Heterologous expression of the avirulence gene product, NIP1, from the barley pathogen Rhynchosporium secalis.
NIP1, the product of the avirulence gene AvrRrs1 from Rhynchosporium secalis, a fungal pathogen of barley, is a small secreted cysteine-rich protein. This protein is essential for the specific recognition of the fungus by host plants carrying the complementary resistance gene Rrs1. Different heterologous expression systems were tested to produce sufficient quantities of NIP1 to allow its utilization in receptor identification and isolation. In addition, protein amounts higher than those produced in fungal cultures are required to determine its 3D structure and to analyze its interaction with a receptor. The most efficient method, the synthesis of a His-tag fusion protein in Escherichia coli combined with a refolding procedure, yielded up to 3 mg of recombinant NIP1 from a 1-liter bacterial culture. After removal of the His-tag, the recombinant protein showed the same physicochemical characteristics as the native NIP1 and, most importantly, full biological activity.
Gierlich
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Department of Biochemistry, Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, Köln, D-50829, Germany.
van 't Slot
K A
KA
Li
V M
VM
Marie
C
C
Hermann
H
H
Knogge
W
W
D000818:Animals; D001203:Ascomycota / Q000235:genetics* / Q000472:pathogenicity*; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D039621:Eukaryotic Initiation Factor-3; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005800:Genes, Fungal; D001467:Hordeum / Q000382:microbiology; D008970:Molecular Weight; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D018411:Spodoptera; D014774:Virulence / Q000235:genetics
D017931:DNA Primers; D039621:Eukaryotic Initiation Factor-3; D005656:Fungal Proteins; C077915:NIP1 protein, S cerevisiae; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins