The Plant Cell, Vol. 12, 1477–1489, August 2000, www.plantcell.org © 2000 American Society of Plant Physiologists

PBF-2 Is a Novel Single-Stranded DNA Binding Factor Implicated in

PR-10a

Gene Activation in Potato

Darrell Desveaux,

a,1

Charles Després,

a,1,2

Alexandre Joyeux,

a,1

Rajagopal Subramaniam,

a

and Normand Brisson

a,3

a

Department of Biochemistry, Université de Montréal, Montréal, Québec, Canada H3C 3J7

Elicitor-induced activation of the potato pathogenesis-related gene

PR-10a

requires a 30-bp promoter sequencetermed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 (

PR-10a

binding factor 2). In thisstudy, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange andDNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and be-comes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is aDNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potentialtranscriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and thecDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner.The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for

PR-10a

expression in vivo, supporting the role of PBF-2 as a transcriptional regulator.

INTRODUCTION

Plants defend themselves against fungal pathogens by a va-riety of mechanisms, including preexisting physical barriersand inducible defenses (Lamb et al., 1989). Attack by anavirulent strain of pathogen results in a rapid localized ne-crosis at the site of infection (termed the hypersensitive re-sponse), which contributes to pathogen limitation (Keen,1992). With a few exceptions, deployment of the inducibledefenses requires massive gene induction (Lamb et al.,1989). Despite the importance of transcriptional activationduring the plant defense response, very little is known aboutthe players involved and the exact mechanisms that lead todefense gene induction.

PR

(pathogenesis-related) genes are among the bestcharacterized genes induced by pathogens. Heterogeneousin structure and function,

PR

genes are subdivided into 11groups (Van Loon et al., 1994). Although the function of cer-tain PR proteins is unknown, some display in vitro antifungalproperties (Schlumbaum et al., 1986; Vigers et al., 1991;Ponstein et al., 1994; Niderman et al., 1995). Genes of the

PR-10

group are present in numerous dicots (Somssich et

al., 1988; Breiteneder et al., 1989; Matton and Brisson,1989; Walter et al., 1990) and monocots (Warner et al.,1992; Moons et al., 1997). Evidence is accumulating thatsome PR-10 proteins might possess ribonuclease activity(Moiseyev et al., 1994; Bufe et al., 1996; Swoboda et al.,1996). More recently, structural and sequential homology be-tween the PR-10 proteins and a group of latex proteins hasbeen described (Osmark et al., 1998). Genes of the

PR-10

group encode small, primarily acidic intracellular proteins withmolecular masses ranging from 15 to 18 kD and have beenshown to be transcriptionally regulated (Linthorst, 1991).

In only two cases have

cis

elements and their

trans

-actingfactors been characterized in the promoters of

PR-10

genes. These studies revealed that the processes of tran-scriptional activation by elicitation differ among the various

PR-10

genes, even in the same species (Korfhage et al.,1994; Rushton et al., 1996; Euglem et al., 1999). In potato, a30-bp elicitor response element (ERE) responsible for induc-tion by the elicitor arachidonic acid is recognized by two nu-clear factors, PBF-1 and PBF-2 (for

PR-10a

binding factors1 and 2), the binding of which to the ERE correlates with theaccumulation of

PR-10a

mRNA, suggesting that they areinvolved in the elicitor-dependent activation of this gene(Matton et al., 1993; Després et al., 1995). The binding ofthese factors to the ERE and the expression of

PR-10a

areboth regulated by phosphorylation (Després et al., 1995).Furthermore,

PR-10a

gene expression and PBF-2 DNAbinding activity are controlled by a functional homolog ofprotein kinase C (Subramaniam et al., 1997). A better under-standing of the mechanisms that control expression of

PR-10a

1

These authors contributed equally to this work.

2

Current address: Plant Biotechnology Institute, Saskatoon, Sas-katchewan, Canada S7K 4P4.

3

To whom correspondence should be addressed at Department ofBiochemistry, Université de Montréal, 2900 Blvd. Edouard-Montpetit,Montréal, Québec, Canada H3T 1J4. E-mail normand.brisson@umontreal.ca.

1478 The Plant Cell

and binding of PBF-2 to the ERE requires that we determinehow this factor interacts with the DNA element to regulategene expression.

This study demonstrates that PBF-2 binds with high affin-ity to the coding strand (CS) and the noncoding strand(NCS) of the ERE. PBF-2 was purified to near homogeneitythrough a combination of anion-exchange chromatographyand DNA affinity chromatography. A 24-kD protein (p24) is aDNA binding component of PBF-2, as indicated by UVcross-linking to the ERE and interference of PBF-2 bindingby p24 antibodies. Interestingly, after purification, PBF-2was also recovered from fresh potato tubers in quantitiescomparable with that from elicited tubers. This suggeststhat PBF-2 is sequestered in an inactive state in the nuclei offresh potato tubers and is activated upon elicitation. Muta-tional analyses demonstrated that PBF-2 is a single-stranded DNA (ssDNA) binding factor that binds with se-quence specificity to the inverted repeat (IR) sequence TGA-CAnnnnTGTCA. In vivo, the TATA-proximal 3

9

half of the EREis critical for

PR-10a

expression. This region contains the corePBF-2 binding site, supporting the role of PBF-2 as a tran-scriptional regulator. A cDNA for p24 was cloned and shownto encode a novel protein with a feature found in many tran-scriptional activators, including a ssDNA binding factor. Re-combinant p24 protein also binds with high affinity to ssDNAand displays the same sequence specificity as purified PBF-2.

RESULTS

PBF-2 Is a ssDNA Binding Factor

Crude extracts of elicited potato tubers were previouslyfound to contain two factors, PBF-1 and PBF-2, that bind the–130 to –105 ERE region of the

PR-10a

promoter (Després etal., 1995). An analysis of the PBF binding site revealed thatthese factors bound with high affinity to ssDNA. A crude nu-clear extract from 18-hr elicited tubers was incubated withlabeled double-stranded and single-stranded CS and NCSof the ERE. As presented in Figure 1, both PBF-1 and PBF-2bound with a greater affinity to ssDNA (lanes 3 and 4) thanto double-stranded DNA (dsDNA) (lane 2). PBF-2 was capa-ble of binding with high affinity to both the CS and NCS(lanes 3 and 4), whereas PBF-1 bound strictly to the NCS(lane 3). Attempts to isolate cDNA clones by screening ex-pression libraries with oligonucleotide probes were unsuc-cessful. Therefore, a purification procedure to isolate PBF-1and PBF-2 from elicited potato tuber discs was devised tofurther characterize these factors.

