Báo cáo khoa học: Analysis of the in planta antiviral activity of elderberry ribosome-inactivating proteins potx

Van Damme4 1 Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium;2Dipartimento di Patologia sperimentale, Universita` di Bologna, Italy;3Rega Inst

Analysis of the in planta antiviral activity of elderberry

ribosome-inactivating proteins

Frank Vandenbussche1, Stijn Desmyter1, Marialibera Ciani2, Paul Proost3, Willy J Peumans1

and Els J M Van Damme4

1

Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium;2Dipartimento di Patologia sperimentale, Universita` di Bologna, Italy;3Rega Institute, Department of Microbiology and Immunology, Katholieke Universiteit Leuven, Belgium;4Department of Molecular Biotechnology, Gent University, Belgium

Although the type-2 ribosome-inactivating proteins (SNA-I,

SNA-V, SNLRP) from elderberry (Sambucus nigra L.) are

all devoid of rRNA N-glycosylase activity towards plant

ribosomes, some of them clearly show polynucleotide–

adenosine glycosylase activity towards tobacco mosaic virus

RNA This particular substrate specificity was exploited to

further unravel the mechanism underlying the in planta

antiviral activity of ribosome-inactivating proteins

Trans-genic tobacco (Nicotiana tabacum L cv Samsun NN) plants

expressing the elderberry ribosome-inactivating proteins

were generated and challenged with tobacco mosaic virus in

order to analyze their antiviral properties Although some

transgenic plants clearly showed antiviral activity, no clear

correlation was observed between in planta antiviral activity

of transgenic tobacco lines expressing the different

ribosome-inactivating proteins and the in vitro polynucleotide– adenosine glycosylase activity of the respective proteins to-wards tobacco mosaic virus genomic RNA However, our results suggest that the in planta antiviral activity of some ribosome-inactivating proteins may rely on a direct mech-anism on the virus In addition, it is evident that the working mechanism proposed for pokeweed antiviral protein cannot

be extrapolated to elderberry ribosome-inactivating proteins because the expression of SNA-V is not accompanied by induction of pathogenesis-related proteins

Keywords: elderberry; polynucleotide–adenosine glycosylase activity; ribosome-inactivating protein; Sambucus nigra; viral protection

Ribosome-inactivating proteins (RIPs; EC 3.2.2.22) are

a heterogeneous family of structurally and evolutionary

related plant proteins sharing a common functional domain

that catalytically removes a specific adenine residue from a

highly conserved, surface-exposed stem-loop structure

found in the large rRNA of prokaryotic and eukaryotic

ribosomes [1,2] At present, they are subdivided on the basis

of the structure of the genes and the corresponding proteins

into holo-RIPs and chimero-RIPs [3] Whereas holo-RIPs

consist exclusively of a single catalytically active protomer

of either one (classical type-1 RIPs) or two smaller

polypeptide chains (e.g maize RIP b-32), chimero-RIPs

are built up of chimeric protomers with an N-terminal

catalytically active domain arranged in tandem with a

structurally and functionally unrelated C-terminal domain (classical type-2 and type-3 RIPs)

Biochemical and molecular studies have shown that the elderberry tree expresses a complex mixture of type-2 RIPs and/or lectins in virtually all tissues In agreement with the chronological order of their discovery, these Sambucus nigra agglutinins (SNAs) are numbered SNA-I to SNA-V The first elderberry lectin was identified in bark tissue and described as a NeuAc(a-2,6)Gal/GalNAc-specific agglutinin (called SNA-I) [4,5] Although already discovered in 1984, SNA-I was recognized as a type-2 RIP only when the corresponding gene was cloned in 1996 [6] Besides SNA-I, elderberry bark contains a second NeuAc(a-2,6)Gal/Gal-NAc-specific agglutinin which shares 77% sequence simi-larity with SNA-I (but has a different oligomeric organization) and was called SNA-I¢ [7] The second lectin identified in elderberry bark was a GalNAc-specific agglu-tinin, called SNA-II with no obvious relation to SNA-I [5] However, molecular cloning of a GalNAc-specific elder-berry bark type-2 RIP (called SNA-V) later revealed that SNA-II consists of subunits that correspond to slightly truncated B chains of this genuine type-2 RIP Both SNA-V and SNA-II are derived from a single precursor, through differential processing [8] It should be mentioned here that SNA-V (or a very closely related paralog) was first described

by Girbes et al [9] as nigrin b After the identification of the bark lectins SNA-I and SNA-II, two other elderberry lectins called SNA-III and SNA-IV were isolated from seeds and

