Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei.

Abstract

Ascochyta blight caused by the ascomycete fungus Ascochyta rabiei, is a major biotic constraint of chickpea (Cicer arietinum L.), resulting in disastrous crop losses worldwide. To study early stages of development and pathogenic mechanisms of the fungus, two binary vectors for the constitutive expression of the red fluorescent protein (DsRed-Express) and the green fluorescent protein (EGFP1) were constructed. Furthermore, we have developed an improved and highly reproducible Agrobacterium tumefaciens-mediated transformation protocol for A. rabiei. Transformation events were confirmed through Southern hybridizations that suggest single-copy integration of reporter genes in majority of the transformants. High level expression of both DsRed and EGFP proteins was obtained both in spores and in mycelia as detected by fluorescence microscopy. Intense fluorescence was used as a highly efficient vital marker to visualize early developmental changes of the fungus. The formation of infection structures like appressoria and germ tubes were observed both in vitro and in planta. This work will be useful to develop methodologies for understanding the mechanisms of Ascochyta-chickpea interaction and functional genomics of A. rabiei towards the isolation of virulence genes.

Full text

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Curr Genet
DOI 10.1007/s00294-010-0305-3
123
TECHNICAL NOTE
Expression of the Xuorescent proteins DsRed and EGFP
to visualize early events of colonization of the chickpea blight
fungus Ascochyta rabiei
Shadab Nizam · Kunal Singh · Praveen K. Verma
Received: 5 February 2010 / Revised: 18 April 2010 / Accepted: 24 April 2010
© Springer-Verlag 2010
Abstract
fungus Ascochyta rabiei, is a major biotic constraint of
chickpea (Cicer arietinum L.), resulting in disastrous crop
losses worldwide. To study early stages of development
and pathogenic mechanisms of the fungus, two binary vec-
tors for the constitutive expression of the red Xuorescent
protein (DsRed-Express) and the green Xuorescent protein
(EGFP1) were constructed. Furthermore, we have devel-
oped an improved and highly reproducible Agrobacterium
tumefaciens-mediated transformation protocol for A. rabiei.
Transformation events were conWrmed through Southern
hybridizations that suggest single-copy integration of
reporter genes in majority of the transformants. High level
expression of both DsRed and EGFP proteins was obtained
both in spores and in mycelia as detected by Xuorescence
microscopy. Intense Xuorescence was used as a highly
eYcient vital marker to visualize early developmental
changes of the fungus. The formation of infection structures
like appressoria and germ tubes were observed both in vitro
and in planta. This work will be useful to develop method-
ologies for understanding the mechanisms of Ascochyta–
chickpea interaction and functional genomics of A. rabiei
towards the isolation of virulence genes.
Ascochyta blight caused by the ascomycete
Keywords
DsRed-Express · EGFP1
Cicer arietinum · Ascochyta rabiei · ATMT ·
Introduction
The necrotrophic fungus Ascochyta rabiei (Pass.) Lab.
(teleomorph: Didymella rabiei) infects all aerial parts of
chickpea (Cicer arietinum L.), the third most important
food legume crop worldwide, resulting in severe grain
yield losses (FAO 2007). There is a high degree of variation
in resistance among chickpea cultivars and complete resis-
tance to A. rabiei has not been found in chickpea (Jayakumar
et al. 2005). The mechanisms by which A. rabiei infects
and colonizes chickpea plants remain poorly understood.
Investigations indicate that during spore germination and
infection, germ tubes secrete a mucilaginous substance to
facilitate attachment to the host surface (Höhl et al. 1990).
The pathogen produces several phytotoxins (solanapyrones
A, B, and C, cytochalasin D and a proteinaceous toxin)
which seems to be responsible for necrosis and cell death
(Höhl et al. 1991; Kaur 1995; Latif et al. 1993; Chen and
Strange 1994; Hamid and Strange 2000). The pathogen can
degrade antimicrobial compounds and suppress their pro-
duction in chickpea (Barz and Welle 1992; Kessmann and
Barz 1986; Höhl et al. 1989; Kraft et al. 1987; Tenhaken
et al. 1991). Genetic transformation of Wlamentous fungi
has paved the way to identify various virulence genes.
