Limits...
Interaction of Medicago truncatula lysin motif receptor-like kinases, NFP and LYK3, produced in Nicotiana benthamiana induces defence-like responses.

Pietraszewska-Bogiel A, Lefebvre B, Koini MA, Klaus-Heisen D, Takken FL, Geurts R, Cullimore JV, Gadella TW - PLoS ONE (2013)

Bottom Line: Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves.Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response.The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.

Show MeSH
Production of AtCERK1 in Nicotiana leaves induces cell death that requires AtCERK1 kinase activity.AtCERK1-sYFP2 (A) and AtCERK1[K349E]-sYFP2 (B) constructs were expressed in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672211&req=5

pone-0065055-g002: Production of AtCERK1 in Nicotiana leaves induces cell death that requires AtCERK1 kinase activity.AtCERK1-sYFP2 (A) and AtCERK1[K349E]-sYFP2 (B) constructs were expressed in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.

Mentions: Notably, heterologous production of AtCERK1-sYFP2 fusion resulted in tissue collapse and desiccation of (nearly) the entire infiltrated region in 20 out of 22 infiltrations 36 hai (Fig. 2A). This CD induction abolished our attempts of precisely characterizing the subcellular localization of AtCERK1 fusion in Nicotiana leaf epidermal cells, although we could detect sYFP2 fluorescence at the cell boundary (unpublished data). On the contrary, we observed clear co-localization of AtCERK1[K349E]-sYFP2 fusion with the PM marker using confocal laser-scanning microscopy analysis (Fig. S1). The PM localization of AtCERK1[K349E] fusion in Nicotiana leaf epidermal cells is in agreement with the reported subcellular localization of AtCERK1 fluorescent fusion in onion (Allium cepa) epidermal cells [31]. Importantly, production of the kinase-inactive variant of AtCERK1 did not result in CD induction, as confirmed with Evans blue staining (Fig. 2B), indicating that biological activity of AtCERK1 in Nicotiana leaves was dependent on its kinase activity.


Interaction of Medicago truncatula lysin motif receptor-like kinases, NFP and LYK3, produced in Nicotiana benthamiana induces defence-like responses.

Pietraszewska-Bogiel A, Lefebvre B, Koini MA, Klaus-Heisen D, Takken FL, Geurts R, Cullimore JV, Gadella TW - PLoS ONE (2013)

Production of AtCERK1 in Nicotiana leaves induces cell death that requires AtCERK1 kinase activity.AtCERK1-sYFP2 (A) and AtCERK1[K349E]-sYFP2 (B) constructs were expressed in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672211&req=5

pone-0065055-g002: Production of AtCERK1 in Nicotiana leaves induces cell death that requires AtCERK1 kinase activity.AtCERK1-sYFP2 (A) and AtCERK1[K349E]-sYFP2 (B) constructs were expressed in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 36 hai. Macroscopic symptoms of cell death were scored 36 hai: only infiltrations that resulted in confluent death of (nearly) the entire infiltrated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
Mentions: Notably, heterologous production of AtCERK1-sYFP2 fusion resulted in tissue collapse and desiccation of (nearly) the entire infiltrated region in 20 out of 22 infiltrations 36 hai (Fig. 2A). This CD induction abolished our attempts of precisely characterizing the subcellular localization of AtCERK1 fusion in Nicotiana leaf epidermal cells, although we could detect sYFP2 fluorescence at the cell boundary (unpublished data). On the contrary, we observed clear co-localization of AtCERK1[K349E]-sYFP2 fusion with the PM marker using confocal laser-scanning microscopy analysis (Fig. S1). The PM localization of AtCERK1[K349E] fusion in Nicotiana leaf epidermal cells is in agreement with the reported subcellular localization of AtCERK1 fluorescent fusion in onion (Allium cepa) epidermal cells [31]. Importantly, production of the kinase-inactive variant of AtCERK1 did not result in CD induction, as confirmed with Evans blue staining (Fig. 2B), indicating that biological activity of AtCERK1 in Nicotiana leaves was dependent on its kinase activity.

Bottom Line: Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves.Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response.The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases.

View Article: PubMed Central - PubMed

Affiliation: Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.

ABSTRACT
Receptor(-like) kinases with Lysin Motif (LysM) domains in their extracellular region play crucial roles during plant interactions with microorganisms; e.g. Arabidopsis thaliana CERK1 activates innate immunity upon perception of fungal chitin/chitooligosaccharides, whereas Medicago truncatula NFP and LYK3 mediate signalling upon perception of bacterial lipo-chitooligosaccharides, termed Nod factors, during the establishment of mutualism with nitrogen-fixing rhizobia. However, little is still known about the exact activation and signalling mechanisms of MtNFP and MtLYK3. We aimed at investigating putative molecular interactions of MtNFP and MtLYK3 produced in Nicotiana benthamiana. Surprisingly, heterologous co-production of these proteins resulted in an induction of defence-like responses, which included defence-related gene expression, accumulation of phenolic compounds, and cell death. Similar defence-like responses were observed upon production of AtCERK1 in N. benthamiana leaves. Production of either MtNFP or MtLYK3 alone or their co-production with other unrelated receptor(-like) kinases did not induce cell death in N. benthamiana, indicating that a functional interaction between these LysM receptor-like kinases is required for triggering this response. Importantly, structure-function studies revealed that the MtNFP intracellular region, specific features of the MtLYK3 intracellular region (including several putative phosphorylation sites), and MtLYK3 and AtCERK1 kinase activity were indispensable for cell death induction, thereby mimicking the structural requirements of nodulation or chitin-induced signalling. The observed similarity of N. benthamiana response to MtNFP and MtLYK3 co-production and AtCERK1 production suggests the existence of parallels between Nod factor-induced and chitin-induced signalling mediated by the respective LysM receptor(-like) kinases. Notably, the conserved structural requirements for MtNFP and MtLYK3 biological activity in M. truncatula (nodulation) and in N. benthamiana (cell death induction) indicates the relevance of the latter system for studies on these, and potentially other symbiotic LysM receptor-like kinases.

Show MeSH