Correspondence to E J M Van Damme, Department of Molecular

Biotechnology, Ghent University, Coupure Links 653, 9000 Ghent,

Belgium Fax: + 32 9264 6219, Tel.: + 32 9264 6086,

E-mail: ElsJM.VanDamme@UGent.be

Abbreviations: PAG, polynucleotide–adenosine glycosylase; PAP,

pokeweed antiviral protein; PR, pathogenesis-related; RIP,

ribosome-inactivating protein; SNA, Sambucus nigra agglutinin; SNLRP,

Sambucus nigra lectin-related protein; TMV, tobacco mosaic virus.

Enzyme: Ribosome-inactivating protein, rRNA N-glycosylase

(EC 3.2.2.22).

(Received 16 January 2004, revised 24 February 2004,

accepted 27 February 2004)

fruits, respectively Molecular cloning revealed that

SNA-IV, which is the most abundant fruit protein, is a dimeric

GalNAc-specific lectin encoded by a truncated type-2 RIP

gene (with a major deletion comprising almost the whole A

chain) [10] Besides the different SNAs, the bark of

elderberry contains an additional protein, SNLRP, which

is both structurally and evolutionary closely related to the

other elderberry type-2 RIPs but possesses a B chain which,

because of several amino-acid substitutions in the

sugar-binding sites, is devoid of carbohydrate-sugar-binding activity [11]

Initially, RIPs were thought to act exclusively on

ribosomes or rRNA through their rRNA N-glycosylase

activity However, using a highly sensitive

HPLC-fluores-cence-based method, Barbieri et al [12] showed that several

RIPs release more than one adenine residue from rRNA

Moreover, they found that saporin L1, a type-1 RIP from

the leaves of soapwort (Saponaria officinalis L.), is capable

of removing multiple adenine residues from various nucleic

acid substrates including herring sperm DNA, mammalian

DNA, genomic viral RNA, rRNA and poly(A) [13–15]

Additional testing of 61 RIPs revealed that all of them

extensively deadenylate herring sperm DNA and that

several are active towards viral genomic RNA [16–20]

On the basis of these findings it has been suggested that

polynucleotide–adenosine glycosylase (PAG) activity may

be responsible for the potent antiviral activity of RIPs

(against both animal and plant viruses) However, it is

still unclear whether the results obtained in these in vitro

studies can be extrapolated to the complex environment of

a cell [16]

Although the antiviral activity of RIPs against plant

viruses is well documented, the underlying mechanism(s) has

not yet been elucidated In principle, three possible

expla-nations can be put forward [3,21] First, RIPs may act

directly on the viral nucleic acids through their PAG activity

Secondly, RIPs may act directly on the host by selectively

killing the infected cells, thus preventing the virus from

replicating and spreading to neighboring cells Finally, RIPs

may act indirectly through activation of the plant’s defence

system As most RIPs with a documented strong in planta

antiviral activity [e.g pokeweed antiviral protein (PAP),

trichosanthin] are able to depurinate both viral nucleic acids

and plant ribosomes, it has been difficult to assess how the

PAG activity contributes to RIP-mediated protection To

further unravel the mode of action of RIPs, the in planta

antiviral activity of a set of type-2 RIPs (SNA-I, SNA-V,

SNLRP) from elderberry (S nigra L.) which are all devoid

of rRNA N-glycosylase activity towards plant ribosomes [9]

