NmDef02, a novel antimicrobial gene isolated fromNicotiana megalosiphon confers high-level pathogenresistance under greenhouse and field conditionsRoxana Portieles1, Camilo Ayra1, Ernesto Gonzalez1, Araiz Gallo1, Raisa Rodriguez1, Osmany Chacon2,Yunior Lopez1, Mayra Rodriguez1, Juan Castillo3, Merardo Pujol1, Gil Enriquez1, Carlos Borroto1,Luis Trujillo1, Bart P.H.J. Thomma4 and Orlando Borras-Hidalgo1,*

1Center for Genetic Engineering and Biotechnology, Havana, Cuba2Tobacco Research Institute, San Antonio de los Banos, Havana, Cuba3INCA, San Jose de las Lajas, Havana, Cuba4Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands

Received 16 July 2009;

revised 10 December 2009;

accepted 12 December 2009.

*Correspondence (fax +537 33 1779;

email orlando.borras@cigb.edu.cu)

Keywords: plant defensin, recombi-

nant proteins, transgenic plants, dis-

ease control.

SummaryPlant defensins are small cysteine-rich peptides that inhibit the growth of a broad

range of microbes. In this article, we describe NmDef02, a novel cDNA encoding a

putative defensin isolated from Nicotiana megalosiphon upon inoculation with the

tobacco blue mould pathogen Peronospora hyoscyami f.sp. tabacina. NmDef02 was

heterologously expressed in the yeast Pichia pastoris, and the purified recombinant

protein was found to display antimicrobial activity in vitro against important plant

pathogens. Constitutive expression of NmDef02 gene in transgenic tobacco and

potato plants enhanced resistance against various plant microbial pathogens, includ-

ing the oomycete Phytophthora infestans, causal agent of the economically impor-

tant potato late blight disease, under greenhouse and field conditions.

Introduction

Upon pathogen challenge, plants activate a defence-

related metabolism producing several metabolites to

defend themselves against these pathogens, including

antimicrobial peptides such as defensins (Bruce and Pick-

ett, 2007). Plant defensins are a family of small (typically

45–54 amino acids), highly stable, basic cysteine-rich pep-

tides that occur ubiquitous throughout the plant kingdom

(Thomma et al., 2002). These peptides play an important

role as part of the plant’s natural defence system by inhib-

iting growth of a broad range of infectious microbes (Bro-

ekaert et al., 1995; Thomma et al., 2002, 2003).

Members of the defensin family are quite diverse with

respect to their amino acid composition, as only the eight

structure-stabilizing cysteines and a glycine residue (posi-

tion 34; numbering relative to type defensin member

Rs-AFP2; Terras et al., 1992) appear to be conserved

among almost all of the family members, which reflects

the diverse biological activities displayed by different plant

defensins (Broekaert et al., 1995; Thomma et al., 2002,

2003; Lay and Anderson, 2005). Furthermore, an aromatic

residue (position 11) that is always followed by a glycine

(position 13), two residues further along the polypeptide

chain, as well as a serine (position 8) and a glutamic acid

(position 29) residue are often found to be conserved (Lay

and Anderson, 2005).

Plant defensins have been isolated from almost all plant

tissues including seeds (Terras et al., 1992; Broekaert

et al., 1995), leaves (Terras et al., 1995), tubers (Moreno

et al., 1994), fruit (Meyer et al., 1996) and floral tissues

(Thomma and Broekaert, 1998; Park et al., 2002; Lay

et al., 2003). While some defensins exhibit constitutive

expression, others are induced by various abiotic and bio-

tic stress factors (Penninckx et al., 1996; Thomma and

Broekaert, 1998; Do et al., 2004). Many defensins have

been proven to display antimicrobial activity (Thevissen

et al., 2007), while others lack apparent antifungal or anti-

bacterial activity (Osborn et al., 1995; Liu et al., 2006).

The alfalfa (alfAFP; Gao et al., 2000) defensin isolated

from seeds of Medicago sativa was found to display

strong activity against the agronomically important fungal

ª 2010 CIGB678 Journal compilation ª 2010 Blackwell Publishing Ltd

Plant Biotechnology Journal (2010) 8, pp. 678–690 doi: 10.1111/j.1467-7652.2010.00501.x

pathogen Verticillium dahliae (Fradin and Thomma, 2006).

This defensin also inhibits the growth of other fungal plant

pathogens such as Alternaria solani and Fusarium culmo-

rum. However, the same defensin lacks significant in vitro

antimicrobial activity against the oomycete Phytophthora

infestans, an economically important pathogen that causes

potato late blight (Gao et al., 2000).

The mode of action of two different plant defensins

from radish (Raphanus sativus) and dahlia (Dahlia merckii),

Rs-AFP2 and Dm-AMP1, respectively, has been studied in

detail (Thevissen et al., 2000, 2004). Both peptides were

found to bind distinct sphingolipids in fungal outer plasma

membranes and, consequently, showed a differential activ-

ity against fungal and yeasts species, including the human

pathogen Candida albicans (Thevissen et al., 2004). The

antimicrobial activity in vitro of plant defensins suggests

an important role in the plant defence response, which is

further evidenced by enhanced disease resistance pheno-

types observed in different plant species heterologously

overexpressing plant defensin genes (Terras et al., 1995;

Gao et al., 2000; Zhu et al., 2007; Sels et al., 2008). Con-

stitutive expression of the radish defensin RsAFP2 was

found to enhance pathogen resistance of various plant

species, such as tobacco plants to the fungal leaf patho-

gen Alternaria longipes (Terras et al., 1995) and tomato to

A. solani (Parashina et al., 2000). Transgenic canola (Bras-

sica napus) constitutively expressing a pea defensin

showed enhanced resistance against blackleg disease

caused by the fungus Leptosphaeria maculans (Wang

et al., 1999). Furthermore, transgenic potato expressing

an alfalfa defensin exhibited robust resistance against

V. dahliae under field conditions (Gao et al., 2000). These

studies indicate that engineering disease resistance in

crops with a range of plant defensins has the potential to

provide protection against various fungal diseases.

