Submitted to: CDFA Pierce's Disease Control Program Research Symposium
Publication Type: Proceedings
Publication Acceptance Date: 10/30/2008
Publication Date: 11/20/2008
Citation: Backus, E.A., Labavitch,, J.M. 2008. Immunohistochemistry of ß 1,4-glucanase, the major enzymatic component of glassy-winged sharpshooter saliva, in probed grape petioles. Proceedings of the CDFA Pierce's Disease Control Program Research Symposium, December 15-16, 2008, San Diego, California. p.3-6.
Interpretive Summary: Glassy-winged sharpshooter (GWSS) is an invasive pest of grape and other crops in California because it is the vector of the Pierce’s Disease bacterium, Xylella fastidiosa (Xf). The actual mechanism by which Xf cells are injected into grape is not known. The objective of this project is to determine whether ß 1, 4-glucanase, the major enzyme in GWSS saliva, occurs together with the few Xf cells that are inoculated into a plant by this vector. If so, vector saliva may be a carrier of the bacteria during inoculation. This year, we developed microscopy protocols for separate detection of glucanase and Xf in grape petioles fed upon by GWSS. We also performed three experiments to monitor feeding of inoculative GWSS, via Electrical Penetration Graph (EPG) technology. Feeding was recorded on healthy grape petiole, followed by histological processing of the fed-upon grape tissues. To date, results show that glucanase is found throughout the solid salivary sheath that encases stylets (the piecing-sucking mouthparts of GWSS that penetrate the plant). However, glucanase was not found diffused into adjoining plant cells along the stylet pathway, as was hypothesized. Glucanase was the major constituent of the deep, narrow sheath branches that enter the xylem, indicating that glucanase is injected into xylem during feeding. If our hypothesis on the role of saliva in inoculation is supported, it suggests that salivary antagonists could interfere with Xf inoculation to grape.
Technical Abstract: The purpose of this project is to determine whether ß 1, 4-glucanase (EGase), the major enzyme in the saliva of glassy-winged sharpshooter (GWSS), co-localizes with the few ‘pioneer’ Xylella fastidiosa (Xf) cells that are inoculated into a plant by this vector. If it does, then this suggests that vector saliva is a carrier of the bacteria during inoculation, and that saliva might aid in this process. This year, we successfully used antibodies to purified EGase in conjunction with fluorescent-labeled secondary antibodies for immunohistological detection of EGase in grape petioles probed by GWSS. Similarly, we used fluorescent-labeled antibody to Xf to detect Xf in mechanically inoculated grape petiole. Protocols for co-localization of both EGase and Xf in the same, insect-probed cells are under development. We also performed three experiments to monitor feeding of inoculative GWSS, via Electrical Penetration Graph (EPG) technology. Feeding was recorded on healthy grape petiole, followed by histological processing of the fed-upon grape tissue containing the salivary sheath, as well as immediately above and below the sheath. To date, results of sectioned salivary sheaths show that EGase occurs throughout the sheath, and is mixed with other autoflourescent salivary components in the sheath trunk and upper sheath branches. EGase occurs in high concentration as virtually the sole sheath constituent in the very narrow, lower sheath branches within vascular tissues, near and in the xylem. Surprisingly, EGase was not found outside the salivary sheath, diffused into adjoining plant cells along the stylet pathway, as was hypothesized. This suggests that enzymatic watery saliva does not diffuse away from GWSS salivary sheaths, but is secreted with and remains bound to the hardening salivary sheath. If our hypothesis on the role of saliva in inoculation is supported, it suggests that salivary antagonists could be engineered into grape for resistance to Xf inoculation.