Earlier, protoplast and polyethylene glycol mediated trans-
formation of A. rabiei were reported (Weltring et al. 1995;
Köhler et al. 1995). Recently, ATMT of A. rabiei has
been reported by expressing hygromycin resistance gene in
the transformants (White and Chen 2006; Mogensen et al.
2006).
Fluorescent reporter proteins are essential tools in study-
ing plant–pathogen interactions. Introduction of new Xuo-
rescent labeling compounds and imaging methods have
now made possible to study fungi in their interaction with
the host using non-destructive methods. The gfp gene from
Communicated by U. Kueck.
S. Nizam and K. Singh contributed equally.
S. Nizam · K. Singh · P. K. Verma (&)
National Institute of Plant Genome Research,
Aruna Asaf Ali Marg, New Delhi 110067, India
e-mail: praveen_verma@nipgr.res.in

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Curr Genet
123
the jellyWsh Aequorea victoria encoding the green Xuores-
cent protein (GFP) is a well-known and widely used Xuo-
rescence marker (Prasher et al. 1992). Enhanced GFP
(EGFP) includes chromophore mutation at amino acid 65
(S65T) that causes a “red shift” from excitation maxima of
395 and 470 nm to a maximum of 488 nm, making it sepa-
rable from the chlorophyll background and optimized
codon usage for yeasts (yEGFP), plants (SGFP) and mam-
mals (EGFP1; Chiu et al. 1996; Haas et al. 1996; Yang
et al. 1996; Cormack et al. 1997). Both SGFP and EGFP1
have been successfully expressed to high levels in a number
of Wlamentous fungi and are widely used for in planta visu-
alization (Lorang et al. 2001). In addition, a novel red Xuo-
rescent protein, DsRed, resembling the structure of GFP
was isolated from reef coral (Discosoma sp) (Matz et al.
1999), which showed potential as a Xuorescent reporter.
The variant DsRed-Express contains nine amino acid sub-
stitutions that improve the maturation and solubility of the
protein (Bevis and Glick 2002). Furthermore, these substi-
tutions reduce the level of residual green emission as well
as the tendency to aggregate. The DsRed-Express protein
has an excitation maximum at 557 nm and an emission
maximum at 579 nm. The codon-optimized DsRed has
been shown to facilitate expression in various Wlamentous
fungi (Mikkelsen et al. 2003; Eckert et al. 2005; Paparu
et al. 2009).
In this paper, we report the construction of two vectors
for ATMT of Wlamentous ascomycetes with the Xuores-
cence markers DsRed-Express and EGFP1 and their
expression in A. rabiei. The early stages of disease estab-
lishment were microscopically visualized that showed vari-
ous infection structures of the fungus. In addition, we also
report the modiWed and highly reproducible protocol for
ATMT of A. rabiei.
Materials and methods
Strains and growth conditions
Ascochyta rabiei (Delhi isolate, D11) was used as a recipi-
ent strain for transformation and was grown in Potato Dex-
trose Agar (PDA) or V8-Juice Agar (Difco, USA) at 20°C.
The LBA4404 strain of A. tumefaciens was used for
ATMT. Introduction of plasmids into Agrobacterium strain
was performed as described by Gelvin and Schilperoort
(1994). The Escherichia coli strain DH5? was used for
cloning and construction of plasmids.
Plasmid construction
The binary vectors were constructed for the ATMT with the
modiWcations of T-DNA region of pCAMBIA1300
(CSIRO, Australia). The 1,503 bp PtrpC-hph cassette was
PCR ampliWed from the vector pDESTR (Abe et al. 2006)
using the primers HPH-F (5?-GCCTCGAGCGAAGA
ACGTTTTCCAATGA-3?) and HPH-R (5?-GCGAATTC
CGTGGAGGTAATAATTGA-3?). The PCR was carried
out using 100 pg of pDESTR plasmid, in 50 ?l reactions
containing 0.5 U of Phusion DNA polymerase (Finzymes,
Finland) and 0.2 ?M primers. Cycling conditions consisted
of an initial denaturation (98°C, 30 s); 34 cycles of denatur-
ation (98°C, 10 s), annealing (55°C, 30 s), and extension
(72°C, 1 min) followed by a Wnal extension (72°C, 7 min).
