Expression, Regulation and Evolutionary patterns of a large family of defensin- Like genes found in the nodules of Medicago truncatula.

University of Minnesota Ph.D. dissertation. May 2011. Major: Plant Biological Sciences. Advisors: Kathryn A. VandenBosch, Kevin A.T. Silverstein. 1 computer file (PDF); ix, 160 pages.A very large number of Defensin-like (DEFL) genes were predicted in analyses of the genome sequences from the model plants Medicago truncatula and Arabidopsis thaliana. DEFLs share common characteristics with antimicrobial defensins, including cysteine-rich sequence motifs, gene structure and genome organization. However, because many of the DEFLs were only recently discovered, comparatively little information is known about their expression, regulation and evolutionary patterns. I explored the expression patterns of DEFLs among various tissues, symbiotic and pathogen treatments in legume M. truncatula and found that the majority of DEFLs are highly expressed in nitrogen-fixing root nodules. A further indepth investigation with various nodule developmental stages and rhizobial mutants indicated that the expression of nodule DEFLs is dependent on the number and morphology of rhizobia in the nodule. I found conserved motifs in the upstream regions that occur uniquely in nodule DEFLs. Promoter deletion assays demonstrated that these cis motifs are required for nodule DEFL expression. Few small regulatory elements known to be involved in the spatial and temporal regulation of various genes were contained within the unique conserved nodule DEFL motifs. Reverse genetic approaches suggested redundancy of function in this large family of genes. Lastly, I examined the evolutionary patterns of nodule DEFLs with four ecotypes of M. truncatula. The presence of expression, sequence variation and signatures of diversifying selection in nodule DEFLs within the Medicago species indicates rapid and recent evolution and suggests that this family of genes is constantly evolving to adapt to different environments and acquiring new functions