Grape: Vitis vinifera

Entering the Regulatory Coordinated Framework

Outline

• PIPRA and CDFA PD/GWSS Board• Grape Industry• Grape Biology• The Problem: Pierce’s Disease• The Solution: Strategies with High Potential

CDFA PD/GWSS Board• CDFA PD/GWSS Board created in 2001 after the

Temecula Valley disaster• Funded from California growers and state and

federal governments money• Fund basic research programs to understand the

fundamental biology of the pathogen: Xylella fastidiosa and the vector: Glassy-Winged Sharpshooter

• Goal develop PD control strategies for the grape industry

PIPRA

• PIPRA = Public Intellectual Property Resource for Agriculture• supports agricultural innovation for both

humanitarian and specialty crop commercial purposes

• bring together intellectual property from over 53 universities, public agencies, and non-profit institutes

www.pipra .org

PIPRA and CDFA PD/GWSS Board

• Since 2005, PIPRA has supported the CDFA PD/GWSS Board in:– Intellectual property management– Policy development for IP management– Research Management Tool (

www.piercesdisease.org)– More recently, Regulatory Assistance for Field

Trial Launch and GM-based strategies for PD control

Categories Total

Wine, Grape and Grape Products contribution to US Economy

$162 Billion

Employment 1.1 Million equivalent full-time employment

Agriculture

Grape Growers 23,486

Grape Bearing Acres 934,750

Farm Gate Grape Sales $3.5 Billion (Highest Value Crop in US/ Largest Fruit Industry in CA)

Taxes $17.1 Billion (9.1 Federal + 8 State and Local)

GRAPE + CITRUS + NURSERIES Industries Revenues

$20.8 Billion (Second after Corn: $26.8 and before Soybean: $18.3)

Categories TotalWine Industry

Direct ImpactWineries 4,929 (2005) in all 50 States

Wineries Sales Revenue $11.4 BillionAdded Value

Distribution Share of American Wine Revenue

$2.7 Billion

Retail and Restaurant Share of American Wine Revenue

$9.8 Billion

Wine-Related Tourism $27.3 MillionWine-Related Tourism Expenditure $3.0 Billion

Grape Production

Product share for grapes in the United States (2000-2006 avg.). Source: USDA Economic Research Service

Grape Distribution• Perennial woody vines in the genus

Vitis, family Vitaceae• According to the USDA data

(http:/plants.usda.gov) 23 Vitis species exist in the United States and Canada

• The most common wild species found in the United States are Summer Grapes, the Frost Grape, the Sweet Winter Grape, the Northern Fox Grape, the Catbird Grape and the Riverbank Grape.

Grape Biology

• Cultivated varieties= propagated by grafting shoots (scions) onto the root of a hardy plant (rootstocks)

• Desirable traits=displayed by the scion not the rootstock

• Self-fruitful => pollination by wind and lesser extent insects

• Fruit = berries; small (<1 inch)http://www.extension.umn.edu/yardandgarden/YGLNews/images/scion&rootstock.jpg

Grape, Papaya & Plum Consumption

• Wine consumption is 2.47 gallons per year (2007)

Total Fresh Processed

Total (lbs)

Fresh Total Canned Juice Dried

Grape 21.14 8.02 13.12 0.14 6.00 6.97Papaya 1.08 1.08 N/APlum 2.03 1.01 1.02 0.02 0.55 0.45

Source: USDA/Economic Research Service. Data last updated February 27, 2009.

Grape Food/Feed Use

Pierce’s Disease : The threat

• Was first discovered by Newton Barris Pierce in 1892 in Anaheim

• Was not a big threat in California until the introduction of the Glassy Winged-Sharpshooter in 1994

• $37.9 million loss in Riverside and San Diego counties in 1998/99 due to vine death and removal of vineyards => triggered the creation of the CDFA PD/GWSS Board which was completely industry-driven in 2000

Glassy-Winged Sharpshooter: The vector

• Originated from Southeast US and Northeast Mexico

• First reported in 1994 in Southern California

• Feeds on xylem fluid of a large number of plants

• Aggressive flyer, traveling greater distances than native sharpshooters

Why is the GWSS a bigger threat?

• Wide host range• Fly further inside the grape fields• Ability to feed on and transmit the pathogen

to older grape wood.

