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: giandomenico.corrado@unina.it

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.

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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

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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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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

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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)

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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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