Purification of PBF-2

Isolated nuclei were lysed in the buffer used for electro-phoretic mobility shift assays (EMSA buffer: 20 mM Hepes-

KOH, pH 7.9, 200 mM NaCl [except where stated], 1.5 mMMgCl

2

, and 0.2 mM EDTA) and loaded directly onto an an-ion-exchange resin (Q Sepharose Fast Flow). Proteins in theflowthrough effluent were collected and precipitated withpolyethylene glycol (PEG). Since both PBF-1 and PBF-2bound the NCS of the ERE (Figure 1), this strand was cou-pled to paramagnetic beads and used for DNA affinity chro-matography.

As indicated in Figure 2A, the crude nuclear extracts con-tained both PBF-1 and PBF-2 (lane 1). However, theflowthrough fraction of the Q Sepharose chromatographycontained only PBF-2 (lane 2). Attempts to recover PBF-1from other Q Sepharose fractions were unsuccessful. Fur-ther purification of PBF-2 was obtained by two rounds ofDNA affinity chromatography (lanes 5 and 8). Neither theflowthrough effluent nor the wash fractions contained PBF-2,which suggests high-affinity binding of PBF-2 to the DNA af-finity columns (lanes 3, 4, 6, and 7). Figure 2B shows the sil-ver stain of proteins recovered in the DNA affinitychromatography steps (lanes 2 and 3) in comparison withthe total proteins in crude nuclear extract (lane 1). The firstround of chromatography purified three main polypeptideswith apparent molecular masses of 105, 48, and 24 kD (lane2). The second round of DNA affinity chromatography fur-ther purified PBF-2 to a single 24-kD protein (lane 3).

To determine whether p24 interacts directly with the DNA,purified PBF-2 was UV cross-linked to radiolabeled NCS,and the DNA probe was subsequently digested. SDS-PAGEof cross-linked proteins and autoradiography revealed a sin-gle band with an apparent molecular mass of 24 kD, con-firming that p24 interacts with DNA (C-L; lane 4).

Table 1 summarizes the purification of PBF-2 from 18-hrelicited tubers as measured by the amount of labeled NCS

Figure 1. PBF-1 and PBF-2 Bind to Single- and Double-StrandedForms of the ERE.

EMSA was conducted in which crude nuclear extract from 18-hrelicited tubers was incubated with double-stranded ERE (lane 2, DS)or with the coding strand (lane 3, CS) or with the noncoding strand(lane 4, NCS) of the ERE. No extract was added in lane 1 (2E). PBF-1and PBF-2 shifts are indicated by arrows.

ssDNA Binding Plant Nuclear Factor 1479

shifted by PBF-2 in EMSA. Q Sepharose anion-exchangechromatography resulted in a 2.6-fold increase in purifica-tion. PEG precipitation served as a concentration step andretained 95% of PBF-2 activity. After DNA affinity chroma-tography, the amount of protein eluted was nonquantifiable;therefore, specific activity is not presented. The yield ofPBF-2 activity increased during purification to a maximumof 1210% after the first affinity step. This suggested thatPBF-2 may be present in tubers but is unavailable for bind-ing before elicitation—an observation that prompted us toinvestigate the possible presence of PBF-2 in fresh tubers.

PBF-2 Is Stored Inactive in the Nuclei of FreshPotato Tubers

Table 2 indicates that a negligible amount of PBF-2 bindingactivity was detected in crude extracts of fresh potato tu-bers. However, after subjecting the extract to Q Sepharosechromatography, PBF-2 binding activity was recovered inquantities comparable with that of elicited tubers. This con-firmed that the increase in PBF-2 binding after elicitationwas mainly the result of either the removal of an inhibitor ora modification of PBF-2 rather than de novo synthesis. Thefirst round of DNA affinity chromatography also resulted inan increase in the yield of PBF-2, similar to that observedwith elicited tubers. This increase in yield may be attributedto a distinct DNA-dependent mechanism. The total yield ofPBF-2 after two rounds of DNA affinity chromatography wasidentical to that obtained from elicited tubers.

p24 Is a Novel Protein with a Feature of Many Transcriptional Activators

Purified p24 was excised and digested with trypsin in-gel.After capillary electrophoresis, two peptides were selectedand sequenced by Edman degradation. The partial aminoacid sequence of the two peptides (SPEFSPLDSGAFK andVEPLPDG) showed no significant similarity to proteins ofknown function according to the BLAST search program(Altschul et al., 1997). However, the large peptide sequencewas found to be encoded by a partial expressed sequencetag (EST) from tomato

carpel tissue extracted 5 days prean-thesis to 5 days postanthesis (AI488224.1). This ESTshowed homology to an Arabidopsis bacterial artificial chro-mosome (BAC) clone (AC002521) from chromosome II thatalso encoded the other peptide sequence of p24. This infor-mation was used to design polymerase chain reaction (PCR)primers. The sequence of a fragment amplified from potatogenomic DNA encoded the large peptide and aligned withthe tomato EST and the Arabidopsis BAC clone (data notshown). The primers also amplified a single fragment froma potato cDNA library constructed from tubers elicitedwith arachidonic acid for 72 hr. Using a PCR-based cDNA

Figure 2. PBF-2 Contains a 24-kD DNA Binding Protein.

(A) EMSA performed with probe of the NCS of the ERE. Crude nu-clear extract from 18-hr elicited tubers (lane 1), the Q Sepharoseflowthrough fraction (lane 2), and the flowthrough, wash, and eluantfractions of the first (lanes 3 to 5, respectively) and second (lanes 6to 8, respectively) DNA affinity chromatography steps. A 50-mg ali-quot of crude extract (lane 1) and 1/100 of the total volume of eachstep of purification (lanes 2 to 8) were examined by EMSA. The in-tensity of the retarded bands in lanes 2 to 8 reflects the total bindingactivity of PBF-2. Arrows at right indicate PBF-1 and PBF-2 shifts.(B) After migration on a 12% SDS–polyacrylamide gel, 100 mg ofprotein from crude extract (lane 1) and 1/4 of the elution volumefrom the first (lane 2) and second (lane 3) DNA affinity chromatogra-phy (Aff.) steps were silver-stained. In lane 4, DNA affinity-purifiedPBF-2 was UV cross-linked to radiolabeled NCS, digested withDNase I, electrophoresed on an SDS–polyacrylamide gel, and visu-alized by autoradiography (C-L). Apparent molecular masses of thethree prominent bands after the first DNA affinity chromatographyprocedure are indicated at right.

1480 The Plant Cell

pooling approach (Israel, 1993), we isolated a cDNA clonefor

p24

. The frequency of this cDNA clone in the 72-hr elic-ited cDNA library was found to be

z

1/210,000, suggestingthat the

p24

gene is not highly expressed after long periodsof elicitation.

The cDNA clone revealed a single open reading frame en-coding a protein of 274 amino acids. As indicated in Figure3, both peptides from the purified p24 were present in theencoded protein. This protein has a predicted molecularmass of 30.3 kD and a pI of 9.16. ESTs encoding proteinswith strong similarity to p24 can be found in the GenBankdatabase from evolutionarily distant plants such as loblollypine, rice, maize, Arabidopsis, and tomato.

