Awc

MN

a

ARRA

KFFCAD

1

wdopodfthwfimaaeys

0d

Microbiological Research 167 (2012) 332– 338

Contents lists available at SciVerse ScienceDirect

Microbiological Research

j our na l ho mepage: www.elsev ier .de /micres

highly efficient Agrobacterium mediated transformation system for chickpeailt pathogen Fusarium oxysporum f. sp. ciceri using DsRed-Express to follow root

olonisation

d. Nazrul Islam, Shadab Nizam, Praveen K. Verma ∗

ational Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110 067, India

r t i c l e i n f o

rticle history:eceived 31 October 2011eceived in revised form 16 January 2012ccepted 6 February 2012

eywords:usarium oxysporumusarium wilthickpea

a b s t r a c t

The soil-borne fungus Fusarium oxysporum f. sp. ciceri (Foc) causes vascular wilt of chickpea (Cicerarietinum L.), resulting in substantial yield losses worldwide. Agrobacterium tumefaciens mediated trans-formation (ATMT) has served as a resourceful tool for plant–pathogen interaction studies and offersa number of advantages over conventional transformation systems. Here, we developed a highly effi-cient A. tumefaciens mediated transformation system for Foc. In addition, a binary vector for constitutiveexpression of red fluorescent protein (DsRed-Express) was used to study developmental stages andhost–pathogen interactions. Southern hybridisation was performed to confirm the transformation eventand the presence of T-DNA in selected hygromycin resistant transformants. Most of the transformants

TMTsRed-Express

showed single copy integrations at random positions. Microscopic studies revealed significant levels offluorescent protein, both in conidia and mycelia. Confocal microscopy of chickpea roots infected with thetransformed Foc showed rapid colonisation. These studies will allow us to develop strategies to deter-mine the mechanisms of Foc-chickpea interaction in greater detail and to apply functional genomics forthe characterisation of involved genes at the molecular level either by insertional mutagenesis or geneknock-out.

. Introduction

Following bean and pea, chickpea (Cicer arietinum L.) is theorld’s third most important grain legume. India is the largest pro-ucer of chickpea in the world and accounts for approximately 69%f the total world chickpea production (FAOSTAT 2009). Chickpealants are affected at various stages of development by a varietyf pathogens. Chickpea generally shows symptoms of an array ofiseases mainly caused by pathogen-like insect pests, nematodes,ungi, bacteria and viruses. Diseases caused by fungi are impor-ant because they are easily propagated and are known to causeuge losses in productivity. Fusarium oxysporum f. sp. ciceri (Pad-ick) Matuo & K. Sato (Foc), one of the most important soil-bornelamentous fungi, causes Fusarium wilt of chickpea resulting inassive decreases in productivity (Jalali and Chand 1992). There is

high degree of variation in resistance among chickpea cultivars,nd complete resistance to Foc has not been found in chickpea. The

xistence of nematode pests, Meloidogyne incognita and Meloidog-ne javanica (Sharma and McDonald 1990; Sharma et al. 1992), inoils infested with Fusarium spp. enhances the incidence rate of

∗ Corresponding author. Tel.: +91 11 26735114; fax: +91 11 26741658.E-mail address: praveen verma@nipgr.res.in (P.K. Verma).

944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.oi:10.1016/j.micres.2012.02.001

© 2012 Elsevier GmbH. All rights reserved.

disease development and severity of Fusarium wilt in grain legumes(Ribeiro and Ferraz 1984; Goel and Gupta 1986; Sharma et al. 1992).The mechanisms by which Foc infects and colonises chickpea plantsremain poorly understood.

Transformation and integration of foreign DNA in most of thefilamentous fungi have been characterised by either homologousor nonhomologous recombination. Nonhomologous recombina-tion (gene tagging or marker gene integration) has been routinelyutilised to understand the morphology, physiology and mode ofinfection. Mutagenesis of a specific gene via homologous recom-bination between the target gene and its mutant allele hasalso been widely used in many filamentous fungi. Therefore, tointroduce foreign DNA into a fungal genome, a highly efficienttransformation system is needed. Previously, a variety of trans-formation systems have been employed to transform differentfilamentous fungi. These systems have included restriction enzymemediated integration (REMI) (Bölker et al. 1995; Granado et al.1997; Sanchez et al. 1998), polyethylene glycol/CaCl2 (PEG/CaCl2)(Sexton and Howlett 2001; Fitzgerald et al. 2003), electroporation(Ward et al. 1989; Ozeki et al. 1994; Kuo et al. 2004), particle

bombardment (Fungaro et al. 1995; Davidson et al. 2000) andAgrobacterium tumefaciens mediated transformation (ATMT) (deGroot et al. 1998; Rho et al. 2001; Michielse et al. 2005; Nizamet al. 2010).

