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ORIGINAL RESEARCH PAPER
A comparison between constitutive and inducible transgenicexpression of the PhRIP I gene for broad-spectrumresistance against phytopathogens in potato
Romel Gonzales-Salazar . Bianca Cecere . Michelina Ruocco . Rosa Rao .
Giandomenico Corrado
Received: 27 January 2017 / Accepted: 29 March 2017
� Springer Science+Business Media Dordrecht 2017
Abstract
Objectives To engineer broad spectrum resistance in
potato using different expression strategies.
Results The previously identified Ribosome-Inacti-
vating Protein from Phytolacca heterotepala was
expressed in potato under a constitutive or a wound-
inducible promoter. Leaves and tubers of the plants
constitutively expressing the transgene were resistant
to Botrytis cinerea and Rhizoctonia solani, respec-
tively. The wound-inducible promoter was useful in
driving the expression upon wounding and fungal
damage, and conferred resistance to B. cinerea. The
observed differences between the expression strate-
gies are discussed considering the benefits and features
offered by the two systems.
Conclusions Evidence is provided of the possible
impact of promoter sequences to engineer BSR in
plants, highlighting that the selection of a
suitable expression strategy has to balance specific
needs and target species.
Keywords Fungal resistance � Potato � Ribosome-
inactivating protein � Solanum tuberosum � Transgenicplants
Introduction
Amain interest of plant biotechnology is the introduc-
tion of resistance traits against fungal pathogens. The
development of the recombinant DNA technology has
allowed considerable progress in increasing plant
resistance against specific biotic stresses (Collinge
2016). Genes to be genetically engineered into plants
have been usually selected considering their role in a
disease resistance pathway or the toxicity of their
products to fungal growth (Punja 2006). Ribosome-
Inactivating Proteins (RIPs) are among the toxic
proteins that plants accumulate at high level to fight
pathogens (Dang and Van Damme 2015; Stirpe and
Gilabert-Oriol 2015). RIPs inhibit protein biosynthesis
in eukaryotes by virtue of their N-glycosidic cleavage
of the rRNA large subunit (Nielsen and Boston 2001).
The use of these enzymes in plant biotechnology has
gained interest not only for their catalytic activity but
also for their antimicrobial effects on a range of
pathogens (Cillo and Palukaitis 2014; Iglesias et al.
2016; Nielsen and Boston 2001; Van Damme et al.
Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-017-2335-0) contains supple-mentary material, which is available to authorized users.
R. Gonzales-Salazar � B. Cecere � R. Rao �G. Corrado (&)
Dipartimento di Agraria, Universita degli Studi di Napoli
‘‘Federico II’’, Portici, NA, Italy
e-mail: [email protected]
M. Ruocco
Istituto per la Protezione Sostenibile delle Piante, CNR,
Portici, NA, Italy
123
Biotechnol Lett
DOI 10.1007/s10529-017-2335-0
2001). Nonetheless, some restrictions on the possible
widespread use ofRIPswere also reported. High-levels
of RIP expression were phytotoxic and produced a
stunted and mottled plant phenotype (Dai et al. 2003;
Gorschen et al. 1997; Lodge et al. 1993), although in
other instances, phenotypic abnormalities were not
present (Desmyter et al. 2003; Maddaloni et al. 1997).
Plants encounter many microbial pathogens during
their lifetime and engineering plants with increased
broad-spectrum disease resistance (BSR) (i.e. against
two or more types of pathogen species) is currently an
important challenge for biotechnologists (Collinge
2016; Dangl et al. 2013). This strategy is highly
desirable as it can complement or enhance classic
R-gene mediated resistance approaches (Sarma et al.
2016). Among the available strategies to generate BSR
in plants (Kou and Wang 2010), the expression of
proteins involved in basal resistance and/or cooperat-
ing in the induction of hypersensitive response has the
advantage of being effective against different species
or strains of fungal pathogens or viruses (Durrant and
Dong 2004). It is expected that a more durable BSR
may be obtained by inducing in plants reactions that
mimic naturally occurring defence mechanisms, such
as cell death at infection sites (Kou and Wang 2010).
Engineering crops for BSR against pathogens can
be achieved using different expression strategies (Dutt
et al. 2014; Punja 2006). Controlled expression
systems provide advantages especially for human
consumption of GM-food (Corrado and Karali 2009).
