Functional Analysis of Medicago truncatula Expressed Sequence Tags Associated
with 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. of
Agronomy & Plant Genetics, 4Dept. of Plant Biology, University of Minnesota,
St. Paul, MN 55108
The annual medic, Medicago truncatula (barrel medic), is the focus of
functional and comparative genomic studies in a number of laboratories world
wide. Attributes including a small, relatively simple diploid genome (5 x
108 bp), short generation time, self fertility, abundant seed production, and
natural variation in well characterized ecotypes make this an attractive
model legume species. Tools for molecular analyses are available such as BAC
libraries, a well defined genetic map, mutant populations, and transformation
by Agrobacterium either through tissue culture or through vacuum infiltration
of seedlings. Functional genomic studies are currently focusing on plant
microbe interactions, particularly nodulation by Sinorhizobium meliloti,
mycorrhizal interactions, and host-pathogen interactions. In our
laboratories, five cDNA libraries were constructed from mRNA isolated from
nitrogen-fixing root nodules, senescent nodules, healthy leaves and
cotyledons, leaves infected with Colletotrichum trifolii, and roots infected
with Phytophthora medicaginis. Over 2,400 clones have been sequenced from
the 5' end from each library. The EST sequence data is available from
GenBank (http://www.ncbi.nlm.nih.gov) and the M. truncatula database
(http://chrysie.tamu.edu/medicago.mtdb). Sequences are being analyzed for
redundancy, homology with known sequences, and placed into functional
categories. 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 from
M. truncatula to compare expression in healthy roots to infected roots,
mature nodules to senescent nodules, healthy leaves to infected leaves, and
roots to nodules. The greatest differences were between roots and nodules in
which 26 ESTs were down-regulated in nodules and 69 ESTs were up-regulated in
nodules, including 44 ESTs of unknown function. In infected roots and
leaves, 11 ESTs were up-regulated in both interactions and 5 ESTs were down
regulated indicating that these ESTs are involved in general host defense
mechanisms. Seven ESTs were regulated similarly in infected tissues and
nodules, adding support to the observation that symbiosis and pathogenesis
share some common molecular processes. To examine if M. truncatula ESTs
arrays can be used to study gene expression in other legumes, macroarrays
were hybridized with probes isolated from nodules of alfalfa, pea, and lupin
and the expression pattern compared with probes from M. truncatula nodules.
The same set of ESTs were expressed in alfalfa nodules as in M. truncatula
nodules, however, hybridization between M. truncatula and pea M. truncatula
and lupin was nonspecific. These results indicate that EST arrays will be a
powerful tool for investigating the gene expression patterns of wild type and
mutant plants, and for identifying changes in gene expression patterns with
plant-microbe interactions and environmental stresses. The similarities
between genes of M. truncatula and alfalfa and common symbiotic and
pathogenic interactions can be employed to facilitate alfalfa improvement.
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