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ORIGINAL ARTICLE
Siela N. Maximova Æ Jean-Philippe Marelli Æ Ann Young
Sharon Pishak Æ Joseph A. Verica Æ Mark J. Guiltinan
Over-expression of a cacao class I chitinase gene in
Theobroma cacao
L.
enhances resistance against the pathogen,
Colletotrichum
gloeosporioides
Received: 1 September 2005 / Accepted: 16 November 2005 / Published online: 16 December 2005
Springer-Verlag 2005
Abstract Theobroma cacao L. plants over-expressing a
cacao class I chitinase gene (TcChi1) under the control
of a modified CaMV-35S promoter were obtained by
Agrobacterium-mediated transformation of somatic
embryo cotyledons. Southern blot analysis confirmed
insertion of the transgene in eight independent lines.
High levels of TcChi1 transgene expression in the
transgenic lines were confirmed by northern blot anal-
ysis. Chitinase activity levels were measured using an in
vitro fluorometric assay. The transgene was expressed at
varying levels in the different transgenic lines with up to
a sixfold increase of endochitinase activity compared to
non-transgenic and transgenic control plants. The in
vivo antifungal activity of the transgene against the fo-
liar pathogen Colletotrichum gloeosporioides was evalu-
ated using a cacao leaf disk bioassay. The assay
demonstrated that the TcChi1 transgenic cacao leaves
significantly inhibited the growth of the fungus and the
development of leaf necrosis compared to controls when
leaves were wound inoculated with 5,000 spores. These
results demonstrate for the first time the utility of the
cacao transformation system as a tool for gene func-
tional analysis and the potential utility of the cacao
chitinase gene for increasing fungal pathogen resistance
in cacao.
Keywords Fungal pathogen resistance Æ Theobroma
cacao Æ Chitinase Æ Transgenic Æ Colletotrichum
gloeosporioides
Introduction
Theobroma cacao L. (cacao) is a small understory tree
endemic to the lowland rainforests of the Amazon
basin (Wood and Lass 1987). Cacao was domesticated
in pre-Columbian times by the Olmec and/or Mayan
civilizations. Its seeds (cocoa beans) were used to pro-
duce beverages for the royalty and in religious cere-
monies (Coe and Coe 1996; Motamayor et al. 2002;
Emch 2003), and also as currency. Today, cacao is
grown throughout the world in the humid tropics, of-
ten in agroforestry ecosystems with other fruit and
commodity crops. Cacao is the main raw ingredient for
the world chocolate indust ry, which is estimated to be
approximately $58 billion per year. Additionally, ca-
cao-growing regions are largely centered in important
biodiversity hotspots, impacting 13 of the world’s most
biologically diverse regions (Piasentin and Klare-Rep-
nik 2004).
For a number of reasons, long-term efforts toward
breeding for disease resistance in cacao has yielded only
limited success. First, the long generation time an d large
size of cacao plants make it difficult to conduct the
necessary long-term and multi-located field trials.
Additionally, because many of the cacao pathogens are
opportunistic, sources of resistance have not co-evolved
for some of the most devastating of the major cacao
diseases. More recently, researchers have begun to apply
the tools of molecul ar genetics to develop genomic re-
sources and molecular markers to help speed up cacao
breeding programs (Bennett 2003). As a tool to study the
functional genomics of cacao candidate res istance genes
and as a possible means to integrate resistance genes into
breeding programs, we have recently developed a genetic
transformation system for cacao (Maximova et al.
2003). The system employs Agrobacterium-mediated
transformation of cotyledon pieces from somatic em-
bryos and a visible marker gene encoding the green
fluorescent protein (GFP) for rapid screening of primary
regenerants. As a first test of this system, we chose to
S. N. Maximova Æ A. Young Æ S. Pishak Æ J. A. Verica
M. J. Guiltinan (&)
The Department of Horticulture, The Pennsylvania State
University, University Park, Chester, PA 16802, USA
E-mail: mjg9@psu.edu
Tel.: +1-814-8637957
Fax: +1-814-8636139
J.-P. Marelli
The Department of Plant Pathology, The Pennsylvania State
University, Chester, PA 16802, USA
Planta (2006) 224: 740–749
DOI 10.1007/s00425-005-0188-6
examine the function of a cacao chitinase gene, by over-
expressing it in transgenic cacao plants and testing the
effect on fungal pathogen resistance.
Chitinases are members of the pathogenesis-related
protein family (PR-proteins), some of which have been
shown to play a role in plant defense by degrading the
chitin of fungal cell walls (Collinge et al. 1993; Neuhaus
1999). In particular, the class I chitinases, which accu-
mulate to high levels in vacuoles in response to wounding
and pathogen infection, have been reported to be
important in these regards. Using transge nic approaches,
chitinase genes from plants and microorganisms have
been introduced into different plant species in order to
enhance resistance against a broad range of fungal
pathogens (Kramer and Mut hukrishnan 1997). Tobacco
has been transformed with chitinases from various other
plants and microorganisms with varied results (Linthorst
et al. 1990; Jach et al. 1995; Vierheilig et al. 1995; Kell-
mann et al. 1996; Carstens et al. 2003; Patil and Widholm
1997). Br oglie et al. (1991) reported enhanced resistance
to Rhizoctonia solani in tobacco plants expressing a bean
chitinase. Other crops transformed with plant chitinases
that resulted in enhanced fungal pathogen resistance in-
clude tomato (Tabaeizadeh 1997), carrot, cucumber,
pickling cucumber (Punja and Raharjo 1996; Raharjo
et al. 1996), rose (Marchant et al. 1998), rice (Liu et al.
