FIRB Future in Research 2010: Cyclic nucleotides in response to biotic stress in plant
Project objectives
The overall objective of the project is the molecular characterization of cNMP dependent signaling pathways that are activated by pathogen attacks in plant. Today numerous physiological processes involving cGMP and cAMP in plants are known. However, the mechanisms with which the cGMP or cAMP-dependent signal transduction takes place are still poorly understood. This project investigates the role of these two cyclic nucleotides in the signaling pathway induced by infection with avirulent pathogen Pseudomonas syringae in Arabidopsis Thaliana.
As the enzymes responsible for the synthesis and degradation of cNMP in plants are still not identified, the role of these secondary messengers has been designed exclusively with a pharmacological approach. The starting point of this project is the production of genetically modified plants that exhibit altered levels of cyclic nucleotides. In particular, plants over-expressing the guanylate cyclase or mammal phosphodiesterase are produced to alternate the levels of cGMP as are plants that express a chimeric protein, called "cAMP-sponge" (CSA) that causes a reduced level of this cyclical nucleotide.
Plants with altered levels of cNMPs are infected with the avirulent pathogen to identify the dependent cNMPs signaling pathways involved in the defense response to the pathogen. As part of the signaling the changes in Ca2, H2O2 and cNMP and alterations in cellular redox state are analyzed. For this purpose plants that express genetically encoded fluorescent probes specific for Ca2 (Cameleon) or for 'H2O2 (hyper) are used.
For the analysis of the dynamics of Ca2 and H2O2 in response to the pathogen attack and in the presence of different levels of cNMP, plants expressing the Cameleon probe or the Hyper probe are crossed with plants engineered with different levels of cNMP.
This series of experiments makes it possible to understand how cAMP and cGMP can lead to a change in the dynamics of other secondary messengers so allowing us to understand the interrelationships between these important signaling molecules.
Studies are also underway to identify possible changes in energy metabolism, proteome, phosphoproteome and transcriptome. The results of this project will allow us to identify genes and the gene products involved in the signaling pathways of the defense response. Understanding the dynamics of secondary messengers such as Ca2+ and H2O2 in the cytoplasm will allow us to evaluate how the action of various secondary messengers orchestrate its behavior. To understand which biological actions are specifically attributable to cyclic nucleotides (cAMP and cGMP) and the mechanisms of signal transduction regulated by these, the challenge is in the study of plant signal transduction.
To understand which biological actions are specifically attributable to cyclic nucleotides (cAMP and cGMP) and the mechanisms of signal transduction are regulated by them, the challenge is in the study of signal transduction of plants in response to biotic and abiotic stresses.
Today, there are several known physiological processes involving cGMP and cAMP but the mechanisms by which the signal transduction cGMP- or cAMP-dependent is implemented are still poorly understood. Therefore, the ultimate aim of the project is to have an overall view of the role played by cNMP in the regulation of other secondary messengers and downstream molecular targets by integrating the data obtained with a detailed and temporal analysis of the molecular events that occur in response to attack by pathogens.
Start/end Date
March 8, 2012 - March 8, 2015 (with a possible 6 months extension)
Principal Investigator
Vittoria Locato
Host Institution
Università degli Studi di Milano - Italy
Other Institutions Involved
- Università degli Studi di Verona
- CNR di Bari
Source of funding
Ministero dell’Istruzione, dell’Università e della Ricerca