gical R

defvetsteAfe

arssdtoltgGrca2oroeGtt2

mtpaeit

2

2

tai(wsmp

2

tw

Md.N. Islam et al. / Microbiolo

ATMT has been successfully applied for the transformation of aiverse array of fungi (Gouka et al. 1999; Chen et al. 2000; Mullinst al. 2001; Covert et al. 2001; Combier et al. 2003). To success-ully transform fungi, A. tumefaciens appears to require the sameirulence genes that are utilised for plant transformation (Bundockt al. 1995; Michielse et al. 2004). A. tumefaciens has an advan-age over other methods because it can easily transform fungaltrains through nonhomologous recombination and T-DNA inser-ion at random sites in a single copy (Chen et al. 2000; Grimaldit al. 2005; Wang et al. 2008; Nizam et al. 2010; Ding et al. 2011).TMT also increases frequency of homologous recombination, a

eature favourable for efficient targeted gene knock-out (Bundockt al. 1999; Zwiers and De Waard 2001; Dobinson et al. 2004).

Transformed organisms carrying fluorescent reporter proteinsre valuable tools for plant pathogen interaction studies and areepeatedly used to observe behaviours of pathogens in plant tis-ues under various physiological conditions. Fluorescent markersuch as red fluorescent protein (DsRed) are beneficial in that theyo not require cofactors or substrates. DsRed is naturally found inhe coloured body parts of the coral Discosoma sp. It is a green flu-rescent protein (GFP) homologue (Wall et al. 2000) and has theongest excitation and emission spectra (558/583 nm) of the wildype fluorescent proteins (Matz et al. 1999). When utilised as a sin-le colour fluorescent marker, DsRed has several advantages overFP. DsRed provides a greater signal-to-noise ratio and is relatively

esistant to photo bleaching (Baird et al. 2000). However, DsRedreates a large amount of insoluble aggregates in transformantsnd exhibits very slow maturation of the chromophore (Baird et al.000). Hence, a substitute of DsRed, DsRed-Express, has been devel-ped by a combination of nine amino acid substitutions throughandom and site-directed mutagenesis of the wild type of red flu-rescent protein that increase its maturation and solubility andnhanced the fluorescence compared with the wild type (Bevis andlick 2002). This codon-optimised DsRed-Express has been shown

o facilitate expression of fluorescent protein in various filamen-ous fungi (Mikkelsen et al. 2003; Eckert et al. 2005; Paparu et al.009; Nizam et al. 2010).

In this study, we report the highly efficient genetic transfor-ation protocol of Foc by A. tumefaciens. In addition, we show

he expression of DsRed-Express (the codon-optimised DsRed) inathogenic fungi Foc. Confocal microscopy was performed to visu-lise various structures of Foc and the initial phases of diseasestablishment in chickpea root. To the best of our knowledge, thiss the first report detailing the A. tumefaciens mediated transforma-ion and the expression of DsRed-Express in Foc.

. Materials and methods

.1. Strains and culture conditions

F. oxysporum f. sp. ciceri strain Delhi isolate was obtained fromhe Indian Agriculture Research Institute (IARI, New Delhi, India)nd maintained in Potato Dextrose Agar (PDA) (Difco, USA). Tonduce conidia formation, Foc was grown on Czapek Dox AgarHimedia, India) with chickpea seed extract. The incubation periodas typically 12 days at 25 ◦C under continuous light. The LBA4404

train of A. tumefaciens was used for ATMT. Introduction of plas-ids containing DsRed-Express into the Agrobacterium strain was

erformed as described by Gelvin and Schilperoort (1994).

.2. Resistance level to hygromycin B

Wild type Foc was grown in the presence of various concentra-ions of hygromycin B to monitor its resistance level. The agar plugas transferred to PDA plates containing various concentrations of

esearch 167 (2012) 332– 338 333

hygromycin B (25, 50, 75 and 100 �g/ml) and incubated at 25 ◦C for7 days.

2.3. A. tumefaciens mediated transformation of Foc

A. tumefaciens strain LBA4404 containing the pBIF-DsRed vector(Nizam et al. 2010) was grown on an LB agar plate supplementedwith 30 �g/ml rifampicin and 100 �g/ml kanamycin at 28 ◦C for2 days. A single colony was picked from the LB agar plate and grownin LB containing 30 �g/ml rifampicin and 100 �g/ml kanamycin at28 ◦C, 150 rpm, for 15 h or until the OD600 reached 0.8–1.0. Fromthe above culture, 25 �l was used to inoculate 25 ml LB contain-ing 30 �g/ml rifampicin and 100 �g/ml kanamycin and incubatedunder the same conditions until the OD600 reached 0.5–0.8. Theculture was pelleted using centrifugation at 5000 rpm for 5 min atroom temperature and washed twice with one volume of Mini-mal Medium (Bundock et al. 1995). The pellet was finally dilutedin 25 ml Induction Medium (IM) (Bundock et al. 1995) with dif-ferent concentrations of acetosyringone (0–400 �M) to acquire anOD600 of 0.15. The culture was incubated at 28 ◦C and 150 rpm for6–7 h until the OD600 reached 0.3–0.4. The Foc conidia were har-vested by adding sterile water to the 12-day-old mycelium grownin Czapek Dox with chickpea seed extract and rubbing the sur-face with the end of a sterile micropipette tip. Conidial suspensionswere filtered through muslin cloth to remove large particles. Theconidial suspension was washed twice with one ml of IM andresuspended in IM to establish a final concentration of 1 × 106