Inducible promoters can limit the accumulation of
toxic, detrimental or noxious proteins in plants as well
as in the environment. For instance, pathogen resis-
tance can be enhanced in plants using pathogen- or
wound-inducible promoters thus avoiding the costly
exogenous treatment required to activate chemical
inducible promoters (Keller et al. 1999; Rizhsky and
Mittler 2001). Although pathogen- and wound-in-
ducible promoters may share some cis- and trans-
acting elements (Rushton et al. 2002; Singh et al.
2002), it is expected that induction by a wide range of
different pathogens can be more easily achieved with
the latter because they are activated virtually by any
biotic stress that lesion plant tissues. However, studies
that evaluate both constitutive and inducible expres-
sion to engineer biotic stress resistance are, to our
knowledge, very scarce.
The aim of this work was to test and compare the
efficacy of the constitutive or inducible accumulation
of an antimicrobial protein to engineer wide-spectrum
disease resistance in potato. Potato is the fourth largest
food crop worldwide and it is vulnerable to diseases
affecting leaves, stems, roots, and tubers. Tetrasomic
inheritance, high level of heterozygosity, low meiotic
recombination and inbreeding depression are the main
factor that burden the introgression of resistance traits
through classic breeding. To this aim, we used the
PhRIP I, a type-I RIP isolated from Phytolacca
heterotepala (Corrado et al. 2005; Di Maro et al.
2007). Previously, we showed that the inducible
expression of the PhRIP I increases resistance against
fungal pathogens and viruses in tobacco (Corrado et al.
2005, 2008). In this work, we generated transgenic
potatoes in which the PhRIP I is under the control of a
constitutive or an inducible promoter in order to
increase resistance against important pathogens that
attack different potato organs. Specifically, we evalu-
ated possible differences between the two strategies of
transgene expression in conferring resistance against
Rhizoctonia solani, a soil-borne fungal pathogen
ubiquitous in potato production, and against Botrytis
cinerea, a very common polyphagous necrotrophic
fungus causing the grey mould on leaves and stems.
Methods and materials
Construction of plant expression vectors
The expression cassette of the pG2935SRIP plasmid
(Corrado et al. 2008), that comprises the PhRIP I
cDNA under the control of the CaMV 35S RNA
promoter and terminator, was excised using EcoRV.
The recipient binary vector pBIN19 (Bevan 1984) was
digested with SmaI and then dephosphorilated with a
calf intestinal alkaline phosphatase (CIAP). After
agarose gel purification with the QIAquick Gel
Extraction Kit (Qiagen), the two fragments were
ligated by a T4 DNA ligase treatment, yielding the
pG35SRIP. All enzymes were purchased from Pro-
mega and were used according to the manufacturer’s
recommendations. For the inducible expression of the
PhRIP I we used the pGIPRIP binary vector (Corrado
et al. 2005). Briefly, in this plasmid the cDNA
encoding the PhRIP I was cloned under the control
of the wound-inducible PGIP promoter from bean
(Devoto et al. 1998). Plasmids were mobilised into
Agrobacterium tumefaciensC58C1Rif (Deblaere et al.
Biotechnol Lett
123
1985) cells by electroporation with a Bio-Rad
MicroPulser, according to the manufacturer’s
instructions.
Potato genetic transformation
Solanum tuberosum cv. Desiree plants grew under
sterile conditions at 24 �C with a 16L:8D cycle for
Agrobacterium tumefaciens-mediated transformation.