In this study, we report on NmDef02, a novel defensin

gene isolated from the tobacco species Nicotiana megalo-

siphon, a non-cultivated wild tobacco species that is gen-

erally used as a parent in genetic tobacco breeding

programs because of its high resistance towards several

important pathogens (Borras-Hidalgo et al., 2006; Collazo

et al., 2006; Chacon et al., 2009). For example, N. mega-

losiphon has been shown to be highly resistant to Pero-

nospora hyoscyami f.sp. tabacina, an obligate biotrophic

oomycete for which species from the genus Nicotiana are

the only hosts and that causes one of the most important

foliar diseases of tobacco (Borras-Hidalgo et al., 2009). We

report on the activity of NmDef02 against several plant

pathogens in vitro. Furthermore, we show that the expres-

sion of this defensin gene in transgenic tobacco and

potato plants provides high-level resistance levels against

several pathogens under greenhouse and field conditions.

Results

In a suppression subtractive hybridization (SSH) library

prepared from N. megalosiphon leaves inoculated with

P. hyoscyami f.sp. tabacina (Borras-Hidalgo et al., 2006,

2009), a 219 bp clone with homology to plant defensin

genes was identified and named NmDef02. Based on the

deduced amino acid sequence, NmDef02 is a 73 amino

acids polypeptide including a putative 27 amino acids

(81 bp) signal peptide (MDRVALVSLCFVYLVLFVAQEIVV-

TEA) (Figure 1a). An amino acid sequence alignment of

plant defensins collected from public databases and pub-

lished reports (Lay and Anderson, 2005; De-Paula et al.,

Figure 1 (a) The DNA coding sequence for the NmDef02 mature peptide (bold letters) and its 27-amino acid signal peptide are shown below the

amino acid sequence. Underlined and in italics are the start and stop codon (*), respectively. (b) Comparison of the putative mature sequence of

NmDef02 and those of other 74 plant defensins retrieved from the SUCEST database and Psd1 (http://compbio.dfci.harvard.edu/tgi/). The

sequence alignment was performed with CLUSTAL X software (Thompson et al., 1997). The signal peptide sequence has been omitted and spaces

have been introduced to maximize the alignment. A conserved glycine and the eight cysteine residues that are invariant among members of the

plant defensin family are included in the consensus sequence dashed in gray and highlighted with capital letters and with asterisks. The serine resi-

due, not conserved in NmDef02, is dashed in black and with lowercase letters. Also, with lowercase letters are indicated the putative aromatic res-

idue (denoted as ‘a’), glycine and a glutamic acid that are conserved in most of the aligned sequences. All the sequences are coded by their

accession number followed by the abbreviated name of plant species from which they were isolated in bold. Classifiers are as follows: At: Arabid-

opsis thaliana, Bv: Beta vulgaris, Bn: Brassica napus, Bo: Brassica oleracea, Ca: Capsicum annuum, Cc: Capsicum chivense, Cp: Citrus paradisi, Dm:

Dahlia merckii, Ew: Eutrema wasabi, Gm: Glycine max, Hs: Heuchera sanguinea, Hv: Hordeum vulgare, Ms: Medicago sativa, Na: Nicotiana alata,

Np: Nicotiana paniculada, Oe: Olea europaea, Os: Oryza sativa, Pi: Petunia integrifolia, Ph: Petunia hybrida, Ps: Pisum sativum, Pp: Prunus persica,

Ppy: Pyrus pyrifolia, Rs: Raphanus sativa, Sa: Sinapis alba, Si: Setaria italica, Sb: Sorghum bicolor, So: Saccharum officinarum, Sol: Spinacia olera-

cea, Sp: Solanum pimpinellifolium, St: Solanum tuberosum, Ta: Triticum aestivum, Vf: Vicia faba, Vu: Vigna unguiculata, Vr: Vigna radiata, Vv: Vitis

vinifera, Zm: Zea mays. (c). Phylogenetic tree of NmDef02 with other representatives members of plant defensins. The phylogenetic tree was gen-

erated using the neighbour joining method (Saitou and Nei, 1987) (MEGA 4.0 (http://www.megasoftware.net)) using the same 74 defensins

sequences from the alignment represented above. NmDef02 is dashed in gray. Numbers represent bootstrap values and protein classifiers are

identical to those used in the multiple alignment.

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein 679

2008) is presented in Figure 1b. As expected, based on

previously published data (Thomma et al., 2002; Lay and

Anderson, 2005), limited sequence conservation is

observed among the 74 polypeptides (Figure 1b). Strictly

conserved are the eight cysteine residues thought to be

involved in disulphide bridge formation, the aromatic resi-

due at position 11 and glycines at position 13 and 34

(numbering relative to Rs-AFP2). Interestingly, the serine

(S) at position 8 that is conserved in many plant defensins

(Lay and Anderson, 2005) is not conserved in NmDef02.

(a)

(b)

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.680

Despite the observation that NmDef02 lands (62%

bootstrap support) within the group of 74 plant defensins

(Figure 1c), NmDef02 is likely to belong to a new defensin

subgroup because only a poor bootstrap support (19%)

links NmDef02 with its nearest Triticum aestivum defensin

homolog (Figure 1c).

(c)

Figure 1 Continued

681

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein

In vitro antifungal activity of recombinant NmDef02

The cDNA sequence coding for the mature part of

NmDef02 was fused in frame to the modified-factor signal

peptide without the last 4 amino acids as previously

described (Spelbrink et al., 2004) and transformed into the

yeast Pichia pastoris. After methanol induction of the

NmDef02 gene for 72 h in shaking flasks cultures, a

recombinant protein with the expected mature NmDef02

size of approximately 5.1 kDa was detected after protein

separation in SDS-PAGE (Figure 2a, lane 3; Figure 2b). The

recombinant protein in the culture supernatant constituted

about 30% of total proteins as judged by densitometry

analysis. Recombinant NmDef02 was not detected in cul-

tures of P. pastoris transformed with pPIC9 (empty vector)

used as control (Figure 2a, lane 2). Further purification of

recombinant NmDef02 from the culture supernatants

yielded a single protein band of the expected size

(Figure 2a, line 4), and purity was assessed by Matrix

assisted laser desorption ⁄ Ionization time-of-flight mass

spectrometry (Figure 2b).

The antimicrobial activity of the recombinant NmDef02

was tested in vitro using several plant pathogens. As

shown in Table 1, the recombinant defensin exhibited

antimicrobial activity displaying different IC50 values rang-

ing from 1 lM in the case of Fusarium oxysporum var.

cubense to 2.8 lM for A. solani after 48 h of incubation.

In the particular case of Phytophthora parasitica var.

nicotianae 4 lM of recombinant NmDef02 was enough to

inhibit hyphal growth after 48 h incubation (Figure 3).

