Chorismate mutase: an alternatively spliced parasitism gene and a diagnostic marker for three important Globodera nematode species
The chorismate mutase gene is widely distributed in both cyst and root-knot nematode species and believed to play a critical role in nematode parasitism. In this study, we cloned a new chorismate mutase gene (Gt-cm-1) from Globodera tabacum and further characterized the gene structure in both G. tabacum and G. pallida, a closely related species of G. rostochiensis. The genomic clones of chorismate mutase genes from these two species were found to contain three introns with the second intron having unusual 5’ and 3’ splice sites. A previous study revealed that the chorismate mutase gene from G. rostochiensis is subject to alternative splicing through retention of intron 2, a process that allows for the generation of multiple mRNA transcripts from a single gene. As expected, we discovered that alternative splicing of the chorismate mutase gene is a conserved event in three Globodera species, supporting an important role of alternative splicing in regulating chorismate mutase gene function in plant parasitism by these nematodes. In addition to the potential suboptimal 5’ and 3’ splice sites and the small size of intron 2, detailed sequence analysis also identified candidate cis-acting elements that might be responsible for regulating intron retention of Globodera chorismate mutase genes. Based on genomic sequence variations observed, we developed TaqMan qPCR assays that provided a highly specific and sensitive identification of each Globodera species, revealing a new application of using the chorismate mutase gene as a valuable diagnostic marker for plant-parasitic nematodes.
KeywordsAlternative splicing intron retention TaqMan qPCR
exonic splicing enhancer
Globodera rostochiensis chorismate mutase gene
Globodera pallida chorismate mutase gene
Globodera tabacum chorismate mutase gene
locked nucleic acids
potato cyst nematode
quantitative real-time polymerase chain reaction
tobacco cyst nematode
We thank Melissa G. Mitchum for providing TCN and for critical review of this manuscript. We thank Eric Grenier for providing DNA from G. pallida populations originated from South Peru, Switzerland and UK, Geert Smant for providing DNA from PCN populations originated from the Netherlands, Guy Bélair for providing DNA from G. rostochiensis populations originated from St.-Amable, Canada, Solke De Boer for providing DNA from PCN populations originated from Newfoundland, Canada, Robert Zemetra for providing G. pallida cysts originated from Idaho, U.S.A., and Eric Davis for providing Heterodera glycines and a second TCN population from North Carolina, U.S.A. This study was supported in part by funding from USDA-ARS and USDA-APHIS.
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