Chemicals were purchased from Duchefa Biochemie,
unless stated otherwise. Co-cultivation was perfomed
on internodal stem explants from 4 to 6 weeks-old
plants growing in Murashige and Skoog medium
supplemented with 30 g sucrose/l and solidified with
9 g Microagar/l (MS30 medium). Explants were cut
one day before co-cultivation and left onMS30 in Petri
dishes. A. tumefaciens carrying the desired construct,
growing in selective LB medium at 28 �C, was
refreshed and used when OD600 was between 0.8 and
1. Explants were co-cultivated for 10 min with
occasional gentle swirling. After washing with sterile
water, explants were blot-dried and transferred to
MS30 plates for 2 days. Explants were then transferred
to P55-I medium (MS30 with 0.1 mg indolacetic/l
acid, 3 mg gibberellic acid/l and 3 mg zeatine ribo-
side/l; pH 5.8) supplemented with 0.25 g cetofaxime/l
and 50 lg kanamycin/ml, solidified using 4 g Phy-
tagel/l (Sigma-Aldrich). Controls for selection were
treated as before but not co-cultivated. Controls for
regeneration were not co-cultivated and placed on
P55-I without selective antibiotics. We performed two
transformation experiments, in each starting with 150
explants (density: 25 explants per Petri dish). Explants
were transferred to fresh selective P55-I medium
every 2 weeks. From 2 months, shoots were trans-
ferred to MS30 medium supplemented with Gam-
borg’s B5 vitamin solution and 50 lg kanamycin/ml.
For bioassays, plants were transferred to soil and
multiplied by tuber for four cycles. To have tubers of
similar size, we used, as untransformed controls,
plants obtained by the in vitro regeneration control of
the genetic transformation experiments.
DNA analysis
Genomic DNA was isolated from leaves by using
previously published procedures (Corrado et al. 2005).
PCR reactions were performed in 50 ll, containingPCR Buffer (ThermoFischer), 0.25 mM. dNTPs,
20 pmol of each primer and 1.25 U di ampliTaq
(ThermoFisher). Primers are given Supplementary
Table 1. The amplification conditions were: one
denaturing step at 94 �C for 5 min, followed by 30
cycles of 94 �C for 45 s, 50 �C for 45 s and 72 �C for
45 s. After the final cycle, a 5 min step at 72 �C was
added. Amplification products were separated by
agarose gel electrophoresis to verify the presence
and size of amplified fragments using the 1 kb? lad-
der as a size marker (ThermoFisher). Southern anal-
ysis was performed digesting 10 lg DNA with 50 U
restriction enzyme for 16 h at 37� C. Fragments were
resolved into a 0.8% (w/v) agarose gel and transferred
onto a Hybond-N? membrane (Amersham) and cross-
linked by UV (150 mJ) using the Gs Gene Linker UV
(Biorad). A probe for the PhRIP I cDNA was prepared
by digesting the pG2935SRIP with HindIII. The DNA
fragments of interest were purified as described above
and labelled with [a-32P]dCTP (50 lCi, 3000 Ci/
mm) using random hexadeoxyribonucleotides accord-
ing to the instructions of the Prime-a-Gene Labeling
System (Promega). Unincorporated nucleotides were
removed by spin-column chromatography (Probe-
Quant G-50; GE Healthcare), according to the man-
ufacturer’s instructions. Hybridization and washing
steps were performed as described (Corrado et al.
2005).
Reverse transcription-PCR (RT-PCR)
The transcription of the transgene was assessed using a
two-steps Reverse Transcription-PCR (RT-PCR).
Total RNA was isolated from leaves as described
(Coppola et al. 2013). DNAse I treatment and first
strand cDNA synthesis were performed as reported
(Coppola et al. 2013) starting from 2 lg total RNA.
RT-PCR were executed in 20 ll with: PCR Buffer
(ThermoFischer), 0.5 mM dNTPs, 30 pmol of each
primer and 1 U di ampliTaq (ThermoFisher).
The thermal cycling program started with a step of
5 min at 50 �C and 10 min at 95 �C, followed by 35
cycles consisting of 20 s at 95 �C, followed by 45 sec
at the temperature of annealing (Ta) for the primer pair
(Supplementary Table 1) and 30 s at 72 �C. A final
10 min extension step at 72 �C ended the reaction.
The amplification of the cDNA coding for the
Elongation Factor 1-a gene (Acc. Num. DQ228326.1)
served as a control for cDNA synthesis and PCR
efficiency in the different samples. The sequences
Biotechnol Lett
123
annealed by the two EF1 primers are localised in two
contiguous exons for the detection of possible con-
taminant DNA in the PCR amplifications. Transgene
expression was detected with the RT-RIP-Fw and RT-
RIP-Rv primers (Supplementary Table 1). For the
analysis of the wound-inducible RIP expression,
leaves were wounded with a haemostat and samples
harvested after 48 h.
