Functional Analysis of Medicago truncatula Expressed Sequence Tags Associatedwith Plant-Microbe Interactions.
Deborah A. Samac1,2, Maria Fedorova3, J. Steven Gantt4,
and Carroll P. Vance1,3
1USDA-ARS-Plant Science Research Unit, 2Dept. of Plant Pathology, 3Dept. ofAgronomy & Plant Genetics, 4Dept. of Plant Biology, University of Minnesota,St. Paul, MN 55108
The annual medic, Medicago truncatula (barrel medic), is the focus offunctional and comparative genomic studies in a number of laboratories worldwide. Attributes including a small, relatively simple diploid genome (5 x108 bp), short generation time, self fertility, abundant seed production, andnatural variation in well characterized ecotypes make this an attractivemodel legume species. Tools for molecular analyses are available such as BAClibraries, a well defined genetic map, mutant populations, and transformationby Agrobacterium either through tissue culture or through vacuum infiltrationof seedlings. Functional genomic studies are currently focusing on plantmicrobe interactions, particularly nodulation by Sinorhizobium meliloti,mycorrhizal interactions, and host-pathogen interactions. In ourlaboratories, five cDNA libraries were constructed from mRNA isolated fromnitrogen-fixing root nodules, senescent nodules, healthy leaves andcotyledons, leaves infected with Colletotrichum trifolii, and roots infectedwith Phytophthora medicaginis. Over 2,400 clones have been sequenced fromthe 5' end from each library. The EST sequence data is available fromGenBank (http://www.ncbi.nlm.nih.gov) and the M. truncatula database(http://chrysie.tamu.edu/medicago.mtdb). Sequences are being analyzed forredundancy, homology with known sequences, and placed into functionalcategories. To identify ESTs associated with plant-microbe interactions,several hundred ESTs were chosen to analyze on nylon filter macroarrays. Plasmid DNA of each EST was purified and 0.5 µl spotted onto nylon membranes. Probes (32P-labeled first strand cDNA) were prepared from mRNA extracted fromM. truncatula to compare expression in healthy roots to infected roots,mature nodules to senescent nodules, healthy leaves to infected leaves, androots to nodules. The greatest differences were between roots and nodules inwhich 26 ESTs were down-regulated in nodules and 69 ESTs were up-regulated innodules, including 44 ESTs of unknown function. In infected roots andleaves, 11 ESTs were up-regulated in both interactions and 5 ESTs were downregulated indicating that these ESTs are involved in general host defensemechanisms. Seven ESTs were regulated similarly in infected tissues andnodules, adding support to the observation that symbiosis and pathogenesisshare some common molecular processes. To examine if M. truncatula ESTsarrays can be used to study gene expression in other legumes, macroarrayswere hybridized with probes isolated from nodules of alfalfa, pea, and lupinand the expression pattern compared with probes from M. truncatula nodules. The same set of ESTs were expressed in alfalfa nodules as in M. truncatulanodules, however, hybridization between M. truncatula and pea M. truncatulaand lupin was nonspecific. These results indicate that EST arrays will be apowerful tool for investigating the gene expression patterns of wild type andmutant plants, and for identifying changes in gene expression patterns withplant-microbe interactions and environmental stresses. The similaritiesbetween genes of M. truncatula and alfalfa and common symbiotic andpathogenic interactions can be employed to facilitate alfalfa improvement.
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