2004), and grape (Yamamoto et al. 2000). Additionally,
tobacco and potato (Lorito et al. 1998), broccoli (Mora
and Earl 2001), and apple (Bolar et al. 2001) were
transformed with an endochitinase derived from the
parasitic fungus Trichoderma harzianum and these plants
exhibited wide-spectrum resistance to the foliar patho-
gens Alternar ia alternata, A. brassicae, A. solani, Botrytis
cinerea, R. solani, and Ven turia inaequalis.
Previously we have reported the regeneration of
multiple transge nic lines carryi ng a class I endochitinase
gene isolated from cacao (Maximova et al. 2003). The
cacao TcChi1 gene (NCBI accession U30324) was iso-
lated by PCR based on tobacco chitinase sequences and
shown to be expressed in cacao fruit in response to fungal
elicitor treatment (Snyder-Leiby and Furtek 1995). Here,
we report on the evaluation of these plants and demon-
strate the enhanced fungal pathogen resistance to Col-
letotrichum gloeosporioides conferred by the chitinase
gene. For the purpose of this study we have developed an
in vivo bioassay using detached young leaves of T. cacao
infected with C. gloeosporioides spores. The symptoms
observed in laboratory conditions were similar to those
observed on infected cacao plants in field conditions,
where C. gloeosporioides attacks primarily young and
soft caca o leaves causing brown lesions surrounded by a
characteristic clear yellow halo (Mohanan et al. 1989).
Our results indicated that the cacao TcChi1 gene
product is active against this fungal pathogen, and that
it is possible to increase the resistance of cacao against
pathogens by over-expression of this gene. Furthermore,
our results demonstrate that the transgenic cacao system
can be used as a tool to study the functions of cacao
genes and perhaps, in the future, for crop improvement.
Materials and methods
Genetic transformation
Plant transformations were performed as previously
described (Maximova et al. 2002, 2003) via Agrobacte-
rium tumefaciens co-cultivation with cacao somatic em-
bryo explants . All transformations were performed with
the cacao genotype PSU-Scavina 6 (S6). S6 plants were
established in a greenhouse at Penn State in 1993 from
clonal material introduced from Mayaguez, PR, USA.
DNA fingerprinting has shown that this clone is not
identical to the bona fide Scavina 6 clone at the Inter-
national Cacao Germplasm collection in Trinidad, so we
have appended the designation PSU to distinguish it
from authentic Scavina 6 (M. Guiltinan, unpubli shed).
For brev ity, we also refer to PSU-Scavina 6 as S6 in this
text. This clone was used as source for explants during
our prior research on somatic embryogen esis and
transformation protocol development (Maximova et al.
2002, 2003 ). The genotype demonstrated the highest
somatic embryogenesis and genetic transformation po-
tential compared to a number of other genotypes. In
brief, staminodes dissected from immature cacao flowers
of S6 were cultured to produce somatic embryos by
methods previously described (Li et al. 1998; Maximova
et al. 2002). Cotyledons from primary somatic embryos
were co-cultivated with A. tumefaciens (AGL1) con-
taining binary plasmids (described below) and further
cultured on 50 mg/I geneticin selection for 2 weeks un-
der conditions conducive for secondary embryogenesis
(Maximova et al. 2002). Transgenic secondary somatic
embryos were selected based on green fluorescence
resulting from the expression of the enhanced green
fluorescence protein (EGFP) marker gene (Clontech,
Palo Alto, CA, USA).
Transformations were performed with pGAM00.0511
(Maximova et al. 2003) (Fig. 1), which contains the ca-
cao TcChi1 chitinase gene (Snyder 199 4; Snyder-Leiby
and Furtek 1995), EGFP, and the neomycin phospho-
transferase II (NPTII) (De-Block et al. 1984) marker
genes, all under the cont rol of high-level constitutive
E12-X CaMV-35S promoter (Mitsuhara et al. 1996). One
control transgenic line was regenerated after transfor-
mation with pGH00.0126 containing the EGFP and
NPTII marker genes only (Maximova et al. 2003).
Additionally non-transformed S6 plants were regener-
ated by somatic embryogenesis (Maximova et al. 2002)
and used as non-transformed controls.
Clonal propagation of transg enic and control plant lines
The individual lines described here originate from
independent transformation events. Multiple individuals
of each line were obtained either through repetitive so-
matic embryogenesis (Maximova et al. 2002) or through
rooted cuttings (Maximova et al. 2005). All control and
741
transgenic plantlets were acclimated to greenhouse
conditions and multiplied by rooted cuttings as previ-
ously described (Maximova et al. 2005). Briefly, single
leaf, semi-hardwood cuttings, with stems approximately
4 cm long, and with leaves pruned to one-third of the
initial length were treated with rooting solution [1:1
mixture of a-naphthalene acetic acid (NAA) and a-in-
dole-3-butyric acid (IBA)]. After hormone treatment,
the cuttings were inserted in wet sand and placed under
intermittent mist (10 s every 10 or 15 min) for 4–6 weeks
at a light inte nsity of approximately 100 lmol/m
2
/s PAR
(85% shade). During the mist ing period the cuttings
were fertilized every 3–4 days with Hoagland’s nut rient
solution (160 ppm N). Rooted cutting with two or more
roots and a growing axillary bud were transplanted to
pots with soil. After transplanting, the plants were re-
moved from the mist and maintained under 50% shade
and a relative humidity of 60–65% at approxim ately
28C. Water an d fertilizer were applied five or more
times daily as needed by drip irrigation. Plants were
grown in the greenhouse for 7–9 months before analysis.