conidia/ml or 1 × 107 conidia/ml. The 100 �l of IM-suspendedAgrobacterium culture was mixed with 100 �l of Foc conidial sus-pension. The mixed suspension (200 �l) was added to Petri platescontaining IM agar and sterile dialysis membranes (10,000 MWCO,SnakeskinTM, Pierce, USA), nylon membranes, or cellophane discs,and co-cultivated under different time and temperature conditions.The filter or membrane was transferred to selection Czapek Doxplates containing hygromycin B (100 �g/ml) as a selection agentfor transformants and 200 �M cefotaxime (to kill Agrobacteriumcells); plates were incubated at 25 ◦C. Subsequently, each transfor-mant was transferred to Czapek Dox plates containing 100 �g/mlhygromycin B and incubated until conidiogenesis. Conidia fromindividual transformants were suspended with sterile water andspread on potato dextrose agar plates (PDA) supplemented withhygromycin B (100 �g/ml). To obtain the monoconidial cultures, asingle germinating conidium from each transformant was pickedand transferred to fresh Czapek Dox plates containing 100 �g/mlhygromycin B (Mullins et al. 2001).

2.4. Molecular analysis of transformants

To analyse the transformation events by Southern blot and PCRassay, DNA from selected hygromycin-resistant transformants andwild type Foc was isolated. For DNA isolation, 100 ml of PotatoDextrose Broth (PDB) containing 100 �g/ml hygromycin B wasinoculated with 105 conidia from each transformant and incu-bated for 4 days at 25 ◦C with gentle shaking. Mycelial cultureswere harvested by filtration through four layers of muslin clothfollowed by washing several times with sterile water. Mycelialmats were ground to a fine powder in liquid nitrogen, and DNAwas extracted using the Gen EluteTM Plant Genomic DNA MiniprepKit (Sigma–Aldrich, USA) according to the manufacturer’s instruc-tions. A 646 bp region of the DsRed-Express gene was PCR amplifiedusing the primers DsRed-F (5′-CGTCATCAAGGAGTTCATGC-3′) andDsRed-R (5′-GCTCCACGATGGTGTAGTCC-3′) (Nizam et al. 2010).

The PCR was conducted using 20 ng of genomic DNA in 25 �lreactions containing 1.5 U of Taq DNA Polymerase (NEB, USA)and 0.2 �M primers. Cycling conditions consisted of an initialdenaturation (94 ◦C, 2 min); 35 cycles of denaturation (94 ◦C, 30 s),

334 Md.N. Islam et al. / Microbiological Research 167 (2012) 332– 338

F stitut( rowin

aabgracE0bU

ig. 1. Fluorescence micrographs of Fusarium oxysporum f. sp. ciceri (Foc) showing conB) germinating macroconidium, (C) chlamydospore (indicated by arrow) and (D) g

nnealing (60 ◦C, 30 s) and extension (72 ◦C, 45 s) followed by final extension (72 ◦C, 10 min). To determine the copy num-er of T-DNA (number of T-DNA insertions throughout the hostenome), the DsRed-Express amplicons were eluted from the gel andadio-labelled with [�-32P] dCTP using the NEBlot Kit (NEB, USA)ccording to the manufacturer’s instructions. Radio-labelled ampli-ons were used as probes. Extracted DNA (10 �g) was digested with

coRI, and the products were separated by electrophoresis on a.7% agarose gel in 1× TAE. The gel was treated with 0.25 M HClefore blotting onto a nylon membrane (Amersham Biosciences,SA). Denaturation, transfer, prehybridisation, hybridisation and

ive expression of DsRed-Express at different developmental stages: (A) microconidia,g hyphae. Each bar represents 10 �m.

high stringency washes for Southern blot analysis were carried outusing standard protocols (Sambrook and Russell 2001).

2.5. Chickpea root infection

Root infection was performed as described by Sarrocco et al.(2007). Three-week-old chickpea cultivar (Pusa 362) was utilised

for studying root colonisation. Conidia were harvested as describedabove in sterile water, and the final concentration was main-tained at 1 × 105 conidia/ml. Plants were inoculated at 25 ◦C (12 hlight/dark) by immersion of roots in spore suspensions for 15 min;

gical Research 167 (2012) 332– 338 335

eeasi

2

(5ptavsgufPtTi

3

3

tgcgtpcwrr(c

3

tcptoieAtpmfoto2wcgp

Fig. 2. Southern blot analysis of wild type Foc and four selected DsRed-Express trans-formants. (A) Illustration of a T-DNA integration event. The line underneath theDsRed-Express gene represents the region used for the probe. The arrow indicatesthe single EcoRI site within the T-DNA. (B) The genomic DNA was digested with EcoRI.