The PCR product thus obtained was gel puriWed, A-tailed
and cloned into the PCR cloning vector pDRIVE (Qiagen,
Germany). The XhoI-EcoRI fragment was excised and
cloned into the same sites in pCAMBIA1300 by replacing
PCaMV35S-hph, to create the pBIF vector. For the con-
struction of DsRed reporter vector, a 3.8 kb EcoRI-HindIII
PgpdA-DsRed-Express-TtrpC cassette was excised from
pPgpdA-DsRed vector (Mikkelsen et al. 2003) and inserted
into the same sites in pBIF, to create the vector pBIF-
DsRed. For the construction of EGFP reporter vector, the
2.4 kb PgpdA-EGFP1-TtrpC cassette was PCR ampliWed
from the vector pIG1783 (Poggeler et al. 2003) using the
primer Gpd-F (5?-GAATTCTGTACAGTGACCGGTG
ACTC-3?) and Trp-R (5?-GCCAAGCTTCCTCTAAAC
AAGTGTACCTGTGC-3?). The PCR was carried out as
mentioned above under similar cycling conditions except
the extension (72°C, 2 min). The PCR product was puriWed,
A-tailed and cloned into pDRIVE. From there it was
excised as an EcoRI-HindIII fragment and cloned into the
respective sites in pBIF, to make the pBIF-EGFP vector.
Agrobacterium tumefaciens-mediated transformation
of Ascochyta
ATMT was performed based on the protocol developed by
Utermark and Karlovsky (2008) for Wlamentous fungus
with modiWcations. The Agrobacterium strain LBA4404
harboring pBIF-DsRed or pBIF-EGFP plasmid was grown
in LB containing 100 ?g/ml kanamycin and 30 ?g/ml
rifampicin, at 28°C and 150 rpm, until the OD600 reached
0.8–1. The culture was washed twice with minimal media
(MM), diluted with induction media (IM) (Bundock et al.
1995) to make the OD600 0.15 and pre-cultivated for 6 h, at
28°C and 150 rpm. The pre-cultivated cell culture was
washed twice with fresh IM and suspended in IM, to make
the OD600 0.3. The spore of A. rabiei from 15-day-old PDA
or V8 agar culture were washed twice with MQ water and
suspended in IM to a Wnal concentration of 1 £ 107spores/
ml. The 100 ?l of IM suspended Agrobacterium culture
was mixed with 100 ?l of A. rabiei spore suspension. The
resulting 200 ?l suspension was added to the sterile dialysis
membranes (10,000 MWCO, SnakeskinTM, Pierce, USA)

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123
or cellophane discs, on IM agar plates and co-cultivated for
72 h at 20°C. The membranes or discs containing the co-
cultivate were inverted and transferred to PDA, containing
50 ?g/ml hygromycin, 200 ?M cefotaxime and 100 ?g/ml
chloramphenicol. Membranes and discs were removed after
an additional 48 h of co-cultivation at 20°C. The putative
transformants that appeared after 7–10 days of transfer
were moved to fresh selection plates and were further
selected by single spore culture on PDA containing hygro-
mycin and timentin to eliminate any Agrobacterium con-
tamination. The transformants were maintained on PDA or
V8 agar, with or without selection to check the stability of
the DsRed-Express, EGFP1 as well as hph genes.
Pycnidiospore germination test
For the germination analysis of pycnidiospore on glass sur-
face, spores were harvested from PDA plates, Wltered
through muslin cloth, suspended in 1 ml of sterile water,
and washed two times. Spore suspensions (25 ?l) with a
concentration of 2 £ 105spores/ml were placed into the
center of the glass slide. It was incubated in the dark under
high humidity at 20°C for several hours. Microscopy was
carried out to monitor germination progress.
Molecular analysis of transformants
For the conWrmation of transformation events, DNA for
PCR and Southern analyses was isolated using the
GenEluteTM Plant Genomic DNA Miniprep Kit (Sigma–
Aldrich, USA). A total of 104 spores of each transformant
was inoculated in 100 ml of potato dextrose broth (PDB)
and incubated for 5 days at 22°C with gentle shaking.