but strikingly differ from each other with respect to their

PAG activity towards tobacco mosaic virus (TMV) RNA,

was analyzed using a TMV/tobacco (Nicotiana tabacum L

cv Samsun NN) model system In addition to the genuine

type-2 RIP, the elderberry lectin SNA-IV, which is

consid-ered a type-2 RIP without an A chain, was included in the

study as a negative control A comparison of the protection

offered by the different ectopically expressed elderberry

RIPs did not reveal a clear correlation between in planta

antiviral activity and PAG activity towards TMV genomic

RNA However, it is evident that the working mechanism

suggested for PAP cannot be extrapolated to elderberry

RIPs because expression of the latter is not accompanied by

induction of pathogenesis-related (PR) proteins

Materials and methods

Plasmid constructions All manipulations were performed according to standard techniques [22] The pGK vector was constructed by replacing the b-glucuronidase gene from the plant transfor-mation vector pGPTV-KAN [23] with the expression cassette of the pFF19 vector [24] The coding sequences of the various RIPs/lectins were amplified by PCR to engineer appropriate restriction sites The restricted PCRproducts were inserted between the cauliflower mosaic virus 35S promoter and polyadenylation signal of the linearized and dephosphorylated pFF19 vector After confirmation of the sequence by dideoxy sequencing [25], inserts were subcloned into the expression cassette of the pGK plant transforma-tion vector The resulting plasmids, pGKsnaI, pGKsnaIV, pGKsnaV and pGKsnlrp, were transferred into Agrobacte-rium tumefaciensGV3101 by electroporation [26]

Transformation ofN tabacum Transformation of tobacco (N tabacum L cv Samsun NN) was performed using the leaf disc cocultivation method [27] Transgenic shoots were selected on Murashige-Skoog medium supplemented with 0.1 mgÆL)1 a-naphthalene acetic acid, 1 mgÆL)1 6-benzylaminopurine, 100 mgÆL)1 cefotaxime, 100 mgÆL)1carbenicillin and 100 mgÆL)1 kana-mycin (Duchefa Biochemie BV, Haarlem, the Netherlands) Transformed plants were kept in a culture room or a greenhouse at 22C, 50% relative humidity, and a 16 h photoperiod until use

Molecular analysis of transformants Total genomic DNA was isolated as described by Goode & Feinstein [28] The presence of the transgenes was investi-gated with PCRusing two internal primers derived from the N-terminal and C-terminal sequence of the various RIPs/ lectins Only the PCR-positive plants were further analysed

at the RNA and protein level

Total RNA was isolated as described by Eggermont et al [29], dissolved in RNase-free water, and quantified spectro-photometrically Approximately 30 lg total R NA was denatured with glyoxal/dimethyl sulfoxide and separated

on a 1.2% (w/v) agarose gel After electrophoresis, RNA was capillary blotted on to Hybond-N+membranes (Amersham Biosciences, Uppsala, Sweden) Membranes were first probed with random-primer-labeled cDNAs encoding the different RIPs/lectins or PR proteins (PR-1, PR-2, PR-3, proteinase inhibitor II) Subsequently the membranes were reprobed with a random-primer-labeled cDNA fragment complementary to the 3¢ end of the tobacco 25S rRNA

To estimate the expression levels of the different RIPs/ lectins, transgenic lines (T1 generation) were analysed for recombinant (r)RIP/lectin content by Western blot densi-tometry To minimize variation, 10 selfed plants of each line were grown under identical conditions (22C, 50% relative humidity, 16 h photoperiod) When plants reached the six-leaf stage, the third and fourth six-leaf were pooled, lyophilized, and ground using mortar and pestle Total protein from

50 mg lyophilized leaf material was extracted in 100 m

Hepes (pH 7.6) The protein concentration was determined

using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA,

USA) using BSA as standard [30] For Western blot analysis,

2.5 lg (V), 7.5 lg (SNLRP) or 15 lg (I,

SNA-IV) total protein was separated by electrophoresis on a 15%

(w/v) SDS/polyacrylamide gel and transferred to an

Immo-bilon-P membrane (Millipore, Bedford, MA, USA) using a

Trans-blot SD semidry transfer cell (Bio-Rad)