These experiments confirmed the efficient antimicrobial

activity and broad spectrum activity of recombinant

NmDef02 in vitro, inhibiting fungi as well as oomycetes.

NmDef02 expression in transgenic tobacco and

potato plants

Because the recombinant NmDef02 produced in yeast exhib-

ited antimicrobial activity in vitro against the five plant patho-

gens that were tested, we generated transgenic tobacco and

potato plants to assess whether transgenes expressing

35S::NmDef02 would acquire resistance against microbial

pathogens in vivo. The relative expression of NmDef02

mRNA was examined by quantitative RT-PCR in 50 individual

35S::NmDef02 homozygous transgenic tobacco and potato

lines before inoculation experiments, to select the best

expressors for future work. Attempts to obtain polyclonal

antiserum that would specifically detect NmDef02 failed

(data not shown), and thus we used quantitative RT-PCR

results to obtain an indication of NmDef02 peptide levels. As

shown in Figure 4, several homozygous tobacco and potato

lines expressing 35S::NmDef02 grown under greenhouse

conditions displayed different expression levels of the

NmDef02 cDNA. The expression levels of NmDef02 were

generally higher in tobacco than in potato transgenic lines

1 2 3 4

54.4 kDa

44.2 kDa

35.1 kDa

14.4 kDa

6.8 kDa

100

75

50

25

0

Rel

ativ

e in

tens

ity (

%)

5131.021 Da

5000 10 000 15 000Mass m/z

(a)

(b)

Figure 2 Expression and partial purification of recombinant NmDef02

from Pichia pastoris culture supernatant. (a) SDS-PAGE (18%) showing

recombinant protein and purification profile of NmDef02. The differ-

ent lanes are: 1. low range marker, 2. Pichia pastoris transformed

with pPIC9 (empty vector), 3. Pichia pastoris transformed with pPIC9

containing NmDef02, 4. Purified NmDef02 using Superdex 75 column.

(b) MALDI-TOF mass spectra of the purified recombinant NmDef02

sample.

682

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.

(Figure 4). Twenty transgenic lines of each species with the

highest relative NmDef02 expression were selected for disease

resistance assays under greenhouse and field conditions.

Enhanced disease resistance of transgenic tobacco

plants under greenhouse conditions

Twenty transgenic 35S::NmDef02 tobacco lines and vector

control plants were inoculated with the oomycete patho-

gens P. parasitica var. nicotianae and P. hyoscyami f.sp. ta-

bacina, respectively. At 5 days after inoculation, slight

disease symptoms produced by P. parasitica var. nicotianae

appeared on the vector control plants whereas no symp-

toms were detected at this stage on the assayed transgenic

lines. However, 10 days after infection, severe disease

symptoms such as leaf wilting and stem rot were observed

in all of the vector control plants, and all of them died

5 days later (Figure 5a). However, only few (6.7%) of the

35S::NmDef02 transgenic lines revealed mild disease symp-

toms while the rest remained healthy (Figures 5a and 6a).

Tobacco transgenic lines inoculated with P. hyoscyami f.sp.

tabacina showed a similar behaviour as upon inoculation

with P. parasitica var. nicotianae. The transgenic tobacco

lines remained without disease symptoms at 10 days after

infection, while the vector control plants died during the

same period (Figures 5b and 6b). Interestingly, we found a

high correlation between the relative expression of

NmDef02 as determined by quantitative RT-PCR and resis-

tance against P. parasitica var. nicotianae and P. hyoscyami

f.sp. tabacina in the various transgenic lines (Figure 6). Lines

4, 5, 8, 28 and 49 with a relatively low NmDef02 expression

(Figure 4a) developed symptoms as a consequence of path-

ogen infection (Figure 6), while non-inoculated control

plants displayed no resistance.

Enhanced disease resistance of transgenic potato

plants

Potato is a very important agronomical crop worldwide that

suffers annual yield losses caused by several pathogens. To

study the response of 35S::NmDef02-transgenic potato

plants to A. solani and P. infestans under greenhouse

conditions, potato plants of transgenic 35S::NmDef02 lines

and vector control plants were inoculated with these patho-

gens. At 10 days post-inoculation, the control plants were

readily infected and disease symptoms were clearly

observed. As a consequence of disease progression, the

vector plants died while the transgenic lines remained

healthy with no obvious disease symptoms. As depicted in

Table 2, non-transformed lines used as controls displayed a

high degree (72.5%) of plants developing symptoms when

compared to the transgenic lines. In a separate experiment,

transgenic plants were also challenged with P. infestans,

causal agent of early blight, using the method described by

Vleeshouwers et al. (1999). Leaves of control plants

displayed typical spreading lesions, with water-soaked areas

and extensive rotting developed at later time points. In

contrast, all plants derived from NmDef02 lines displayed

resistance to P. infestans infection. At 10 days post-inocula-

tion, the lesion growth rate (LGR) found in potato transgenic

lines averaged from 0 to 1.9 mm ⁄ day, respectively. In

contrast to the NmDef02 transformed lines, inoculation of

non-transformed plants resulted in LGR of 5.9 mm ⁄ day

(Table 2).

Similar to tobacco transgenic lines, the level of disease

resistance correlated with the relative level of NmDef02

expression as determined by quantitative RT-PCR also in

potato transgenic lines. Potato transgenic lines 1, 3, 8, 9,

13, 20, 24, 25, 26, 36, 37 and 38 accumulated the high-

est transcript levels and displayed the highest degree of

resistance to both diseases (Table 2). According to these

results and similar to tobacco, the expression of NmDef02

(a) (b)

Figure 3 NmDef02 inhibits microbial growth in vitro. Purified

NmDef02 was evaluated for in vitro antimicrobial activity. Pre-germi-

nated zoospores of Phytophthora parasitica var. nicotianae

(3 · 104 zoospores ⁄ mL) were grown for 48 h at 23 �C in culture

medium in the absence (a) or presence (b) of 4 lM of NmDef02.

Table 1 Antimicrobial activity of NmDef02 on five plant

pathogens

Plant pathogens IC50* (lM)

Phytophthora infestans 1.4

Phytophthora parasitica var. nicotianae 4

Alternaria solani 2.8

Fusarium oxysporum var. cubense 1

Verticillium dahliae 2

*Protein molar concentrations (lM) required for 50% growth inhibition

(IC50) after 48 h of incubation were determined from the dose–response

curves (per cent growth inhibition vs. protein concentration). Five replicates

(N = 10) per experiments were performed.

