between attacin, cecropin and lysozyme has been suggested [8] because of the complementary mode
of action of these three proteins against bacterial membranes. Minimum inhibitory concentration
MIC of attacin E against different strains of E. coli was 8 mM [8], whereas only 1 mM or less of
cecropins was lethal [4]. While MICs of cecropins have been determined against a variety of phyto-
pathogenic bacteria [11], to our knowledge, no information is available about the activity of
attacin E against plant pathogens.
Introduction of antibacterial protein genes from insects to enhance plant resistance was previously
suggested by Jaynes et al. [12] and Casteels et al. [13]. In particular, expression of cecropin B gene
and synthetic analogues has been widely analyzed. Although enhanced resistance to bacteria was re-
ported in some cases [11,14], susceptibility was not modified in others [15,16]. Degradation of ce-
cropins by intercellular fluids IF [16 – 18] could explain the lack of resistance. In comparison with
cecropins, nothing is known about activity of attacins and their stability in planta or in IF. The
first transgenic plants expressing the attacin E gene, driven by the potato proteinase inhibitor II
[14] or by the CaMV
35
S promoter [19], were obtained from apple rootstock and displayed en-
hanced resistance to fire blight [20]. In this report, we describe for the first time the successful trans-
formation of pear with the attacin E gene and its expression in transgenic lines.
2. Materials and methods
2
.
1
. Plant material The Passe Crassane PC cultivar was chosen
for transformation experiments because of its high susceptibility to fire blight. For the in vitro fire
blight resistance test, the resistant cv. Old Home OH and non-transformed PC were used as con-
trols. Shoots were propagated in vitro as previ- ously reported [1].
2
.
2
. Plasmid constructions Two binary expression vectors, pFAJ3000 and
pFM3002, were used: pFAJ3000 [21] contained an nptII-based
expression cassette
as selectable
marker and a uidA-intron expression cassette ob- tained as a 3 kbp HindIII fragment from p35S
GUS INT [22], adjacent to the right border; pFM3002 derived from pFAJ3000 by replacement
of the uidA-intron cassette by an attacin E expres- sion cassette excised from pLDB11[23] as a
HindIII fragment, containing the attacin E gene [12] driven by a derivative of the cauliflower mo-
saic virus promoter Ca
2
MV
35
S containing a tandem duplication of 250 bp of upstream se-
quence [24] and the nopaline synthase transcrip- tional terminator sequence Fig. 1. The binary
vectors were transferred by electroporation to su- pervirulent Agrobacterium tumefaciens strain EHA
101 [25].
2
.
3
. Transformation experiments and propagation of transgenic lines
Five hundred fully expanded leaves were excised from shoots 2 weeks after subculture and transfor-
mation experiments were carried out as previously reported [2], using A. tumefaciens strain EHA101
containing either pFM3002 or pFAJ3000. Trans- genic shoots were subcultured on basal medium
with kanamycin 100 mgl as selective agent, then rooted as described in [2].
2
.
4
. E6idence of transformation DNA isolation from leaves of in vitro shoots
and polymerase chain reaction PCR experiments were carried out as previously described [2]. In
order to check the presence of attacin E or uidA
Fig. 1. Schematic representation of attacin E construct used in transformation experiments with pFM3002. The external re-
gion to the T-DNA borders is identical to that of pFAJ3000. Components are represented by boxes. RB and LB, right and
left borders; Ca
2
MV
35
S, derivative of the cauliflower mosaic virus promoter containing a tandem duplication of 250 bp of
upstream sequences; pNos and tNos, promoter and termina- tion signal respectively of the nopaline synthase gene; nptII,
coding region of the neomycinphosphotransferase gene kanamycin resistance; tOcs, termination signal of the oc-
topine synthase gene.
gene, specific primers were designed: 5-CT- TACGCTCAACTCCGATG-3 forward and 5-
AATCCGAAGTTAGGCTCCC-3 reverse for amplification of a 530 bp fragment in attacin E
gene,
5-GGTGGGAAAGCGCGTTACAAG-3 forward
and 5-GTTTACGCGTTGCTTC-
CGCCA-3 reverse for amplification of a 1.2 kb fragment in uidA gene. Annealing was performed
at 58°C.
2
.
5
. Control of the ploidy le6el Ploidy level in the transgenic lines and control
was estimated by flow cytometry. Nuclei were isolated from leaves by manual chopping with a
razor blade directly into Brown et al. buffer [26] with 2 4,6 diamino 2 phenyl indole dihy-
drochlorode Cheminex, then filtered through a 4 mm nylon mesh and analysed with a cytometer
Cell analyzer II; Partec, Germany. Pea leaf nu- clei were used as internal reference.
2
.
6
. Determination of expression le6els
2
.
6
.