To collect tissue samples for the different assays, 2–6
trees per individual line were used.
Fluorescent imaging of GFP
Fluorescent GFP images of transgenic leaves were ob-
tained as previousl y described (Maximova et al. 2003).
Images were captured using a Nikon SMZ-4 stereo-
dissecting microscope equipped with an epi-fluorescence
attachment, a 100 W mercury light source, and a 3-CCD
video camera system (Optronics Engineering , Goleta,
CA, USA). The fluorescence imaging filters used were a
520–560 nm emission filter and a 450–490 nm excitation
filter. Fluorescence intensity of each image was mea-
sured as the mean integrated pixel value (mIPV) using
NIH Image 1.6 image processing and analysis software.
Mean IPV (ranging from 0 to 255 each) was calculated
by dividing the sum of the intensity values of all pixels in
a given image by the total number of pixels in that im-
age. To ensure equal imaging conditions of all mea-
surements, all images were acquired at 30·
magnification with a 30 s exposure time with no changes
in lighting setup. Using a set of representative samples
ranging from the lowest to the brightest fluorescence,
exposure time was determined to result in a minimum of
99% of all pixels below the saturation point (255) in the
brightest image. Three individual leaves from three trees
per line were measured for a total of nine measurements
per line. The means of replicate mIPV ± SE measur e-
ments were calculated and significant differences were
established by Fisher Protected LSD test at the P<0.05
level of significance.
Southern genomic blot analysis
Genomic DNA was isolated from mature cacao leaves
as previously described using a modified CTAB extrac-
tion protocol for latex-containing plants (Michiels et al.
2003) and additionally purified by CsCl gradient ultra-
centrifugation (Ausubel et al. 2001). Per tissue sample,
5 lg of genomic DNA was incubated overnight with 60
units of EcoRI or 50 units of SphI (Promega, Madison,
WI, USA). DNA was separated on 1% agarose gels and
transferred to nylon membranes (Hybond-N+, Amer-
sham Bioscience, Piscataway, NJ, USA) in 10· sodium
chloride/sodium citrate (SSC). Following transfer, DNA
was fixed to the membranes via UV crosslinking
(120 mJ). Me mbranes were prehybridized in Expres-
sHyb solution (Clontech) supplemented with 100 lg
salmon sperm DNA for 3 h at 60C. The blots were
hybridized with 1.2 kb TcChi1 gene probe (Fig. 1) gen-
erated by PCR from pGAM00.0511 as described by
Maximova et al. (2003). Using Megaprime DNA label-
ing kit (Amersham Bioscience), 50 ng of the probe was
labeled with a-32P-dCTP, denatured by boiling, quen-
ched on ice, and added directly to the prehybridization
mix. Hybrid izations were carried out at 60C for 20–
24 h. Membranes were washed twice at 55Cin2· SSC,
0.5% sodium dodecyl sulfate (SDS) for 20 min, and
twice at 55Cin1· SSC, 0.5% SDS for 20 min.
Radiographic imaging was performed via storage
phosphorimaging (Molecular Dynamics, Sunnyvale,
CA, USA).
Northern blot analysis
Total RNA was extracted from mature cacao leaves
using a modified protocol for highly viscous samples rich
in polyphenols and polysaccharides (Zeng and Yang
2002). Additionally, RNA wa s purified following the
Qiagen RNeasy clean up protocol (QIAGEN, Valencia,
E12Ω
pro
Cacao
Chitinase
CaMV
35S 3
,
E12Ω
pro
NPT II
CaMV
35S 3
,
E12Ω
pro
EGFP
CaMV
35S 3
,
RB LB
SphI
SphI
SphI
SphI
EcoRI
EcoRI
EcoRI
EcoRI
EcoRI
T-DNA region of pGAM00.0511
______
Hybridization
probe (1.2 Kb)
2.6 0.3 0.7 1.0 0.3 1.4 0.3
Fig. 1 Illustration of the T-DNA region of pGAM00.0511 binary
plasmid containing the TcChi1 transgene (Maximova et al. 2003).
Components of this vector include: E12-X 35S promoter (Mitsu-
hara et al. 1996), the cacao genomic DNA sequence encoding a
basic chitinase class I (TcChi1) (Snyder 1994; Snyder-Leiby and
Furtek 1995), the CaMV-35S terminator sequence, the NPTII
kanamycin selectable marker gene (De-Block et al. 1984), and the
EGFP gene encoding green fluorescent protein (Clontech). The
modified CaMV-35S derivative, E12-X promoter, drives all
transgenes. Only restriction enzyme recognition sites important
for the Southern Blot analysis (Fig. 1) are shown at the top of the
map and are abbreviated as follows: E, EcoRI; Sh, SphI. The
1.2 kb restriction fragment containing the TcChi1 coding region of
the cacao endochitinase (underlined) was used as probe for the
Southern and northern analyses. RB and LB indicate the right and
left borders of the Ti plasmid T-DNA region, respectively. Number
of base pairs between restriction sites is indicated above the map in
kb. The drawing is not to scale to allow labeling
742
CA, USA). Twenty micrograms of total RNA was
denatured for 30 min at 55C with 1:1 (v/v) sample and
glyoxal/DMSO loading dye (Amersham Bioscience).