Western analysis
Total soluble proteins isolation from leaves of plants
growing in greenhouse, SDS-PAGE and blotting on
nitrocellulose membrane were performed according to
previously published procedures (Coppola et al.
2015). Western analysis was carried out as described
(Corrado et al. 2005).
Botrytis cinerea assay
B. cinerea was grown on potato/dextrose/agar (PDA)
plates and cultured for approx. 2 weeks at 24 �C with
light. Fungal spores were harvested by flooding the
plates and filtered with sterile fine-mesh bags and
adjusted 1 9 108 spore/ml with sterile distilled water
using a hemocytometer. Tubers were sown in clay pots
with sterile soil. We used three tubers per pot and five
pots per genotype, for a total of 15 plants per thesis.
Forty days after sowing, one or two healthy, fully
expanded leaves per plants without visible signs of
physical injuries were inoculated with 10 ll spore
suspension (108 spores/ml). Plants were maintained in
a growth chamber at 18 �C, 16L:8D photoperiod, with
a relative humidity higher than 95%. Observations
were conducted 2, 4, 7 and 9 days following inocu-
lations. Disease development was evaluated as num-
ber of inoculation points developing a lesion and by
measuring the size of lesion. The infected area was
calculated by measuring the average of two mutually
perpendicular lengths for each lesion with a digital
calibre. Statistical differences were evaluated by One-
way Analysis of Variance (ANOVA) followed by a
Tukey post hoc procedure (p\ 0.05) on lesion area.
Genotype, time, and genotype 9 time effects on
lesion area were evaluated with Two-way Independent
ANOVA procedures with a post hoc (Duncan’s
grouping) test for genotype. Calculation were per-
formed with SPSS 20 software (SPSS Inc., Chicago,
Illinois, USA).
Rhizoctonia solani assay
R. solani was kindly provided by the ‘‘Biologia e
protezione dei sistemi agrari e forestali’’ Section of the
Dipartimento di Agraria (Universita di Napoli ‘‘Fed-
erico II’’, Naples, Italy). The isolate was cultured on
PDA plates and tested for pathogenicity on the potato
cultivar ‘‘Desiree’’. To prepare soil inoculum, R.
solani was cultured in sterile wheat bran in water (1:2
w/v) at 24 �C for 1 week, collected on filter paper,
dried and pulverised. Tubers were gently brushed,
washed with tap water, rinsed five times with sterile
water and blot-dried. For the bioassay, sterile soil was
added with R. solani inoculum (3% w/w), thoroughly
mixed and distributed to previously bleached pots. We
used two tubers per pot and ten pots per genotypes, for
a total of 20 tubers per thesis. Pots containing only
sterile soil were sown and used as a control. The fresh
weight of the tubers of each experimental group was
not statistically different. Plants were left to grow in a
growth chamber at 24 �C with a 16L:8D photoperiod
and 80% RH. The emergence of potato shoots was
visually monitored every 2 days. After 20 and 30 days
from sowing, disease symptoms were also checked.
The number of healthy shoots for each genotype was
compared between R. solani treated and untreated
tubers (control). Statistical differences were evaluated
by One-way Analysis of Variance (ANOVA) followed
by a Tukey post hoc procedure (p\ 0.05) on raw data.
Results
Potato genetic transformation
For the constitutive expression of the PhRIP I in
potato, we cloned the expression cassette of the
pG2935SRIP plasmid (Corrado et al. 2008) into the
binary vector pBIN19 (Bevan 1984), yielding the
pG35SRIP. In this plasmid the PhRIP I cDNA is under
the control of the constitute 35S RNA promoter. For
wound-inducible expression, we used the pGIPRIP
(Corrado et al. 2005), in which the PhRIP I gene is
under the control of the bean polygalacturonase-
inhibiting protein gene promoter (PGIP) (Fig. 1a).