Md.N. Islam et al. / Microbiolo

ach plant was then placed in an enclosed plastic box. Two lay-rs of wet blotting paper were kept inside of the box to maintain

high level of humidity. Colonisation of the DsRed transformedtrains was microscopically observed in roots 1–6 days after thenoculation.

.6. Microscopy

A Nikon 80i fluorescent microscope equipped with DICNomarski optics) and the red-shifted TRITC filter (excitation45 nm, emission 620–660 nm) was used to visualise fungal mor-hology growing on plates and to determine in planta colonisationo see fluorescent protein expression. To study fungal morphologynd germination processes on a glass surface, conidia were har-ested from PDA plates as described above. A total of 20 �l conidialuspension (1 × 105 conidia/ml) was placed onto the centre of alass slide and incubated at 25 ◦C for several hours (12 h light/dark)nder high humidity conditions. An inverted laser-scanning con-ocal microscope, Leica TCS SP2 AOBS (Leica Microsystems, Exton,A), was used for confocal microscopy. The He–Ne laser (excita-ion 543 nm, emission 560–615 nm) was used for imaging DsRed.o observe the DsRed signal in different layers of chickpea root,mages were scanned in multiple planes.

. Results

.1. A. tumefaciens mediated transformation of Foc

Prior to completing the transformation, the sensitivity of wildype strains of Foc to hygromycin B was determined by growing fun-al cultures on PDA plates in the presence (or absence) of varyingoncentrations of hygromycin B. Seven days after inoculation, therowth of Foc was completely inhibited at a hygromycin concen-ration of 100 �g/ml (Suppl. Fig. S1). To transform Foc, we used theBIF-DsRed vector (Nizam et al. 2010). Co-cultivation of A. tumefa-iens cells (carrying DsRed-Express) with conidia from F. oxysporumas performed in the presence of varying amounts of acetosy-

ingone (AS). The co-cultivation led to the emergence of coloniesesistant to hygromycin B after 3–4 days of membrane transferdialysis membrane, cellophane discs or nylon membrane) fromo-cultivated medium to selection plate.

.2. Optimisation of transformation conditions

To optimise transformation efficiency, we have determinedhe influence of the following factors: concentration of AS, initialonidial concentration, type of membrane, co-cultivation tem-erature and incubation time. Acetosyringone was required forransformation as no transformants were obtained in the absencef acetosyringone. Consequently, the number of transformantsncreased with increasing amounts of AS (100 and 200 �M); how-ver, the number of transformants remained nearly constant atS concentrations of 200 and 400 �M. We observed that an ini-

ial conidial concentration of 1 × 106 conidia/ml was sufficient toroduce transformants. During membrane experiments, the maxi-um number of transformants (17) was obtained using cellophane,

ollowed by dialysis membranes (11). Only two transformants werebtained from plates containing nylon membranes. In experimentso determine the role of temperature, most transformants (18) werebtained at 25 ◦C, and the number of transformants at 22 ◦C and8 ◦C were 14 and 9, respectively. The number of transformants

as significantly higher (19) at an incubation time of 48 h when

ompared to 36 h (10). However, it was very difficult to distin-uish separate colonies when plates were incubated for prolongederiods (72 h) because the transformants overlapped and mixed

Blots were hybridised using reporter-gene specific fragments. Numbers on the leftindicate the position and size of molecular size markers (M – �-HindIII Marker, WT– wild type).

with each other. By conducting these experiments, we have estab-lished that the ideal conditions for the transformation of Foc isco-cultivation in the presence of 200 �M acetosyringone on cel-lophane for 48 h at 25 ◦C; these conditions produce approximately16–19 transformants/105 conidia/Petri plate (90 mm).

3.3. Expression analysis of DsRed-Express in Foc

To evaluate the expression of DsRed-Express in the trans-formed F. oxysporum, mycelia from transformants were directlyharvested from the selection plate and observed with fluorescencemicroscopy. The red fluorescent mycelia were easy to detect inall transformants of Foc. Additionally, no auto-fluorescence wasdetected in the wild type strain. The level of fluorescent proteinexpression varied among individual transformants. To further anal-yse germination and fungal morphology, conidia from 12-day-oldplates were harvested and studied at different time intervals on aglass slide. Fluorescence and confocal microscopy studies clearlyindicate the expression of DsRed in different stages of fungal devel-opment (i.e., conidia, germ tube and fungal hyphae) as shown inFig. 1 and Supplementary Fig. S2.