683

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein

strongly increased the resistance of potato to A. solani

and P. infestans under greenhouse conditions.

Finally, to determine the performance of NmDef02-trans-

genic plants in natural infections under field conditions, tri-

als were conducted in a potato field in Havana during the

cold and wet season when P. infestans poses a significant

problem for potato cultivation with a high inoculum pres-

sure. As shown in Figure 7, NmDef02 lines displayed high

levels of field resistance against P. infestans when

compared with control plants of the parental cultivar

(Desiree). The relative area under the disease progress curve

(RAUDPC) value in NmDef02 lines was significantly lower

than that of vector controls (Figure 7d), which is consistent

with results obtained in greenhouse experiments and con-

firm the in vivo activity of NmDef02 to control P. infestans.

Discussion

Numerous plant defensins have been purified from several

plant species and plant tissues that show a wide range of

in vitro biological activities including a-amylase activity,

ion-channel blocking and antifungal and antibacterial

activity (Thomma et al., 2002; Lay and Anderson, 2005).

In this study, we isolated a novel plant defensin induced

by the tobacco species N. megalosiphon during the inter-

action with the blue mould pathogen P. hyoscyami f.sp.

tabacina (Borras-Hidalgo et al., 2009). We isolated a cDNA

clone encoding the plant defensin, designated NmDef02,

from a subtractive cDNA library (Borras-Hidalgo et al.,

2006). Interestingly, phylogeny studies including a collec-

tion of 73 proteins showed that NmDef02 belongs to a

new subgroup of plant defensins. Thus, we conclude that

NmDef02 is a novel member of the plant defensin family.

Defensins have been shown to accumulate in tissues,

especially seeds, as part of normal development or matura-

tion, as a constitutive defence against pathogens (Thomma

et al., 2002). Plant defensins consisting of 45–54 amino

acids have a net positive charge and characteristic sequence

conservation (Thomma et al., 2003). In general, plant

defensins possess potent and broad-spectrum growth-inhib-

itory activity against fungi. The antimicrobial activity of

NmDef02 in this study was investigated with recombinant

NmDef02 produced in P. pastoris (Figure 2). The recombi-

nant NmDef02 defensin exhibited antimicrobial activity

against several important common plant pathogens; P. infe-

stans, P. parasitica var. nicotianae, A. solani, F. oxysporum

var. cubense and V. dahliae (Table 1). The activity was rang-

ing in IC50 value from 1 to 4 lM.

The important role played by antimicrobial proteins in

plant defence has been well documented (Broekaert et al.,

(a)

0

2

4

6

8

10

12

14

16

18

Con

trol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Transgenic lines

Rel

ativ

e ex

pres

sion

(b)

0

2

4

6

8

10

12

14

16

18

Con

trol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50

Transgenic lines

Rel

ativ

e ex

pres

sion

Figure 4 Relative expression of NmDef02

defensin gene in tobacco (a) and potato (b)

transgenic lines. Bars represent mean values

and standard error of the results obtained

from three replicates. Real-time PCR was

used to measure the relative expression of

transcript level of NmDef02 defensin gene,

when compared to the constitutively expres-

sed 26S rRNA gene as an endogenous

control.

684

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.

1995; Francois et al., 2002). Transgenic plants have the

potential to provide a generic resistance against different

pathogens and are likely to reduce dependence on chemi-

cal pesticides. The independent transgenic plants showed

a range of NmDef02 expression levels, which might be

because of position effects, the effects of transgene copy

number and ⁄ or silencing processes. The relative expression

level of NmDef02 in transgenic lines ranged between 1

and 15 (Figure 4). Strong evidences supporting the func-

tion of NmDef02 in plant defence derives from the

enhanced resistance of transgenic tobacco (Figure 5) and

potato plants (Figure 7) constitutively expressing NmDef02.

Analysis of the disease resistance of tobacco (Figure 6)

and potato (Table 2) transgenic lines revealed enhanced

resistance against P. parasitica var. nicotianae, P. hyoscy-

ami f.sp. tabacina, A. solani and P. infestans, respectively,

compared to wild type or transformants containing vector

only. There was a significant correlation between the lev-

els of NmDef02 and the degree of disease resistance

observed in the transgenic lines. We interestingly showed

for the first time a high level of potato resistance to

P. infestans using a plant defensin under greenhouse and

field conditions (Figure 7). Gao et al. (2000) used a plant

defensin to increase potato resistance to V. dahliae, but

(a)

_____________________ __________

(b)

_____________________ ___________ ____________________ NmDef02 transgenic tobacco

NmDef02 transgenic tobacco

Vector control

Vector control

NmDef02 transgenic tobacco

Figure 5 Greenhouse evaluation of NmDe-

f02 transgenic tobacco for resistance against

Phytophthora parasitica var. nicotianae (a)

and Peronospora hyoscyami f.sp. tabacina

(b). Phenotype of vector control and patho-

gen-infected transgenic tobacco plant in the

greenhouse at 10 days after inoculation.

685

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein

P. infestans resistance was not observed in the potato

transgenic lines evaluated in field condition. Additionally,

Solis et al. (2007) reported the isolation of a defensin

gene, lm-def, isolated from the Andean crop ‘maca’ (Lepi-

dium meyenii) with activity against the pathogen P. infe-

stans. However, the activity of the defensin was not

evaluated in transgenic plants.

All tobacco and potato transgenic plants were healthy

and showed no morphological or developmental abnor-

malities even in the lines with the highest NmDef02 accu-

mulation. Thus, the expression level of NmDef02 in

tobacco and potato plants was sufficient to obtain anti-

fungal activity without any toxic effects to the host plant.

Finally, the present investigation shows that the expression

of the NmDef02 defensin in transgenic potato plants dem-

onstrated the efficacy of defensins in imparting disease

resistance in crop plants and indicated that they are good

candidates for crop improvement.

Experimental procedures

Identification and cloning of full-length cDNA

NmDef02 cDNA was isolated from an SSH library prepared from

N. megalosiphon leaves inoculated with P. hyoscyami f.sp. tabacina

according to Borras-Hidalgo et al. (2006). The full-length coding

region for NmDef02 gene was obtained according to specifications

in the SMART RACE II kit (BD Clontech, Palo Alto, CA, USA) and

submitted to GenBank (accession number: FJ948813).