RNA samples were separated on 1.5% agarose gels,
transferred to nylon membranes (Hybond-N+, Amer-
sham Bioscience) in 10· SSC, and fixed to the mem-
branes via UV crosslin king (120 mJ). The probe
preparation for the northern blot was identical to that
for the Southern blots describe d above. Probe labeling,
hybridization, washing, and radiographic imaging were
also performed as described above. Sizes of hybridizing
fragments were determined by comparison with mobility
of known RNA markers (RNA ladder, Ambion, Inc.,
Austin, TX, USA). Hybridizing band intensity was
quantified by phosphorimaging (Molecular Dynamics,
Amersham Bioscience) using ImageQuant software
(Molecular Dynamics, Amersham Bioscience). All
measurements were within the linear range of the
phosphorimager.
In vitro endochitinase activity assay
Total protein extracts from two leaves from two differ-
ent trees per line (total of four extracts/line) were ex-
tracted as follows. Fr ozen leaves were placed in liquid
nitrogen with polyvinyl pyrrolidone (PVP) (0.07 gm of
PVP per gm of leaf tissue) and ground to a fine powder.
The powder was then added to ice-cold extraction buffer
[50 mM sodium acetate, pH 5.0, 2 mM EDTA, 3 mM
sodium metabisulfite, 5 mM dithiothreitol (DTT), and
250 mM sodium chloride, 7 ml per gm of leaf tissue] in
sterile 30 ml Oakridge tubes, then swirled briefly to
suspend the powder. Samples were centrifuged at
10,000g at 4C for 15 min and the supernat ants trans-
ferred to fresh tubes. Total protein content was quanti-
fied using Bio-Rad standard protein microassay (Bio-
Rad Laboratories, Richmond CA, USA). Endochitinase
activity was quantified using a fluorometric assay (Mora
and Earl 2001 ). For each individual sample, the assay
contained 20 lg of total protein in a final volume of
100 ll. To the extracts, 50 ll of substrate solution
[0.2mM methylumbelliferyl B-
D-N,N¢,N¢¢-triacetylchito-
trioside (Sigma M-5369) in 100 mM sodium acetate, pH
5.0] was added and reactions were incubated on an
orbital shaker (200 rpm), at 37C for 40 min after which
50 ll of STOP buffer (0.2 M sodium carbonate) was
added. Each sample was assayed in triplicate. Back-
ground fluorescence of each sample was determined by
assays identically prepa red except that the STOP buffer
was added prior to the 37C incubation. Standard curves
were determined using a series of dilutions of 1 lM4-
methylumbelliferone (4-MU) (Sigma M-1508) in 0.2 M
sodium carbonate (STOP buffer) prepared by the man-
ufacturer’s instructions. All treatment and control
samples, including the standard curve dilution set, were
incubated together in a black, 96-well microtiter plate
(#3650, Corning, Inc., Corning, NY, USA). Fluores-
cence generated by reaction byproduct (4-MU) was re-
corded at 360/485 gm ex/em with a fluorescence plate
reader (FluoroCount Packard BioScience, Meriden, CT,
USA) using the FluoroCount software package. To
normalize the fluorescence intensity value of each sam-
ple, the respective background fluorescence values were
subtracted. Based on the standard curves, endochitinase
activity was calculated as nmols of 4-MU generated per
min per lg of total protein. Means of all technical and
biological replica tes were calculated and variation was
established by paired comparisons among all possible
pairs using Fisher Protected LSD test at the P<0.05
level of significance.
In vivo evaluation of antifungal activity of transgenic
cacao plants against C. gloeosporioides
Colletotrichum gloeosporioides was isolated from in-
fected roots of a 2-month-old S6 cacao plant. The
identity of the pathogen was determined by culture
morphology on PDA media and sequence homology of
the ITS region to reference sequences of isolates of C.
gloeosporioides available from NCBI database (J.-P.
Marelli, data not shown). C. gloeosporioide s single spore
cultures were grown on 2% water agar for 2–4 weeks at
30C under a 12:12 h day:night cycle. When acervul i
were formed on the entire surface of the plate, 3 ml of
sterile water was poured over the culture and the conidia
were harvested by scraping the surface of the culture
with a glass rod. The resulting conidia suspension was
filtered through sterile miracloth (Calbiochem, La Jolla,
CA, USA) and diluted to 500,000 conidia/ml with sterile
distilled water.
For each bioassay, two large fully expand ed light
green and soft cacao leaves were collected from control
and transgenic lines established in the greenhouse from
rooted cuttings (corresponding to stage C leaves as de-
fined in the Guiltinan lab, http://guiltinan-
lab.cas.psu.edu/Research/Cocoa/leaves/stages.jpg). All
leaves collected were 7–10 days old and appeared to be
at the same developmental stage. Leaf discs were cut to
fit into 100·15 mm
2
Petri dishes (VWR, West Chester,
USA) containing two layers of sterile filter paper soaked
in 2.5 ml steri le distilled water. Ten areas (1.5 mm
2
)
were sampled on each leaf (five on each side of the main
vein). Immediately before inoculation, the selected areas
were wounded with eight insect pin needles bunched and
taped together. Single drops of 10 ll of inoculum (5,000
conidia) were placed on eight of the ten wounded areas
and the last two areas were left as water-only controls
without infection. Wounding and inoculation steps were
performed under a magnifying lens to improve visuali-
zation. Plates were incubated at 30C in a 12:12 h
light:dark cycle under fluorescent light for 4 days. Ima-
ges of the cont rol and infected areas after 4 days of
incubation were captured using Nikon SMZ-4 dissecting
microscope and a 3-CCD video camera system (Op-
tronics Engineering). The area of necrosis was measured
using Scion Image for Windows (version beta 4.0.2,
743
Scion Corporation). The average area was calculat ed
and the data were statistically analyzed using the SAS
software (version 9.1, SAS Institute, Inc., Cary, NC,
USA). The mean areas of necrosis per genotype were
separated using a t test LSD.