3.4. Molecular analysis of transformants

To confirm the presence of the transgene in transformants, ini-tial screening was conducted by performing PCR amplification ofa 646 bp amplicon using DsRed-Express specific primers. IsolatedDNA was used as the template, and all of the hygromycin B resistanttransformants contained the transgene. Furthermore, to determinethe copy number and integration site of T-DNA, genomic DNA wasisolated from selected transformants exhibiting varying expressionlevels as well as wild type Foc. The restriction enzyme EcoRI wasused to digest genomic DNA, and it cuts the T-DNA construct once

in such a way that the smallest hybridised fragment with the DsRedprobe should be greater than 3.8 kb (Fig. 2A). The purified ampli-con of DsRed-Express was used as a probe for hybridisation. Theexistence of different band sizes suggests random integration of

336 Md.N. Islam et al. / Microbiological Research 167 (2012) 332– 338

F g confl pi andm

tmi

3

tort

ig. 3. Infection assay of DsRed-transformed Foc in chickpea root by laser-scanninater stage of germination showing germ tube and penetration into the root after 3 d

erged images after z-stacking using the laser-scanning confocal microscope.

he T-DNA gene. The results indicate that most of the transfor-ants have integration of a single copy; however, multiple copy

ntegration was observed in one of the transformants (Fig. 2B).

.5. Confocal microscopy studies of Foc-chickpea root interaction

After 1, 3, 5 and 6 days post-inoculation (dpi), the function of

he red fluorescent marker was evaluated by confocal microscopyf the infected roots. Because the auto-fluorescence of the chickpeaoot was very low at the emission wavelength of DsRed-Express,he position of infecting hyphae within the host tissues was easily

ocal microscope. (A) Attachment of conidia with the root observed after 1 dpi, (B) (C and D) massive colonisation after 5 and 6 dpi. The photographs (B–D) represent

visualised. The germinating conidia were observed on the surfaceof the chickpea root on the first dpi (Fig. 3A). Further, the conidiaexhibited a definite germ tube, grew on the surface of the plantand penetrated directly into the root on 3 dpi (Fig. 3B). Severecolonisation into different sections of the chickpea root was clearlyvisualised on days 5 and 6 post inoculation (Fig. 3C and D).

4. Discussion

Fusarium wilt is a devastating disease that affects a wide rangeof legumes. To manage and prevent the disease, plant–pathogen

gical R

iptstpmhtgetrmDn2

cwpumFotcttttcToglAaotoc

otordgpcflAvrpbt(i

Ftppa

Md.N. Islam et al. / Microbiolo

nteraction studies are key to developing a strategy to controlathogen infection. The fluorescent reporter proteins are usefulools for monitoring plant–pathogen interactions within host tis-ues under varying physiological conditions. We aim to focus onhe establishment of Agrobacterium-mediated transformation of aathogenic strain of Foc with the DsRed-Express fluorescent proteinarker gene to study and visualise its behaviour directly in the

ost tissues. Previously, different formae specials of F. oxysporumransformation using Agrobacterium with a fluorescence markerene was used to monitor infection (Mullins et al. 2001; Takkent al. 2004; Michielse et al. 2009). However, at the present time,here are no reports regarding the transformation of F. oxyspo-um that is pathogenic to chickpea. Therefore, to transform andonitor the fluorescent protein in Foc, binary plasmids containingsRed-Express under the control of the gpdA promoter of Aspergillusidulans and carrying a hygromycin cassette were used (Nizam et al.010).

To perform the transformation, we checked the optimal con-entration of hygromycin B for the survival of wild type Foc. Itas found that 100 �g/ml hygromycin B was sufficient to com-letely restrict the growth of wild type Foc. This concentration wassed for the selection of transformants. Moreover, we also opti-ised the conidiation of Foc on different types of media. Because

oc is pathogenic to chickpea, the most substantial sporulation wasbserved on the Czapek Dox with chickpea seed extract. To fur-her optimise the transformation efficiency, various co-cultivationonditions were tested in five independent experiments. Ace-osyringone was found to be necessary for transformation as noransformant was observed at 0 �M AS; however, 10, 17 and 18ransformants were generated at 100, 200 and 400 �M AS, respec-ively. It was observed that the initial concentration of 1 × 106

onidia/ml is better for transformation than 1 × 107 conidia/ml.ransformation efficiency decreases with increasing concentrationf conidia because fungal colonies merge with each other, and sin-le transformants are difficult to distinguish from one another. Aonger co-cultivation period is also not required for F. oxysporum.fter 2 days of co-cultivation, transformants grew substantially,nd excessive mycelial growth complicated subsequent isolationf individual transformants. The optimal combination of condi-ions for transformation of Foc was co-cultivation in the presencef 200 �M of acetosyringone for 48 h at 25 ◦C and with an initialoncentration of 1 × 106 conidia/ml.

