Alternative splicing: a novel mechanism of regulation identified in the chorismate mutase gene of the potato cyst nematode Globodera rostochiensis.
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 Escherichia 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.
Lu
Shun-Wen
SW
Department of Plant Pathology and Plant-Microbe Biology, Cornell University, Ithaca, NY 14853, USA.
Tian
Duanhua
D
Borchardt-Wier
Harmony B
HB
Wang
Xiaohong
X
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002826:Chorismate Mutase / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D006790:Host-Parasite Interactions; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000469:parasitology; D020033:Protein Isoforms; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000469:parasitology*; D020798:Two-Hybrid System Techniques; D014431:Tylenchoidea / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D020033:Protein Isoforms; D002826:Chorismate Mutase