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Title: Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis

item Borchardt Wier, Harmony
item Wang, Xiaohong

Submitted to: Molecular and Biochemical Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/20/2008
Publication Date: 8/22/2008
Citation: Lu, S., Tian, D., Borchardt Wier, H., Wang, X. 2008. Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis. Molecular and Biochemical Parasitology. 162:1-15.

Interpretive Summary: The potato cyst nematode (PCN) Globodera rostochiensis (a.k.a. the golden nematode) is a quarantined pest representing one of the most devastating pests of potatoes. In order to successfully parasitize a host root, the nematode secretes proteins originated from its esophageal gland cells to transform plant cells into a specialized site for the nematode to feed. Understanding the function of nematode parasitism genes encoding these gland secretions will be very useful for developing novel nematode control strategies. This paper describes the isolation and functional characterization of a new parasitism gene, the chorismate mutase gene, from G. rostochiensis. This study has discovered the first example of alternative splicing in plant-parasitic nematodes and suggested an important role of this nematode-secreted chorismate mutase in plant parasitism. Knowledge developed from this study may lead to the development of molecular diagnostic methods for PCN species identification and the development of novel resistance in transgenic potatoes against PCN infection.

Technical Abstract: Chorismate mutase (CM) secreted from the stylet of plant-parasitic nematodes plays an important role in plant parasitism. We isolated and characterized a new nematode CM gene (Gr-cm-1) from the potato cyst nematode, Globodera rostochiensis. The Gr-cm-1 gene was found to exist in the nematode genome as a single-copy gene that has two different alleles, Gr-cm-1A and Gr-cm-1B, both of which could give rise to two different mRNA transcripts of Gr-cm-1 and Gr-cm-1-IRII. In situ mRNA hybridization showed that the Gr-cm-1 gene was exclusively expressed within the subventral oesophageal gland cells of the nematode. Gr-cm-1 was demonstrated to encode a functional CM (GR-CM-1) potentially having a dimeric structure as the secreted bacterial *AroQ CMs. Gr-cm-1-IRII, generated by retention of intron 2 of the Gr-cm-1 pre-mRNA through alternative splicing (AS), would encode a truncated protein (GR-CM-1t) lacking the CM domain with no CM activity. The quantitative real-time reverse transcription-PCR assay revealed that splicing of the Gr-cm-1 gene was developmentally regulated; Gr-cm-1 was up-regulated whereas Gr-cm-1-IRII was down-regulated in early nematode parasitic stages compared to the preparasitic juvenile stage. Low temperature SDS-PAGE analysis revealed that GR-CM-1 could form homodimers when expressed in E. coli and the dimerization domain was retained in the truncated GR-CM-1t protein. The specific interaction between the two proteins was demonstrated in yeast. Our data suggested that the novel splice variant might function as a dominant negative isoform through heterodimerization with the full-length GR-CM-1 protein and that AS may represent an important mechanism for regulating CM activity during nematode parasitism.