Results
The objective of this study was to evaluate the potential
function of the TcChi1 gene product in pathogen defense
and specifically to determine if it plays a role as an
antifungal defense protein. This gene was chosen for the
study because it was previously described to be ex-
pressed in fruit pericarp in response to wounding and
fungal cell wall elicitors (Snyder 1994). Hence it is likely
to be an important pathogenesis response protein in
cacao, although a direct functional analysis was not
demonstrated. We studied the function of TcChi1 gene
by constitutively over-expressing it at lev els much higher
than the endogenous gene, using a very highly active,
modified vira l promoter. Furthermore, we reasoned that
in the future, acceptance of transgenic cacao by the
public could be facilitated if cacao genes were reintro-
duced into cacao, rather than using genes isolated from
a different species.
Generation of transgenic plant lines
Co-cultivation was carried out with cacao somatic em-
bryo cotyledons and A. tumefaciens strain AGL 1 con-
taining binary vectors with the EGFP and NPTII genes
with and without the cacao TcChi1 gene (Fig. 1).
Transgenic somatic embryos from nine independent
transformation events were cultured to maturity, then
converted to plants, and acclimated to greenhouse con-
ditions. Rooted cuttings of the T0 plants were used to
produce plants (clonal lines) for replica ted experiments.
Consistent with our previous report (Maximova et al.
2003), no visible phenotypic differences were observed
between the TcChi1 transgenic plants and control
transgenic plants (without the TcChi1 gene) or non-
transgenic S6 control plants.
GFP fluorescence intensity variation in transgenic cacao
As an early screen to identify high-level EGFP-express-
ing plants, the intensity of green fluorescence in mature
leaves from different transgenic lines was measured and
compared to that of cont rol leaves (Fig. 2). All plants
evaluated were propagated via somatic embryogenesis
and grown for 9–10 months after acclimation to green-
house conditions. Consistent with our previous obser-
vations, the fluorescence intensity of individual leaf
samples varied significantly among the transgenic lines
(Maximova et al. 2003). The intensities, expressed as
mean integrated pixel values, ranged from 25.1±SE
2.4 mIPV for line 61.8 which was only slightly above the
background (14.8 ±SE 0.045) seen in non-transgenic
plants, to 215±SE 15.8 mIPV for line 47.1. One EGFP-
expressing line containing the EGFP and NPTII genes
only (Fig. 2, GFP lacking the TcChi1 gene) was selected
as a control and it showed EGFP expression at levels
similar to the highest TcChi1-containing line.
Transgene integration and expression
To investigate T-DNA integration and copy number in
the GFP-expressing plants, genomic DNA from one
control non-transformed S6 and all transgenic lines were
evaluated by Southern blot hybridization (Fig. 3a, b).
As a control, plasmid pGAM00.0511 DNA containing
the TcChi1 gene was diluted to equimolar equivalents
and included in the analysis (Fig. 3a, b). DNA was di-
gested with EcoRI (Fig. 3a) and SphI (Fig. 3b), blotted,
then hybridized to the 1.2 kb cacao TcChi1 probe
(Fig. 1). In all plant genomic DNA samples, the
endogenous TcChi1 gene was detected as a large frag-
ment above 12.2 kb (Fig. 3a, b). Only this single band
was detected in the control S6 (Fig. 3a) and GPF lines
(Fig. 3a, b), consistent with the previous report by
Snyder (1994), concluding that the TcChi1 gene was
single copy. All but one of the transgenic plant lines
(56.2) transformed with the TcChi1 gene vector exhib-
ited additional hybridization bands indicating the pres-
ence of the transgene (Fig. 3a, b). Line 44.1 digested
with EcoRI showed no hybridizing bands (Fig. 3a), but
the digest with SphI resulted in a 3 kb hybridizing band
Relative GFP Fluorscence
(Mean Intergrated Pixel Value)
S6
GFP
31.1
39.2
44.1
47.1
55.1
55.3
61.8
Genotype (Transgenic Line #)
0
25
50
75
100
125
150
175
200
225
250
Fig. 2 Evaluation of EGFP fluorescence of control and transgenic
cacao leaves. Three young fully expanded light green leaves from
each of three trees (total of nine leaves per line) were observed
under a fluorescence stereomicroscope equipped with a digital
CCD camera. The fluorescence intensities of the digital images were
measured and expressed as the mean integrated pixel values
(mIPV). Mean IPVs ± SE of control and transgenic leaves were
calculated. The genotypes measured were: PSU-Scavina 6 (S6),
control untransformed plants of identical genotype used for all in
transformations, EGFP, control plants transformed with the EGFP
and NPTII genes only (pGH00.0126, Maximova et al. 2003), and a
series of transgenic plant lines, indicated with various numbers,
which contained the EGFP, NPTII, and the TcChi1 genes
744
(Fig. 3b). From the map of TcChi1 (Fig. 1), EcoRI was
expected to cut in the middle of the T-DNA, 2.9 kb
away from the right border, and SphI was predicted to
cut 1.9 kb away from the right border. The analysis of
the SphI blot resulted in the discovery of additional SphI
site on pGAM00.0511 DNA outside of the T-DNA
borders that generated a 3 kb hybridizing fragment.