Strikingly, a stretch of 11 glutamine residues interruptedonly by one proline residue can be found in the N-terminalhalf of the protein. Such glutamine stretches are part of theproline/glutamine class of transcriptional activation do-mains. Polyglutamine stretches have been shown to activatetranscription in human HeLa cells (Gerber et al., 1994) and inplant cells (Schwechheimer et al., 1998).

p24 Is a DNA Binding Component of PBF-2

To confirm that p24 is a DNA binding component of PBF-2,antibodies were raised against the large peptide (SPEFS-PLDSGAFK). As demonstrated by the protein gel blot in Fig-ure 4A, the p24 antibodies detected a protein with anapparent molecular mass of 24 kD in the fraction elutedfrom the first round of DNA affinity chromatography (lane 2,

1

PBF-2). However, the antibody did not cross-react withany proteins in a purified potato tuber extract that did notproduce a PBF-2 shift (lane 1,

2

PBF-2).Furthermore, if p24 is a component of PBF-2, then incu-

bation of p24 antibody with an extract of the first round ofDNA affinity chromatography should either produce a super-shift or interfere with PBF-2 binding in EMSA. As shown inFigure 4B, a decrease in PBF-2 binding was observed (lane2,

1

PBF-2 Ab) compared with a reaction mixture containingan equal amount of preimmune serum (lane 1,

1

PI). Theseresults support our earlier observation that a 24-kD proteinfrom the purified PBF-2 factor can be cross-linked to the

Table 1.

Purification of PBF-2 from Elicited Potato Tuber Discs

Fraction Total Protein (mg) Total Activity

a

(pmol of DNA) Specific Activity (pmol/mg) Purification (fold) Yield (%)

Crude nuclear 17.4 2.2 1.3

3

10

2

1

1.0 100Q Seph.

b

10.4 3.5 3.4

3

10

2

1

2.6 160PEG

c

10.0 3.4 3.4

3

10

2

1

2.6 150Aff. 1

d

—

e

26.7 — — 1210Aff. 2

f

— 7.0 — — 320

a

Total activity determined by measuring labeled probe bound by PBF-2 in EMSA and calculating total picomoles of DNA bound in each fraction.

b

Q Seph., Q Sepharose Fast Flow anion exchange chromatography.

c

PEG, protein precipitated by 25% PEG 8000.

d

Aff. 1, first round of DNA affinity chromatography.

e

Nonquantifiable.

f

Aff. 2, second round of DNA affinity chromatography.

Table 2.

Purification of PBF-2 from Fresh Potato Tubers

Fraction Total Protein (mg) Total Activity

a

(pmol of DNA) Specific Activity (pmol/mg) Purification (fold) Yield (%)

Crude nuclear 19.6 0.1 5.1

3

10

2

3

1.0 100Q Seph.

b

11.6 2.2 1.9

3

10

2

1

37.3 2,200PEG

c

11.1 2.1 1.9

3

10

2

1

37.3 2,100Aff. 1

d

—

e

26.1 — — 26,100Aff. 2

f

— 7.1 — — 7,100

a

Total activity determined by measuring labeled probe bound by PBF-2 in EMSA and calculating total picomoles of DNA bound in each fraction.

b

Q Seph., Q Sepharose Fast Flow anion exchange chromatography.

c

PEG, protein precipitated by 25% PEG 8000.

d

Aff. 1, first round of DNA affinity chromatography.

e

Nonquantifiable.

f

Aff. 2, second round of DNA affinity chromatography.

ssDNA Binding Plant Nuclear Factor 1481

ERE (Figure 2B, lane 4) and confirm that p24 is a DNA bind-ing component of PBF-2.

PBF-2 and Recombinant p24 Protein Bind to ssDNA with Sequence Specificity

A comparison of the NCS and CS of the ERE reveals a com-mon 14-base IR sequence (TGACAnnnnTGTCA) present inthe same orientation on both strands. As demonstrated inFigure 1, PBF-2 bound both the CS and NCS of the EREwith high affinity. Therefore, the common IR sequence mayrepresent the core binding site of PBF-2. Mutational analy-ses within the IR were undertaken to test this hypothesis.Figure 5A represents mutations of the NCS tested for theireffect on PBF-2 binding. As demonstrated in Figure 5B, mu-tant oligonucleotides containing only IR1 (TGACA; m3) orIR2 (TGTCA; m4) were not bound by PBF-2. Similarly, muta-tions affecting simultaneously both IR1 and IR2 led to astrong reduction (m1) or an abolishment (m2) of PBF-2 bind-ing. These results suggest that both IR1 and IR2 contributeto the interaction of PBF-2 with the ERE. The mutant m5, inwhich the majority (75%) of nucleotides outside the IR se-quence are altered, resulted in a net increase in PBF-2 bind-ing. This increase may have been the result of stabilizationof the PBF-2 complex afforded by the presence of sur-rounding G/C-rich sequences.

The interaction of PBF-2 withm5 suggests that no specific sequences of the ERE outsidethe IR sequence are required for PBF-2 binding.

To demonstrate that the protein encoded by the

p24

clone possesses characteristics similar to those of PBF-2,we expressed the

p24

cDNA as a histidine tag fusion proteinand tested whether it could bind the single-stranded ERE.However, repeated attempts to express the full-length

p24

coding region in

Escherichia coli

failed to yield enough pro-tein for EMSA, suggesting that the cDNA or the encodedprotein was toxic to these cells. A truncated version of theprotein (Rp24) containing a deletion of the 67 N-terminalamino acids showed much less toxicity to

E. coli

. As dem-onstrated in Figure 5C, Rp24 bound to the single-strandedERE in vitro, confirming that the cDNA clone encodes a DNAbinding protein. Furthermore, the recombinant proteinbound to mutant ERE sequences with the same specificityas PBF-2 purified from tubers, providing further evidencethat the cloned cDNA encodes p24.

PBF-2 Preferentially Binds the Single-Stranded Form of the IR Sequence

IR sequences may promote the self-annealing of two identi-cal DNA strands or the annealing of IR on the same strand toform a hairpin structure. To examine this problem, the IR ofthe NCS was sequentially mutated two nucleotides at a timeas presented in Figure 6A. Most of these mutants would dis-rupt the IR sequence and destabilize a double-stranded

structure. DNA with a hairpin structure is known to havegreater electrophoretic mobility than does linear DNA. Fur-thermore, the migration pattern of hairpin structure formedby short ssDNA on nondenaturing gels is dependent on thelength of the stem (Baumann et al., 1989). As demonstratedin Figure 6B, the NCS mutants displayed electrophoreticmobilities characteristic of hairpin formation. Mutations re-sulting in a stem of three nucleotides (m6, m7, m11, andm12) displayed the slowest mobilities, whereas those mu-tants that decreased the stem by only one nucleotide (m8and m10) migrated at a rate that was intermediate betweenthe full-length five-nucleotide stem of the wild type and thethree-nucleotide stem.

