Regulation of senescence-specific proteases during the nitrogen-fixing symbiosis in
Medicago truncatula
Works under the direction of Pr. Pierre FRENDO and Dr. Eric BONCOMPAGNI
The interaction between legumes and soil bacteria rhizobia induces the formation of a new
organ, the root nodule. Inside the nodule, the bacteria are able to reduce the atmospheric
nitrogen in ammonia which is transported and used by the plant partner. In exchange, the plant
provides energy to the bacteria. In Medicago truncatula nodules, this symbiotic interaction is
characterized by the apparition of a senescence zone in which there is a rupture of the
interaction. Cysteine proteases are typical markers of plant senescence, either in leaves or in
nodules. Two genes are induced at the nodule N2 fixation / senescence transition, encoding
papain-like and legumain proteases: the Cysteine protease 6 (CP6) and the Vacuolar Processing
Enzyme (VPE), respectively. The goal of the project is to analyze the regulation of two cysteine
proteases, CP6 and VPE, which are specifically induced during the nodule senescence process
(Pierre et al., 2014).
The phD project that will be develop is focus on:
- The analysis of the activity of recombinant CP6 and VPE will be performed; substrate
specificity and post-translational regulation by the redox state will be analyzed on the
recombinant proteases produced in yeast Pichia pastoris. In addition to transcriptional
regulations, plant CPs are known to be highly regulated by modulation of their activity through
post-translational modifications (PTMs) such as NO-mediated S-nitrosylation. The
importance of each Cys residue in both proteases will be evaluated by targeted mutagenesis.
Then, transgenic roots of M. truncatula overexpressing WT or selected mutated proteins will
be produced in order to confirm and specify the regulatory role of NO in the regulation of
nodule senescence.
- The transcriptional regulation of the two protease genes will be investigated. Analysis of the
cis-regulatory element present on the two gene promoters will be performed by constructing
series of promoter fragments which will allow to determine the DNA sequence responsible for
the nodule senescence specificity of expression under developmental and stress-induced
senescence.
- Furthermore, in silico and protein-DNA interaction analyses (tested by ChIP-qPCR) will be
performed to characterize the putative transcription factors involved in the transcriptional
regulation of the two cysteine proteases. This project should lead to a better understanding of
the regulation of two cysteine proteases, and to the characterization of transcription factors
involved in the regulation of developmental-induced senescence in the nitrogen-fixing
symbiosis.
References :
Pierre, O., Hopkins, J., Combier, M., Baldacci, F., Engler, G., Brouquisse, R., Hérouart, D., and
Boncompagni, E. (2014). Involvement of papain and legumain proteinase in the senescence
process of Medicago
10.1111/nph.12717.
truncatula nodules. New
Phytologist.
202(3),
849-63
DOI: