Expression and functional characterization of a single chain Fv antibody directed against secretions involved in plant nematode infection process.
Expression in plants of antibodies directed against proteins essential for pathogenesis could provide an alternative approach to engineer new resistance traits into crops. Salivary secretions of the root-knot nematode Meloidogyne incognita are known to play a key role during this nematode infection process. From a hybridoma cell line producing an IgM monoclonal antibody specific to these secretions, we have constructed a synthetic gene that encodes an antigen-binding single-chain Fv protein (scFv). The scFv gene was created by polymerase chain reaction amplification of variable domain encoding regions from the IgM antibody. The cloned scFv was initially expressed in Escherichia coli as a 33-kDa protein which could be purified to near homogeneity by immobilized metal affinity chromatography. The produced scFv is fully functional since it shows the same specificity towards a crude extract of M. incognita infective larvae as the corresponding parental IgM. Transient expression assays with tobacco leaf protoplasts using different targeting signals resulted in a high intracellular accumulation of scFv, especially when fused to the tetrapeptide KDEL retention signal. Activity analysis and stability characterization of this scFv in tobacco protoplast represent the first step before plant transformation in order to test a new form of resistance to root-knot nematode in plants.
Rosso
M N
MN
Laboratoire de Biologie des Invertébrés, INRA, Antibes, France.
Schouten
A
A
Roosien
J
J
Borst-Vrenssen
T
T
Hussey
R S
RS
Gommers
F J
FJ
Bakker
J
J
Schots
A
A
Abad
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007075:Immunoglobulin M / Q000235:genetics; D007135:Immunoglobulin Variable Region; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000276:immunology; D014431:Tylenchoidea / Q000276:immunology*
D000909:Antibodies, Helminth; D007128:Immunoglobulin Fragments; D007075:Immunoglobulin M; D007135:Immunoglobulin Variable Region; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C055966:immunoglobulin Fv; C073762:lysyl-aspartyl-glutamyl-leucine