Immuno-detection of the recombinant proteins was performed as

described by Desmyter et al [31] using affinity-purified

polyclonal rabbit antibodies raised against native SNA-I,

SNA-II or SNLRP as the primary antibody

Purification of recombinant proteins

rRIPs were isolated using a combination of classical protein

purification techniques and affinity chromatography (except

for SNLRP, which possesses no sugar-binding activity) For

the purification of recombinant SNA-I, leaves of SNA-I

transformants were homogenized in a solution of 1 gÆL)1

ascorbic acid using a Waring blender After centrifugation

at 3000 g for 10 min, 1 gÆL)1 CaCl2 was added to the

supernatant, and the pH adjusted to 9.0 with 0.5MNaOH

The extract was cleared by centrifugation at 8000 g for

10 min and filtered through Whatman 3MM filter paper

The filtrate was subsequently adjusted to pH 2.8 with 0.5M

HCl and loaded on to a column (2.6 cm· 10 cm; 50 mL

bed volume) of S Fast Flow (Amersham Biosciences,

Uppsala, Sweden) equilibrated with 20 mM acetic acid

After loading, the column was washed with 20 mMacetic

acid until the A280fell below 0.01, and the bound proteins

were eluted with 300 mL 0.1MTris/HCl (pH 8.7)

contain-ing 0.5MNaCl This partially purified protein fraction was

adjusted to pH 7.0 with 0.5M NaOH and loaded on a

column (1.6 cm· 5 cm; 10 mL bed volume) of fetuin–

Sepharose 4B equilibrated with 0.2M NaCl After the

column had been washed with 0.2MNaCl, the bound lectin

was desorbed with 20 mM 1,3-diaminopropane (pH 9.0)

and stored at)20 C until use

For the purification of recombinant IV and

SNA-V, leaves of SNA-IV and SNA-V transformants were

homogenized in 20 mMcitric acid (pH 3.0) using a Waring

blender The crude extract was cleared by centrifugation at

3000 g for 10 min and filtered through Whatman 3MM

filter paper (NH4)2SO4was added to a final concentration

of 1.5Mand the pH adjusted to 7.0 with 0.5MNaOH The

extract was then applied to a column (2.6 cm· 10 cm;

50 mL bed volume) of galactose–Sepharose 4B equilibrated

with 1.5M(NH4)2SO4 After the column had been washed

with 1.5M(NH4)2SO4until the A280fell below 0.01, bound

proteins were desorbed with 0.2M galactose in 1.5M

(NH4)2SO4 The affinity-purified proteins were loaded on

a column (1.6· 10 cm; 20 mL bed volume) of

phenyl-Sepharose (Amersham Biosciences) equilibrated with 1.5M

(NH4)2SO4 After the column had been washed with 1.5M

(NH4)2SO4, the protein was desorbed with 20 mM

1,3-diaminopropane (pH 9.0) Fractions (2.5 mL each) were

collected and analysed by SDS/PAGE Peak fractions

containing the RIPs/lectins were pooled and stored at

)20 C until use

For the purification of rSNLRP, leaves from SNLRP

transformants were homogenized in 50 m acetic acid

using a Waring blender After adjustment of the pH to 3.0 with 0.5MHCl, the homogenate was centrifuged at 3000 g for 10 min and filtered through Whatman 3MM filter paper The cleared filtrate was then loaded on a column (2.6 cm· 10 cm; 50 mL bed volume) of S Fast Flow (Amersham Biosciences) equilibrated with 20 mM acetic acid After the column had been washed with 100 mL

25 mMsodium formate (pH 3.8), the bound proteins were desorbed with a linear gradient (500 mL) of increasing NaCl concentration (from 0 to 0.5M) Fractions (5 mL each) were collected and analysed by SDS/PAGE Peak fractions containing SNLRP were pooled, concentrated by freeze-drying, and loaded on a column (2.6 cm· 70 cm;

350 mL bed volume) of Sephacryl 100 equilibrated with

10 mM Tris/HCl (pH 7.5) for gel filtration Finally, frac-tions (2.5 mL each) were collected and analysed with SDS/ PAGE Peak fractions containing SNLRP were pooled and stored at)20 C until use

Protein sequencing Purified recombinant proteins were analysed by SDS/ PAGE and transferred to ProBlottTMmembranes (Applied Biosystems, Foster City, CA, USA) using a Trans-blot SD semidry transfer cell Proteins were excised from the blots and sequenced on a Procise 491 cLC protein sequencer (Applied Biosystems)

