The functional categories of genes which are downregulated include genes involved in flavonoid synthesis, defense response, stress response, and cell wall modifications. This suggests that the lesion affects the regulation of multiple genetic pathways despite the mild morphological phenotype. We have also identified significant differences in gene expression between sunn roots and wild type roots even in the absence of bacteria, providing further evidence that SUNN gene has effects
on plant development outside of its effects on nodulation.
Impacts
Cloning of the SUNN gene is already aiding the understanding of the nodulation process. An ortholog of the gene has been extensively studied in a non-nodulating plant, Arabidopsis thaliana. This adds evidence to support the theory that nodulation may not use unique genes, but rather organize common genes into unique pathways. If this is true, engineering of non-nodulating plants so that they can fix nitrogen from the air may be technically feasible.
Publications
- Schnabel, E., Journet, E.P., Carvalho-Niebel, F. Duc, G., and Frugoli, J. (2005) The Medicago truncatula SUNN gene encoding a CLV1-like leucine-rich repeat receptor kinase regulates both nodule number and root length, submitted to Plant Molecular Biology
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Progress 01/01/03 to 12/31/03
Outputs
This project is a year and a half old, with a three year duration. We have made significant progress on the two project objectives. Objective 1. Positional cloning of the M. truncatula sunn supernodulation mutation and verification of cloning by rescuing the mutation: We have confirmed that the isolated candidate gene with a mutation in the sunn background is indeed the gene responsible for the phenotype through the identification of two other alleles arising from separate mutagenesis experiments, eliminating the need to rescue the mutation. We have confirmed that the gene is expressed in both shoots and roots of plants, including in the sunn mutant. We are presently preparing the manuscript on the final cloning. Objective 2. Characterize the effect of the sunn mutation on gene expression at a large scale by comparison to wild type via microarray analysis: All of the microarray hybridizations have been completed, allowing the expression level of at least 1800 genes to
be compared over 5 time points in the nodulation process in both wild type and sunn mutants. Statistical analysis of this very large data set in ongoing. Preliminary results indicate differences in gene expression between sunn roots and wild type roots even in the absence of bacteria. This suggests that the SUNN gene has effects on plant development outside of its effects on nodulation.
Impacts
Cloning of the SUNN gene is already aiding the understanding of the nodulation process. An ortholog of the gene has been extensively studied in a non-nodulating plant, Arabidopsis thaliana. This adds evidence to support the theory that nodulation may not use unique genes, but rather organize common genes into unique pathways. If this is true, engineering of non-nodulating plants so that they can fix nitrogen from the air may be technically feasible.
Publications
- Schnabel, E., Kulikova, O., Penmetsa, V., Bisseling, T., Cook, D., Frugoli, J., (2003) An integrated physical, genetic and cytogenetic map around the sunn locus of M. truncatula, Genome, 46(4):665-72.
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Progress 08/15/02 to 12/31/02
Outputs
Although the project is only 5 months old, we have made significant progress on the first year's goals. We have narrowed the area containing the SUNN gene down to a window of approximately 400 kb, of which over 250 kb have been sequenced as part of the public genome effort. We have isolated a candidate gene with a mutation in the SUNN background and are in the process of making transgenic plants to look for rescue of the SUNN phenotype. We have harvested tissue from nodulating and uninoculated plants of both wild type and SUNN at 5 time points, in 3 replicates. We then used this RNA to generate cDNA for use as probes in microarray experiments. Using a 6000 clone array, we have hybridized most of the time points at least twice and several time points are close to the 6 replicates of each spot with dye swap required for proper statistical analysis.
Impacts
Cloning of the SUNN gene is already aiding the understanding of the nodulation process. The candidate gene has been extensively studied in a non-nodulating plant, suggesting that nodulation may not use unique genes, but rather organize common genes into unique pathways. If this is true, engineering of non-nodulating plants so that they can fix nitrogen from the air may be technically feasible.
Publications
- Schnabel, E., Kulikova, O., Bisseling, T., Cook, D., Frugoli, J., (2003) An integrated physical, genetic and cytogenetic map around the sunn locus of M. truncatula, Genome, in press.
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