Disease
TM
Block :
Genetically Engineered Plants with Disease Resistance
Chandrika Ramadugu and Dean W. Gabriel, Integrated Plant Genetics, Inc., 12085 Research Drive, Alachua, FL 32615
NSF Phase II Award # 011131
Introduction
Abstract
Citrus production for the major producing
countries in the world is estimated at
approximately 62 million tons or $5.878
billion. One of the leading diseases
affecting this industry is citrus canker,
which seriously limits citrus production in
Asia and S. America (among other places)
and now threatens Florida citrus. In
heavily infested areas, 50% or more of the
fruit fail to develop and fall from the tree
prematurely. Canker causes such losses to
grapefruit, sweet orange and lime that
these simply cannot be grown in many
parts of Asia and the Middle East. There is
no cure, and resistance cannot be
genetically introgressed by breeding.
Citrus
canker
results
in
premature
fruit-drop
(left).
A potentially food-grade technology for controlling plant pathogens involves cloned single chain
variable region antibody fragments (SCFVs) expressed in plants (“plantibodies”). We obtained
monoclonal antibodies (MAbs) that had been selected to bind two functionally significant domains of
a pathogenicity protein required for citrus canker disease. The pathogenicity protein is literally
injected by the pathogen into citrus cells to cause canker. The MAbs were used to clone the
corresponding SCFVs in various combinations to form monovalent, bivalent and bispecific SCFVs.
The SCFV clones were used to transform grapefruit seedlings. Initial tests on some of the SCFVs
indicated that they delayed and suppressed canker symptoms but did not confer immunity. However,
Western blot analyses revealed that the translational expression levels were below the threshold limit
of detection.
Material and Methods
IPG's Disease Block technology is based on expression of
antibody fragments (ScFvs) in transgenic plants. The ScFvs
are directed against a protein signal, PthA, that is injected by
the bacterial pathogen into the plant cell cytoplasm, and is
then translocated into the plant cell nucleus to cause disease.
Results
Over 50 putative transformed grapefruit seedlings
were selected on medium containing kanamycin.
IPG NSF Phase II Project: citrus canker resistance
Plants are genetically altered express the ScFvs by use of
Agrobacterium tumefaciens to inject a piece of engineered DNA
into the citrus genome. Grapefruit cultivar "Flame" was
used. Southern, Northern and Western blot analyses were
performed to confirm transformation and gene expression.
4 weeks --- 26% “flame” grapefruit survive;
1/2 of these are transformed
The Molecular Basis of Canker Disease:
Disease Block TM
1. Contagious canker bacteria (Xanthomonas citri)
enter through stomata or wounds.
Plant Cell
Microbe
2. They contact the plant cell wall and draw tight.
Avr/Pth +
?
3. They inject a protein signal, PthA, that causes cell
division and some epidermal cell death.
Nuclear Import of
Avr/Pth Protein
hrp
genes
4. The loss of intercellular space creates a capillary
effect to draw water from xylem.
-pH
-Salt
-Plant
Signals
-Nutrient
level
5. Xanthomonas makes xanthan gum, which absorbs
water and swells, allowing egress from the
epidermis.
Nucleus
Intercept
Block Function
Program m ed
Cell Response
--Division
--Host HR
--Nonhost HR
--Water soaking
(nutrient export?)
6. Wind-blown rain, high pressure sprays, insects &
people do the rest.
Surviving seedlings were then grafted onto nontransgenic rootstock, allowed to grow for at least six
months, and then evaluated for transformation by
PCR, Southern blots and Northern blots (not shown).
Most DNA insertions were single copy events, and most
plants with inserts showed good transcriptional levels
of the transgenes (~60%). Transformants were then
challenged by inoculations with the citrus canker
pathogen. A few plants exhibited resistance, as shown:
Canker challenge test: 16 days post-inoculation
ScFv Gene Constructs and Protein Products
VL
P
L
VHA
Monovalent, monospecific
P
VH
ScFv
P
VL
VHB
L
Control—(Duncan grapefruit)
VLA
P
L
Results and Discussion
When challenged by inoculation, several transformants exhibited
canker resistance as shown, but the resistance was gradually, if
only partially, overcome by the canker pathogen. The resistant
plants did not appear to be overcome by fast-growing pathogenic
variants, but rather the resistance simply appeared to slow the
disease process. Western blot analyses revealed that the level of
translation of all ScFv transgenes was below the threshold limit
of detection.
The expression levels of ScFvs are often below the threshold limit
of detection in plants, an issue that has recently been addressed
in various ways, including addition of promoter enhancer
sequences, targeting or retaining the ScFv to the endoplasmic
reticulum, and also use of full-length MAbs in place of the ScFvs
(De Jaeger et al., 1999; Vine et al., 2001; Xu et al., 2002). Fulllength MAbs have the advantage of a greater avidity of binding
than the ScFvs, but they tend to accumulate in the apoplastic
space (outside the cell wall) or in the plant cell membrane,
neither of which is suitable for the interception of the
pathogenicity signal protein, PthA. ScFvs have the advantage of
being expressed in the cytosol, where they have the best chance of
encountering and blocking PthA.
We are currently in the process of performing Biacore analyses
to assess the binding avidity of the full length MAbs as compared
to the ScFvs. We are also currently engineering new gene
constructs to increase translation of the ScFvs in the cytosol.
Vine, N. D., P. Drake, A. Hiatt, and J. K. C. Ma. 2001. Assembly and plasma
membrane targeting of recombinant immunoglobulin chains in plants with a
murine immunoglobulin transmembrane sequence. Plant Molecular Biology
45:159-167.
VLB
L
At least 2,000X fewer X. citri
cells released
De Jaeger, G., E. Buys et al., 1999. High level accumulation of single-chain
variable fragments in the cytosol of transgenic Petunia hybrida. European
Journal of Biochemistry 259:426-434.
Bivalent, bispecific
If X. citri cannot break the epidermis,
it cannot spread.
Control Grapefruit
5,000 cfu/ml
References
Bivalent, monospecific
VH
Disease BlockTM
Grapefruit
(J605)
50,000 cfu/ml
X. citri, 5,000 cfu/ml
Disease BlockTM--(Flame grapefruit)
Xu, H., F. U. Montoya, et al.,. 2002. Combined use of regulatory elements
within the cDNA to increase the production of a soluble mouse single-chain
antibody, scFv, from tobacco cell suspension cultures. Protein Expression and
Purification 24:384-394.