Cecilia B. Agüero

An engineered innate immune defense protects grapevines from Pierce disease

We postulated that a synergistic combination of two innate immune functions, pathogen surface recognition and lysis, in a protein chimera would lead to a robust class of engineered antimicrobial therapeutics for protection against pathogens. In support of our hypothesis, we have engineered such a chimera to protect against the Gram-negative Xylella fastidiosa (Xf), which causes diseases in multiple plants of economic importance. Here we report the design and delivery of this chimera to target the Xf subspecies fastidiosa (Xff), which causes Pierce disease in grapevines and poses a great threat to the wine-growing regions of California. One domain of this chimera is an elastase that recognizes and cleaves MopB, a conserved outer membrane protein of Xff. The second domain is a lytic peptide, cecropin B, which targets conserved lipid moieties and creates pores in the Xff outer membrane. A flexible linker joins the recognition and lysis domains, thereby ensuring correct folding of the individual domains and synergistic combination of their functions. The chimera transgene is fused with an amino-terminal signal sequence to facilitate delivery of the chimera to the plant xylem, the site of Xff colonization. We demonstrate that the protein chimera expressed in the xylem is able to directly target Xff, suppress its growth, and significantly decrease the leaf scorching and xylem clogging commonly associated with Pierce disease in grapevines. We believe that similar strategies involving protein chimeras can be developed to protect against many diseases caused by human and plant pathogens.

Overexpression of a thaumatin-like protein gene from Vitis amurensis improves downy mildew resistance in Vitis vinifera grapevine

Downy mildew is a highly destructive disease in grapevine production. A gene encoding pathogenesis-related (PR) thaumatin-like protein was isolated from the downy mildew-resistant grapevine “Zuoshan-1,” a clonal selection from wild Vitis amurensis Rupr. The predicted thaumatin-like protein (VaTLP) has 225 amino acids and it is acidic, with a calculated isoelectric point of 4.8. The full length of the VaTLP gene was transformed into somatic embryogenic calli of V. vinifera ‘Thompson Seedless’ via Agrobacterium tumefaciens. Real-time RT-PCR confirmed that the VaTLP gene was expressed at a high level in the transgenic grapevines. Improved resistance of the transgenic lines against downy mildew was evaluated using leaf disks and whole plants inoculated with Plasmopara viticola, the pathogen causing grapevine downy mildew disease. Bioassay of the pathogen showed that both hyphae growth and asexual reproduction were inhibited significantly among the transgenic plants. Histological analysis also confirmed this disease resistance by demonstrating the inhibition and malformation of hyphae development in leaf tissue of the transgenic plants. These results indicated that the accumulation of VaTLP could enhance resistance to P. viticola in transgenic ‘Thompson Seedless’ grapevines.

Overexpression of 9-cis-Epoxycarotenoid Dioxygenase Cisgene in Grapevine Increases Drought Tolerance and Results in Pleiotropic Effects

9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme involved in the biosynthesis of abscisic acid (ABA), which is associated with drought tolerance in plants. An osmotic-inducible VaNCED1 gene was isolated from a drought-resistant cultivar of Vitis amurensis and constitutively overexpressed in a drought-sensitive cultivar of Vitis vinifera. Transgenic plants showed significantly improved drought tolerance, including a higher growth rate and better drought resistant under drought conditions, compared to those of wild-type (WT) plants. After water was withheld for 50 days, the upper leaves of transgenic plants remained green, whereas most leaves of WT plants turned yellow and fell. Besides the increase in ABA content, overexpression of VaNCED1 induced the production of jasmonic acid (JA) and accumulation of JA biosynthesis-related genes, including allene oxide cyclase (AOC) and 12-oxophytodienoate reductase (OPR3). Moreover, transgenic plants possessed advantageous physiological indices, including lower leaf stomatal density, lower photosynthesis rate, and lower accumulation of proline and superoxide dismutase (SOD), compared to those of WT plants, indicating increased resistance to drought stress. Quantitative real time polymerase chain reaction (RT-qPCR) analysis revealed that overexpression of VaNCED1 enhanced the expression of drought-responsive genes, such as ABA-responsive element 1 (ABRE1), ABRE binding factors 2 (ABF2), plasma membrane intrinsic proteins 2 (PIP2), C-repeat/DRE-Binding Factor 4 (VvCBF4) and ABA-insensitive 5 (ABI5). Although the development of transgenic plants was delayed by 4 months than WT plants, because of seed dormancy and abnormal seedlings, the surviving transgenic plants provided a solid method for protection of woody plants from drought stress.