Expression in yeast, purification and anti-microbial

assay of recombinant NmDef02

The DNA sequence coding for the mature NmDef02 sequence was

cloned without signal peptide into the pPIC9 expression vector

in-frame with the secretion signal a-mating factor, downstream of

the alcohol oxidase I promoter. A sequence of KEX2 sites compro-

mising 12 nucleotides was placed ahead of the 141 bp NmDef02

cDNA for cleaving the signal peptide. While the sequence encod-

ing, the STE13 cleavage sites (Glu–Ala–Glu–ala) in pPIC9 was

deleted because STE13 protease cleavage of Glu-Ala repeats is not

efficient and these repeats are not necessary for cleavage by KEX2.

A stop codon and an EcoRI restriction site were added at the

3¢-end. The NmDef02 fragment was isolated from pGEM-T plasmid

by digestion with XhoI and EcoRI enzymes and ligated into the

EcoRI- and XhoI-digested pPIC9. After transformed into Escherichia

coli JM109 host strain, the plasmid pPIC9 ⁄ NmDef02 was isolated

and linearized with SalI to favour integration at the his4 locus of

P. pastoris (GS115; Invitrogen, Carlsbad, CA, USA) genome, and

transformed into yeast by electroporation (according to the manual

of the Pichia expression kit; Invitrogen). The N-terminal sequencing

of recombinant NmDef02 and mass spectroscopy experiments con-

firmed that the recombinant NmDef02 was expressed correctly.

The supernatant was precipitated by saturated ammonium

sulphate, and the pellet was resolved in 20 mM Tris–HCl (pH 7.5)

buffer. After desalting and concentrating by ultrafiltration, the

(a)

1

2

3

4

5

6

7

8

9

10

Con

trol 4 5 8 15 16 17 18 19 27 28 32 33 34 35 39 40 41 42 43 49

Transgenic lines

Stem

dis

ease

rat

ing

(b)

0

10

20

30

40

50

60

70

80

90

100

Con

trol 4 5 8 15 16 17 18 19 27 28 32 33 34 35 39 40 41 42 43 49

Transgenic lines

Plan

ts w

ith s

ymto

ms

(%)

Figure 6 Evaluation of different NmDef02

tobacco lines in greenhouse condition after

inoculation with Phytophthora parasitica var.

nicotianae (a) and Peronospora hyoscyami

f.sp. tabacina (b) at 10 days after inocula-

tion. One hundred plants were used per

transgenic line. Each bar represents mean

values with standard errors.

686

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.

sample was applied to Superdex 75 column pre-equilibrated with

buffer of 50 mM Tris–HCl (pH 7.5) and 0.15 M NaCl on AKTA

Prime system (Amersham-Pharmacia, Uppsala, Sweden). The frac-

tion containing recombinant NmDef02 was collected and pre-

pared for subsequent studies. The molecular mass of the protein

was estimated by SDS-PAGE in 18% (w ⁄ v) polyacrylamide gels

(Laemmli, 1970) stained with Coomassie Blue (Bio-Rad, Hercules,

CA, USA) and determined by MALDI-TOF mass spectrometry.

Densitometry analysis was used to estimate the per cent of pro-

tein secreted using Bio-Rad GS-700 Image Densitometer and

Molecular Analyst Software. Finally, the purified NmDef02 con-

centration was estimated according to Pace et al. (1995).

The antimicrobial activity of NmDef02 was measured in an

in vitro assay using 96-well microtiter plates. Fifty microlitres of

defensin dilution was added to each well of the microtiter plate

containing 50 ll of spore suspension from each microorganism

prepared in potato dextrose medium (Difco, Sparks, MD, USA) at

the following concentrations: P. infestans, 2500 sporangia ⁄ mL;

P. parasitica var. nicotianae, 3 · 104 sporangia ⁄ mL; A. solani,

2 · 106 spores ⁄ mL; F. oxysporum var. cubense, 40000 spor-

es ⁄ mL; V. dahliae, 104 spores ⁄ mL were harvested from potato

dextrose medium (Difco) agar plates, by washing the plate with

water. The microbial cultures were incubated at 28 �C for

48 h. The IC50 values were determined by spectrophotometry

and microscopic analysis of hyphal growth after 48-h incubation

at room temperature (Broekaert et al., 1990). Values were

obtained from data collected in at least three independent

experiments.

Construction of transformation vectors and

production of transgenic plants

The signal sequence and coding region of the NmDef02 defensin

peptide cloned in the pBluescript vector were digested with NcoI

and SmaI and ligated into the pBP-Omega8 vector between cauli-

flower mosaic virus (CaMV) 35S promoter and the nopaline syn-

thase terminator (nos T). The resulting CaMV 35S

promoter ⁄ NmDef02 ⁄ nos T fusions were cloned into the binary T-

DNA pCambia 2300 plasmid (kindly provided by Prof. Richard Jef-

ferson, CAMBIA, Australia) for tobacco transformation using the

HindIII and PstI restriction sites. One construct was transferred to

Agrobacterium tumefaciens strain At2260 by the liquid nitrogen

method (Hofgen and Willmitzer, 1988). The tobacco transforma-

tion protocol according to Ayala et al. (2009) was followed. For

construction of potato transformations, the vector and all the

cloning procedures were the same used above for tobacco except

that the pCambia3300 was used as binary vector. The potato

transformation was performed according to Soto et al. (2007).

Regenerated tobacco and potato plants, all 5 cm in height, were

transferred to pots containing black turf and rice husk (4 : 1) and

grown in growth chambers at 23 �C.

Relative gene expression of NmDef02 defensin in

transgenic plants

Total RNA was extracted from leaves of homozygous (T6) tobacco

(45 day-old plants) and potato (vegetatively propagated) (30 day-

old plant) transgenic lines using the RNeasy kit (Qiagen, Valencia,

CA, USA), including an on-column DNAse treatment (Qiagen)

according to manufacturer’s instructions. Poly (A)+ RNA was iso-

lated using the Dynabeads� mRNA Purification kit (Dynal A.S.,

Oslo, Norway), according to the manufacturer’s instructions. The

cDNA was synthesized using an oligo-(dT) primer and the Super-

Script III reverse transcriptase kit (Invitrogen) according to the

manufacturer’s instructions. Fifty transgenic lines from each spe-

cies were evaluated. Quantitative real-time PCR was conducted

using a Rotor-Gene 3000 PCR machine (Corbett, Sydney, Austra-

lia) with the QuantiTect SYBR Green PCR kit (Qiagen). The primer

sequences from NmDef02 defensin were: forward 5¢-AAGCT-

TATGCGTGAGTGCAAGGCTC-3¢ and reverse 5¢-CTGCAGTTAGC

ACTCGAATATAC-3¢. Real-time PCR was used to measure the rel-

ative expression of NmDef02 transcript levels, when compared to

the constitutively expressed 26S rRNA gene as an endogenous

control. Real-time PCR conditions were as follows: an initial 95 �Cdenaturation step for 15 min followed by denaturation for 15 s at

95 �C, annealing for 30 s at 60 �C, and extension for 30 s at

72 �C for 40 cycles and analysed on the Rotor-Gene 3000 soft-

ware (Corbett). All experiments were repeated twice. Finally, 20

lines from each species with the highest expression of NmDef02

defensin, inferred by quantitative PCR, were selected for disease

testing.

