Identification And Characterization Of Nodule-Specific Genes Of Medicago truncatula

1. Identification And Characterization Of Nodule-Specific Genes Of Medicago truncatula Jing Ye , Fares Z. Najar , Hongshing Lai, Steve Kenton, and Bruce A. Roe   Department of Chemistry and Biochemistry, Stephenson Research and Technology Center, University of Oklahoma, 101 David L. Boren Blvd, Norman, Oklahoma, 73019

2. Abstract • Legumes, through a symbiotic relationship with rhizobial bacteria, create a nitrogen- fixing novel organ known as the root nodule. During the formation of nodules, genes in both bacteria and in plant are involved. Although there has been significant progress in identifying and characterizing the bacterial genes involved in this process, the plant-specific nodule genes have been more elusive. As our sequencing of the Medicago truncatula genome progresses, we now have determined approximately 350 out of the 1183 sequenced BACs contain genes homologous to known nodule-specific genes. The analysis of these genomic regions reveals that five genes on chromosome 2 may belong to the NIP subfamily of the MIP/aquaporin super family as they are similar to nodulin-26 gene in Glycine max (soybean) and have the conserved -NPA- (-Asn-Pro-Ala-) motif. Six genes similar to the early nodulin 55-2 precursor in soybean are clustered within 100Kbp on chromosome 7 and seven genes homologous to lipoxygenase gene in Sesbania rostrata are similarly clustered on chromosome 8. Thus, although nodule-specific genes are dispersed on different chromosomes, multiple copies of these genes often occur in clusters that likely result from recent duplications.

3. Bacteriods begin to fix nitrogen Flavonoids released by the legume attract rhizobia to the root hairs Introduction • Medicago truncatula has been chosen to be sequenced as a model legume because of its small genome size, comparatively simple genome structure, short seed-to-seed generation time, high-efficiency transformation and nitrogen fixation by the formation of the nodule in symbiosis with rhizobial bacteria. The nodulation process is as follows: Nod factor released by rhizobia From http://www.microbiologyonline.org.uk/forms/rhizobium.pdf

4. To understand legume-rhizobium symbiosis, genes directly or indirectly involved in this process in legumes and in rhizobium need to be identified and characterized. The entire genomes of two rhizobia, i.e. Sinorhizobium meliloti and Mezorhizobium loti have been sequenced, which has helped to understand the roles and regulation of many genes required for effective symbiosis in rhizobium. • The advances in legume genomic sequencing also will help us understand legume-controlled aspects of the nitrogen fixing symbiosis. • By analyzing the genomic data of Medicago truncatula, we can identify nodule-specific genes ,their location on different chromosomes and some properties of their genomic organization.

5. Preliminary result of data analysis • All available nodule-specific protein sequences were retrieved from Genbank and a database for comparing all Medicago truncatula ORFspredicted by Genscan was constructed. • We found that approximately 350 BACs out of the 1183 sequenced BACs contain known nodule-specific genes. • These genes are encoded on every chromosomes • Some nodule-specific genes occur in clusters while others do not.

6. How are these genes located on different chromosomes NOD26 100cM ENOD70 *: Some nodule-specific genes are on chromosome 6 but their positions are not confimed ∆: gene clusters MtN21 NOD20 NOD21 NOD16 NOD552 Serine-threonine protein kinase * Nodulation receptor kinase ∆ Thioredoxin Calcium binding protein Ca2+ and calmodulin-dependent protein bZIP with a Ring-finger motif Lipoxygenase Cyclin Nodule autoregulation receptor-like protein ∆ 0cM Receptor-like protein kinase ∆ S-receptor kinase-like protein 1 2 3 4 5 6 7 8 LGB2

7. Summary of nodule-specific genes on different Medicago truncatula chromosomes

8. Genomic organization on BAC mth2-17p16 From http://dna8.genome.ou.edu/cgi-bin/gbrowser (gene2) (gene5) (gene4) (gene3) • Five genes are clustered within 23 Kb on chromosome 2 BAC mth2-17p16 (AC140547) that are similar to the soybean nodulin-26 gene • Nodulin-26 belongs to the NIP subfamily of the MIP/aquaporin super family that contains a highly conserved –NPA (-Asn-Pro-Ala-) motif • Nodulin 26 transports water, glycerol, formamide and possibly ammonia (gene1)

9. ClustalW result of mth2-17p16 • From the phylogenetic tree, we can see that gene140547.10 is more similar to NOD26. The other four genes might have resulted from gene140547.10 The common -NPA- motif is found in 5 genes with NOD26 homology

10. BAC mth2-123f14 contains 7 genes homologous to lipoxygenase gene in Sesbania rostrata From the phylogenetic tree, we can see that gene AC149580.11 is more similar to lipoxygenase in Sesbainia rostrata From http://dna8.genome.ou.edu/cgi-bin/gbrowser

11. Possible duplication events resulting in the present day nodule-specific gene clusters Ancestor 1 duplication Ancestor 2 Multiple duplications Ancestor N Different duplication events Nodule-specific gene clusters

12. Summary • By comparing the genomic data of Medicago truncatula with known nodule-specific genes, we identified many nodule-specific genes dispersed on different chromosomes. • Three nodule-specific gene clusters are homologous to NOD26, NOD552 and lipoxygenase, respectively. • These nodule-specific genes likely arose from several recent gene duplications.