Final Report Summary - OXYLIPIN SIGNALLING (Oxylipin signalling in plant defence against pathogens)
Project context and objectives
Oxylipin synthesis is triggered by various biotic and abiotic stresses as well as also taking place during specific developmental stages (Howe and Schilmiller, 2002). It is known that oxylipins regulate gene expression, impact plant cell death and display direct antimicrobial activity (Almeras et al., 2003; Rusterucci et al., 1999 and Prost et al., 2005). The best-characterised oxylipin is jasmonic acid (JA) with its well-defined role in developmental and defence signalling (Turner et al., 2002; Truman et al., 2007). Ample evidence also implies that a fundamental role is played in plant immunity by the 9-LOX pathway-derived oxylipins. Oxylipins of the 9-LOX pathway, like Hydroxyoctadecatrienoic acid (9-HOT), have been shown to accumulate after pathogen inoculation (Jalloul et al., 2002), and possess direct antifungal and antioomycete activities in vitro (Prost et al., 2005).
In a recent study, Arabidopsis thaliana seedlings were grown in-vitro in the presence of distinct oxylipins to study their effect on the plant's physiology, development and defence system (Vellosillo et al., 2007). Among the different oxylipins (9-LOX- and 13-LOX-derived) tested, the 9-LOX derivate 9-HOT was shown to be one of the most potent inducers of a characteristic root waving phenotype as well as an inhibitor of lateral root formation. In the same study, it was further established that the molecular events triggered by the application of 9-HOT resemble the plant's response to pathogen infection including focal accumulation of callose, production of ROS, and transcriptional activation of plant genes that are in part also induced after inoculation with the bacterial plant pathogen Pseudomonas syringae. The fact that the application of 9-HOT induces a prominent root-waving phenotype was used to identify approximately 70 9-HOT insensitive mutants (non-responding to oxylipins, NOXY) within a mutagenised A. thaliana population (Vellosillo et al., 2007 and Carmen Castresana, personal communication).
The objective of the project was to identify and functionally characterise genes involved in oxylipin signalling with a focus on the still elusive 9-LOX pathway. The approximate 70 NOXY mutants that were identified in a forward genetic screen are likely to represent crucial signalling elements of the 9-LOX pathway. This report outlines the progress so far of the project, which has aimed to identify the mutated NOXY genes and to perform their functional characterisations. The project is structured into the seven objectives:
- Group the approximate 70 NOXY mutants in complementation groups and identify representative noxy mutations by map based cloning.
- Perform an in-depth phenotypic analysis of the NOXY mutants and the transgenic NOXY over-expressing Arabidopsis lines, with a focus on testing their resistance to biotrophic and necrotrophic pathogens, and testing their responsiveness to various oxylipins.
- Carry out genetic analysis by crossing NOXY mutants with defined defence mutants in order to place the NOXY genes within the signalling network that controls plant immunity.
- Conduct a gene expression analysis to elucidate the conditions of NOXY transcript activation and to identify potentially co-regulated signalling partners.
For an interesting subset of NOXY mutants:
- Determine the sub-cellular localisation of NOXY proteins (in vivo imaging of fluorescently labelled fusion proteins by confocal microscopy and western blot analysis).
-Examine the biochemical function of NOXY proteins (enzymatic assays and identification of binding partners).
- Analyse 9-LOX activity and the accumulation of 9-LOX derivatives in the roots and leaves of NOXY mutants.
Project results
Phenotypic analysis enabled 70 NOXY mutants to be grouped into 38 groups that shared common traits such as leaf shape, altered pathogen resistance and resistance to the herbicide isoxaben. Eight noxy mutants were tested for their basal resistance to the virulent strain of the oomycete Hyaloperonospora arabidopsis.
Oxylipin synthesis is triggered by various biotic and abiotic stresses as well as also taking place during specific developmental stages (Howe and Schilmiller, 2002). It is known that oxylipins regulate gene expression, impact plant cell death and display direct antimicrobial activity (Almeras et al., 2003; Rusterucci et al., 1999 and Prost et al., 2005). The best-characterised oxylipin is jasmonic acid (JA) with its well-defined role in developmental and defence signalling (Turner et al., 2002; Truman et al., 2007). Ample evidence also implies that a fundamental role is played in plant immunity by the 9-LOX pathway-derived oxylipins. Oxylipins of the 9-LOX pathway, like Hydroxyoctadecatrienoic acid (9-HOT), have been shown to accumulate after pathogen inoculation (Jalloul et al., 2002), and possess direct antifungal and antioomycete activities in vitro (Prost et al., 2005).
In a recent study, Arabidopsis thaliana seedlings were grown in-vitro in the presence of distinct oxylipins to study their effect on the plant's physiology, development and defence system (Vellosillo et al., 2007). Among the different oxylipins (9-LOX- and 13-LOX-derived) tested, the 9-LOX derivate 9-HOT was shown to be one of the most potent inducers of a characteristic root waving phenotype as well as an inhibitor of lateral root formation. In the same study, it was further established that the molecular events triggered by the application of 9-HOT resemble the plant's response to pathogen infection including focal accumulation of callose, production of ROS, and transcriptional activation of plant genes that are in part also induced after inoculation with the bacterial plant pathogen Pseudomonas syringae. The fact that the application of 9-HOT induces a prominent root-waving phenotype was used to identify approximately 70 9-HOT insensitive mutants (non-responding to oxylipins, NOXY) within a mutagenised A. thaliana population (Vellosillo et al., 2007 and Carmen Castresana, personal communication).
The objective of the project was to identify and functionally characterise genes involved in oxylipin signalling with a focus on the still elusive 9-LOX pathway. The approximate 70 NOXY mutants that were identified in a forward genetic screen are likely to represent crucial signalling elements of the 9-LOX pathway. This report outlines the progress so far of the project, which has aimed to identify the mutated NOXY genes and to perform their functional characterisations. The project is structured into the seven objectives:
- Group the approximate 70 NOXY mutants in complementation groups and identify representative noxy mutations by map based cloning.
- Perform an in-depth phenotypic analysis of the NOXY mutants and the transgenic NOXY over-expressing Arabidopsis lines, with a focus on testing their resistance to biotrophic and necrotrophic pathogens, and testing their responsiveness to various oxylipins.
- Carry out genetic analysis by crossing NOXY mutants with defined defence mutants in order to place the NOXY genes within the signalling network that controls plant immunity.
- Conduct a gene expression analysis to elucidate the conditions of NOXY transcript activation and to identify potentially co-regulated signalling partners.
For an interesting subset of NOXY mutants:
- Determine the sub-cellular localisation of NOXY proteins (in vivo imaging of fluorescently labelled fusion proteins by confocal microscopy and western blot analysis).
-Examine the biochemical function of NOXY proteins (enzymatic assays and identification of binding partners).
- Analyse 9-LOX activity and the accumulation of 9-LOX derivatives in the roots and leaves of NOXY mutants.
Project results
Phenotypic analysis enabled 70 NOXY mutants to be grouped into 38 groups that shared common traits such as leaf shape, altered pathogen resistance and resistance to the herbicide isoxaben. Eight noxy mutants were tested for their basal resistance to the virulent strain of the oomycete Hyaloperonospora arabidopsis.