Because the NCS displayed the characteristics of a hair-pin structure, it is possible that PBF-2 interacts with dsDNAin the hairpin stem. In such a case, mutations that destabi-lize the hairpin structure should lead to a reduction in PBF-2binding. Results presented in Figure 6C show that mutationsthat destabilized the hairpin structure (m6, m7, m11, andm12) increased PBF-2 binding over that of the wild type.Mutants with a stem only one residue short of wild type (m8and m10) and the mutation not affecting the stem (m9) hadPBF-2 binding capacities similar to the wild type. These re-sults confirm that PBF-2 preferentially binds the single-stranded form of its core binding site.

The 3

9

IR Sequence of the ERE Is Critical for

PR-10a

Activation

Earlier studies demonstrated that the ERE is sufficient andnecessary for the activation of

PR-10a

. The studies alsoshowed a strong correlation between activation of

PR-10a

and binding of PBF-2 to the ERE (Després et al.,1995).

Figure 3. Sequence of the p24 Protein.

The protein sequence represents the single open reading framefound in the p24 cDNA. Sequences of the two peptides obtainedfrom the purified p24 protein are underlined. The polyglutaminestretch is shaded. The GenBank accession number for the sequenceis AF233342.

1482 The Plant Cell

Those findings are supported in this study by the observa-tion that PBF-2 is present in fresh tubers but is unavailablefor binding to DNA. Only upon elicitation is PBF-2 able tobind to the ERE and promote

PR-10a

expression.Because the IR sequence of the ERE is required for PBF-2

binding, we were interested in testing the role of the IR onthe expression of

PR-10a

. Mutations in this promoter region,presented in Figure 7A, were constructed and the expres-sion of

PR-10a

was monitored by way of

b

-glucuronidase(GUS) activity in transient expression assays in potato pro-toplasts. Earlier results had shown that the expression levelsof GUS in electroporated protoplasts matched closely thoseobtained after elicitor treatment of transgenic potato tuberscarrying deletions in the

PR-10a

promoter (Matton et al.,1993; Després et al., 1995). As indicated in Figure 7B, muta-tions m13 to m17 of the 5

9

half of the ERE resulted in eitherthe same as wild type or increased gene expression. Onlythe two mutations, m18 and m19 of the 3

9

half of the IR se-quence (TGTCA), decreased GUS activity to 20 and 40% ofwild type, respectively. Therefore, the 3

9

IR of the ERE, butnot the 5

9

IR, is critical for

PR-10a

activation in vivo.The DNA binding results demonstrated that mutations in

both IR1 and IR2 are required to decrease PBF-2 binding(Figures 5 and 6). However, the in vivo results indicated thatonly the 3

9

IR of the ERE is sufficient for

PR-10a

expression.This apparent contradiction is reconciled by the model out-

lined in Figure 8. Figure 8A shows the structure of the EREthat may be present in vivo. In this model, PBF-2 interactswith

IR1 on the NCS and IR2 on the CS. Similarly, as indi-cated in Figure 8B, PBF-2 could interact in vitro with both IR1and IR2 of the NCS or the CS on bent DNA. This model sug-gests that PBF-2 would have easier access to its binding siteif the DNA does not form a stable hairpin, which is corrobo-rated by the evidence presented in Figure 6. Furthermore, ac-cording to the model, m18 and m19, which reducedexpression in vivo (Figure 7), are analogous to m1 and m2. Aspredicted, m1 and m2 interfered with PBF-2 and Rp24 bind-ing, respectively, in vitro (Figure 5).

DISCUSSION

In this study, PBF-2 was purified to near homogeneity andshown to contain a protein with an apparent molecular massof 24 kD. p24 can be cross-linked to DNA, and antibodiesraised against a p24 peptide interfere with PBF-2 binding inEMSA, indicating that p24 is a DNA binding component of

Figure 4. Anti-p24 Antibodies Interfere with PBF-2 Binding.

(A) Protein gel blot of a 400 mM NaCl anion-exchange chromatogra-phy fraction that does not produce a PBF-2 shift (2PBF-2; lane 1)and of an extract from 250 g of 9-hr elicited tubers after the firstround of DNA affinity chromatography that does produce a PBF-2shift (1PBF-2; lane 2), as detected with anti-p24 peptide antibodies.Equal amounts of protein were loaded in each lane. Mobility of mo-lecular mass standards is indicated at left in kilodaltons.(B) EMSA of protein eluted from the first round of DNA affinity chro-matography in the presence of p24 antibodies (1p24 Ab; lane 2) andof preimmune serum (1PI; lane 1) containing an equivalent amountof protein. A preincubation was conducted overnight at 48C followedby a 15-min incubation with radioactive NCS probe. The PBF-2 shiftis indicated (arrowhead).

Figure 5. PBF-2 Binds Specifically to the IR Sequence of the ERE.

(A) Oligonucleotides are shown in the 59 to 39 orientation. The IR se-quences of the CS and NCS are underlined, and IR1 and IR2 are in-dicated. m1 to m5 represent mutants of the NCS. The commonsequence between wild-type NCS and mutated NCS oligonucle-otides is indicated by dashed lines; mutant nucleotides are repre-sented by lowercase boldface letters.(B) EMSA of proteins eluted from the first round of DNA affinity chro-matography, performed with the oligonucleotides presented in (A).The PBF-2 shift is indicated (arrowhead).(C) EMSA of purified recombinant p24 protein (Rp24) with the oligo-nucleotides presented in (A). The Rp24 shift is indicated (arrowhead).

ssDNA Binding Plant Nuclear Factor 1483

PBF-2. The sequence of a cDNA clone encoding p24 indi-cates that it is a novel protein with a polyglutamine stretch,found in many transcriptional activators. Furthermore, bothPBF-2 and Rp24 bind preferentially to ssDNA with the samesequence specificity.

PBF-2 was also recovered from nuclear extracts of freshpotato tubers in amounts comparable with those recoveredfrom elicited tubers, but anion-exchange chromatographywas required to uncover its binding activity. Two proteinswith apparent molecular masses of 48 and 105 kD copuri-fied with p24 up to the first round of DNA affinity chromatog-raphy, suggesting that p24 may form part of a multiproteincomplex. Support for this comes from a gel filtration chro-matography procedure, in which PBF-2 eluted at a molecu-lar mass of z100 kD (A. Joyeux, unpublished results). Theloss of the 48- and 105-kD proteins after the second roundof DNA affinity chromatography may have resulted from thehigh salt concentration (1 M) used to elute the first DNA af-finity column, which could irreversibly dissociate the pro-teins from p24.

