Secreted proteins in Medicago species : identification, gene expression and functional analysis
Creators: name: Kusumawati, Lucia
Publisher: name: The Australian National University
Year: 2010
Subjects: subject: QK495.L52 K87 2010
subject: Medicago
subject: Medicago Genetics
subject: Plant proteins
subject: Nitrogen Fixation
subject: Root-tubercles

description: Previous studies have shown that plant secreted proteins are involved in many physiological processes, including plant-microbe interactions and plant development. A process involving both plant-microbial interactions and alteration of plant development is the formation of nitrogen fixing nodules by rhizobia. So far very little is known about the role of secreted proteins in nodule formation. The aim of this thesis was to identify and characterize secreted proteins from Medicago species and to test their function during nodule development. In the first part of this thesis, suspension cultures of Medicago species were used to isolate and identify secreted proteins because of the ease of collecting secreted proteins in culture supernatant. There were 39 proteins identified in the cultures derived from M. sativa, M. truncatula 2HA (a highly embryogenic line) and M. truncatula sickle (a super nodulating ethylene-insensitive mutant). N-terminal secretion signals were detected in 34 proteins and five other proteins were predicted to be secreted via a non-classical (ER-independent) route. All samples possessed defence related proteins including PR proteins. The glycoprotein, SIEPIL, was found only in M. sativa. Three secreted proteinases were identified in M. truncatula including a serine carboxypeptidase found only in 2HA. Some proteins were unique to a cell culture line. Quantitative real time RT-PCR was used to determine mRNA expression of selected genes corresponding to proteins found only in 2HA or sickle or in both. The results correlated well with the proteomic data. For instance, a GDSL-lipase gene known to be ethylene regulated was found only in 2HA. The PR1a protein, encoded by a well recognised ethylene-regulated gene, was found in 2HA but not sickle. These experiments indicate that the suspension culture systems established here are useful to avoid contamination from cytoplasmic proteins and to identify new secreted proteins in Medicago, and should have application in other plant systems. Seven of the identified secreted proteins were selected to be examined further during nodule development in M. truncatula. These were subtilisin-like serine protease (MtSBT), serine carboxypeptidase (MtSCP), MtN5, non-specific lipid transfer protein (MtnsLTP), early nodulin2-like (MtENOD2-like), FAD-binding domain containing protein and rhicadhesin receptor. Promoter:GUS fusions of six of the seven genes encoding secreted proteins were strongly expressed in the vascular bundle of transgenic hairy roots. All six genes encoding secreted proteins were expressed in 14-day old nodules, although their expression varied in intensity and cell type. The expression levels of the selected seven genes were quantified in intact Sinorhizobium-inoculated and control plants using quantitative real time RT-PCR. The expression level of only one gene, MtN5, was up-regulated significantly in inoculated root segments compared to controls. Transgenic hairy roots over-expressing MtN5 formed significantly less nodules compared to control hairy roots. It is proposed that MtN5 has a role in the Rhizobium-legume symbiosis.