Mycelial balls were harvested by Wltering through two lay-
ers of muslin cloth and ground in liquid nitrogen to a Wne
powder. It was then used for the isolation of genomic DNA
according to the manufacturer’s instructions. An internal
646 bp region of DsRed-Express gene was PCR ampliWed
using the primers DsRed-F (5?-CGTCATCAAGGAGTT
CATGC-3?) and DsRed-R (5?-GCTCCACGATGGTGTA
GTCC-3?) whereas 720 bp EGFP1 gene was PCR ampli-
Wed using the primers EGFP-F (5?-ATGGTGAGCAAGG
GCGAGGAGCTG-3?) and EGFP-R (5?-TTACTTGTAC
AGCTCGTCCATGC-3?). The PCR was carried out using
10 ng of genomic DNA in 25-?l reactions containing 1 U of
Taq DNA Polymerase (NEB, USA) and 0.2 ?M primers.
Cycling conditions consisted of an initial denaturation
(94°C, 2 min); 34 cycles of denaturation (94°C, 30 s),
annealing (60°C, 30 s), and extension (72°C, 45 s) followed
by a Wnal extension (72°C, 7 min). The DsRed-Express and
EGFP1 amplicons were radio-labeled with [?-32P] dCTP
using the NEBlot Kit (NEB, USA) according to the
manufacturer’s instructions and used as probe to determine
number of T-DNA integrations. Southern blot analysis was
performed with EcoRI digested genomic DNA (10 ?g) that
were size-fractionated on 0.7% agarose gels and transferred
to Hybond-NTM nylon membranes (Amersham Biosci-
ences, USA). Hybridization and high stringency washing
was carried out using standard protocols (Sambrook and
Russell 2001).
Plant inoculation
The leaves of 3-week-old Cicer arietinum L. variety Pusa-
362 (Indian Agricultural Research Institute, New Delhi)
were inoculated with 10 ?l of spore suspensions
(2 £ 105spore/ml) of A. rabiei. The infected plants were
incubated in a closed chamber at 20°C and high humidity,
under natural illumination for several days. Infection pro-
gress was monitored by microscopy.
Microscopy
For Xuorescence microscopy, a Nikon 80i Xuorescence
microscope, equipped with DIC (Nomarski microscopy)
and Wlters FITC (excitation 425 nm, emission 480–520 nm)
and the red shifted TRITC Wlter (excitation 545 nm, emis-
sion 620–660 nm) was used both to screen colonies of
transformants growing on plates for Xuorescent protein
expression and for observation of fungal colonization in
plant tissues. For confocal microscopy, an inverted confo-
cal laser scanning microscope Leica TCS SP2 AOBS
(Leica Microsystems, Exton, PA) was used. For imaging
EGFP, the Argon laser (excitation 488 nm, emission
515–530 nm) was used while He–Ne laser (excitation
543 nm, emission 560–615 nm) was used for imaging
DsRed.
Results
Construction of pBIF-DsRed and pBIF-EGFP vectors
and transformation of Ascochyta rabiei
In order to transform Ascochyta using A. tumefaciens,
binary vectors were constructed using the backbone of
pCAMBIA1300. The hygromycin cassette of the vector
(PCaMV35S-hph) was replaced by a compact and modiWed
hygromycin cassette of the plasmid pDESTR (Abe et al.
2006), having hygromycin phosphotransferase (hph) gene
under the control of tryptophan (trpC) promoter of Asper-
gillus nidulans. The resulting plasmid was termed as pBIF
(binary vector for fungal transformation). The reporter
genes DsRed-Express and EGFP1, under the control of
glyceraldehyde-3-phosphate dehydrogenase (gpdA) pro-
moter and trpC terminator of A. nidulans were then cloned

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in pBIF to generate the plasmids pBIF-DsRed and pBIF-
EGFP, respectively (Fig. 1).
Prior to transformation, the sensitivity of wild-type
A. rabiei to hygromycin was determined by transferring
fungal cultures onto PDA plates containing various concen-
trations of hygromycin (i.e. 50, 100, 150 and 200 ?g/ml).