The fluorescent reporter gene, DsRed-Express, under the controlf the gpdA promoter was able to induce significant expression ofhe red fluorescent protein (DsRed) in Foc. The primary screeningf hygromycin-resistant transformants was conducted by fluo-escence microscopy, and DsRed-Express protein expression wasetected in early developmental stages of Foc (i.e., conidia anderm tube). F. oxysporum is unique in its asexual reproduction as itroduces three kinds of asexual spores viz. microconidia, macro-onidia, and chlamydospores (Nelson 1981). The expression ofuorescent protein was observed in all three types of spores (Fig. 1).dditionally, the expression of the fluorescent protein was highlyariable among different transformants, and the stability of fluo-escent marker remains constant even after removal of selectiveressure. Integration of T-DNA in the transformants was confirmedy Southern hybridisation, and most of the transformants con-ained a single, integrated copy of T-DNA at a random positionFig. 2). This result supports the earlier finding of T-DNA insertionn filamentous fungi (Chen et al. 2000; Grimaldi et al. 2005).

Chickpea root infection analysis with the DsRed-transformedoc confirmed the germination of conidia during 1 and 2 dpi by

he development of infection hyphae (Fig. 3). The major form ofenetration was observed to be directly through the apex. Norominent appresorium or appresorium-like structures were visu-lised during the penetration of Foc in our studies. This finding

esearch 167 (2012) 332– 338 337

also correlates with earlier reports viz. Ramírez-Suero et al. (2010)and Ruiz-Roldán et al. (2010) regarding the direct penetration ofF. oxysporum f. sp. medicaginis and F. oxysporum f. sp. lycopersici,respectively. Whether the pathogen penetrates the root only atthe apex or through differentiated tissue is still controversial, andthe invasion of F. oxysporum may depend upon different forma spe-cialis. There are various findings for each f. sp. of F. oxysporum, viz.F. oxysporum f. sp. asparagi (Smith and Peterson 1983) seems topenetrate the root only at the apex while f. sp. lini (Kroes et al.1998; Turlier et al. 1994), f. sp. pini (Farquhar and Peterson 1989)and f. sp. lycopersici (Bishop and Cooper 1983) appear to invadeboth at the apex and through differentiated tissues. However, ithas also been reported that F. oxysporum f. sp. radicis-lycopersicipenetrates portions of the root at random locations other than roottips whereas F. oxysporum (pathogenic on Eucalyptus) specificallypenetrates through root hairs or at the junction of epidermal cells(Salerno et al. 2004). DsRed transformation allows researchers totrace the growth of Foc within host structures for more detailedstudy by visualising the fluorescence of the marker protein.

The present study has described the optimal conditions for effi-cient transformation of Foc using A. tumefaciens. This techniquewill help us to perform various applications including the tar-geted disruption of specific genes, ectopic complementation ofloss-of-function strains and over-expression. Moreover, the strongexpression of DsRed fluorescent protein in transformants will allowus to monitor the mechanisms of host colonisation. We believe thatthe successful and highly reproducible transformation of Foc willaid our further study of fungal genomics because ATMT is advanta-geous over protoplast-based and other transformation techniques(de Groot et al. 1998).

Acknowledgements

This work is supported partially by research grant(BT/AB/01/01/2008) provided by the Department of Biotechnology,Government of India and a core grant from the National Instituteof Plant Genome Research, New Delhi. The Foc Delhi isolate wasprovided by Dr. S.C. Dube, Division of Plant Pathology, IndianAgricultural Research Institute, New Delhi. S.N. acknowledges theUniversity Grant commission for the fellowships.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.micres.2012.02.001.

References

Baird GS, Zacharias DA, Tsien RY. Biochemistry, mutagenesis, and oligomeriza-tion of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA2000;97:11984–9.

Bevis BJ, Glick BS. Rapidly maturing variants of the Discosoma red fluorescent protein(DsRed). Nat Biotechnol 2002;20:83–7.

Bishop CD, Cooper RM. An ultrastructural study of root invasion in three vasculardiseases. Physiol Plant Pathol 1983;22:15–27.

Bölker M, Böhnert HU, Braun KH, Görl J, Kahmann R. Tagging pathogenicity genesin Ustilago maydis by restriction enzyme-mediated integration (REMI). Mol GenGenet 1995;248:547–52.

Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ. Trans-kingdom T-DNAtransfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J1995;14:3206–14.

Bundock P, Mroı̌czek K, Winkler AA, Steensma HY, Hooykaas PJJ. T-DNAfrom Agrobacterium tumefaciens as an efficient tool for gene targeting inKluyveromyces lactis. Mol Gen Genet 1999;261:115–21.

Chen X, Stone M, Schlagnhaufer C, Romaine CP. A fruiting body tissue method for effi-cient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ

Microbiol 2000;66:4510–3.

Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in thesymbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS MicrobiolLett 2003;220:141–8.

