Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5

TY - JOUR

T1 - Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5

AU - Madsen, Esben B

AU - Antolín-Llovera, Meritxell

AU - Grossmann, Christina

AU - Ye, Juanying

AU - Vieweg, Syndi

AU - Broghammer, Angelique

AU - Krusell, Lene

AU - Radutoiu, Simona

AU - Jensen, Ole N

AU - Stougaard, Jens

AU - Parniske, Martin

N1 - © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

PY - 2011/2/1

Y1 - 2011/2/1

N2 - Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

AB - Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots.

U2 - 10.1111/j.1365-313X.2010.04431.x

DO - 10.1111/j.1365-313X.2010.04431.x

M3 - Journal article

C2 - 21265894

SN - 0960-7412

VL - 65

SP - 404

EP - 417

JO - Plant Journal

JF - Plant Journal

IS - 3

ER -