Structural and functional characterization of a novel, host penetration-related pectate lyase from the potato cyst nematode Globodera rostochiensis.
SUMMARY The cell wall, a strong extraprotoplasmic layer surrounding plant cells that mainly consists of a variety of polysaccharides, constitutes a major barrier for potential parasites. Plant-parasitic nematodes are well equipped to overcome this barrier as they produce and secrete cell-wall-degrading enzymes. Expression profiling of various life stages of the potato cyst nematode Globodera rostochiensis revealed a novel pectate lyase gene (Gr-pel2, 759 bp). The Gr-PEL2 protein showed highest similarity to pectate lyases from the facultative plant-parasitic nematodes Bursaphelenchus mucronatus and B. xylophilus and the soil-inhabiting saprophytic Streptomyces and Frankia species (i.e. 40-42% identity and 58-60% similarity), whereas only a remote relatedness to the previously identified Gr-PEL1 was observed (i.e. 28% identity and 43% similarity). Transient expression of Gr-pel2 in leaves of Nicotiana benthamiana resulted in severe malformations of the infiltrated tissues, not relating to maceration and soft rot symptoms. Ca(2+) is known to be essential for pectate lyase activity, and the most likely calcium-binding site was identified in the Gr-PEL2 protein by combining homology modelling of the three-dimensional structure, site-directed mutagenesis and transient expression in leaves. A highly charged cleft in Gr-PEL2, which is likely to be involved in substrate binding and which is also significantly more hydrophobic in Gr-PEL1, was shown to be essential for protein activity. Our results underline the broad spectrum of pectate lyases and cell-wall-degrading enzymes necessary for successful parasitism by cyst nematodes.
Kudla
Urszula
U
Laboratory of Nematology, Wageningen University, Binnenhaven 5, 6709 PD Wageningen, the Netherlands.
Milac
Adina-Luminita
AL
Qin
Ling
L
Overmars
Hein
H
Roze
Erwin
E
Holterman
Martijn
M
Petrescu
Andrei-Jose
AJ
Goverse
Aska
A
Bakker
Jaap
J
Helder
Johannes
J
Smant
Geert
G