This fragment was detected in seven of the eight
chitinase transgenic lines analyzed, suggesting transfer
and insertion of a portion of the plasmid backbone
DNA into the plant geno me. Similar results have been
reported by a number of authors who also observed
transfer of DNA outside of the T-DNA borders
(Kononov et al. 1997; Matzke and Matzke 1998). To-
gether, these results indicate that each of the transgenic
lines contain the TcChi1 transgene, although in most
cases it appears that the integration sites may include
multiple and, possibly, longer than expected insertions.
In order to assess the expression of the TcChi1 gene
in the transgenic plants, northern blot analysis was
performed with total RNA isolated from leaves
(Fig. 3c). No TcChi1 transcript was detected in the
control S6 and GFP plants, suggesting that the TcChi1
gene is not expressed in cacao leaves at levels detectable
by this method. However, high levels of the transgene
mRNA were observed in transgenic TcChi1 plants, with
the exception of line 55.3.
Increased levels of chitinase activity in TcChi1
transgenic plants
In order to evaluate if TcChi1 over-expression contrib-
uted to an increase of chitinase activity in the leaves of
the transge nic plants, an in vitro endochitinase activity
assay was used (Fig. 4). Light green, opaque, fully ex-
panded (longer than 4.5 in.) leaves were harvested from
all plants and total protein was extracted. S6 and GFP
lines were used as controls. The chitinase activity levels
of the two co ntrols were detectable, but were very low
and not significantly different from each other, indicat-
ing the endogenous chitinas e activity in cacao leaves.
Protein extracts isolated from the TcChi1 transgenic
leaves exhibited a three to seven fold increase in total
chitinase activity compared to the controls. All of the
TcChi1-containing plants showed statistically significant
increases in chitinase activity compared to control plants
(P<0.001) (Fig. 4).
In vivo evaluation of antifungal activity of transgenic
cacao plants against C. gloeosporioides
To assess the in vivo functionality of the TcChi1 protein
in the transgenic lines, we developed an excised leaf
challenge assay aga inst the pathogenic fungus C. gloeo-
sporioides. This species has been reported as a pathogen
of economic importance for cacao in India and Vene-
zuela (Wood and Lass 1987). The strain of C. gloeo-
sporioides used in this study was isolated from infected
cacao plants at The Pennsylvania State University,
University Park, PA, USA. When young cacao leaves
were wound inoculated with this strain, they exhibited
pathogenicity, severe lesions and necrosis in 4 days
Fig. 3 Southern (a and b) and northern (c) analysis of DNA and
RNA isolated from leaf tissue of control and transgenic cacao
plants. Nucleic acids were isolated from leaves of plants as follows:
lane 2 non-transgenic PSU-Scavina 6 (S6), lane 3 transgenic plants
transformed with pGH00.0126 control vector without chitinase
(GFP) (Maximova et al. 2003), lanes 4–11 eight transgenic lines
transformed with pGAM00.0511 (Maximova et al. 2003; Fig. 1)
(lines 31.1, 39.2, 44.1, 47.1, 55.1, 55.3, 56.2, and 61.7 in lanes 4–
11,respectively). For the Southern analysis, DNA from
pGAM00.0511 (lane 1) and genomic DNA from transgenic and
non-transgenic cacao leaves were digested with EcoRI and SphIin
different reactions (a and b, respectively). The blots were hybridized
to the TcChi1 genomic restriction fragment (see Fig. 1). In b, the S6
control was not included. Sizes and positions of molecular weight
markers are indicated on the right side of the gels in kb. c Northern
blot analysis. Total RNA (20 lg) isolated from all plant samples
was separated by electrophoresis and blotted onto nylon mem-
branes, followed by hybridization with the same TcChi1 probe as
the Southern blots (see Fig. 1). The gel was stained with ethidium
bromide prior to transfer (top panel) and autoradiogram was
captured by phosphorimager analysis after hybridization (bottom
panel). Size of TcChi1 transcript is indicated (1.6 kb)
745
(Fig. 5d, e). When leaves from S6 and GFP control
plants were inoculated, we observed rapid formation of
lesions (Fig. 5d, e) compared to mock inoculated leaves
(Fig. 5a, b). On the contrary, plants over-expressing the
TcChi1 gene showed reduced necrosis after inoculation
(Fig. 5f) compared to the inoculated control plants
(Fig. 5d, e). Lesion formation on the transgenic plants
was similar to mock inoculat ed control and transgenic
plants. To measure lesion sizes, digi tal ima ges were ta-
ken an d image analysis was performed. The mean lesion
sizes of multiple replicates are presented in Fig. 6. The
analysis established that the transgenic plants repro-
ducibly showed enhanced resistance, as measured by the
significantly smaller lesion formation compared to either
of the control plant lines (Fig. 6). The reduction of the
lesion size correlated with the increase of in vitro protein
activity (Fig. 6). Two of the lines with the least chitinase
activity showed the least reductions in lesion sizes, while
two lines with much higher levels of chitinase activity
showed the smallest lesion sizes. Taken together, our
results indicate that the high expression of the TcChi1
gene in the transgenic plants contributes to enhancing
resistance to infection in our in vitro assay.