The Polyglutamine Tract of p24 Is Found in Many Transcriptional Activators

The cDNA clone encoding p24 was isolated from a cDNA li-brary constructed from elicited potato tubers. The predictedmolecular mass of the encoded protein differs from theapparent molecular mass of p24 purified from tubers, sug-gesting that the protein may be processed. The fact that weobserved strong specific DNA binding activity with a trun-cated form of the recombinant protein suggests that tuber-purified p24 may indeed be derived from a precursor proteinencoded by the p24 cDNA.

Figure 6. PBF-2 Preferentially Binds the Single-Stranded Form ofthe IR Sequence.

(A) The sequences of wild-type (NCS) and mutated NCS oligonucle-otides (m6 to m12) are presented in the 59 to 39 orientation with theIR sequence underlined and IR1 and IR2 indicated. The common se-quence between wild-type and mutated oligonucleotides is indi-cated by dashed lines; mutant nucleotides are represented bylowercase boldface letters.(B) Differential mobility of the NCS oligonucleotides presented in (A)under EMSA conditions. 20,000 counts min21 (cpm) of each oligonu-cleotide probe was loaded, and the gel was exposed for 1 hr. WT,wild type.(C) EMSA of extract from the first round of DNA affinity chromatog-raphy after incubation with radiolabeled oligonucleotides presentedin (A) as probes. PBF-2 shifts are indicated.

Figure 7. The 39 IR Sequence of the ERE Is Required for PR-10aExpression.

(A) Mutations within the ERE sequence (m13 to m19) were con-structed and fused to the GUS reporter gene. Wild-type double-stranded ERE is presented with the IR sequence in boldface. Thecommon sequence between wild-type and mutated oligonucle-otides is indicated by dashed lines, and mutant nucleotides are rep-resented by lowercase boldface letters for the CS of the ERE.(B) The ERE mutants presented in (A) were fused to a GUS reportergene and electroporated into potato leaf protoplasts. The histogramrepresents the GUS activity of each ERE mutant relative to the wild-type pLP9 construct (WT [wild type] 5 1.0). Transfection efficiencieswere corrected by coelectroporating a luciferase reporter gene. Re-sults are for two batches of protoplasts prepared at different times,each plasmid having been electroporated three times with eachbatch of protoplasts. Error bars indicate the standard deviation ofthe mean.

1484 The Plant Cell

The most striking feature of the p24 protein sequence isthe homopolymeric stretch of 11 glutamine residues. Suchstretches are found in many transcription factors and areknown to activate transcription in human HeLa cells (Gerberet al., 1994). Glutamine-rich activation domains are alsoknown to activate transcription preferentially from proximalpromoter positions in concert with an enhancer element(Seipel et al., 1992). Similarly, PBF-2 acts from a proximal el-ement (2135 to –105) of the PR-10a promoter, and distalenhancer elements (2155 to –135 and –670 to –441) are re-quired for high expression of PR-10a (Després et al., 1995).Recently, a fusion protein containing the GAL4 DNA bindingdomain, the VP16 acidic activation domain, and a stretch of51 glutamine residues activated transcription 14-fold morethan did the GAL4/VP16 fusion in plant cells (Schwechheimeret al., 1998). Studies in animal cells have shown that fusionproteins with a tract of 10 glutamines displayed the mosttranscription, whereas proteins with .26 glutamine resi-dues showed progressively less transcription (Gerber et al.,1994). These observations strongly suggest that the glu-tamine stretch found in p24 has the potential to activatetranscription.

More recently, the glutamine-rich domain of the Drosoph-ila melanogaster GAGA factor (GAF) has been shown to bindssDNA. Furthermore, the glutamine-rich domain of GAF pro-motes the stabilization of melted dsDNA (Wilkins and Lis,1999). Similarly, the p24 glutamine stretch may help stabilizessDNA in the ERE.

Single-Stranded ERE Mimics the Double-StrandedPBF-2 Binding Site

In vitro results indicate that the core PBF-2 binding site isthe IR sequence TGACAnnnnTGTCA. In vivo (i.e., dsDNA),the 39 half of the ERE is required for optimal gene expres-sion (Figure 7). This region contains the PBF-2 core bindingsite with IR1 (TGACA) on the NCS and IR2 (TGTCA) on theCS. Because PBF-2 binds with greater affinity to ssDNA, it isproposed to bind melted ERE in vivo, as presented in Figure8A. In this model, PBF-2 contacts its core binding site onboth the CS and NCS of the ERE. In vitro, however, PBF-2would bind a bent form of the ssDNA, which would mimicthe dsDNA binding site, as presented in Figure 8B.

The symmetry of the IR sequence in the ERE makes it apossible site for protein dimerization (Lamb and McKnight,1991). However, PBF-2 binding displays the properties ofmonomeric binding. In vitro, removal of either half of the IRabolished PBF-2 binding and did not result in an intermedi-ate shift corresponding to a monomer. Furthermore, cross-linking of PBF-2 to the NCS of the ERE followed by SDS-PAGE without digestion of the DNA probe resulted in a bandat z30 kD (p24/DNA complex) rather than the 48 kD pre-dicted for dimer formation (data not shown). Finally, in vivo,only the 39 half of the ERE is critical for PR-10a expression,rather than both halves.

Elicitor-Induced Activation of PBF-2

As indicated in Table 2, PBF-2 is present in the nuclei offresh tubers in an inactive form. Activation of PBF-2 may re-semble the model proposed for the salicylic acid–inducedtranscription from the as-1 element of the cauliflowermosaic virus (Jupin and Chua, 1996). Binding of the cellularfactor SARP (salicylic acid response protein) to the as-1 ele-ment correlates with the activation of transcription after sali-cylic acid treatment. SARP, in turn, is proposed to besequestered by the inhibitory protein SAI (salicylic acid in-hibitor) and released by treatment with salicylic acid. Disrup-tion of protein–protein interactions in extracts of untreatedleaves leads to a strong increase in SARP binding activity.Furthermore, the increase in SARP DNA binding activity ob-served after treatment with salicylic acid does not involve denovo synthesis of protein. Interestingly, the DNA binding ac-tivity was sensitive to phosphatase treatment, similar to thatobserved with PBF-2 (Després et al., 1995), which suggests arole for phosphorylation in salicylic acid–induced gene activa-tion. Phosphorylation events also play a critical role in activa-ting PR-10a and establishing the defense response in potato(Subramaniam et al., 1997). p24 may also be kept inactive inthe form of a precursor in fresh tubers. Unfortunately, thishypothesis could not be tested because anti-p24 antibodiesdid not detect the protein in crude nuclear extracts fromeither fresh or elicited tubers.

The presence of PBF-2 activity in the nuclei of fresh po-tato tubers to the extent recovered from elicited tubers makes

Figure 8. Proposed Model of PBF-2 Binding in Vivo and in Vitro.

