Polygalacturonase-inhibiting protein sequence, structural, and functional analyses with implications in plant disease resistance

Plant-pathogen interactions result in degrees of plant resistance or susceptibility depending on, in part, numerous biotic factors from both players. Microbe and/or pest challenges to plants often involve the plant cell wall, an outer compartment with roles in cell support and signaling. This dissertation examines the specific interplay between a class of pathogen virulence factors that can degrade the pectin component of cell walls, polygalacturonases (PGs), and plant proteins that limit PG activity and serve as defense factors, polygalacturonase-inhibiting proteins (PGIPs). PGIPs' role in plant defense response pathways is detailed in Chapter 1. PGIPs are common to all angiosperms examined and have diversified into small gene families in many lineages. The sequence variation of 237 PGIPs from 114 angiosperm species was examined here to identify regions of diversity and to attempt to predict the consequent implications on PGIP structure. The majority of divergent residues lie within the beta-sheet shown to interact with PGs. This variation indicates that structural consideration must be taken when selecting PGIPs for inhibition of specific PGs.;Pierce's disease (PD) of grapevines is caused by the bacterium Xylella fastidiosa (Xf), which resides in the xylem vessels, systemically infecting vines and eventually causing plant death. The PG of Xf is necessary for systemic vine colonization. Simulated interaction models of XfPG and various PGIPs highlight residues that help stabilize the complex between the pear fruit PGIP (pPGIP) and XfPG; these same complexes are less electrostatically feasible in reactions modeled with a grape PGIP. Transgenic grapevines expressing pPGIP were previously shown to provide increased PD protection, presumably through XfPG inhibition. We developed recombinant expression systems to compare angiosperm PGIPs' inhibition of XfPG through in vitro and in vivo assays to find an optimal inhibitor to exploit for PD protection.;Given that pPGIP expression imparts increased PD resistance in greenhouse trials, we sought to evaluate the pPGIP-expressing grapevines in a commercially relevant setting. Field examinations in two agricultural areas -- Solano and Riverside Counties, CA -- were established to test pPGIP efficacy against mechanical inoculations or natural infections of Xf, respectively. PGIPs, as cell wall proteins, are found in the xylem sap and are, therefore, able to cross graft junctions from PGIP-expressing rootstocks into target scions. We confirmed that pPGIP from transgenic rootstocks can be recovered in scion leaves that do not express pPGIP. Grafted grapevines were tested in the field alongside the own-rooted transgenic and control vines for PD resistance. PD Symptoms at the Solano site appeared after two years of annual mechanical inoculations and were reduced in 'Thompson Seedless' vines expressing pPGIP. Symptoms at the Riverside site show similar trends with pPGIP affording some protection, though unintended diseases have confounded results from that site.;The transgrafting strategy used here, delivering defense factors via transgenic rootstocks, has promising commercial applications within the evolving regulatory climate for genetically modified plant products. The phylogenetic and structural analyses of PGIPs detailed in Chapter 2 provide a framework to study the predicted efficacy of a PGIP to inhibit numerous PGs from important pathogens. Such candidate PGIPs can then be examined using the techniques refined in Chapter 3, with promising PGIPs tested in expanded trials, as was started with grapevines and pPGIP in Chapter 4.