Discussion
Fungal diseases constitute a major challenge to the mil-
lions of cacao farmers throughout the tropical regions
where T. cacao is grown. Despite efforts at breeding for
resistance, a large proportion of the potential cacao crop
is lost yearly to several major pathogens. Understanding
mechanisms by whic h cacao responds to pathogen
infection could lead to the development of molecular
markers of key defense genes, which could be applied to
accelerate breeding programs. Analysis of transgenic
plants provides a powerful tool for functional studies of
defense genes in cacao. We investigated transgenic
expression of a known plant defense response gene
encoding a class I chitinase and have demonstrated the
effectiveness of its encoded protein in inhibiting the
growth of a cacao leaf pathogen. While similar studies
have been reported for other plant species, this is the first
report of such an analysis in T. cacao and, to our
knowledge, for any tropical tree spec ies.
Our results demonstrate that the transgene was inte-
grated into the cacao genome in varying copy numbers
and expressed to varying degrees, in each of the inde-
pendent lines studied. Measurement of EGFP expression
levels and protein activity in the transgenic plants re-
vealed that there was little correlation between the
EGFP fluore scence and the expression of the linked
chitinase transgene. However, as expected, we observed
a good correlation between chitinase activity and fungal
pathogen resistance. When evaluated for fungal patho-
gen resistance, the two lines with lower protein activity
(55.1 and 55.3) also developed significantly larger lesions
than the two lines with higher protein activity (56.6 and
61.7). This supports our hypothesis that the TcChi1 gene
product acts as an antifungal defense protein in cacao.
Similarly, the two control lines (S6 and GFP) that
contained undetectable levels of TcChi1 mRNA in
leaves also displayed lower chitinase activity than any of
the chitinase expressing transgenic lines. Consistent with
the hypothesis that the TcChi1 gene product functions in
defense, the two control lines develop ed the largest le-
sion sizes of all lines tested. These results are consistent
with an earlier report where TcChi1 expression was de-
tected in the fruit pericarp in response to wounding and
ethylene, which indirectly implicated a role for this gene
in defense (Snyder 1994). From our data we can con-
clude that the TcChi1 transgene product contributes
significantly to defense against C. gloeosporioides in
laboratory condition s.
Colletotrichum spp. are a hemibiotrophic fungi
(Mendgen and Hahn 2002) that cause anthracnose in a
variety of plant species including Solanaceae plants (to-
mato, pepper) and many tropical cultivated crops such as
mango, papaya, avocado, coffee, and coconut. After pe-
netrating the host, the fungus initially grows intracellu-
larly and esta blishes as a biotroph for one or a few days
(O’Connell et al. 2000). In a later phase, secondary nar-
rower hyphae are formed that kill the host cells and
proliferate necrotrophically. Cacao anthracnose, caused
by C. gloeosporioides, is one of the major cacao problems
in India and has been reported to cause significant dam-
age to trees in Brazil (Wood and Lass 1987) and in
Venezuela, where the variety ‘Porcelana’ is very suscep-
Genot
y
pe (Trans
g
enic Line #)
a
a
c
0
.00005
.0001
.00015
.0002
.00025
.0003
.00035
.0004
.00045
de
ef
e
bc
c
f
cde
Relative Chitinase Activity
(nmoles MU/min/µg protein)
S6
GFP
33.1
39.2
44.1
47.1
55.1
53.3
56.2
61.7
Fig. 4 Endochitinase protein activity assay of control and trans-
genic cacao plants. Total protein was extracted from control non-
transgenic PSU-Scavina 6 (S6), control pGH00.0126 (GFP)
(Maximova et al. 2003) and eight transgenic pGAM00.0511
(TcChi1 chitinase) lines (Maximova et al. 2003). Four leaves from
each line were collected (two from each of two clonal replicate
plants). Total protein was extracted from each sample, and these
were assayed in triplicate (total of 12 measurements per line). The
total relative chitinase activity was analyzed by fluorometric assay
using methylumbelliferyl as a substrate. The fluorescence of the 4-
methylumbelliferone (MU) reaction product was recorded at 360/
485 gm ex/em. Means of all 12 replicate measurements from each
line ± SE are presented. Mean separation was performed with
Fishers Protected LSD test at P<0.05 level of significance.
Different letters (a, b, c, d, e, f) indicated significant difference
746
tible to this disease (Ceden
˜
o and Carrero 2003). C. glo-
eosporioides attacks primarily young and soft cacao
leaves causing brown lesions surrounded by a charac-
teristic clear yellow halo (Mohanan et al. 1989). With the
development of the disease, the lesions expand and
gradually coalesce to form large blighted areas that can
lead to defoliation. The disease also affects mature and
young fruits. The symptoms on mature fruit appear as
dark sunken lesions that expand and agglomerate but
rarely damage the beans. Infection of the cherelles (young
fruits) causes wilt and death, but the mummified fruits
remain attached to the truck (Mohanan et al. 1989).
