Role of a calcium-binding protein in the saliva of Nephotettix cincticeps in ingestion from the sieve tubes

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Green rice leafhoppers (Nephotettix cincticeps) secrete saliva during feeding. In this study, we found an 84 kDa calcium-binding protein (NcSP84) in the saliva. The NcSP84 was produced in the largest lobes in the primary salivary glands. The NcSP84 was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. The nucleotide and amino acid sequences of NcSP84 did not share homology with any sequences in public databases. Motif search predicted that this protein had multiple EF-hands, the most common motif found in Ca^<2+>-binding proteins. As predicted, NcSP84 exhibited Ca^<2+>-binding activity. The mobility of purified NcSP84 bound to Ca^<2+> declined during SDS-PAGE, depending on the concentration of CaCl_2 present. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca^<2+> ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes. This is the first finding of gene cloning of an EF-hand Ca^<2+>-binding protein in the saliva of a vascular-feeding insect