Enzyme assay The N-glycosylase activity of crude tobacco extracts or recombinant proteins towards rRNA was determined as described previously using rabbit reticulocyte ribosomes as substrate [32]

The PAG activity of the native RIPs was determined by measuring the adenine released from the substrate as described by Brigotti et al [33] Reactions were performed

in Eppendorf tubes containing 1 pmol RIP and 5 lg substrate (TMV genomic RNA) in 50 lL PAG buffer (50 mM sodium acetate, 100 mM KCl, pH 4.0) After

40 min incubation at 30C, the adenine released was quantified by LC/MS on a Waters Alliance/ZQ apparatus (Waters Corporation, Milford, MA, USA)

TMV bioassays Based on the molecular analysis, five transgenic lines per construct (SNA-I, SNA-IV, SNA-V, SNLRP) were ran-domly chosen to study their level of protection against TMV infection Wild-type and transgenic (T1-generation) tobacco plants were grown to the six-leaf stage in a culture room at

22C under a 16 h photoperiod Plants were mechanically inoculated by rubbing the upper two fully expanded leaves with a virus suspension [1 lgÆmL)1TMV in 0.1MKH2PO4, 2% (w/v) polyvinylpyrrolidone, pH 7.2] in the presence of carborundum After infection, plants were maintained in

a greenhouse at 22C, 50% relative humidity, and a 16 h photoperiod Four days after infection the total number of lesions on both leaves was counted To minimize variation,

a complete randomised block design was used Experiments were repeated three times Before analysis, the dataset was transformed to the logarithmic scale The transformed data

were analysed using analysis of variance, and means were

compared using a Tukey’s HSD multiple range test

(a¼ 0.05)

Induction of PR proteins

The possible constitutive expression of PRproteins in

transgenic plants expressing the different RIPs/lectins was

checked by Northern blot analysis Total RNA was

isolated, separated by agarose gel electrophoresis, and

blotted Blots were hybridized with specific probes for PR-1,

PR-2, PR-3 and proteinase inhibitor II [34]

Results and discussion

PAG activity towards TMV genomic RNA

To corroborate the possible relation between the PAG and

antiviral activity of RIPs, native elderberry RIPs were tested

for their depurinating activity towards TMV genomic RNA

(Table 1) Of the RIPs tested, both SNA-V and SNLRP

exhibited weak PAG activity towards TMV genomic RNA,

whereas SNA-I showed no activity under the same

experi-mental conditions This complete lack of PAG activity for

SNA-I is most probably due to its intrinsically lower

enzymatic activity As previously shown, SNA-I exhibits a

markedly lower PAG activity on different nucleic acid

substrates than other type-2 RIPs (possibly as a result of its

complex tetrameric structure) [35,36] As none of the

elderberry RIPs display enzymatic activity towards plant

ribosomes in rRNA N-glycosylase activity assays [9], they

are ideal candidates for assessing whether the PAG activity

is involved in RIP-mediated protection

Expression of SNA-I, SNA-IV, SNA-V and SNLRP

in transgenic tobacco

Tobacco leaf discs were transformed with A tumefaciens

GV3101 harboring the various pGKrip/lectin vectors The

RIP/lectin-expressing plants are hereafter referred to as

RIP/n where RIP denotes the RIP/lectin expressed and n

the number No visible phenotypic aberrations were

observed in any of the RIP/lectin-expressing transformants,

indicating that these elderberry type-2 RIPs exert no

cytotoxic effects in planta

All plants obtained after transformation were analysed by

PCRto confirm the presence of the RIP/lectin coding

sequence in the tobacco genome Only those plants that

were positive in the PCRanalysis were withheld for further

analysis Expression of the RIP/lectin transgenes was

examined by analysing all transgenic lines (T0generation)

