ARS | Publication request: Gene characterization of two digestive serine proteases in orange blossom wheat midge (Sitodiplosis mosellana)

Submitted to: The Canadian Entomologist
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 6, 2010
Publication Date: January 26, 2011
Citation: Arrueta, L., Shukle, R.H., Wise, I.L., Mitapalli, O. 2011. Gene characterization of two digestive serine proteases in orange blossom wheat midge (Sitodiplosis mosellana). The Canadian Entomologist. 142:(6)532-545.

Interpretive Summary: The orange blossom wheat midge is the most important insect pest of wheat on the prairie provinces of Canada and is an emerging pest of wheat on the north-central plains of the United States, with the potential of causing increased yield loss in wheat across the central plains. Alternate hosts of this pest include barley and rye. Larvae destroy the developing kernels in the wheat head and damaged kernels can affect the baking quality of flour. Only one gene for resistance to this pest has been identified in wheat and there is deep concern about the development of biotypes capable of overcoming this resistance. Further, to date there is no knowledge about the digestive enzymes used by this pest in its attack of the wheat kernel. We have identified and molecularly characterized digestive proteases used by the pest in feeding on the wheat kernel. Knowledge gained from this study will help breeders and scientists facing the challenge of protecting wheat devise alternative genetically engineered strategies to ensure durable resistance to this pest. The agricultural community (crop producers, commodity groups and consumers) will benefit from the resulting improved control that will increase yield, quality and sustainability of production without increasing cost.

Technical Abstract: Two full length cDNA sequences, encoding digestive serine proteases (designated as SmPROT-1 and SmPROT-2), were recovered from the midgut of the wheat midge, Sitodiplosis mosellana in an ongoing EST project. The deduced amino acid sequences shared homology with digestive serine proteases from insect and non-insect species, including conserved regions such as the catalytic triad, active pocket and conserved structural motifs. Secretion signal peptides in both proteases at the N-terminals indicate these proteins could function as midgut digestive serine proteases. A phylogenetic analysis grouped SmPROT-1 and SmPROT-2 with trypsin-like and chymotrysin-like serine proteases, respectively. Quantitative real time PCR analysis showed that SmPROT-1 and SmPROT-2 were expressed predominantly in the midgut compared to other tissues (fat body and salivary glands). Expression analyses revealed mRNA levels were highest for the feeding instars (1st -and 2nd- instar larvae) than for neonates, 3rd-instars, pupae or adults. These results provide new insights into the biology of S. mosellana and are discussed in the context of developing alternate control strategies.