Biology ETDs

Author

Arinder Arora

Publication Date

12-1-2015

Abstract

Pierce's Disease (PD) is a deadly grapevine disease, which is caused by a Gram negative bacterium, Xylella fastidiosa and transmitted by leafhoppers commonly known as sharpshooters, including the glassy-winged sharpshooter (GWSS, Homalodisca vitripennis). We developed paratransgenic control of PD using a commensal bacterium Pantoea agglomerans E325. The GWSSs acquired enhanced green fluorescent (EGFP)-expressing P. agglomerans from an artificial feeding system and the bacteria persisted within the insect foregut for two weeks. P. agglomerans was selected as the paratransgenic control agent based on its persistence within the sharpshooter foregut, a niche also inhabited by X. fastidiosa. P. agglomerans lines secreting two antimicrobial peptides (AMPs), melittin and scorpine-like molecule (SLM), were generated to accomplish transmission blockage. These AMPs were selected after testing their toxicity to P. agglomerans and X. fastidiosa. Our experiments indicated that both melittin and SLM are more toxic to X. fastidiosa compared to P. agglomerans. Paratransgenic GWSSs, which have acquired an AMP-expressing P. agglomerans prior to X. fastidiosa acquisition, displayed a lower competence to acquire the pathogen in comparison to control insects. These paratransgenic sharpshooters failed to transmit X. fastidiosa to naive grapevines. This is the first instance, wherein paratransgenesis has been demonstrated to block PD transmission. We established melittin and SLM expression within the paratransgenic sharpshooters via Western blot and confirmed that transmission blockage was as a direct result of AMPs expressed by P. agglomerans within the insect gut. Subsequently, we also engineered calcium-alginate based microparticles to reduce environmental contamination during field application of genetically modified bacteria. The insects were able to acquire the bacteria from plants painted with P. agglomerans - containing micropaticles. Amongst the tested microparticles (engineered using 1%, 2% and 3% alginate) the ones engineered using 1% alginate resulted in the highest P. agglomerans acquisition (51.8%) by the sharpshooters. These microparticles will be a means to take the technology from a lab to the field. Active molecules, which target specifically the pathogen only, have an advantage that they won't affect the natural flora and fauna. Keeping this in mind we have engineered antibodies that bind to X. fastidiosa surface protein mopB using ribosomal display methodology. These antibodies bind specifically to X. fastidiosa surface protein. In the future we can express these antibodies individually or as chimeras in combination with AMPs via P. agglomerans. Expression of two active molecules will also decrease the rate of resistance development and will increase the useful life span of this technology in the field.

Project Sponsors

USDA NIFA

Language

English

Keywords

Paratransgenesis, Pierce's Disease, Grape, Xylella fastidiosa, Sharpshooter, Homalodisca vitripennis, AMPs, Antibodies, Microencapsulation

Document Type

Dissertation

Degree Name

Biology

Level of Degree

Doctoral

Department Name

UNM Biology Department

First Advisor

Loker, Eric

First Committee Member (Chair)

Durvasula, Ravi

Second Committee Member

Adema, Coenraad

Third Committee Member

Takacs-Vesbach, Cristina

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