3 gical R

C

D

d

D

D

E

FF

F

F

G

G

G

G

G

J

K

K

M

M

M

M

M

38 Md.N. Islam et al. / Microbiolo

overt SF, Kapoor P, Lee M, Briley A, Nairn CJ. Agrobacterium tumefaciens-mediatedtransformation of Fusarium circinatum. Mycol Res 2001;105:259–64.

avidson RC, Cruz MC, Sia RAL, Allen BM, Alspaugh JA, Heitman J. Gene disruption bybiolistic transformation in serotype D strains of Cryptococcus neoformans. FungalGenet Biol 2000;29:38–48.

e Groot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM. Agrobacteriumtumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol1998;16:839–42.

ing Y, Liang S, Lei J, Chen L, Kothe E, Ma A. Agrobacterium tumefaciens mediatedfused egfp-hph gene expression under the control of gpd promoter in Pleurotusostreatus. Microbiol Res 2011;166(4):314–22.

obinson KF, Grant SJ, Kang S. Cloning and targeted disruption, via Agrobacteriumtumefaciens-mediated transformation, of a trypsin protease gene from the vas-cular wilt fungus Verticillium dahliae. Curr Genet 2004;45:104–10.

ckert M, Maguire K, Urban M, Foster S, Fitt B, Lucas J, et al. Agrobacteriumtumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp.with the reef coral gene with the reef coral gene DsRed and the jellyfish genegfp. FEMS Microbiol Lett 2005;253:67–74.

AOSTAT, 2009. Statistical Database. http://www.fao.org.arquhar ML, Peterson RL. Pathogenesis in Fusarium root rot of primary roots of Pinus

resinosa grown in test tubes. Can J Plant Pathol 1989;11:221–8.itzgerald AM, Mudge AM, Gleave AP, Plummer KM. Agrobacterium and PEG-

mediated transformation of the phytopathogens Venturia inaequalis. Mycol Res2003;107:803–10.

ungaro MHP, Rech E, Muhlen GS, Vainstein MH, Pascon RC, de Queiroz MV,et al. Transformation of Aspergillusnidulans by microprojectile bombardmenton intact conidia. FEMS Microbiol Lett 1995;125:293–8.

elvin SB, Schilperoort RA. Plant molecular biology manual. 2nd ed. Dordrecht, TheNetherlands: Kluwer Academic Publishers; 1994.

oel SR, Gupta DC. Interaction of Meloidogyne javanica and Fusarium oxysporum f.sp. ciceri on chickpea. Indian Phytopathol 1986;39:112–4.

ouka RJ, Gerk C, Hooykaas PJ, Bundock P, Musters W, Verrips CT, et al. Transforma-tion of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologousrecombination. Nat Biotechnol 1999;17:598–601.

ranado JD, Kertesz-Chaloupkova K, Aebi M, Kues U. Restriction enzyme-mediatedDNA integration in Coprinus cinereus. Mol Gen Genet 1997;256:28–36.

rimaldi B, de Raaf MA, Filetici P, Ottonello S, Ballario P. Agrobacterium-mediatedgene transfer and enhanced green fluorescent protein visualization in the myc-orrhizal ascomycete Tuber borchii: a first step towards truffle genetics. CurrGenet 2005;48:69–74.

alali BL, Chand H. Chickpea wilt. In: Singh US, Mukhopadhyay AN, Kumar J, ChaubeHS, editors. Plant diseases of international importance. Diseases of cereals andpulses, vol. 1. Englewood Cliffs, NJ: Prentice Hall; 1992. p. 429–44.

roes JMLW, Sommers E, Lange W. Two in vitro assays to evaluate resistance inLinum usitatissimum to Fusarium wilt disease. Eur J Plant Pathol 1998;4:725–36.

uo CY, Chou SY, Huang CT. Cloning of glyceraldehyde-3-phosphate dehydrogenasegene and use of the gpd promoter for transformation in Flammulina velutipes.Appl Microbiol Biotechnol 2004;65:593–9.

atz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, et al. Flu-orescent proteins from non bioluminescent Anthozoa species. Nat Biotechnol1999;17(10):969–73.

ichielse CB, Hooykaas PJJ, van den Hondel CAMJJ, Ram AFJ. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet2005;48:1–17.

ichielse CB, Ram AFJ, Hooykaas PJJ, Van den Hondel CAMJJ. Role of bacterialvirulence proteins in Agrobacterium-mediated transformation of Aspergillusawamori. Fungal Genet Biol 2004;41:571–8.

ichielse CB, Wijk R, Reijnen L, Manders EMM, Boas S, Olivain C, et al. The nuclear

protein Sge1 of Fusarium oxysporum is required for parasitic growth. PLOSPathogens 2009;5(10):e100637.

ikkelsen L, Sarrocco S, Lübeck M, Jensen DF. Expression of the red fluorescentprotein DsRed-express in filamentous ascomycete fungi. FEMS Microbiol Lett2003;223:135–9.

esearch 167 (2012) 332– 338

Mullins E, Romaine CP, Chen X, Geiser D, Raina R, Kang S. Agrobacterium tumefaciens-mediated transformation of Fusarium oxysporum: an efficient tool for insertionalmutagenesis and gene transfer. Phytopathology 2001;91:173–80.