Growth of A. rabiei was completely inhibited for 14 days at
50 ?g/ml hygromycin, although some growth occurred
after prolonged incubation. The same concentration of
hygromycin was suYcient enough to completely inhibit the
growth of A. rabiei in PDB grown for 6 days at 22°C with
gentle shaking. To ensure that recovered transformants
were not false positive, fungal colonies were selected on
hygromycin for the Wrst 10 days of post co-cultivation. For
the ATMT of A. rabiei, the starting material was taken
either from PDA or V8 agar plates. Induced LBA4404
strain of Agrobacterium harboring the plasmid (pBIF-
DsRed or pBIF-EGFP) was co-cultivated with A. rabiei
spores on IM agar plates containing either dialysis mem-
brane or cellophane discs. The co-cultivation was extended
for either 60 h or 72 h at 20°C. Maximum number of trans-
formants was recovered from cellophane discs (8–12 trans-
formants per plate) as compared to dialysis membrane (2–5
transformants per plate). Moreover, spores from PDA
plates resulted in slightly higher number of transformants
(10–14 transformants per plate) as compared to spores from
V8 agar plates (8–10 transformants per plate). Furthermore,
it was observed that co-cultivation for 72 h resulted in
higher number of transformants as compared to 60 h. The
morphology of transformants did not show any anomalies
with respect to untransformed A. rabiei.
Expression of DsRed-Express and EGFP1 in A. rabiei
In order to ensure the expression of reporter proteins, fun-
gal mycelia were directly taken from the selection plates
and visualized under Xuorescence microscope using the
appropriate Wlters for DsRed and EGFP. Expression of
DsRed-Express and EGFP in the transformed A. rabiei was
clearly evident as red and green Xuorescent mycelia
(Fig. 2). However, no auto-Xuorescence was detected in
untransformed fungi. The DsRed or EGFP Xuorescence
was observed in all the hygromycin-resistant fungal colo-
nies, however, the level of Xuorescence was variable. Two
transformants each of the DsRed and EGFP that showed
high Xuorescence were selected for further microscopy.
The germination analysis of transformed A. rabiei pycni-
diospores was performed on glass slides. Microscopic stud-
ies of germinating spores showed formation of germ tubes
and appressoria-like structures (Fig. 3). All the germinating
spores showed bright red or green Xuorescence under Xuo-
rescence (Fig. 2) as well as laser scanning confocal micro-
scope (Fig. 3). The Xuorescence was observed both in the
spores and in the germ tubes, conWrming the expression of
DsRed and EGFP during the early phases of A. rabiei
germination.
Molecular characterization of transformants
The presence of T-DNA was initially conWrmed through
PCR by amplifying 646 and 720 bp regions of the DsRed-
Express and EGFP1 genes, respectively. Southern hybrid-
izations were performed with the randomly chosen Wve
A. rabiei DsRed and EGFP transformants along with the wild
type to determine the frequency and randomness of T-DNA
integration. The genomic DNA was digested with EcoRI
that cuts once in the T-DNA region of DsRed and EGFP
transformants (Fig. 4a). The PCR-ampliWed 646 and 720 bp
regions of the DsRed-Express and EGFP1 genes, respec-
tively, were used as probes (Fig. 4a). The binary vectors
pBIF-DsRed and pBIF-EGFP contain a single EcoRI site,
located within the T-DNA region itself, such that smallest
hybridizing fragment of EcoRI digested DsRed or EGFP
transformant DNA would contain approximately 3.8 or
Fig. 1 Schematic representation of binary vectors constructed in this
study. T-DNA region of transformation vectors showing relative posi-
tion of selectable marker, promoter sequences, reporter genes, termina-
tor sequences and the relevant restriction enzyme sites. The plasmid
pBIF-DsRed is having 2.3 kb gpdA promoter (Mikkelsen et al. 2003)
as compared to pBIF-EGFP, which is having a smaller 0.9 kb gpdA
promoter (Poggeler et al. 2003). All plasmids were constructed using
the binary vector pCAMBIA1300 as a backbone

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2.4 kb of T-DNA, respectively (Fig. 3B). The presence of
diVerent sized bands showed random integration of the
reporter genes. In majority of the transformants, single
bands were observed which conWrm single-copy integration
of the T-DNA region, while multi-copy integration events
were also observed in few transformants (Fig. 4b).
