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
Co-production of MtNFP and MtLYK3 induces cell death in Nicotiana leaves.A, The following MtNFP and MtLYK3 constructs were expressed alone or co-expressed in Nicotiana leaves: mock infiltration (1); MtNFP untagged+MtLYK3 untagged (2); MtNFP-sYFP2+MtLYK3-sYFP2 (3); MtLYK3-sYFP2 (4); MtLYK3 untagged (5); MtNFP-sYFP2 (6); MtNFP untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, MtDMI2-sYFP2 construct was expressed alone or co-expressed with either MtNFP-mCherry or MtLYK3-mCherry construct in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3672211&req=5

pone-0065055-g001: Co-production of MtNFP and MtLYK3 induces cell death in Nicotiana leaves.A, The following MtNFP and MtLYK3 constructs were expressed alone or co-expressed in Nicotiana leaves: mock infiltration (1); MtNFP untagged+MtLYK3 untagged (2); MtNFP-sYFP2+MtLYK3-sYFP2 (3); MtLYK3-sYFP2 (4); MtLYK3 untagged (5); MtNFP-sYFP2 (6); MtNFP untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, MtDMI2-sYFP2 construct was expressed alone or co-expressed with either MtNFP-mCherry or MtLYK3-mCherry construct in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.

Mentions: Agrobacterium tumefaciens GV3101::pMP90 and LBA4404 strains were transformed with the respective constructs via electroporation. The LBA4404 strain was used only in the experiments that compared the effect of different Agrobacterium strains on cell death (CD) induction upon MtNFP and MtLYK3 co-production. All results presented in Figures 1–5, Figure S1 and S3, and Tables 1–3 were obtained with Agrobacterium GV3101::pMP90 strain. Agrobacterium-mediated transformation of Nicotiana was performed essentially as described [42], except that Agrobacterium cultures were grown in LB medium supplemented with 25 µg/mL of rifampicin and 50 µg/mL of kanamycin. Resuspended cells were incubated at room temperature for at least 1 h before being infiltrated into fully expanded leaves of green house-grown plants using needleless syringes. Agrobacterium transformants carrying the respective construct were resuspended in the infiltration medium to desired OD600: all MtNFP and MtNFP[ΔInR]-sYFP2 constructs - OD600 = 0.4; all MtLYK3 and AtCERK1 constructs - OD600 = 0.7; MtDMI2-sYFP2 - OD600 = 1.0. Then, they were mixed 1∶1 with GV3101::pMP90 transformants carrying: pCambia1390 vector with an empty CaMV 35Sp::35S terminator cassette (for separate expression), a desired MtNFP construct or a desired MtLYK3 construct before being infiltrated into Nicotiana leaves. All experiments included mock infiltration with GV3101::pMP90 transformants carrying pCambia1390 vector with an empty CaMV 35Sp::35S terminator cassette, and a positive control (co-expression of WT MtNFP-FP and WT MtLYK3-FP constructs). Cell death induction upon separate expression or co-expression of each (pair of) constructs was analyzed between 24 and 72 hai in at least three independent experiments, every time using three different plants. In case of no macroscopic symptoms, three leaves were stained with Evans blue to confirm the lack of CD.


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)

Co-production of MtNFP and MtLYK3 induces cell death in Nicotiana leaves.A, The following MtNFP and MtLYK3 constructs were expressed alone or co-expressed in Nicotiana leaves: mock infiltration (1); MtNFP untagged+MtLYK3 untagged (2); MtNFP-sYFP2+MtLYK3-sYFP2 (3); MtLYK3-sYFP2 (4); MtLYK3 untagged (5); MtNFP-sYFP2 (6); MtNFP untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, MtDMI2-sYFP2 construct was expressed alone or co-expressed with either MtNFP-mCherry or MtLYK3-mCherry construct in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0065055-g001: Co-production of MtNFP and MtLYK3 induces cell death in Nicotiana leaves.A, The following MtNFP and MtLYK3 constructs were expressed alone or co-expressed in Nicotiana leaves: mock infiltration (1); MtNFP untagged+MtLYK3 untagged (2); MtNFP-sYFP2+MtLYK3-sYFP2 (3); MtLYK3-sYFP2 (4); MtLYK3 untagged (5); MtNFP-sYFP2 (6); MtNFP untagged (7). Macroscopic observation (left panel) and subsequent Evans blue staining (right panel) are depicted 48 hai. Bar is 1 cm. B, MtDMI2-sYFP2 construct was expressed alone or co-expressed with either MtNFP-mCherry or MtLYK3-mCherry construct in Nicotiana leaves. Macroscopic observations (left panel) and subsequent Evans blue stainings (right panel) are depicted 48 hai. Bars are 1 cm.
Mentions: Agrobacterium tumefaciens GV3101::pMP90 and LBA4404 strains were transformed with the respective constructs via electroporation. The LBA4404 strain was used only in the experiments that compared the effect of different Agrobacterium strains on cell death (CD) induction upon MtNFP and MtLYK3 co-production. All results presented in Figures 1–5, Figure S1 and S3, and Tables 1–3 were obtained with Agrobacterium GV3101::pMP90 strain. Agrobacterium-mediated transformation of Nicotiana was performed essentially as described [42], except that Agrobacterium cultures were grown in LB medium supplemented with 25 µg/mL of rifampicin and 50 µg/mL of kanamycin. Resuspended cells were incubated at room temperature for at least 1 h before being infiltrated into fully expanded leaves of green house-grown plants using needleless syringes. Agrobacterium transformants carrying the respective construct were resuspended in the infiltration medium to desired OD600: all MtNFP and MtNFP[ΔInR]-sYFP2 constructs - OD600 = 0.4; all MtLYK3 and AtCERK1 constructs - OD600 = 0.7; MtDMI2-sYFP2 - OD600 = 1.0. Then, they were mixed 1∶1 with GV3101::pMP90 transformants carrying: pCambia1390 vector with an empty CaMV 35Sp::35S terminator cassette (for separate expression), a desired MtNFP construct or a desired MtLYK3 construct before being infiltrated into Nicotiana leaves. All experiments included mock infiltration with GV3101::pMP90 transformants carrying pCambia1390 vector with an empty CaMV 35Sp::35S terminator cassette, and a positive control (co-expression of WT MtNFP-FP and WT MtLYK3-FP constructs). Cell death induction upon separate expression or co-expression of each (pair of) constructs was analyzed between 24 and 72 hai in at least three independent experiments, every time using three different plants. In case of no macroscopic symptoms, three leaves were stained with Evans blue to confirm the lack of CD.

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