Nelson PE. Life cycle and epidemiology of Fusarium oxysporum. In: Mace ME, BellAA, Beckman CH, editors. Fusarium wilt diseases of plants. New York: AcademicPress, Inc.; 1981. p. 51–80.

Nizam S, Singh K, Verma PK. Expression of the fluorescent proteins DsRed and EGFPto visualize early events of colonization of the chickpea blight fungus Ascochytarabiei. Curr Genet 2010;56:391–9.

Ozeki K, Kyoya F, Hizume K, Kanda A, Hamachi M, Nunokawa Y. Transforma-tion of intact Aspergillus niger by electroporation. Biosci Biotechnol Biochem1994;58:2224–7.

Paparu P, Macleod A, Dubois T, Coyne D, Viljoen A. Efficacy of chemical andfluorescent protein markers in studying plant colonization by endophytic non-pathogenic Fusarium oxysporum isolates. Bio Control 2009;54:709–22.

Ramírez-Suero M, Khanshour A, Martinez Y, Rickauer M. A study on the susceptibil-ity of the model legume plant Medicago truncatula to the soil-borne pathogenFusarium oxysporum. Eur J Plant Pathol 2010;126:517–30.

Rho H, Kang S, Lee Y. Agrobacterium tumefaciens mediated transformation of theplant pathogenic fungus Magnaporthe grisea. Mol Cells 2001;12:407–11.

Ribeiro CAG, Ferraz S. The interaction between Meloidogyne javanica and Fusarium-oxysporum f. sp. Phaseoli in Beans (Phaseolus vulgaris). Fitopatologia Brasileira1984;8:439–46.

Ruiz-Roldán MC, Köhli M, Roncero MIG, Philippsen P, Di Pietro A, Espeso E. Nucleardynamics during germination, conidiation and hyphal fusion of Fusarium oxys-porum. Eukaryotic Cell 2010;9:1216–24.

Salerno MI, Gianinazzi S, Arnould C, Gianinazzi-Person V. Ultrastructural and cellwall modification during infection of Eucalyptus viminalis roots by a pathogenicFusarium oxysporum strain. J Gen Plant Pathol 2004;70:145–52.

Sambrook J, Russell DW. Molecular cloning – a laboratory manual. 3rd ed. New York:Cold Spring Harbour Laboratory Press; 2001.

Sanchez O, Navarro RE, Aguirre J. Increased transformation frequency and taggingof developmental genes in Aspergillus nidulans by restriction enzyme-mediatedintegration (REMI). Mol Gen Genet 1998;258:89–94.

Sarrocco S, Falaschi N, Vergara M, Nicoletti F, Vannacci G. Use of Fusarium oxysporumf. sp. Dianthi transformed with marker genes to follow colonization of carnationroots. J Plant Pathol 2007;89:47–54.

Sexton AC, Howlett BJ. Green fluorescent protein as a reporter in the Bras-sica – Leptosphaeria maculans interaction. Physiol Mol Plant Pathol 2001;58:13–21.

Sharma SB, Smith DH, McDonald D. Nematode constraints of chickpea and pigeonpeaproduction in the semi-arid tropics. Plant Disease 1992;76:868–74.

Sharma SB, McDonald D. Global status of nematode problems of peanut, pigeon-pea, chickpea, sorghum, and pearl millet and suggestion for future work. CropProtection 1990;9:453–8.

Smith AK, Peterson RL. Examination of primary roots of asparagus infected by Fusar-ium. Scanning Electron Microsc 1983;3:1475–80.

Takken FL, Van Wijk R, Michielse CB, Houterman PM, Ram AF, Cornelissen BJ. A one-step method to convert vectors into binary vectors suited for Agrobacteriummediated transformation. Curr Genet 2004;45:242–8.

Turlier MF, Eparvier A, Alabouvette A. Early dynamic interactions between Fusar-ium oxysporum f. sp. lini and the roots of Linum usitatissimum as revealed bytransgenic GUS-marked hyphae. Can J Bot 1994;72:1605–12.

Wall MA, Socolich M, Ranganathan R. The structural basis for red fluorescence in thetetrameric GFP homolog DsRed. Nat Struct Biol 2000;7:1133–8.

Wang J, Guo L, Zhang K, Wu Q, Lin J. Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea. Bioresour Technol 2008;99:8524–7.

Ward M, Kodama KH, Wilson LJ. Transformation of Aspergillus awamori and A. nigerby electroporation. Exp Mycol 1989;13:289–93.

Zwiers LH, De Waard MA. Efficient Agrobacterium tumefaciens mediated genedisruption in the phytopathogen Mycosphaerella graminicola. Curr Genet2001;39:388–93.