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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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MtNFP and MtLYK3, or AtCERK1 (co-)production in Nicotiana leaves induces defence-like responses.A, Kinetics of cell death development in Nicotiana. Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in Nicotiana leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of NbHIN1, NbPR1 basic, NbACRE31, and NbACRE132 expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]-3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of NbHIN1 (white columns), NbPR1 basic (grey columns), NbACRE31 (hatched columns), and NbACRE132 (black columns) normalized by one reference gene, MtEF1 α. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.
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pone-0065055-g004: MtNFP and MtLYK3, or AtCERK1 (co-)production in Nicotiana leaves induces defence-like responses.A, Kinetics of cell death development in Nicotiana. Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in Nicotiana leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of NbHIN1, NbPR1 basic, NbACRE31, and NbACRE132 expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]-3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of NbHIN1 (white columns), NbPR1 basic (grey columns), NbACRE31 (hatched columns), and NbACRE132 (black columns) normalized by one reference gene, MtEF1 α. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.

Mentions: Subsequently, we investigated whether co-production of MtNFP and MtLYK3 or production of AtCERK1 in Nicotiana leaves was associated with an accumulation of phenolic compounds and/or induction of defence-related gene expression, two established hallmarks of plant defence response, including that induced by COs and/or PGN [54]–[56]. We started by analysing the kinetics of CD development. To this end, Agrobacterium transformants carrying MtNFP-3xFLAG, MtLYK3-3xFLAG or AtCERK1-3xFLAG construct were (co-) infiltrated at different time-points in adjacent circles in Nicotiana leaves, and CD development was monitored between 24 and 48 hai. In case of co-production of MtNFP and MtLYK3 fusions, macroscopic symptoms of CD were first observed around 36 hai (Fig. 4A) as a type of flaccidity and the appearance of small patches of collapsed tissue (these were more pronounced on the abaxial side of the leaf). Forty-eight hai, 30 out of 31 infiltrations showed pronounced tissue desiccation of the entire infiltrated region (Fig. 1A). Compromised membrane permeability preceded tissue collapse and often occurred over the entire infiltrated region approximately 33 hai (Fig. 4A). Compromised membrane permeability of leaf regions producing AtCERK1 fusion was observed already approximately 27–30 hai, and pronounced macroscopic symptoms of CD developed 36 hai (Fig. 2A, Fig. 3 C, 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)

MtNFP and MtLYK3, or AtCERK1 (co-)production in Nicotiana leaves induces defence-like responses.A, Kinetics of cell death development in Nicotiana. Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in Nicotiana leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of NbHIN1, NbPR1 basic, NbACRE31, and NbACRE132 expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]-3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of NbHIN1 (white columns), NbPR1 basic (grey columns), NbACRE31 (hatched columns), and NbACRE132 (black columns) normalized by one reference gene, MtEF1 α. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672211&req=5

pone-0065055-g004: MtNFP and MtLYK3, or AtCERK1 (co-)production in Nicotiana leaves induces defence-like responses.A, Kinetics of cell death development in Nicotiana. Agrobacterium transformants carrying either MtNFP-3xFLAG or MtLYK3-3xFLAG construct were co-infiltrated into Nicotiana leaves at five different time points (1–5). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 42 hai (region 1), 39 hai (region 2), 36 hai (region 3), 33 hai (region 4) and 30 hai (region 5). Mock infiltration (region 6) was done concomitantly with the infiltration of region 1. Bar is 1 cm. B, Changes in leaf autofluorescence upon MtNFP and MtLYK3 co-production. Leaf regions co-producing MtNFP-3xFLAG and MtLYK3-3xFLAG fusions were analyzed between 24 and 48 hai (here depicted 36 hai) using a stereoscope. Note the decrease in chlorophyll content, as indicated by the decrease of far-red autofluorescence of chlorophyll (left panel), and enhanced accumulation of blue light-excited autofluorescence (right panel) within the infiltrated region. Bar is 1 cm. C, Accumulation of phenolic compounds. The following fusions were (co-)produced in Nicotiana leaves: MtNFP-3xFLAG (1); MtLYK3-3xFLAG (2); MtNFP-3xFLAG+MtLYK3-3xFLAG (3); MtNFP-3xFLAG+MtLYK3[G334E]-3xFLAG (4); AtCERK1-3xFLAG (5); or AtCERK1[K349]-3xFLAG (6). Macroscopic observations (left panel) and subsequent UV-excited autofluorescence of ethanol/lactophenol-cleared (right panel) leaf regions are depicted 36 hai (except for 5–30 hai). Bars are 1 cm. D, Induction of NbHIN1, NbPR1 basic, NbACRE31, and NbACRE132 expression in response to separate production or co-production of: MtNFP-3xFLAG (NFP), MtLYK3-3xFLAG (LYK3), MtLYK3[G334E]-3xFLAG (LYK3[G334E]), and AtCERK1-3xFLAG (CERK1). Leaf samples were collected 24 hai and induction of gene expression was analyzed using qRT-PCR. Histograms represent induction of NbHIN1 (white columns), NbPR1 basic (grey columns), NbACRE31 (hatched columns), and NbACRE132 (black columns) normalized by one reference gene, MtEF1 α. Induction of each gene was normalized to that caused by mock infiltration, and then calculated as % induction relative to the induction observed upon co-production of MtNFP and MtLYK3 fusions. Bars represent standard deviation of the mean. At least two technical replicates from two biological replicates were analyzed.
Mentions: Subsequently, we investigated whether co-production of MtNFP and MtLYK3 or production of AtCERK1 in Nicotiana leaves was associated with an accumulation of phenolic compounds and/or induction of defence-related gene expression, two established hallmarks of plant defence response, including that induced by COs and/or PGN [54]–[56]. We started by analysing the kinetics of CD development. To this end, Agrobacterium transformants carrying MtNFP-3xFLAG, MtLYK3-3xFLAG or AtCERK1-3xFLAG construct were (co-) infiltrated at different time-points in adjacent circles in Nicotiana leaves, and CD development was monitored between 24 and 48 hai. In case of co-production of MtNFP and MtLYK3 fusions, macroscopic symptoms of CD were first observed around 36 hai (Fig. 4A) as a type of flaccidity and the appearance of small patches of collapsed tissue (these were more pronounced on the abaxial side of the leaf). Forty-eight hai, 30 out of 31 infiltrations showed pronounced tissue desiccation of the entire infiltrated region (Fig. 1A). Compromised membrane permeability preceded tissue collapse and often occurred over the entire infiltrated region approximately 33 hai (Fig. 4A). Compromised membrane permeability of leaf regions producing AtCERK1 fusion was observed already approximately 27–30 hai, and pronounced macroscopic symptoms of CD developed 36 hai (Fig. 2A, Fig. 3 C, 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