Optimizing Grape Rootstock Production and Export of inhibitors of

X. fastidiosa PG Activity

Alan Bennett, Ann Powell, John Labavitch Rachell Booth, Dan King and Zac Chestnut

(Poster 58)

Rationale for achieving transgenic protection against Xf

This project is based on observations made over the past several years by various teams in their studies of PD development in grapevines.

UCD Plant Pathology Ph.D. student Caroline Roper (guided by Kirkpatrick & Labavitch):

Cloned one EGase gene and the PG gene

She expressed both the PG gene and the EGase gene in E. coli

Isolated protein from the E. coli and

Demonstrated that the proteins had the predicted enzymatic activities. The PG digested pectin. The EGase digested xyloglucan as well as a cellulose substrate!

Caroline knocked out Xf’s PG gene, creating a PG-less Xf.

Tn903 kan-2

Pgforw Pgrev

metA PG (pglA) Conserved hypothetical

1.6 kB

2.7 kB

MW WT PG-

Wild Type Xf Fetzer PG (-) Xf Fetzer H2O control

Pathogenicity Results18 weeks post-inoculation

Without its PG, X. fastidiosa apparently does not cause PD!

PG is a pectin-degrading enzyme. Pectins are cell wall-localized polysaccharides. What aspect of PG-catalyzed cell wall degradation is important for PD development?

Pit “membranes” are natural filters that normally separate one vessel from neighboring vessels or living parenchyma cells.

From: Taiz and Zeiger “Plant Physiology”

From: Zimmermann “Xylem Structure and the Ascent of Sap”

Pit membranes are really not membranes, they are primary cell walls and, therefore, are likely to contain:

Pectins (PG substrates) and

Xyloglucans (substrates for EGase)

Thus, one might imagine that the roles of X. fastidiosa’s PG and EGase are to break down the pit membranes and thus allow the pathogen to spread throughout a grapevine.

Ca-bound pectin XyloglucanDr. Qiang Sun used monoclonal antibodies to identify some of the grapevine pit membrane polysaccharides.

In fact, grapevine pit membranes do contain pectins and xyloglucan.

Pérez-Donoso and Greve introduced PG and EGase into explanted, healthy grape stems and Sun did the microscopy to show that the enzymes digested holes in pit membranes. (for more PM studies, see Sun et al. poster, #60)

Control Enzyme treated

Pit membrane (face-on view)

UntransformedPD

TransgenicPD

When PD develops on needle-inoculated control vines, symptoms are well developed in ca. 3 months. Symptom development on pear PGIP-expressing transgenics is substantially less.

Controls Transgenics

0

5

10

15

20

25

0 8 24

time

A27

6 n

m Xf PG

Xf PG + PGI P

Roper expressed the Xf PG in E. coli and she and Greve tested the expressed protein for PG activity. It was a PG (based on generation of reducing sugars when digesting pectin) and is inhibited by pear PGIP.

An important aspect of this proposal comes from the work of Agüero et al. (2005):

They reported that the PGIP expressed in transgenic grapes that were used as rootstocks was transported across the graft junction and into non-transgenic scions via the xylem.

Given these observations, the RSAP recommended that a potentially useful strategy for improving grapevine resistance to PD would be to generate PGIP-expressing rootstocks that could mobilize enough PGIP into scions to interrupt PG-assisted systemic movement of Xf.

Objective 1: Define a path for commercialization of a PD control strategy using PGIPs, focusing on IP and regulatory issues associated with the use of PGIPs in grape rootstocks.

Objective 2: Identify plant PGIPs that maximally inhibit X. fastidiosa PG.

Objective 3: Assemble transcription regulatory elements, Xf-inducible promoters and signal sequences that maximize PGIP expression in and transport from roots.

Objective 4: Create PGIP-expressing rootstocks and evaluate their PD resistance.

Objective 1: Define a path for commercialization of a PD control strategy using PGIPs, focusing on IP and regulatory issues associated with the use of PGIPs in grape rootstocks.

PIPRA (the Public Intellectual Property Data base for Agriculture) is led by Co-PI Alan Bennett. He and his colleagues are devising a strategy that involves the use of more readily licensed promoters, vectors etc. for use in transforming grapevines.

This particular advance will be of value in grape germplasm improvement well beyond the scope of our project and problems with PD!

pPIPRA Vectors with Maximum FTO: Marker Excision Vectors

LBGene of Interest

CassetteRB

RRS

RRS

Recombinase Cassette

Selectable Marker Cassette-Cyt Dea

Selectable Marker Cassette-DEF/Atwbc19Recombinase Module

pPIPRA

Bacterial lR

OripVS1

Col

E1

Module: Recombinase, Transposon

For asexually propagated plants (grape, citrus)

Construct allows excision of selectable markers after insertion of gene of interest (selected PGIP). For more vector information, see Bird et al. poster, #54.

1. Selectable markersUniversity of Tennessee,University of Kentucky

3. Constitutive and tissue Specific PromotersUniv California,Cornell Univ., AgriFood Canada, public domain

2. Recombinase systemUSDA and Rockefeller University

System comprised of multiple patented technologies – all from PIRPA members

RRS

Recombinase Cassette

Selectable Marker Cassette-Cyt Dea

Selectable Marker Cassette-DEF/Atwbc19LB

Gene of Interest Cassette

RRS

RB

Identification of useful promoters (Gilchrist, Lincoln et al.). For more information, see Lincoln et al. poster (56).

Identification of optimized signal sequences (Dandekar et al., see Ibañez et al. poster,#42; also Gilchrist et al.).

In addition, we must identify the best PGIPs. That is the PGIPs most effective in inhibiting Xf PG (Poster 58):

Expression of useful amounts of Xf PG (Booth)

Selection and screening of PGIPs (Powell and Chestnut)

Modeling of PG-PGIP interactions and impact on PG activity (King)

Observed phenotype of transformed, uninoculated grapevine

Other than the PGIP biochemical change and impacts on PD susceptibility of scions, we anticipate no observable phenotype. Transformed plants are to be used as rootstocks!

Xf challenge method described and compared to vector inoculation

Tests of potential utility of currently available pear PGIP-expressing transgenics. Data from Agüero et al. do not adequately test the concept of this strategy.

Assay of susceptibility to be used:

GFP-tagged Xf (Lindow et al.)

Use of Inoculation test devised by Gilchrist et al. (uses 1/1000th of the usual Xf inoculum, thus not overwhelming what may be useful levels of pathogen resistance)

Assessment of visible symptoms and Xf titers and movement from the point of inoculation (qPCR)

Comparison of challenged transgenic and non-transgenic grapevines.

In our case, this means challenged scions grafted onto transgenic vs. non-TG rootstocks.

Visible symptoms, Xf titers and spread from inoculation point (GFP-Xf, qPCR)

Xf accumulation and localization in scions on TG vs. non-TG rootstocks as above

Assessment of the value and applicability of the TG approach taken

In addition to the specific issue of PD susceptibility of scions on the TG rootstocks, evaluations of the viticultural characteristics of the grafted vines and grape/wine quality must be made.

Other potential benefits and spin-offs:PGIP has been shown to provide pathogen protection (gray mold: B. cinerea), may provide protection against insect pests, nematodes“Rootstock strategy” may be applicable for control of Xf-caused diseases of other important crops