at the RNA and protein level Transcription products of the predicted size were detected in most transformants but never in wild-type plants (data not shown) Western blot analysis of crude leaf extracts confirmed that most of these transformants contained immunoreactive bands As only a limited number of transformants could be analysed in the viral bioassay, five lines per construct were randomly chosen for a more detailed analysis To estimate the expression levels of the RIPs/lectins, transgenic lines (T1 generation) were analysed for rRIP/lectin content by Western blot densitometry (Fig 1, Table 2) The highest expression levels were observed for the SNA-V lines, in which the RIP accounted for 0.8–5.0% of the total soluble leaf protein All other lines exhibited markedly lower expression levels of recombinant RIPs/lectins, varying between 0.03% and 1.7% of the total soluble protein To verify whether the rRIPs are enzymatically active, crude extracts from four different RIP/lectin-expressing transformants (SNA-I/4, SNA-IV/3, SNA-V/4 and SNLRP/2) were analysed for the presence of rRNA N-glycosylase activity The SNA-IV transformant was included as a negative control As shown

in Fig 2A, the characteristic Endo fragment was only detected in the SNA-V and SNLRP transformants (lanes 6 and 8), but not in the SNA-I and SNA-IV transformants (lanes 2 and 4) The lack of enzymatic activity in the SNA-I

Table 1 RIP-catalysed release of adenine (pmol per pmol RIP per

40 min) from TMV genomic RNA.

a

Statistically significantly at P<0.02.

Fig 1 Western blot analysis of total soluble protein from wild-type and transgenic tobacco plants Approximately 15 lg (SNA-I, SNA-IV), 7.5 lg (SNLRP) or 2.5 lg (SNA-V) total soluble protein was separ-ated in a 15% (w/v) SDS/polyacrylamide gel and transferred to a nylon membrane Membranes were probed with polyclonal rabbit antibodies raised against the different RIPs/lectins Sample loading was as fol-lows: lane –, wild-type; lane +, pure native R IP/lectin; lanes 1–5, R IP/ 1–5; lane 6–12, serial dilution (400–6.25 ng) of native pure RIP/lectin.

Table 2 Expression levels (% of total soluble protein) of recombinant elderberry RIPs/lectins in transgenic tobacco plants in the different line numbers.

SNA-IV 0.03 1.0 1.7 0.4 0.4

transformants was presumably due to the lower expression

levels in these transformants and the intrinsically lower

enzymatic activity of SNA-I

Purification and characterization of recombinant proteins

Recombinant SNA-I, SNA-IV, SNA-V and SNLRP were

purified from leaves of transgenic tobacco plants, and their

purity confirmed by SDS/PAGE and Western blot analysis

Staining of the gels with Coomassie Brilliant Blue yielded

virtually identical migration patterns for the native and

recombinant proteins (Fig 3), indicating that the rRIPs/

lectins were electrophoretically pure Moreover, the

pres-ence of high-molecular-mass bands in the lanes with

unreduced rSNA-I suggests that it adopts the same

[A-s-s-B-s-s-B-s-s-A]2structure as native SNA-I However, the presence of a faster migrating band in unreduced

rSNA-I suggests that the intermolecular disulfide bridge formation between the two [A-s-s-B] pairs is less efficient in tobacco than in elderberry Upon reduction, the high-molecular-mass bands of both rSNA-I and SNA-I disappeared giving rise to two polypeptides of nearly identical mass All other RIPs (SNA-V, SNLRP) yielded a banding pattern typical

of type-2 RIPs (i.e characterized by the presence of two distinct polypeptides of
30 and 35 kDa, respectively), indicating that they are composed of [A-s-s-B] protomers

As antibodies to SNA-II were used to detect SNA-V, no antibodies directed against the A chain were present and accordingly only the B chain of SNA-V could be visualized

in the Western blot analysis In contrast with elderberry bark where
95% of the SNA-V precursor is converted into SNA-II and only 5% in SNA-V, the SNA-V-expressing tobacco plants contained little or no rSNA-II, indicating that in tobacco the precursor of rSNA-V is exclusively converted into the genuine type-2 RIP SNA-V The molecular structure of the recombinant RIPs/lectins was further analysed by gel filtration on a Superose 12 column

As expected, the native and recombinant proteins were eluted at the same position (data not shown) Furthermore N-terminal sequencing of the recombinant proteins con-firmed that tobacco cells successfully recognize and cleave the signal peptide as well as the internal linker peptide at exactly the same positions as in the parent plant (data not shown) Analysis of the rRNA N-glycosylase activity using rabbit ribosomes as a substrate demonstrated that both purified rSNA-V and rSNLRP exhibited strong enzymatic activity (Fig 2B, lanes 6 and 8) whereas no activity could be detected for the purified rSNA-IV (lane 4), which served as a negative control However, in contrast with the results of the enzymatic assays with crude extracts, a clear signal was also observed for purified rSNA-I (lanes 2) These results clearly show that all rRIPs are enzymatically active Similar results were recently also reported for SNA-If and SNA-I¢ [37,38]