(A) In vivo, PBF-2 binds a melted ERE recognizing IR1 of the NCSand IR2 of the CS. The nucleotides of IR1 and IR2 are indicated inboldface. dsERE, double-stranded ERE.(B) In vitro, PBF-2 binds to both IR1 and IR2 of ssDNA with its corebinding site oriented the way it is found in vivo. The nucleotides ofIR1 and IR2 in both CS and NCS are indicated in boldface.

ssDNA Binding Plant Nuclear Factor 1485

PBF-2 activation a potentially rapid response to pathogeninfection. In addition to activating PR-10a, PBF-2 could acti-vate the transcription of other factors involved in the de-fense response, such as the WRKY proteins. The factorsWRKY1 and WRKY3 bind the promoters of two parsley PR-10a homologs and are transcriptionally activated by elicita-tion (Rushton et al., 1996). Interestingly, the promoters ofboth these proteins contain GTCA motifs, which have beenshown to be important for WRKY1 promoter activity in vivo(Euglem et al., 1999). A possible defense pathway induced byelicitation may involve the phosphorylation-dependent activa-tion of proteins such as PBF-2 followed by the transcriptionalactivation of other factors involved in the defense response.

Transcriptional Activation of PR-10a by PBF-2

Sequence-specific ssDNA binding proteins have beenshown to participate in the regulation of transcription.Among the best characterized are the FUSE binding protein,the heterogeneous ribonucleoprotein K, and the cellular nu-cleic acid binding protein, all of which bind the promoter ofthe c-myc protooncogene and stimulate its expression(Takimoto et al., 1993; Duncan et al., 1994; Michelotti et al.,1995). Other examples of ssDNA binding proteins that regu-late transcription include single-strand D-box binding factor(ssDBF), which represses estrogen-induced transcription ofthe chicken apo VLDL II gene (Smidt et al., 1995); CBF-A,which represses SMa-A transcription in C2 myotubule cells(Kamada and Miwa, 1992); and MEF-1/Pur a and MyEF-2,which enhance and repress transcription of the myelin basicprotein, respectively, in mouse cells (Haas et al., 1993, 1995;Muralidharan et al., 1997). In both prokaryotes and eukary-otes, DNA in non-B conformations, such as melted DNA,Z-DNA, and cruciform structures, forms as a consequence oftranscriptionally induced supercoiling in vitro and in vivo (Liuand Wang, 1987; Giaver and Wang, 1988; Horwitz and Loeb,1988; Dröge and Nordheim, 1991; Dayne et al., 1992; Dai etal., 1997). Torsional stress that accumulates in upstream se-quences as a result of transcription can be relieved by un-winding, generating single strands. This process is facilitatedby the presence of A/T-rich sequences (Kowalski et al., 1988).Proteins such as PBF-2 with a greater affinity for ssDNA thanfor dsDNA may stabilize melted forms of helically unstablesites, thereby relieving transcriptionally produced superhelicaltorsion, similar to the model proposed for the c-myc promoter(Duncan et al., 1994; Bazar et al., 1995; Michelotti et al., 1996).

Accumulation of small amounts of PR-10a mRNA is in-duced by wounding and increases greatly after elicitation(Matton and Brisson, 1989). The transcriptional activity in-duced by wounding could result in upstream negative su-percoiling and possible localized melting of the ERE, 80% ofwhich is A or T residues. When elicited, activated PBF-2could recognize, bind, and stabilize the melted ERE, therebyamplifying the PR-10a transcriptional activity. The overalldestabilization of DNA would increase the potential for the for-

mation of site-specific loops or bends (Kahn et al., 1994) thatcould act as hinges, allowing for interaction of distal cis-regu-latory elements with the downstream promoter. Analogous tothe c-myc system, multiple ssDNA binding factors could bindnon-B-DNA of the PR-10a promoter to regulate transcription.Preliminary results suggest that SEBF, a nuclear factor shownto bind a repressor region of the PR-10a promoter (Després etal., 1995), is also a sequence-specific ssDNA binding factor(B. Boyle and N. Brisson, unpublished results).

Concluding Remarks

PBF-2 represents a novel DNA binding factor that potentiallycould exploit non-B-DNA to activate gene expression. It mayfunction in concert with potato homologs of other transcrip-tion factors implicated in the defense response that recognizedsDNA motifs such as the WRKY proteins (Rushton et al.,1996; Euglem et al., 1999), TGA6 (Xiang et al., 1997; Zhang etal., 1999), AHBP-1b (Kawata et al., 1992; Zhang et al., 1999),and other leucine zipper proteins (Izawa et al., 1993; Foster etal., 1994). The TGAC sequence found in the 59 IR sequence ofPR-10a is a W-box type element known to bind the WRKYproteins (Rushton et al., 1996). The increased in vivo activityobserved when this sequence was mutated (Figure 7) sug-gests that a repressor site may have been mutated. In pars-ley, WRKY2 mRNA decreases in response to elicitor,suggesting that this factor may have a negative regulatoryrole (Rushton et al., 1996). A potato WRKY2 homolog couldpotentially bind the TGAC sequence of the ERE and act to in-hibit PR-10a transcription. The inhibitor might stabilize ds-DNA, which would be less favorable for PBF-2 binding. Theregulation of PR-10a at the ERE is likely to involve a combina-tion of activation and derepression events, as proposed forthe Arabidopsis PR-1 gene (Lebel et al., 1998).

METHODS

Plant Material

Potato tubers (Solanum tuberosum cv Kennebec) were obtainedfrom the Québec Ministry of Agriculture “Les Buissons” ResearchStation (Pointe-aux-Outardes, Québec). Tubers were stored in thedark at 48C and brought to room temperature 24 hr before use. Leafmesophyll protoplasts were isolated from 7- to 8-week-old potatoplants (cv Kennebec) grown in growth chambers as described byMagnien et al. (1980).

Preparation of Crude Nuclear Extracts

Potatoes for fresh extracts were washed in water, surface-sterilizedin 70% ethanol for 5 min, rinsed with distilled water, dried, andweighed. Nuclear protein extracts from elicited potato tubers wereprepared as described previously (Marineau et al., 1987; Després etal., 1995), with slight modifications. One kilogram of fresh tubers or

1486 The Plant Cell

elicited tuber discs was homogenized in a blender at maximumspeed for 1 min in 1 liter of ice-cold buffer containing 1 M hexyleneglycol, 20 mM 2-mercaptoethanol, 10 mM Pipes-KOH, pH 6.0, 10mM MgCl2, 0.5 mM spermidine, and 0.15 M spermine. After the firstcentrifugation, the nuclear pellets were rinsed with 50 mL of EMSAbuffer (20 mM Hepes-KOH, pH 7.9, 200 mM NaCl, 1.5 mM MgCl2,and 0.2 mM EDTA) and resuspended in 16 mL of the same buffer.The nuclei were lysed by sonication, incubated on ice for 30 min, andcentrifuged for 60 min at 9000g at 48C. The supernatant was recov-ered and used as crude extract.