Potato genetic transformation experiments indicated a
significant decrease of the transformation efficiency
for the construct in which the PhRIP I is constitutively
expressed (Table 1). Specifically, the largest
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123
difference between the two vectors was evident in the
number of plantlets that were able to root in the
antibiotic selective medium (Table 1). The nine
rooted plants obtained with the pG35SRIP (hereafter
named COST) and ten plants obtained with the
pPGIPRIP vector (hereafter named IND) were
transferred to soil and screened by PCR. The analysed
plants were all positive for the PhRIP I transgenic
sequence and did not display any obvious phenotypic
abnormalities compared to the regenerated untrans-
formed controls both in vitro and in vivo conditions
(data not shown). Two lines for each construct were
Fig. 1 T-DNA region of the binary vectors employed and
Southern blot analysis of the transgenic lines. Schematic
representation of the T-DNA region of the pG35SRIP (panel
A) and pGIPRIP (panel B) vectors showing some unique
restriction sites. Arrows represent cis-controlling elements, grey
boxes coding sequences and dark grey boxes terminators. A
dashed line underlines the region hybridized in the Southern
analysis. RB, T-DNA right border sequence; nos pro, nopaline
synthase gene promoter; npt II, neomycin phosphotransferase
gene; nos ter, poly(A) addition sequence of the nos gene; 35S
Pro, CaMV 35S RNA gene promoter; PGIP Pro, bean
polygalacturonase gene I promoter; PhRIP I, Phytolacca
heretotepala RIP I cDNA sequence; and LB, T-DNA left border
sequence. Southern blot analysis of the potato lines for the
constitutive (COST) or the inducible (IND) expression of the
PhRIP I gene. DNA of COST genotypes (panel C) and of the
IND genotypes (panel D) was digested with XbaI or HindIII,
respectively, along with DNA from untrasformed potato plants
(De). Numbers at the right margin indicate fragment sizes in
kilobase pairs
Biotechnol Lett
123
analysed in more details. Genomic DNA was hybri-
dised with a probe for the PhRIP I cDNA and Southern
blot analysis indicated in the different lines the
presence of two to three transgenic copies (Fig. 1b, c).
Expression analysis of the transgene
The expression analysis of the transgene in the COST
genotypes, evaluated by RT-PCR, indicated that the
PhRIP I was transcribed. Western analysis showed an
immune-reactive product of the expected size (Fig. 2).
Analysis of the inducible expression in the IND
genotypes were performed using fully expanded
leaves wounded with a hemostat. RNA was isolated
form the wounded area of the leaves. We also analysed
the possible expression of the RIP in the area
surrounding the lesions caused by B. cinerea 5 days
following inoculation, when the damage caused by the
fungus was clearly visible. The RT-PCR analysis
indicated that the PGIP promoter drives the expression
of the PhRIP I upon wounding and in the areas around
the B. cinerea infection, without a detectable back-
ground in untreated plants (Fig. 3).
Resistance to Botrytis cinerea
In vivo bioassay indicated that the expression of the
PhRIP I increases the resistance to B. cinerea in
potato. A representative example of leaves taken from
plants inoculated with B. cinerea is reported in Fig. 4.
The analysis of the repeated measures indicated that
the genotype, time, and genotype 9 time interaction
significantly affected the size of the lesion and while
differences were not present between the two trans-
genic types (Supplementary Table 2). At 9 days
following inoculation, lesions of untransformed con-
trols were so extended to hinder a consistent quantifi-
cation of the damaged area (Fig. 5a). One-way
ANOVA 7 days following inoculation indicated that
both the COST and the IND lines were significantly
more resistant than control plants (Supplementary
Fig. 1). However, a noticeable decrease in the number
of inoculation points developing lesions was evident
Table 1 Summary information on the genetic transformation experiments
Stem explants Regenerated shootsa Rooted plants KR/RSb TFc
pG35SRIP 275 120 9 7.5 3.3
pGIPRIP 300 120 30 25 10.0
Regeneration control 50 24 24
Selection control 50 14 0
a Number of regenerated shoots deriving from independent calli that were transferred for rooting (each explant can have two
independent calli)b Percentage of kanamycin resistant (KR) plants on regenerated shoots (RS)c Transformation frequency (TF) (percentage of kanamycin resistant plants on explants employed)
Fig. 2 Expression analysis of the COST genotypes. a RT-PCRanalysis of the PhRIP I expression in four COST lines. C-water
negative control; C? PCR positive control (pG35SRIP DNA);
De: Desiree cDNA. Numbers at the left margin indicate marker
sizes in basepairs. b Western blot analysis of the PhRIP protein
using an anti-Phytolacca RIP polyclonal antibody. Numbers at
the left margin indicate marker sizes in kDa
Biotechnol Lett
123
for the COST genotypes, compared both with IND
genotypes and the control Desiree variety (Fig. 5b).
