Enhancing the Innate Immune Response in Plants

The economic viability of orchard and vineyard crops in many areas of the world is threatened by the determinative spread of destructive diseases. Without exception all of these crops are grown as composite genetic systems with the fruit producing scion cultivars grafted onto specific rootstock genotypes. The rootstock genotype provides a locally adapted vigorous root system that sustains productivity and quality. Rootstocks have been shown to promote vigor, early flowering, compact architecture, disease and pest resistance, tolerance/resistance to environmental stresses like drought and salinity and improved mineral nutrition. A single rootstock having all of these traits in would be outstanding, unfortunately current rootstocks for many fruit crops fall short of this ideal. Our concept is to change this by engineering rootstocks using Agrobacterium-mediated transformation to provide resistance to biotic and abiotic stress. We proposed this concept over a decade ago when we reported the first RNAi mediated resistance to a major bacterial disease, crown gall. Crown gall is the number one bacterial disease for walnuts in California. The predominant rootstock, Paradox, is exquisitely sensitive to this disease. Consequently the walnut industry has grown increasingly vulnerable to this disease in recent years. We demonstrated that expression of self complimentary versions of the iaaM and ipt genes from Agrobacterium tumefaciens resulted in formation of dsRNA that was processed to siRNA which blocked expression of oncogenes from incoming T-DNA of infecting wild type Agrobacterium. This resulted in the loss and elimination of the tumor phenotype (Escobar et al., PNAS 98:13437-13442, 2001; Plant Sci. 163: 591-597, 2002). We demonstrated that a high degree of resistance could be engineered into rootstocks grafted to wild type scion. The resistance was restricted to the rootstock and not transmissible to the wild type scion that was still susceptible to crown gall. These lines showed dramatic levels of resistance to a very broad range of Agrobacterium tumefaciens strains (Escobar et al., Mol Plant Pathol. 4: 57-65, 2003). We are currently field testing transgenic Paradox walnut lines that show immunity to crown gall. 
We have expanded this concept to include building grapevines resistant to Pierce’s Disease (PD), a vector-transmitted disease that limits the cultivation of grapevines in many areas of the world. The causative agent of PD is the gram-negative bacterium Xylella fastidiosa (Xf). This bacterium is deposited into the xylem tissue by the feeding action of an insect vector, the Glassy Winged Sharpshooter (GWSS) that efficiently transmits the disease and is of great concern to the $60 billion wine industry of California. The virulence of the bacterium is associated with its colonization of xylem elements and its ability to degrade pit pore membranes, allowing it to move and colonize adjacent water conducting elements. The growth and productivity of grapevines is compromised by growth and movement of Xf and its ability to occlude the water-conducting vessels. Our strategy is based on developing and testing proteins to be produced in transgenic rootstocks to 1) limit movement of Xf, and 2) clear the bacteria. We have successfully demonstrated the expression of one such protein, polygalacturonase inhibitory protein (PGIP) from pear fruit. PGIP was able to protect the plant, presumably by limiting the movement of Xf (Agüero et al. Mol Plant Pathol. 6: 43-51, 2005). Recently, it has been shown that Xf expresses polygalacturonase (PG), a virulence factor that degrades the pectin in pit pore membranes in grapevines, allowing the bacterium to pass between xylem vessels. We also showed that expression of PGIP in grapevine rootstocks is associated with secretion of this protein into the xylem and its movement through the graft union via xylem sap into the tissues of the grafted wild type scion grapevine (Aguero et al. Mol Plt Path. 6: 43-51, 2005). Xf is xylem-limited, therefore expression of transgenic therapeutic PGIP and potential antimicrobial proteins must be targeted to xylem tissue in order to prevent and control PD infestations. Signal peptides control entry of virtually all proteins to the secretory pathway in both eukaryotes and prokaryotes. We and others have characterized proteins naturally secreted to the xylem of grapevines which are excellent sources of potential signal peptides (Agüero et al. Am. J Enol. Viticult. 59: 301-311, 2008). Our goal is to use signal sequences from grapevine xylem proteins to deliver therapeutic proteins into the xylem of transgenic rootstocks, thus conferring resistance to PD in the entire plant without modifying the scion or affecting the fruit. This approach has yielded at least four PGIP-transgenic lines of grapevine with good tolerance to PD that are currently being field tested.
We have taken a structure-based approach to develop a chimeric anti-microbial proteins for rapid destruction of Xf (Kunkel et al. Crit. Rev. Immunol. 27: 233-245, 2007).  The designed chimeric anti-microbial protein has two functional domains: 1) a surface recognition domain (SRD) that specifically targets the bacterium’s outer membrane and 2) a lytic protein to lyse the membrane and kill Xf. In this chimera, human neutrophil elastase (HNE) is the SRD that recognizes MopB, the major outer membrane protein of Xf. The second domain is cecropin B(CECB), a lytic peptide that targets and lyses Gram-negative bacteria (Kunkel et al. Crit. Rev. Immunol. 27: 233-245, 2007). We have combined HNE and CECB using a flexible linker to allow both components to bind simultaneously to their respective targets. We have evaluated 11of 36 transgenic grapevines expressing HNE-CECB in the greenhouse for clearance of Xf, and obtained good evidence that at least four of the 11 evaluated lines show good tolerance to Xf infection. Magnetic resonance imaging (MRI) of infected stem sections further revealed that fewer vessels were clogged in the transgenic compared to control plants, indicating clearance of the infected bacteria. Our strategy of combining a pathogen recognition element and a pathogen killing element in the HNE-CECB chimeric molecule is a novel innate immune concept and has several immediate and long term impacts. The strategy is based on the fundamental principle of innate immunity in which pathogen clearance occurs in three sequential steps: pathogen recognition, activation of anti-microbial processes, and pathogen destruction. We are using this approach against other diseases.