1
. RNA extraction, Northern analysis and semi-quantitati6e re6erse transcription PCR
analysis Total RNA 50 – 100 mg was extracted as de-
scribed by Verwoerd et al. [27] from 0.5 g of young leaves excised from in vitro shoots. Purifica-
tion was achieved by addition of 2-butoxyethanol in order to precipitate polysaccharides [28].
For Northern blot analysis, 10 mg of total RNA were denatured and separated by electrophoresis
on a 1.2 agaroseformaldehyde gel in MOPS buffer, then transferred to Hybond™-N nylon
membranes Amersham, UK as described by Sambrook et al. [29]. Filter was prehybridized for
2 h then hybridized overnight at 50°C in sodium dodecyl sulfate SDS-formamide buffer [30], with
50 ng of an attacin E DNA probe. Attacin E DNA was obtained by digestion of pFM3002 with
HindIII, then
32
P-labelled with the ‘Ready to go’ kit Pharmacia, purified on TE MIDI Select-D
G-50 columns 5-3 Inc., USA. After hybridiza- tion, the filter was washed according to manufac-
turer’s recommendations and finally exposed for 4 days on X-OMAT
TM
film with one intensifying screen at − 80°C.
Reverse transcription RT was carried out from 1 mg total RNA according to Rosati et al. [31]. In
order to evaluate relative differences in cDNAs between transgenic lines, comparative kinetic anal-
ysis was conducted by PCR as suggested by Horikoshi et al. [32]. Initial amounts of PCR
substrates were adjusted for each line on the basis of an equivalent amplification of a cDNA from a
member of the constitutively expressed gene family encoding the alpha subunit of translation elonga-
tion factor 1 EF
1
-a [33]. In order to evaluate differences among lines proportional to differences
in initial amounts, we limited the amplification to 20 cycles. EF
1
-a PCR reactions were carried out on 2 ml reverse transcription products. Degener-
ated EF
1
-a primers forward, 5-ATTGTGGT- CATTGGYCAYGT-3; and reverse, 5-CCAAT
CTTGTAVACATCCTG-3, custom
designed from multiple sequence alignments [31], were used
to amplify a 702 bp fragment. In a second step, comparative amplification was carried out with
attacin E primers on equivalent total cDNA amounts, adjusted in a volume of 2 ml, using the
minimal number of cycles required to distinguish differences. The PCR reactions were run with
products from Eurogentec Seraing, Belgium and 1 mM primers in a Minicycler MJ Research,
USA. The thermocycler program was: 94°C, 5 min, 20 cycles of: 94°C, 30 s; 58°C, 1 min; 72°C,
1 min; 72°C, 15 min for specific attacin E and EF
1
-a amplifications. After gel electrophoresis on 0.8 wv agarose-ethidium bromide gel and de-
tection by ultraviolet light fluorescence, amplified products were blotted onto a Hybond™-N nylon
membrane following standard procedures of the manufacturer Amersham, UK. The filter was
prehybridized for 2 h at 42°C in SDS-formamide buffer [30], then hybridized overnight under the
same conditions, with attacin E and constitutively expressed EF
1
-a probes prepared as already de- scribed. After hybridization and washing, mem-
branes were exposed for 5 – 24 h to X-OMAT
TM
film with one intensifying screen at − 80°C.
2
.
6
.
2
. Protein extraction and Western analysis Protein extraction was carried out from 100 mg
of leaves excised from in vitro shoots for each transgenic line according to Schuster and Davies
[34], with the following modifications: 1 mM phenylmethylsulfonyl fluoride PMSF, an in-
hibitor of serine proteases, was added to the ex- traction buffer 0.7 M sucrose, 0.5 M Tris, 50 mM
EDTA, 0.1 M KCl, 2 vv b-mercaptoethanol.
Fig. 2. PCR analysis of transgenic pear plants. Gel elec- trophoresis of attacin E and uidA gene PCR products; lane C,
non-transformed plant; lanes P1 and P2, plasmids pFM3002 and pFAJ3000; lanes 1, 5 and 20, attacin E transgenic lines 1,
5 and 20; lane G, GUS transgenic line; lane L, molecular weight marker. Fragment sizes are indicated in kb.
severity was assessed by estimating the progression of necrosis from the inoculated leaf and apex to
the base using a scale of 0 – 3: 0, no damage; 1, necrotic apex; 2, less than half of the shoot ne-
crosed; 3, more than half of the shoot necrosed. For each line, susceptibility was calculated as the
average of all replicates about 75 replicates per line. Shoots of non-transformed lines of PC and
OH were used as controls, and a transgenic PC line expressing the b-glucuronidase and the NPTII
enzymes was also inoculated in order to estimate the effect of the expression of the transgenes that
were assumed to lack antibacterial activity.
Resistance levels were compared pairwise be- tween transgenic lines and non-transformed PC
using the non-parametric H test of Kruskall and Wallis. Data analysis was carried out using the
SASSTAT
6.06 software, NPAR1WAY procedure.
3. Results