Table 2 Evaluation of different NmDef02 potato lines in green-

house condition after inoculation with Alternaria solani and

Phytophthora infestans

Control and

transgenic

lines

Plants with

symptoms produced

by A. solani (%)*

Lesion growth rate

(LGR) produced by

P. infestans (mm ⁄ day)†

Vector control 72.5 a 5.9 a

1 0.0 c 0.0 c

3 0.0 c 0.0 c

7 0.0 c 1.2 b

8 0.0 c 0.0 c

9 0.0 c 0.0 c

10 1.2 b 0.2 c

12 2.3 b 1.6 b

13 0.0 c 0.0 c

17 0.5 c 0.0 c

20 0.0 c 0.0 c

23 2.0 b 1.9 b

24 0.0 c 0.0 c

25 0.0 c 0.0 c

26 0.0 c 0.0 c

36 0.0 c 0.0 c

37 0.0 c 0.0 c

38 0.0 c 0.0 c

40 0.0 c 0.0 c

44 2.5 b 1.5 b

48 0.0 c 0.8 c

*Mean with the same letter are not different (P < 0.05) according to

Tukey test.†LGR was estimated and analysed with ANOVA using SPSS (IBM Company,

Chicago, IL, USA). LSD = 0.05 (P < 0.05).

687

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein

Disease resistance assay for transgenic plants

The microbes P. parasitica var. nicotianae race 0, P. hyoscyami

f.sp. tabacina, A. solani and P. infestans used in this study were

isolated from naturally infected tobacco and potato plants in

Havana fields. Each isolate was identified by specialists at the

Plant Health Institute, Havana. The inoculum production and inoc-

ulation were performed according to previously published reports

(Csinos, 1999; Vleeshouwers et al., 1999; Borras-Hidalgo et al.,

2006; Dita et al., 2006).

Black shank assay

Plants were inoculated by aseptically pushing the infested tooth-

picks into stems 2–3 cm above the soil line or into root systems

near the base of the plant. Greenhouse temperature was ranging

from 15 to 25 �C during the tests. Development of stem lesions

was evaluated using a linear scale of 1–10, where 1 was no dis-

ease and 10 was a dead plant. Ratings were taken on stems

10 days post-inoculation (Csinos, 1999).

Blue mold assay

The plants were inoculated by placing several 10-ll droplets of a

5 · 103 spores ⁄ mL suspension onto leaf panels. The plants were

then placed in moistened, black plastic bags overnight to promote

infection. Bags were removed the following day, and plants were

scored 10 days following spraying for symptoms: necrosis and ⁄ or

chlorosis on any leaves was scored as positive, while no visible

symptoms on all leaves were scored as negative. The percentage

of plants with disease symptoms was determined (Borras-Hidalgo

et al., 2006).

Potato early blight assay

The plants were inoculated by spraying to run off with a suspen-

sion of 103 conidia ⁄ mL. To enhance drop adherence at the inocu-

lation site, the conidial suspension was supplemented with gelatin

(1% w ⁄ v; SigmaG-8150; Sigma Chemical Co., St Louis, MO,

USA). After inoculation, plants were kept for 24 h in a moist

chamber at 25 �C, 12-h photoperiod. After this time, plants were

transferred to greenhouse conditions. The percentage of plants

with symptoms was determined at 10 days post-inoculation (Dita

et al., 2006).

Potato late blight assay

The third to sixth leaves from the top were detached from several

plants, and the petioles were fitted in water-saturated florist foam

(Oasis; V.L. Smithers A ⁄ S, Denmark) and placed in plastic trays

lined with wet filter paper. Ten-microlitre droplets containing 500

zoospores were applied to the lower side of the leaves. A total of

2–10 droplets were placed on each leaf, depending on its size.

The trays were tightly fitted with a transparent plastic cover and

(d) 80

70

60

50

40

30RA

UD

PC

20

10

0

Desiree (control)

NmDef02: Line 9

NmDef02: Line 1

NmDef02: Line 13

NmDef02: Line 37

NmDef02: Line 3

NmDef02: Line 20

NmDef02: Line 38

NmDef02: Line 8

NmDef02: Line 24

NmDef02: Line 36

(a) (b) (c)

Figure 7 Field trails of NmDef02 transgenic potato (cultivar ‘Desiree’) for resistance against Phytophthora infestans. Phenotype of vector control

(a) and transgenic potato plant lines (b, c) in a potato production area with a high late blight incidence at 35 days after planting. Relative area

under the late blight progress curve was determined (d). Each bar represents mean values and standard error.

688

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.

placed under fluorescent light in a regulated growth chamber

(15 �C for a 16-hr photoperiod). Inoculation spots were examined

for disease symptoms and necrosis daily for 10 days. The largest

length and width (perpendicular to the length) of each lesion

were measured, and the ellipse area (A = 1 ⁄ 4 · p · length ·width) was calculated. Analysis of LGR was estimated by linear

regression over time (Vleeshouwers et al., 1999).

One hundred plants of each of the homozygous transgenic

tobacco (T6) and potato lines (vegetatively propagated) were used

in the experiments under greenhouse conditions. Subsequently,

field evaluation of potato expressing NmDef02 was made. To

determine the performance of NmDef02 under field conditions,

trials were conducted in a potato production area in Havana with

a high inoculum pressure where P. infestans is a significant prob-

lem for potato production each year. During cold and wet season

of 2008, a total of 10 transgenic NmDef02 lines and transgenic

control lines (cultivar ‘Desiree’ transformed with the vector) were

evaluated by planting 100 tubers of each transgenic homozygous

line in the potato production area with a high late blight inci-

dence. The tubers were planted with a random design to look at

positional effects in the field. The RAUDPC was calculated accord-

ing to Kirk et al. (2001). Data were analysed by analysis of vari-

ance or general linear model procedures of SAS (SAS Institute,

Cary, NC, USA). Significant difference among means was deter-

mined by Fisher’s least significant difference mean separation at

P = 0.05.