Resistance to Rhizoctonia solani
Tubers of the selected transgenic lines were analysed
for resistance to the infection of R. solani in compar-
ison with control plant. For each genotype, we
evaluated the variation in the number of healthy shoot
between untreated and treated tubers. To ease the
comparison between genotypes, results are expressed
as percentage relative to the untreated samples. In our
bioassay conditions, shoots clearly emerged 10 days
post inoculation. Although differences between trea-
ted and untreated tubers were visible since the early
time points, we analysed the data 20 days following
sowing, when we did not observe anymore the
appearance of new shoots in all the theses. The
statistical analysis at this time point indicated that the
COST genotypes were resistant to R. solani because
the fungal treatment did not cause a reduction in the
number of healthy sprouts (Fig. 6). The IND
Fig. 3 Expression analysis of the IND genotypes. RT-PCR
analysis of the PhRIP I expression in the IND lines 2 and 17.
Total RNA was isolated from leaf sectors of untreated,
mechanically wounded, and Botrytis cinerea-infected potato
plants. After first-strand cDNA synthesis, PCRs were carried out
with the FoEF1stb and ReEF1stb for the constitutively
expressed Elongation Factor 1-a gene as a control for the
cDNA synthesis efficiency and to detect possible contamination
of genomic DNA in the PCR reactions (panel A). The primers
RT-RIP-Fw and RT-RIP-Rv were employed to detect the PhRIP
I transcript (panel B). M molecular marker (1 kb plus,
ThermoFishser); G genomic DNA of the Desiree cultivar; De
Desiree cDNA; C- water negative control; C? PCR positive
control (pG35SRIP DNA). Numbers at the left margin indicate
marker sizes in base pairs
Fig. 4 Bioassay against Botrytis cinerea. A representative
sample of leaves of the transgenic lines (COST and IND) and the
untransformed control (De)
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123
genotypes did not show difference compared to the
susceptible Desiree variety. Measures at 20 and
30 days did not show a significant difference between
times (data not shown). At the end of the bioassay,
leaves of the surviving stems of the IND genotypes
treated with R. solani were mechanically wounded.
The RT-PCR expression analysis confirmed the tran-
scription of the PhRIP I transgene following induction
by wounding (data not shown).
Discussion
Progress in understanding the genetic elements that
reinforce disease resistance in plants has provided the
opportunity to engineering pathogen control in crops
(Collinge et al. 2010). Genes conferring race-specific
or BSR have been long used in plant biotechnology
and typically, the gene of interest has been expressed
constitutively. Nonetheless, the controlled expression
in plants of a compound that increases resistance
against biotic stresses provides the advantages of
reducing the effect on non-target organisms and the
unnecessary accumulation of toxic proteins in edible
organs and in the environment. It is therefore needed
to increase the understanding of the possibilities
offered by different expression systems, in order to
select the best strategy according to specific needs
(Dutt et al. 2014).
Our work indicated that both the constitutive and
the inducible promoter enabled the expression of the
PhRIP I in potato without obvious phenotypic abnor-
malities. For plant transformation, we employed two
different widely used binary vectors and it is very
unlikely that both plasmids would affect plant phys-
iology in the same way. Moreover, the resistant
phenotype was scored in two independent transfor-
mants for each construct, in order to get rid of
confounding factors related to the transgenic insertion.
Differently from our previous report on tobacco
(Corrado et al. 2005), we obtained lines that express
the gene of interest under the 35S RNA CaMV
promoter. A significant difference was present in the
number of COST transformants, obtained with
pG35SRIP construct. The strong reduction of the
Fig. 5 Enhanced resistance to Botrytis cinerea. a Time course
of the lesion development (mean lesion area and its standard
error) on the different potato lines (see also Supplementary
Fig. 1; Supplementary Table 2 for the statistical analysis).