Microscopic studies of Ascochyta–chickpea interaction
Chickpea infection using the DsRed transformed A. rabiei
were performed on aerial parts of the plants as described in
“Materials and methods”. The inoculated plants were kept
in high humidity in transparent plastic boxes. Fluorescence
microscopy was performed after 1, 2, 5 and 7 days post-
inoculation (dpi). Germination of spores was observed
within 1 and 2 dpi on chickpea leaves (Fig. 5). The spores
on the surface of the plants swelled prior to, and during
germ tube emergence. The germ tube grew on the surface
of the plant and formed a typical appressorium (Fig. 5A,
B). The penetration of the fungus was direct as well as
through stomatal openings. Interestingly, many incidences
of epiphyllous growth and pycnidium formation on chick-
pea leaves around the trichomes were also observed
(Fig. 5C, D). Severe infection was found after 5 and 7 dpi
(Fig. 5E). Visible necrotic lesions on the infected leaves
were also observed. These lesions were microscopically
examined which showed massive fungal colonization. The
EGFP-transformed A. rabiei was also used for in planta
visualization but the level of Xuorescence was not as prom-
inent as DsRed to analyze various infection structures.
Discussion
Microscopy techniques have been of great value for study-
ing infection structures, developmental processes and
plant–pathogen interactions.
microscopy has improved our understanding of the interac-
tion between fungus and its host. Therefore, the early
events during in planta colonization of A. rabiei in chickpea
were studied using the expression of Xuorescence markers.
ATMT has been reported to be an eYcient tool for the
genetic transformation of Wlamentous fungi. Evidences for
ATMT of A. rabiei has been reported by expressing hygro-
mycin resistance gene (White and Chen 2006; Mogensen
et al. 2006). Recently, a more generalized protocol for
ATMT of Wlamentous fungi was reported (Utermark and
Karlovsky 2008). Therefore, we have modiWed the protocol
Recently,
Xuorescence
Fig. 2 Expression of DsRed-Express and EGFP1 in A. rabiei. a Expression of DsRed-Express and b EGFP1 in spore (I), hyphae (II) and mycelia
(III) of transformants, and in wild type (IV)

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Curr Genet
123
Fig. 3 Spore germination assay
of DsRed transformed A. rabiei
through confocal laser scanning
microscope. A Formation of
germ tube (g), B formation of
appressorium (a) and C late
stage of germination
Fig. 4 Southern blot analysis of
A. rabiei wild type and randomly
chosen, DsRed-Express
and EGFP1 transformants.
a Illustration of T-DNA integra-
tion event. Dark lines indicate
T-DNA region. The line under-
neath the DsRed-Express and
EGFP1 gene represents the
region used as probe. The arrow
indicates the single EcoRI site
within the T-DNA. b The geno-
mic DNA was digested with
EcoRI. Blots were hybridized
using reporter-gene speciWc
fragments. Numbers
on the left indicate the position
and size of molecular size
markers. (Marker = ? HindIII,
WT wild type.)

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Curr Genet
123
for the ATMT of A. rabiei which appeared simple and
highly reproducible.
There are various reporter proteins that have been
expressed to signiWcant levels in many Wlamentous fungi.
Among these, DsRed-Express and EGFP1 are the most
widely used. Therefore, in order to facilitate the expression
of Xuorescent proteins in A. rabiei, binary plasmids con-
taining DsRed-Express and EGFP1 under the control of
gpdA promoter of Aspergillus nidulans and a hygromycin
cassette were constructed. To test whether the constructs
were functional, ATMT of A. rabiei was performed. It was
found that cultures, both from PDA and V8 agar plates can
be used as the starting material for transformation. How-
ever, A. rabiei pycnidiospores from PDA agar plates
resulted in slightly higher number of transformants as com-
pared to V8 agar plates. Using the modiWed protocol we
were able to get on an average of 10–14 transformants per
90 mm plate. Since this eYciency was observed using both
the constructs, therefore, we assume it to be a more repro-
ducible protocol for ATMT of Ascochyta. Moreover, we
speculate that using hypervirulent strains of Agrobacterium
(i.e. strains having C58 chromosomal background) instead
of common strain LBA4404 for the ATMT of A. rabiei,
will result in more number of transformants.