Acknowledgements

We are grateful to the Cuban State Council for providing

support of this research. The authors would like to thanks

Dr Muluneh Tamiru Oil for comments and critical reading

of the manuscript. The authors thank to anonymous

reviewers and editor for useful suggestion.

References

Ayala, M., Gavilondo, J., Rodrıguez, M., Fuentes, A., Enrıquez, G.,

Perez, L., Cremata, J. and Pujol, M. (2009) Production of

Plantibodies in Nicotiana plants. in: Methods in Molecular

Biology. Recombinant Proteins from Plants (Faye, L. and

Gomord, V., eds), pp. 103–134. New York: Humana Press.

Borras-Hidalgo, O., Thomma, B., Collazo, C., Chacon, O.,

Borroto, C.J., Ayra, C., Portieles, R., Lopez, Y. and Pujol, M.

(2006) EIL2 transcription factor and glutathione synthetase are

required for defense of tobacco against tobacco blue mold.

Mol. Plant Microbe Interact. 19, 399–406.

Borras-Hidalgo, O., Thomma, B., Silva, Y., Chacon, O. and Pujol,

M. (2010) Tobacco blue mould disease caused by Peronospora

hyoscyami f. sp. tabacina. Mol. Plant Pathol. 11, 13–18.

Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A. and

Vanderleyden, J. (1990) An automated quantitative assay for

fungal growth inhibition. FEMS Microbiol. Lett. 69, 55–60.

Broekaert, W.F., Terras, F.R., Cammue, B.P. and Osborn, R.W.

(1995) Plant defensins: novel antimicrobial peptides as

components of the host defense system. Plant Physiol. 108,

1353–1358.

Bruce, T.J. and Pickett, J.A. (2007) Plant defence signalling

induced by biotic attacks. Curr. Opin. Plant Biol. 10, 387–392.

Chacon, O., Hernandez, I., Portieles, R., Lopez, Y., Pujol, M. and

Borras-Hidalgo, O. (2009) Identification of defense-related

genes in tobacco responding to black shank disease. Plant Sci.

177, 175–180.

Collazo, C., Ramos, P.L., Chacon, O., Borroto, C.J., Lopez, Y.,

Pujol, M., Thomma, B.P.H.J., Hein, I. and Borras-Hidalgo, O.

(2006) Phenotypical and molecular characterization of the

tomato mottle taino virus – Nicotiana megalosiphon interaction.

Physiol. Mol. Plant Pathol. 67, 231–236.

Csinos, A.S. (1999) Stem and root resistance to tobacco black

shank. Plant Dis. 83, 777–780.

De-Paula, V.S., Rasear, G., Medeiros, L., Miyamoto, C.A.,

Almeida, M.S., Kurtenbach, E., Almeida, F.C.L. and Valente,

A.P. (2008) Evolutionary relationship between defensins in the

Poaceae family strengthened by the characterization of new

sugarcane defensins. Plant Mol. Biol., 68, 321–335.

Dita, M.A., Brommonschenkel, S.H., Matzuoka, K. and Mizubuti, E.

(2006) Components of resistance to early blight in four potato

cultivars: effect of leaf position. J. Phytopathol. 153, 1–6.

Do, H.M., Lee, S.C., Jung, H.W., Sohn, K.H. and Hwang, B.K.

(2004) Differential expression and in situ localization of a

pepper defensin (CADEF1) gene in response to pathogen

infection, abiotic elicitors and environmental stresses in

Capsicum annuum. Plant Sci. 166, 1297–1305.

Fradin, E.F. and Thomma, B.P.H.J. (2006) Physiology and

molecular aspects of Verticillium wilt diseases caused by

V. dahliae and V. albo-atrum. Mol. Plant Pathol. 7, 71–86.

Francois, I.E., De Bolle, M.F., Dwyer, G., Goderis, I.J., Woutors, P.F.

and Verhaert, P.D. (2002) Transgenic expression in Arabidopsis

of a polyprotein construct leading to production of two different

antimicrobial proteins. Plant Physiol. 128, 1346–1358.

Gao, A.G., Hakimi, S.M., Mittanck, C.A., Wu, Y., Woerner, B.M.,

Stark, D.M., Shah, D.M., Liang, J. and Rommens, C.M. (2000)

Fungal pathogen protection in potato by expression of a plant

defensin peptide. Nat. Biotechnol. 18, 1307–1310.

Hofgen, R. and Willmitzer, L. (1988) Storage of competent cells

for Agrobacterium transformation. Nucleic Acids Res. 16, 9877.

Kirk, W.W., Felcher, K.J., Douches, D.S., Coombs, J.M., Stein,

J.M., Baker, K.M. and Hammerschmidt, R. (2001) Effect of host

plant resistance and reduced rates and frequencies of fungicide

application to control potato late blight. Plant Dis. 85, 1113–

1118.

Laemmli, U.K. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature, 227, 681–

685.

Lay, F.T., Schirra, H.J., Scanlon, M.J, Anderson, M.A. and Craik,

D.J. (2003) The three-dimensional solution structure of NaD1, a

new floral defensin from Nicotiana alata and its application to

homology model of the crop defensin alfAFP. J. Mol. Bio., 325,

175–188.

Lay, F.T. and Anderson, M.A. (2005) Defensins – components of

the innate immune system in plants. Curr. Protein Pept. Sci. 6,

85–101.

Liu, Y.J., Cheng, C.S., Lai, S.M., Hsu, M.P., Chen, C.S. and Lyu,

P.C. (2006) Solution structure of the plant defensin VrD1 from

mung bean and its possible role in insecticidal activity against

bruchids. Proteins, 63, 777–786.

689

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

NmDef02, a novel antimicrobial protein

Meyer, B., Houlne, G., Pozueta-Romero, J., Schantz, M.L. and

Schantz, R. (1996) Fruit-specific expression of a defensin-type

gene family in bell pepper. Plant Physiol. 112, 615–622.

Moreno, M., Segura, A. and Garcia-Olmedo, F. (1994)

Pseudothionin-St1, a potato peptide active against potato

pathogens. Eur. J. Biochem. 223, 135–139.

Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I.,

Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B. and

Broekaert, W.F. (1995) Isolation and characterisation of plant

defensins from seeds of Asteraceae, Fabaceae,

Hippocastanaceae and Saxifragaceae. FEBS Lett. 368, 257–262.

Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995)

How to measure and predict the molar absorption coefficient

of a protein. Protein Sci. 4, 2411–2423.

Parashina, E.V., Serdobinskii, L.A., Kalle, E.G., Lavorova, N.V.,

Avetisov, V.A., Lunin, V.G. and Naroditskii, B.S. (2000) Genetic

engineering of oilseed rape and tomato plants expressing a

radish defensin gene. Russ. J. Plant Physiol. 47, 417–423.

Park, H.C., Kang, Y.H., Chun, H.J., Koo, J.C., Cheong, Y.H., Kim,

C.Y., Kim, M.C., Chung, W.S., Kim, J.C. and Yoo, J.H. (2002)

Characterization of a stamen specific cDNA encoding a novel

plant defensin in Chinese cabbage. Plant Mol. Biol. 50, 59–69.

Penninckx, I.A., Eggermont, K., Terras, F.R., Thomma, B.P., De

Samblanx, G.W., Buchala, A., Metraux, J.P., Manners, J.M. and

Broekaert, W.F. (1996) Pathogen-induced systemic activation of

a plant defensin gene in Arabidopsis follows a salicylic acid-

independent pathway. Plant Cell, 8, 2309–2323.

Saitou, N. and Nei, M. (1987) The neighbor-joining method:

a new method for reconstructing phylogenetic trees. Mol. Biol.

Evol. 4, 406–425.

Sels, J., Mathys, J., De Coninck, B.M.A., Cammue, B.P.A. and

De Bolle, M.F.C. (2008) Plant pathogenesis-related (PR)

proteins: a focus on PR peptides. Plant Physiol. Biochem. 46,

941–950.

Solis, J., Medrano, G. and Ghislain, M. (2007) Inhibitory effect of

a defensin gene from the Andean crop maca (Lepidium

meyenii) against Phytophthora infestans. J. Plant Physiol. 164,

1071–1082.

Soto, N., Enrıquez, G.A., Ferreira, A., Corrada, M., Fuentes, A.,

Tiel, K. and Pujol, M. (2007) Efficient transformation of potato

stems segments from cultivar Desiree, using phosphinothricin as

selection marker. Biotecnol. Apl. 24, 139–144.

Spelbrink, R.G., Dilmac, N., Allen, A., Smith, T.J., Shah, D.M. and

Hockerman, G.H. (2004) Differential antifungal and calcium

channel-blocking activity among structurally related plant

defensins. Plant Physiol. 135, 2055–2067.

Terras, F.R., Schoofs, H.M., De Bolle, M.F., Van Leuven, F., Rees,

S.B., Vanderleyden, J., Cammue, B.P. and Broekaert, W.F.

(1992) Analysis of two novel classes of plant antifungal proteins

from radish (Raphanus sativus L.) seeds. J. Biol. Chem. 267,

15301–15309.

Terras, F.R., Eggermont, K., Kovaleva, V., Raikhel, N.V., Osborn,

R.W., Kester, A., Rees, S.B., Vanderleyden, J., Cammue, B.P.

and Broekaert, W.F. (1995) Small cysteine-rich antifungal

proteins from radish: their role in host defense. Plant Cell, 7,

573–588.

Thevissen, K., Cammue, B.P.A., Lemaire, K., Winderickx, J.,

Dickson, R.C., Lester, R.L., Ferket, K.K., Van Even, F., Parret,

A.H. and Broekaert, W.F. (2000) A gene encoding a sphingolipid

biosynthesis enzyme determines the sensitivity of Saccharomyces

cerevisiae to an antifungal plant defensin from dahlia (Dahlia

merckii). Proc. Natl Acad. Sci. USA, 97, 9531–9536.

Thevissen, K., Warnecke, D.C., Francois, I.E.J.A., Leipelt, M.,

Heinz, E., Ott, C., Zahringer, U., Thomma, B.P.H.J., Ferket, K.K.

and Cammue, B.P.A. (2004) Defensins from insects and plants

interact with fungal glucosylceramides. J. Biol. Chem. 279,

3900–3905.

Thevissen, K., Kristensen, H.H., Thomma, B.P.H.J., Cammue,

B.P.A. and Francois, I.E.J.A. (2007) Therapeutic potential of

antifungal plant and insect defensins. Drug Discov. Today, 12,

966–971.

Thomma, B.P.H.J. and Broekaert, W.F. (1998) Tissue-specific

expression of plant defensin genes PDF2.1 and PDF2.2 in

Arabidopsis thaliana. Plant Physiol. Biochem. 36, 533–537.

Thomma, B.P.H.J., Cammue, B.P.A. and Thevissen, K. (2002) Plant

defensins. Planta, 216, 193–202.

Thomma, B.P.H.J., Cammue, B.P.A. and Thevissen, K. (2003)

Mode of action of plant defensins suggests therapeutic

potential. Curr. Drug Targets Infect. Disord. 3, 1–8.

Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and

Higgins, D.G. (1997) The CLUSTAL X windows interface:

flexible strategies for multiple sequence alignment aided by

quality analysis tools. Nucleic Acids Res. 25, 4876–4882.

Vleeshouwers, V.G.A.A., van Dooijeweert, W., Keizer, L.C.P.,

Sijpkes, L., Govers, F. and Colon, L.T. (1999) A laboratory assay

for Phytophthora infestans resistance in various Solanum

species reflects the field situation. Eur. J. Plant Pathol. 105,

241–250.

Wang, Y.P., Nowak, G., Culley, D., Hadwiger, L.A. and Fristensky,

B. (1999) Constitutive expression of pea defense gene DRR206

confers resistance to blackleg (Leptosphaeria maculans) disease

in transgenic canola (Brassica napus). Mol. Plant Microbe

Interact. 12, 410–418.

Zhu, Y.J., Agbayani, R. and Moore, P.H. (2007) Ectopic expression

of Dahlia merckii defensin DmAMP1 improves papaya resistance

to Phytophthora palmivora by reducing pathogen vigor. Planta,

226, 87–97.

690

ª 2010 CIGBJournal compilation ª 2010 Blackwell Publishing Ltd, Plant Biotechnology Journal, 8, 678–690

Roxana Portieles et al.