Purification of PBF-2

Q Sepharose chromatography was performed as a batch procedureby adding crude nuclear extract from 1 kg of potato tubers to 15 mLof Q Sepharose Fast Flow resin (Amersham Pharmacia Biotech, Baied’Urfé, Québec) preequilibrated at 200 mM NaCl with the EMSAbuffer. After incubation for 15 min on ice, the slurry was passedthrough glass wool to separate the resin from the flowthrough frac-tion containing PBF-2. One volume of 50% polyethylene glycol (PEG)solution was added to the flowthrough fraction, and proteins wereprecipitated overnight at 2208C. The next day, the mixture wasthawed and centrifuged at 48C for 60 min at 9000g. The pellet wasresuspended in 4 mL of EMSA buffer and centrifuged at 48C for 15min at 11,000g. Aliquots of the supernatant were stored at 2808Cwith 10% glycerol, and the remaining solution was used immediatelyfor DNA affinity chromatography.

The DNA sequence 59-CTAGACCATTTTTGACATTTGTGTCATTTT-ATCTAG corresponding to the noncoding strand (NCS) of the elicitorresponse element (ERE) was chemically synthesized with biotin atthe 39 position (Keystone Labs, Camarillo, CA). This biotinylated oli-gonucleotide was coupled to streptavidin-coated paramagneticbeads (Sigma; 1 mg of streptavidin per 1 mL of beads) by incubatingin EMSA buffer containing 2 M NaCl for 15 min. Coupling was moni-tored by adding a trace of biotinylated oligonucleotide labeled withphosphorus-32. The DNA-coupled affinity beads were equilibratedby three washes with 200 mM NaCl EMSA buffer. The extract fromthe equivalent of 250 g of starting material (1 mL of PEG-precipitatedQ Sepharose fraction) was incubated with 500 mL of affinity beadsfor 15 min at room temperature. For the first round of DNA affinitychromatography, EDTA was added directly to the binding reaction toa final concentration of 100 mM to inhibit nucleases. The beads werewashed twice with 500 mL of 200 mM NaCl EMSA buffer. The boundproteins were then eluted twice with 100 mL of EMSA buffer contain-ing 1 M NaCl. The eluent was immediately equilibrated to 200 mMNaCl by adding 800 mL of EMSA buffer devoid of NaCl. The secondround of DNA affinity chromatography was identical to the first ex-cept that EDTA was omitted from the 15-min incubation period. Ali-quots were stored at 2808C with 10% glycerol added. Proteins ineach fraction were separated by 12% SDS-PAGE and visualized bysilver staining (Oakley et al., 1980).

For peptide sequencing, p24 was purified from 10 kg of elicitor-treated potato tuber discs. Sequencing was performed by the Har-vard Microchemistry Facility (Cambridge, MA).

Cloning of p24

A partial tomato expressed sequence tag (EST) sequence(AI488224.1) coding for the p24 large peptide (SPEFSPLDSGAFK)

and an Arabidopsis bacterial artificial chromosome (BAC) clone(AC002521) coding for both peptides were aligned and polymerasechain reaction (PCR) primers were designed to flank the large pep-tide. The primers derived from the tomato EST sequence hadsequences of 59-ATATACAAAGGGAAGGCAGCT and 59-GATAGA-TCCAATTTCAGTCAC. These primers were first used to amplifypotato genomic DNA. A single DNA fragment of z550 bp was ampli-fied, cloned, partially sequenced, and shown to share 99% identityover 109 bp with the tomato sequence and to code for the large pep-tide (data not shown).

A modified version of the method described by Israel (1993) wasused to screen a potato cDNA library made in lambda ZAP (Strat-agene, La Jolla, CA) from mRNAs isolated from potato tubers elicitedwith arachidonic acid for 72 hr (Matton and Brisson, 1989). Approxi-mately 960,000 plaque-forming units (pfu) were used to infect Esch-erichia coli XL-1 Blue (Stratagene), and portions were placed in a 96-well microtiter plate at 10,000 pfu per well in 100 mL. The phageswere amplified for 8 hr at 378C. A portion from each well was mixedwith an equal volume of water, and 3 mL from each sample wasadded to a PCR reaction containing 2.5 mM MgCl2, 50 mM KCl, 10mM Tris-HCl, pH 9.0, 0.01% gelatin, 0.1% Triton X-100, 200 mMdeoxyribonucleotide triphosphates, and 0.4 units of Taq polymerase(Roche Molecular Biochemicals, Laval, Québec) with 10 pmol eachof the primers described above. The DNA was amplified in a stan-dard PCR reaction, with an annealing temperature of 538C, ina Whatman/Biometra T-gradient PCR apparatus. Approximately336,000 pfu from a positive well was transferred to another 96-wellplate at 4000 pfu per well, amplified, and analyzed by PCR as de-scribed above. This process was repeated a total of four times, withz3000 and 250 pfu per well in the third and fourth screens, respec-tively. Isolation of the p24 cDNA clone was done by hybridization ofplaques from a positive well with the genomic DNA fragment de-scribed above. A positive clone was excised by using the ExAssistsystem (Stratagene), according to the manufacturer’s instructions.The clone was sequenced on both strands by using Thermoseque-nase (Amersham Pharmacia Biotech) in a cycle-sequencing reactionthat was then processed in a Lycor automated sequencer (Lycor,Lincoln, NE).

Expression and Purification of Protein Fusions

The p24 coding region was amplified by using PCR primers (59-CCAAAAATCTCTTGGATCCATGTCC and 59-CCAGAACTCGAGATT-CCATTC) that inserted a BamHI site immediately preceding the ATGand a XhoI site after the stop codon, respectively. The PCR productwas purified and inserted into the BamHI and XhoI sites of the pET-21a vector (Novagen, Madison, WI), creating a fusion protein with aT7 tag at the N terminus and a histidine tag at the C terminus. Thetruncated form of the p24 protein was produced by using the sameXhoI primer and a PCR primer (59-TTAACATGTCGCGGATCCGAT-TATTTTG) inserting a BamHI site 67 amino acids from the N ter-minus. These constructs were made in XL-1 blue E. coli cells(Stratagene) and then transferred in the expression strain BL21pLysS (Novagen). A single colony from the latter was then grown at378C, and the culture was induced for 3 hr by isopropyl-b-D-thioga-lactopyranoside. The cells were then harvested and resuspended in0.1 volume of START buffer (20 mM Na2HPO4, pH 7.2, and 500 mMNaCl). After treatment with a freeze/thaw cycle and sonication, thesamples were centrifuged at 11,000g for 1 hr at 48C. One volume of50% PEG 8000 was added to the supernatants, and the precipitated

ssDNA Binding Plant Nuclear Factor 1487

proteins were centrifuged for 1 hr at 11,000g. Pellets were washedand resuspended in START buffer. Fusion proteins were purified byusing the HiTrap affinity columns and a fast protein liquid chroma-tography apparatus (Amersham Pharmacia Biotech), according tomanufacturer’s instructions. The columns were charged with 100mM NiSO4, and the samples were loaded in START buffer. The fusionproteins were eluted in START buffer containing 50 mM EDTA. Tenpercent glycerol was added to the samples for long-term storage at–808C. For EMSA, the eluted samples were first diluted 1:1 in EMSAbuffer without NaCl.