Fig 2 rRNA N-glycosylase activity of crude extracts from five different

RIP/lectin-expressingtransformants (A) or purified recombinant RIPs/

lectins (B) towards rabbit reticulocyte ribosomes The arrow indicates

the position of the Endo fragment released from the rRNA (–) and

(+) indicate no treatment and aniline treatment, respectively Sample

loading was as follows: lanes 1 and 2, SNA-I/4; lanes 3 and 4, SNA-IV/

3; lanes 5 and 6, SNA-V/4; lanes 7 and 8, SNLRP/2.

Fig 3 SDS/PAGE (left hand panel) and Western blot (right hand panel) analysis of purified native and recombinant RIPs/lectins under nonreducing(A) and reducing(B) conditions Sample loading was as follows: lane 1, SNA-I; lane 2, I; lane 3, SNA-IV; lane 4, rSNA-IV; lane 5, SNA-V; lane 6, rSNA-V; lane 7, SNLR P; lane 8, rSNLR P Molecular mass markers were phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20 kDa) and lysozyme (14 kDa).

The results presented here and elsewhere thus indicate that

tobacco is not only capable of correctly processing and

assembling monomeric (SNLRP) but also dimeric (SNA-I¢,

SNA-V) and even tetrameric (SNA-I, SNA-If) type-2 RIPs

TMV bioassays

To assess whether and, if so, to what extent the elderberry

RIPs provide in planta protection against viruses

(non)trans-genic control plants and RIP-expressing tobacco plants (T1

generation) were challenged with TMV For each RIP, five

transgenic lines were randomly chosen to be analysed in the

bioassays As SNA-IV lacks the catalytically active A chain,

it is expected to confer no protection against viruses and

hence serves as a negative control To minimize variation, a

complete randomised block design was used, with each

block consisting of a nontransgenic control plant

(wild-type), a transgenic control plant (empty-vector) and five

RIP-expressing transgenic plants (RIP/1–5) Three

inde-pendent experiments were set up for each RIP

Experimen-tal data were combined to calculate the mean number of

lesions, which were compared using a Tukey’s HSD

multiple range test (a¼ 0.05) Significant differences

between control and RIP-expressing plants were only

observed for SNA-V (SNA-V/1, SNA-V/3, SNA-V/4)