Interestingly, Colletotrichum sp. M1 has also been
described as the most frequently encountered endophyte
of T. cacao mature and old leaf samples collected from
five lowland sites with mixed forest cover acro ss Panama
(Arnold et al. 2003). The same authors also reported
that endophyte-free T. cacao seedlings inoculated with
mixture of endophytes representing three common
endophyte genera of T. cacao including Colletotrichum,
Xylaria, and Fusarium/Nectria demonstrated increased
resistance to black pod disease (Phytophthora sp.).
Hence the possibility that Colletotrichum spp. in T. ca-
cao could behave as beneficial endophytes as well as
fungal pathogens is not surprising given that different
species, or even different genotypes of the same species,
could have alternatively pathogenic or symbiotic/mutu-
alistic effects. Furth ermore, it is important to acknowl-
edge that because of their typical behavior as a
hemibiotrophic fungi and also due to their very broad
host range, Colletotrichum spp. are commonly employed
as experimental organisms for the studies of fungal
biotrophy (Perfect et al. 1999; Mendgen and Hahn
2002), the switch between biotrophy and necrotrophy
(Dufresne et al. 2000), and also endophytic versus
pathogenic behavior (Freeman and Rodriguez 1993).
The recent description of the interactions between C.
destrictivum and Arabidopsis thaliana was a milestone in
the investigations of plant–fungal interactions (O’Con-
nell et al. 2004). The protocols developed for genetic
transformation of Colletotrichum spp. with GFP via
protoplast transf ormation (Dumas et al. 1999) and
Agrobacterium-mediated transformation (O’Connell
et al. 2004) have also made it possible to conduct cyto-
logical studies of the penetration and colonization pro-
cesses at the cellular level. Accordingly, we consider that
the fast and easy Colletotrichum leaf disk infection assay
developed for this study not only provided the necessary
evidence for the antifungal activity of the TcChi1 but
Fig. 5 Representative images of in vivo leaf infection assay of
control and transgenic lines. Two young, fully expanded cacao
leaves from each line were collected and wounded at ten different
areas per leaf (five on each side of the leaf main vein, total of 20
areas per line). Sixteen areas per line were inoculated with
Colletotrichum gloeosporioides (5,000 conidia per inoculated area).
The necrosis of the control and infected areas was evaluated 4 days
after inoculation. Top row non-inoculated plants, bottom row
inoculated plants. Images include non-inoculated: control non-
transgenic Scavina 6 (S6) (a), control GFP (b), chitinase 56.2 line (c),
and C. gloeosporioides inoculated: control non-transgenic S6 (d),
control transgenic GFP (e), and transgenic chitinase 56.2 line (f)
0
2
4
6
8
10
12
14
16
18
0 0.0001 0.0002 0.0003 0.0004 0.0005 0.0006
Relative Chitinase Activity
(nmoles MU/min/µg protein)
Leaf Necrosis (mm
2
)
55.3
y = -0.0072x + 13.787
R
2
= 0.753
S6
GFP
55.1
56.2
61.7
Fig. 6 Correlation of Colletotrichum resistance with TcChi1 chitin-
ase activity. Young, fully expanded cacao leaves from control non-
transgenic PSU-Scavina 6 (S6), control transgenic GFP, and four
transgenic pGAM00.0511 (chitinase) lines were wounded prior to
inoculation with Colletotrichum gloeosporioides as described in
Fig. 5. Disease necrosis was evaluated 4 days after inoculation. The
area of necrosis was measured using Scion Image for Windows
(version beta 4.0.2, Scion Corporation). The average areas of
necrosis were calculated. Correlations were established between the
average necrotic areas and total chitinase activities as presented in
Fig. 4
747
could also potentially be utilized as a tool for future
research of the interactions between cacao and hemi-
biotrophic fungi, representative of which is the impor-
tant Crinipellis perniciosa fungus.
In conclusion, although functional genomic analysis
is a difficult and time-consuming process in cacao trees,
this experimental approach allows us to make specific
and strong inferences about gene function that would be
impossible in any other way. Information gained by this
method is very useful in developing markers for breeding
programs and for screening of germplasm collections.
For example, with this knowledge in hand, it would be
possible to screen cacao germplasm collections for
genotypes with elevated levels of TcChi1 gene or for
expression of the gene in unique tissue-specific manners.
The selected genotypes could further be utilized in
breeding programs. Furthermore, based on its known
expression pattern, it is likely that the TcChi1 gene
contributes to fungal pathogen resistance in pods (cacao
fruit) and would map to the location of QTL markers
associated with resistance. If so, we could develop
molecular markers for the TcChi1 gene to be used in
marker-assisted selection breeding programs.
More directly, our results suggest that the TcChi1 gene
can confer enhanced resistance in transgenic plants when
over-expressed. In the specific example presented in this
manuscript, the DNA constructions were not designed
for commercial deployment due to the incorporation of
the EGFP marker gene. To ensure these genotypes were
not released, none of the cacao plants developed in this
research have been moved outside of our laboratories and
greenhouses, excluding the possibility of field-testing.
Although public acceptance has limited the applications
of transgenics to cacao improvement, in the future it is
possible that this gene could be used to enhance fungal
pathogen resistance in commercial varieties of cacao.
Furthermore, the utility of the TcChi1 gene in other plant
species has yet to be demonstrated and this is currently
being tested in our laboratory.
Acknowledgements We would like to thank Gabriela Antunez de
Mayolo Wilmking for her contribution to the construction of
vector pGAM00.0511 and Sara Milillo and Amanda Thompson for
their technical assistance with performing and data analysis of the
chitinase protein activity assay.
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