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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.

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Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.Agrobacterium transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD600 [MtNFP-3xFLAG] = 0.125 and OD600 [MtLYK3-3xFLAG] = 0.2 (A, B); OD600 [AtCERK1-3xFLAG] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
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pone-0065055-g003: Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.Agrobacterium transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD600 [MtNFP-3xFLAG] = 0.125 and OD600 [MtLYK3-3xFLAG] = 0.2 (A, B); OD600 [AtCERK1-3xFLAG] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.

Mentions: An influx of extracellular Ca2+ causes an increase in the cytosolic [Ca2+] that is required for MAMP (including COs)-induced activation of a MAPK cascade, ROS production, and gene expression. Thus, Ca2+ influx is postulated to occur very early in the plant defence signalling pathway [51]–[52], possibly immediately upon the activation of the PM-localised MAMP receptors [53] We wanted to know whether an influx of extracellular Ca2+ was similarly involved in CD induction upon MtNFP and MtLYK3 co-production or separate production of AtCERK1. To this end, MtNFP-3xFLAG and MtLYK3-3xFLAG fusions or AtCERK1-3xFLAG fusion were (co-)produced in adjacent regions in Nicotiana leaves. Twelve hours later, parts of the infiltrated leaf regions were syringe-infiltrated with 5 mM lanthanum chloride (an established inhibitor of the PM-localized calcium channels) or water, and the CD development was monitored between 24 and 72 hours after the first infiltration (with Agrobacterium). Notably, in 24 out of 30 leaf regions co-producing MtNFP and MtLYK3 fusions, compromised membrane permeability and tissue collapse were first (i.e. between 36 and 42 hai) localized only (or mostly) outside the lanthanum chloride-treated regions (Fig. 3A). Later on (i.e. 60 hai), 26 out of 30 parts of leaf regions treated with lanthanum chloride showed confluent death of the entire infiltrated region (unpublished data). Similar delay of the CD development was observed 33 hai in 11 out of 21 leaf regions producing AtCERK1 fusion and treated with lanthanum chloride (Fig. 3C). On the contrary, control treatment with water did not affect the development of confluent CD upon (co-)production of MtNFP and MtLYK3 fusions or AtCERK1 fusion (Fig. 3B, D).


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)

Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.Agrobacterium transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD600 [MtNFP-3xFLAG] = 0.125 and OD600 [MtLYK3-3xFLAG] = 0.2 (A, B); OD600 [AtCERK1-3xFLAG] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated 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-g003: Lanthanum chloride delays the cell death development upon MtNFP and MtLYK3, or AtCERK1 (co-)production.Agrobacterium transformants carrying the following constructs were (co-)infiltrated at a final concentration: OD600 [MtNFP-3xFLAG] = 0.125 and OD600 [MtLYK3-3xFLAG] = 0.2 (A, B); OD600 [AtCERK1-3xFLAG] = 0.2 (C, D). Twelve hai parts of the infiltrated regions were syringe-infiltrated with 5 mM lanthanum chloride (circled in red) or water (circled in white). Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 42 hai for leaf regions co-producing MtNFP and MtLYK3 fusions (A, B), and 33 hai for leaf regions producing AtCERK1 fusion (C, D). Cell death development was scored 42 hai (A, B) or 33 hai (C, D): only infiltrations that showed the lack of tissue collapse and no compromised membrane permeability in the lanthanum chloride- or water-treated region were scored and are presented (right panel) as a fraction of total infiltrations performed. Bars are 1 cm.
Mentions: An influx of extracellular Ca2+ causes an increase in the cytosolic [Ca2+] that is required for MAMP (including COs)-induced activation of a MAPK cascade, ROS production, and gene expression. Thus, Ca2+ influx is postulated to occur very early in the plant defence signalling pathway [51]–[52], possibly immediately upon the activation of the PM-localised MAMP receptors [53] We wanted to know whether an influx of extracellular Ca2+ was similarly involved in CD induction upon MtNFP and MtLYK3 co-production or separate production of AtCERK1. To this end, MtNFP-3xFLAG and MtLYK3-3xFLAG fusions or AtCERK1-3xFLAG fusion were (co-)produced in adjacent regions in Nicotiana leaves. Twelve hours later, parts of the infiltrated leaf regions were syringe-infiltrated with 5 mM lanthanum chloride (an established inhibitor of the PM-localized calcium channels) or water, and the CD development was monitored between 24 and 72 hours after the first infiltration (with Agrobacterium). Notably, in 24 out of 30 leaf regions co-producing MtNFP and MtLYK3 fusions, compromised membrane permeability and tissue collapse were first (i.e. between 36 and 42 hai) localized only (or mostly) outside the lanthanum chloride-treated regions (Fig. 3A). Later on (i.e. 60 hai), 26 out of 30 parts of leaf regions treated with lanthanum chloride showed confluent death of the entire infiltrated region (unpublished data). Similar delay of the CD development was observed 33 hai in 11 out of 21 leaf regions producing AtCERK1 fusion and treated with lanthanum chloride (Fig. 3C). On the contrary, control treatment with water did not affect the development of confluent CD upon (co-)production of MtNFP and MtLYK3 fusions or AtCERK1 fusion (Fig. 3B, D).

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
Related in: MedlinePlus