It was observed that both PgpdA-DsRed-Express and
PgpdA-EGFP1 reporter constructs were able to drive the
expression of the red Xuorescent protein DsRed and the
green Xuorescent protein EGFP, respectively. The expres-
sion was also observed in the early stages of A. rabiei spore
germination like germ tubes and appressoria. These results
Fig. 5 Infection assay of DsRed
transformed A. rabiei on
chickpea leaves. A Germination
of pycnidiospore by the forma-
tion of appressorium (a), B later
stage of germination showing
germ tube (g) and appressorium
(a) C epiphyllous growth,
D germination and colonization
around trichomes and,
E massive colonization of the
fungus around the necrotic
lesion after 7 dpi. Bar 10 ?m

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123
suggest that both the PgpdA and PtrpC gene promoters are
functional in A. rabiei. Furthermore, expression of the Xuo-
rescent proteins was stable and did not disappear after sub-
sequent culturing on plates having no selection pressure.
Our results support that ATMT is more stable as compared
to other transformation techniques in fungi. This is possibly
because Agrobacterium inserts the DNA usually as a single
copy into the genome, resulting in a higher level of mitotic
stability of the transformants (Michielse et al. 2005). The
conWrmation of the transformation event was performed
using PCR and Southern hybridization. Southern analysis
showed that most of the DsRed or EGFP transformants
contained a single copy of the T-DNA molecule integrated
in random positions, similar to A. tumefaciens transforma-
tion systems of other Wlamentous fungi (Michielse et al.
2005). However, we did not observe any relation between
copy number and Xuorescence. To the best of our knowl-
edge, this is the Wrst report in which the in vivo expression
of Xuorescent proteins DsRed-Express and EGFP1 is dem-
onstrated in A. rabiei.
Infection analysis of the DsRed transformed A. rabiei on
chickpea leaves conWrmed the germination of spores during
1 and 2 dpi by the formation of infection structures (Fig. 5).
The spore initially forms a germ tube and later develops an
appressorium (Fig. 5A, B). In context to earlier reports we
also observed the formation of a typical appressorium,
which is the Wrst step in the penetration of the host (Höhl
et al. 1990). The predominant form of penetration was
found to be direct through cuticle. Occasionally, penetra-
tion through stomatal openings was also observed. Since
the appressoria of A. rabiei are not melanized (Tenhaken
1992), therefore, we assume the penetration of the cuticle
likely through the enzymatic degradation rather than
mechanical force. One interesting Wnding was the inci-
dences of hyphal spread on the leaf surface of the plants
(Fig. 5C). In addition, a unique observation was the pycnid-
ium formation on chickpea leaves around the trichomes
(Fig. 5D). This was visualized both on glandular as well as
non-glandular trichomes. However, the exudates secreted
from glandular trichomes of chickpea are acidic in nature
(pH 0.4–1.3) which inhibit A. rabiei spore germination
(Armstrong-Cho and Gossen 2005).
The present study has identiWed optimal conditions for
the eYcient transformation of A. rabiei using ATMT. The
method is highly reproducible and is suitable for functional
genomics of the fungus for various applications including
the targeted disruption of speciWc genes, ectopic comple-
mentation of loss-of-function strains and over-expression.
It will also facilitate investigations into the molecular
mechanisms of virulence in A. rabiei. In addition, the
strongly expressing DsRed or EGFP Xuorescent proteins in
transgenic A. rabiei will allow further studies on mecha-
nisms during host colonization and also suggests use of
these reporter proteins to investigate interactions of other
phytopathogenic fungi with their hosts.
Ethical standards
We declare that the experiments comply with the current
laws of the country.
Acknowledgments
vided by Department of Biotechnology, Government of India. Authors
acknowledge Dr. L. Mikkelsen, The Royal Veterinary and Agricultural
University, Denmark, for providing pPgpdA-DsRed, Dr. T. Sone,
Hokkaido University, Japan, for providing pDESTR vector and Dr. U.
Kück, Ruhr-University Bochum, Germany, for providing pIG1783
vector. The Ascochyta rabiei, (Delhi isolate, D11) was provided by
Dr. S.C. Dube, Division of Plant Pathology, Indian Agricultural
Research Institute, New Delhi. S.N. and K.S. acknowledge University
Grant Commission and Council of ScientiWc and Industrial Research
for the fellowships, respectively.
This work is supported by research grant pro-
ConXict of interest
We have no conXict of interest.
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