|Andrews, Kim -|
|Greve, L. -|
|Labavitch, John -|
|Alhaddad, Hasan -|
Submitted to: Journal of Insect Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 24, 2012
Publication Date: May 31, 2012
Citation: Backus, E.A., Andrews, K.B., Shugart, H., Greve, L.C., Labavitch, J.M., Alhaddad, H. 2012. Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa. Journal of Insect Physiology. 58(7):949-959. Interpretive Summary: Xylella fastidiosa is a bacterial plant pathogen that causes Pierce’s disease of grape and other scorch diseases in important crops. The bacteria live exclusively in plant xylem cells and inside the foregut of its vectors, xylem-feeding insects such as the glassy-winged sharpshooter, Homalodisca vitripennis. Exactly how the insect injects bacteria from its foregut into xylem cells is not known, but is thought to involve the insect’s saliva. Bacteria form a dense layer in the insect’s foregut, anchored by a polysaccharide matrix. We hypothesized that sharpshooters secrete saliva that contains ß-1,4-endoglucanase (EGase; commonly called cellulase), an enzyme that may dissolve the X. fastidiosa matrix, thereby loosening bacteria from the foregut cuticle. Proteins purified from dissected sharpshooter salivary glands contained EGase, and were used to raise antibodies to EGase. Antibody-based staining of sharpshooter salivary deposits in grape petioles showed that gelling saliva containing EGase was injected into a xylem cell, from which it was transported in the xylem fluid flow. Thus, we identified part of the mechanism of inoculation of X. fastidiosa to plants, because an enzyme that may dissolve the matrix that cements bacteria to insect foregut cuticle was secreted in vector saliva and is injected into xylem during feeding. Understanding the inoculation mechanism will aid development of grape varieties resistant to inoculation of X. fastidiosa by sharpshooter vectors.
Technical Abstract: Certain hemipteran insects such as the glassy-winged sharpshooter, Homalodisca vitripennis, subsist entirely on xylem fluid, notwithstanding the poor nutrition of such food. Among many adaptations enabling xylem-feeding are aspects of the insect’s salivation that may also allow these insects to transmit plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierce’s disease of grape and other scorch diseases in important crops. Bacteria colonize the foregut of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored by an exopolysaccharide (EPS) matrix with a ß-1,4-glucan backbone. A recently published hypothesis for the mechanism of X. fastidiosa inoculation during vector feeding is that salivation is combined with egestion (outward fluid flow) to carry bacteria into the xylem. We hypothesized that sharpshooters secrete saliva that contains ß-1,4-endoglucanase (EGase; commonly called cellulase), an enzyme that can potentially hydrolyze the ß-1,4-linked glucan backbones in bacterial exopolysaccharide as well as plant cell wall hemicellulose, xyloglucan, and cellulosic glucans. Proteins purified from dissected sharpshooter salivary glands contained EGase, and its digestive activity against model ß-1,4-glucan polysaccharides was demonstrated in vitro. Furthermore, immunohistology of sharpshooter salivary sheaths in planta showed that EGase-containing gelling saliva was injected into a xylem vessel and in one case was carried by the transpiration stream away from the injection site. Thus, we demonstrated that ß-1,4-endoglucanase, which may digest polysaccharides in cell walls and exopolysaccharide that cements X. fastidiosa to insect foregut cuticle, is secreted in vector saliva and is injected into xylem during feeding.