(Table 3) Interestingly, a direct correlation was observed

between the expression level of rSNA-V and the level of

protection against TMV in the different SNA-V lines Only

the three lines with the highest protection levels showed a

significant reduction in lesion numbers compared with

control plants Although these results show that the type-2

RIP SNA-V is capable of conferring local protection against

TMV infection, the level of protection is markedly lower

than that of type-1 RIPs On average, the three highest

expressing SNA-V lines showed a reduction in lesion

numbers of 39% Using the same bioassay, Desmyter et al

[31] showed that IRIP, a type-1 RIP from Iris hollandica L.,

provides high levels of protection for transgenic tobacco

plants, with an average reduction in lesion numbers of 75%

Moreover, all five IRIP-expressing lines exhibited similar

protection levels The observation that SNA-V provides

protection only at very high expression levels (> 3.7% of the

total soluble protein) suggests that the antiviral mechanism

of SNA-V differs from that of IRIP Like most type-1 RIPs,

IRIP exhibits rRNA N-glycosylase activity towards both

animal and plant ribosomes [39], and accordingly is capable

of depurinating in planta part of the ribosomes in

IRIP-expressing tobacco on TMV infection (as could be

demon-strated by an in planta depurination assay) [40] Thus, the

strong in planta antiviral activity of IRIP is presumably due

to its direct depurinating activity towards both viral nucleic

acids and plant ribosomes and hence relies on a direct effect

on both the invading viruses and the infected cells As

SNA-V does not act on plant ribosomes, the elderberry RIP can

only act on the virus itself and not on the infected cells, which

may explain why it provides less protection against viral

infection than IRIP or other type-1 RIP

Although both SNA-V and SNLRP exhibited similar

depurination rates on TMV genomic RNA (Table 1), only

the highest SNA-V-expressing lines showed a significant

reduction in TMV lesion numbers However, as mentioned

above, the SNA-V lines showed markedly higher expression

levels than any of the other RIP-expressing tobacco lines These results suggest that in planta depurination of viral nucleic acids requires high cellular RIP concentrations Similar results were recently reported for the cap-specific depurination by the type-1 RIP PAP from pokeweed [41] Using a filter-binding assay, the authors demonstrated that PAP has a nearly fourfold lower affinity for capped RNAs than for rRNA As a consequence, PAP is only expected to significantly interact with capped RNAs at high cellular concentrations and/or high capped RNA levels It is likely therefore that the ectopically expressed SNLRP exerts no protection in planta because its expression level remains below the threshold concentration required for activity

Analysis of PR protein expression in transgenic tobacco plants

As according to previous reports the ectopic expression of PAP and various PAP mutants resulted in constitutive expression of some PRproteins [42,43], it seemed worth

Table 3 Susceptibility of wild-type and transgenic tobacco plants to TMV infection The mean number of lesions was calculated for each RIP separately and compared using a Tukey’s HSD multiple range test (a ¼ 0.05) Inhibition percentages were calculated by the following formula: 100 · (number of lesions wild-type – number of lesions transformant)/number of lesions wild-type When the number of lesions of the transformant was as high or higher than the wild-type value, the inhibition percentage was set to zero.

Tobacco line

Number

of plants

Number of lesions

Inhibition (%) Wild-type 26 151 ± 67 A 0 Empty vector 26 140 ± 47 A 7 SNA-I/1 26 113 ± 56 A 25 SNA-I/2 25 133 ± 68 A 12

SNA-I/4 26 122 ± 57 A 19

Wild-type 24 149 ± 60 A 0 Empty vector 24 152 ± 27 A 0 SNA-IV/1 24 158 ± 56 A 0 SNA-IV/2 24 145 ± 69 A 3 SNA-IV/3 24 131 ± 63 A 12 SNA-IV/4 24 112 ± 37 A 25 SNA-IV/5 24 123 ± 36 A 17 Wild-type 28 146 ± 77 A 0 Empty vector 28 141 ± 62 A 3

Wild-type 24 208 ± 128 A 0 Empty vector 24 167 ± 99 A 20

SNLRP/2 24 197 ± 115 A 5 SNLRP/3 24 179 ± 93 A 14

while to check whether the same phenomenon occurs in

tobacco plants that synthesize one of the elderberry type-2

RIPs/lectins Therefore, the presence of mRNAs encoding

PR-1, PR-2, PR-3 and proteinase inhibitor II was verified

by Northern blot analysis In none of the transgenic plants

could any of these mRNAs be detected (data not shown),

indicating that constitutive expression of neither the type-2

RIPs SNA-V, SNA-I and SNLRP nor the lectin SNA-IV

was accompanied by an increase in acidic or basic PR

proteins Similar results were recently also reported for

transgenic tobacco plants expressing IRIP or SNA-I¢

[31,37,38] In spite of enhanced protection levels against

TMV infection, none of these plants constitutively

accu-mulated PRproteins The results presented here and

elsewhere thus clearly indicate that the proposed mode of

action of PAP (i.e through activation of the plant’s defence

system) cannot be generalized to other RIPs

Acknowledgements

This work was supported in part by grants from the Katholieke

Universiteit Leuven and DG6 Ministerie voor Middenstand en

Landbouw-Bestuur voor Onderzoek en Ontwikkeling P P is a

Postdoctoral Fellow of the Fund for Scientific Research-Flanders The

work performed in Bologna was partially supported by Progetto

Strategico Oncologia n.74 (DD 19Ric, 09/01/02) from Ministero

Istruzione Universita` e Ricerca, Italy The TMV strain was a gift from

Professor M Ho¨fte (Laboratory of Phytopathology, Ghent University,

Belgium).

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