Important Vectors of Xf

Glassy-winged sharpshooterHomalodisca coagulata

Blue-green sharpshooter Graphocephala atropunctata Redheaded sharpshooter

Carneocephala fulgida

Green SharpshooterDraeculacephala minerva

Pierce’s Disease

Gram negative rod fastidious

Insect vectored

Xylem limited

PD symptoms similar to waterstress

Pierce’s Disease

Oleander Leaf Scorch Coffee Leaf Scorch

Almond Leaf Scorch

Maple Leaf ScorchMaple Leaf Scorch

Elm Leaf ScorchOak Leaf Scorch

Phony Peach FruitNo leaf scorching

CVC fruit and stunted growthNo leaf Scorching

Leaf scorch symptom

Pierce’s Disease Symptoms

Berry desiccation

“Green Islands” Irregular periderm development

“Matchstick” petioles

Pierce’s Disease Symptoms

Chronically infected vines have delayed growth and stunted shoots

Varela et al. 2001

Specialty Crop PD Threat on Crop Production Value ($)

Grape 4.0 Billion

Almond 2.8 Billion

Citrus 1.1 Billion

Stone Fruit 1.0 Billion

PD Threatens Other Crop Industries

Summary

• Grape Industry is an important for the US Agricultural Economy

• The Pierce’s Disease is threatening the California Grape/Wine Industry since 2000

• The CDFA PD/GWSS Board has invested $270 Million of research funding to find a solution

• Major research findings are finally giving some hope to the industry

Strategies with high potential of commercialization

2 Main strategies

• Barrier Crop: Use the GM Crop as a barrier crop border to protect grapevine crop

• GM Grape Rootstocks: Develop GM-Grape rootstock with PD-Resistance => Non-GM Grape scions can still be grafted with their own wine and grape qualities

2 Development Strategies

• 1TDNA Plant Transformation with Gene of Interest and Plant Selection Gene:– Gene of Interest conferring PD resistance from:

baculovirus, bacteria, grape and heterologous plants

– Selectable Markers: kanamycin, hygromycin and visual marker GUS

PIPRA’s recombinase-based marker-free transformation platform

• Recombinase-based marker excision system => GM Vitis marker free

Figure 1. Negative selection construct to test efficacy Par-A mediated recombinase activity in tobacco and grape.

Genomic DNA

Selectable Marker Cassette

RB

Recombinase Cassette

RRS

RRS

Gene of Interest Cassette: GUS

LB

DsRed fluorescece protein Marker Cassette

A. Recombinase-based Marker Excision Module

B. Recombinase-mediated Excision

Genomic DNA

RB

RRS

Gene of Interest Cassette: GUS

LB

Selectable Marker Cassette

Recombinase Cassette

RRS

RRS

DsRed fluorescece protein Marker Cassette

Lost DNA

Diffusible Signaling Factor (DSF)• DSF = small fatty acid that accumulates when Xf

is high density in xylem

• Strategy = altering DSF levels in a grape rootstock to decrease Xf virulence by a process called pathogen confusion

• Gene of Interest = rpfF/B from Xylella or Xanthomonas

Poly-Galacturonase Inhibiting Protein (PGIP)

• Xf Polygalacturonase = Cell Wall Degrading Enzyme allowing movement and dispersion of Xf between xylem vessels in the plant

• Strategy = Expressing PG-Inhibiting Proteins directly in a grape rootstock to inhibit Xf PGs

• Gene of Interest = PGIP from heterologous plant

Anti-Programmed Cell Death (PCD)

• Symptoms = Activation of Programmed Cell Death Pathways (PCD)

• Strategy = Suppression of PD Symptoms by constitutively expressing anti-PCD genes

• Gene of Interest = anti-PCD genes from bacoluvirus, bacteria, grape or heterologous plants

Comparison of constructs

Discussion• Short-term objectives:

– First field trials planned for Spring 2010• What information needed for field permit and interstate

movement?

– Regulatory Road-Map• Evaluate the different GM strategies in terms of Cost/Time

– TDNA – Recombinase-based Marker-free System– GM Rootstock– GM Grapevines as Border Barrier

Discussion

• Long-term Objectives:– Data requirements for deregulation of a TDNA vs.

Marker-free developed strategy?

– Data requirements for deregulation of a GM Grapevine vs. GM Rootstock?