b Percentage of the number of B. cinerea inoculation points that
developed a lesion. Different letters represent statistically
different group (Tukey; p\ 0.05)
Fig. 6 Enhanced resistance to Rhizoctonia solani. (For each
line, the graph reports the variation in the number of healthy
shoots emerged following R. solani infection. The percentage
was calculated as ratio between the number of shoots of treated
and untreated tubers. Different letters represent statistically
different group (Tukey; p\ 0.05)
Biotechnol Lett
123
transformation efficiency at the rooting stage could be
explained by a phytotoxic effect of the constitutive
PhRIP I accumulation. This hypothesis is also sup-
ported by the relative low expression level of PhRIP I
of the COST genotypes, when compared to other type
of transgenic potato plants obtained in our laboratory
(data not shown). It is not possible to exclude that, if
present, strong expressing COST lines were counter-
selected, because we aimed for potato plants without
phenotypic defects (Dai et al. 2003; Wang et al. 1998).
In tobacco, another member of the Solanaceae family,
low levels of Pokeweed Antiviral Protein (a RIP from
Phytolacca Americana) did not associate to pheno-
typic abnormalities (Lodge et al. 1993). The use of the
wound-inducible PGIP promoter allowed driving the
expression of a PhRIP I in potato upon mechanical
wounding and pathogen-derived leaf damage, without
a detectable background expression level in
unwounded plants. The natural arrangements and
spacing of the cis-elements occurring in inducible
promoters of higher plants is considered optimal to
combine good inducibility with low background
expression (Rushton et al. 2002).
To our knowledge, our work is the first parallel
comparison of the constitutive and inducible trans-
genic resistance in plants. The constitutive expression
of the PhRIP I conferred a BSR in potato. Leaves and
tubers were significantly more resistant to different
fungal pathogens, indicating the usefulness of RIP
proteins in controlling very different pathogens. The
resistance level against B. cinerea provided by the
inducible promoter was similar to the protection
obtained with the CaMV 35S RNA promoter. How-
ever, a difference was present in the number of
inoculation points that evolved in lesions, as only the
COST genotypes displayed a significant reduction of
this parameter compared to the control genotypes.
PhRIP I gene expression analysis indicated that in the
IND genotypes transcription starts only after a direct
damage of the tissue. The interval between pathogen
attack and the onset of the wound-inducible expres-
sion should explain the difference obtained between
the COST and IND genotypes, implying that at the
early stage of infection, there is the possibility for fast
growing fungi as B. cinerea to spread into non
expressing/non-resistant areas. Nonetheless, consid-
ering the different time points analysed, the disease
progression did not show a statistical difference
between the transgenic types, indicating that, for some
interactions, an inducible expression of a cytotoxic
protein is a feasible option to obtain a resistance level
similar to a constitutive expression. The analysis of the
potato resistance to R. solani indicated that only the
COST genotypes were resistant, while the IND lines
did not display a significant difference when compared
with the control plants. R. solani penetrates young,
susceptible tissue, ultimately stopping the expansion
of the infected stems, while fast growing sprouts can
emerge from the soil before R. solani infects the root
tip. It is therefore possible that infected tips of the IND
lines do not have the possibility to mount a
suitable defence.
In conclusion, we obtained BSR against important
necrotrophic pathogens over-expressing the PhRIP I in
potato, a plant species that can greatly take advantage
from the speed and accuracy provided by the recom-
binant DNA technology. In addition, a wound
inducible expression system demonstrated its useful-
ness in controlling a RIP protein, which may have
adverse side-effects on plants when present at high
level (Nielsen and Boston 2001). The inducible
expression was sufficient to abort the progression of
a compatible interaction between leaves and a
necrotrophic pathogen. On the other hand, the delay
between infection and onset of transgene expression is
likely to be significant for young developing sprouts.
Our work provided evidence that is significant to
choose the appropriate expression strategy to engineer
plants with increased broad-spectrum disease
resistance.
Supporting information Supplementary Table 1—Primers
employed and their main features.
Supplementary Table 2—Analysis of variance of the mea-
sures of the lesion area produced by B. cinerea as a function of
the genotype (Desiree, COST and IND) and time (2, 4 and
7 days following inoculations). Post-hoc test on lesions
indicated that the COST and IND genotypes are not different
(p = 0.125).
Supplementary Fig. 1—Statistical analysis of the severity of
the symptoms 7 days following inoculation. The graph reports
mean values and its standard error of the lesion area. Different
letters represent statistically different groups (Tukey;
p\ 0.05).
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