Electrophoretic Mobility Shift Assays

Double-stranded ERE was prepared as described previously (Despréset al., 1995). Single-stranded synthetic oligonucleotides for the –130to –105 region of the ERE were labeled by using T4 polynucleotidekinase. Specific activities ranged from 5 3 104 to 105 counts min21

(cpm) ng21 and were adjusted by adding cold DNA to give all sam-ples the same value for use in comparing the different mutant oligo-nucleotides. Reaction mixtures contained 1 mL (20,000 cpm) of end-labeled oligonucleotide and 40 mL of DNA affinity or 10 mL of QSepharose extract or 10 mg of nuclear proteins of the crude extractwith a final EDTA concentration of 50 mM. Reactions were performedat room temperature for 15 min and subsequently loaded onto 5.4%polyacrylamide gels (29:1 acrylamide:bisacrylamide in 100 mM Tris-HCl,pH 8.0, 100 mM borate, and 2 mM EDTA). After electrophoresis, thegels were blotted onto Whatman 3MM paper and autoradiographedat 2808C on Kodak (Rochester, NY) XAR films. Quantitation of DNAbound by PBF-2 was assessed by liquid scintillation counting of theretarded band excised from the gel or by measuring the density ofthe shifted probe on developed films by using a densitometry pack-age from Molecular Dynamics (Sunnyvale, CA).

UV Cross-Linking

Bluescript plasmids (Stratagene) containing wild-type oligonucle-otides were internally labeled with Klenow and radioactive a-32P-dTTP. The cloned oligonucleotide was excised from the plasmid bydigestion with XbaI and purified. The gel-purified oligonucleotide washeat-denatured and used as a probe in EMSA. Reaction mixtureswere irradiated for 30 min with a UV transilluminator (Fotodyne, SanDiego, CA) before EMSA. The retarded complexes were excisedfrom the gel and subjected to electroelution followed by digestionwith DNase I according to standard protocol (Sambrook et al., 1989).Cross-linked proteins were then separated by 12% SDS-PAGE. Thegels were subsequently blotted onto Whatman 3MM paper and au-toradiographed at 2808C.

Protein Gel Blot of PBF-2

A synthetic peptide corresponding to the sequence SPEFSPLDS-GAFK of the p24 protein was synthesized (Sheldon BiotechnologyCentre, Montréal, Québec). An antiserum was prepared by immuniz-ing female New Zealand white rabbits with the synthesized p24 pep-tide. For protein gel blots, DNA affinity-purified proteins from 250 g ofpotato tubers were precipitated with 5 volumes of cold acetone, re-suspended in SDS sample buffer, and separated by SDS-PAGE.Protein gel blots were performed as described previously (Constableand Brisson, 1992) with p24 antibody used at 1000-fold dilution. The

blots were developed with the electrochemiluminescent detectionsystem (Amersham Pharmacia Biotech) according to the manufac-turer’s instructions.

b-Glucuronidase Reporter Gene Constructs

All DNA manipulations were performed according to standard proce-dures (Sambrook et al., 1989). The mutant EREs were constructedfrom the vector pLP9, which contains the –135 to –47 region of the EREcoupled to the –27 to 1145 region of the PR-10a gene by the se-quence 59-TCGAATTCCTGCAGG. To create the pLP9 vector, the plas-mid pDPM686 (Matton et al., 1993) was cut with the restriction enzymePstI in the 15-bp linker region, blunt-ended, and ligated to an 8-bpoligonucleotide at the vector, resulting in a 19-bp full-length se-quence from –47 to –27 (59-TCGAATTCCGGTCGACCGG).

PCR site-directed mutagenesis was used to create the ERE mu-tants. The two wild-type primers were 59-GCGAAGCTTGATTCT-AGATAAAATGACACAAATGTCAAAAATGG and 59-CCACCCGGG-GATCCAGCTTTGAAC. The reaction mixtures contained 50 ng oftemplate DNA (pLP9), 25 pmol of each primer, 200 mM dNTPs, 50mM KCl, 10 mM Tris-Cl, pH 9.0, 0.01% gelatin, 0.1% Triton X-100,2.5 mM MgCl2, and 0.4 units of Taq DNA polymerase (Roche Molec-ular Biochemicals, Laval, Québec) in a total reaction mixture volumeof 30 mL. PCR reactions were performed at an annealing temperatureof 508C, and the PCR products were digested with HindIII andBamHI and ligated into the digested pLP9 vector. The entire insert ofeach mutant was sequenced to ensure that no additional mutationshad been introduced in the sequence.

Protoplast Isolation and Electroporation

Leaf mesophyll protoplasts were electroporated with CsCl-purifiedsupercoiled plasmid DNA as previously described (Matton et al.,1993). Luciferase plasmid pWB216 (Barnes, 1990) containing the lu-ciferase gene under control of the cauliflower mosaic virus 35S pro-moter was coelectroporated with 40 mg of reporter plasmids.Protoplasts were pelleted, resuspended in 100 mL of cell culture lysisreagent (Promega, Nepean, Ontario), and frozen immediately. Lu-ciferase activity was determined by adding 50 mL of Luciferase As-say Reagent (Promega) to 5 mL of freshly thawed protoplast extractsand without delay measuring photon emission (EG and G BertholdLumat LB9507 Luminometer, Bad Wildbad, Germany) for 1 min. b-Gluc-uronidase (GUS) activity was measured as described previously(Matton et al., 1993) and standardized by using luciferase activity asan indicator of transformation efficiency.

ACKNOWLEDGMENTS

We thank Louise Paquet for preparation of the pLP9 reporter plasmidand optimizing the GUS reporter gene assays. We also thank LouiseCournoyer for preparing in vitro grown plants and Barbara Otryskofor the gift of certified potato tubers. We are also very grateful toFranz Lang and his group for sequencing the p24 cDNA in its entirety.This work was supported by research grants from the Natural Scienceand Engineering Research Council (NSERC) of Canada and from theFonds pour la Formation de Chercheurs et l’Aide à la Recherche,

1488 The Plant Cell

Québec. D.D., C.D., and A.J. were recipients of NSERC postgraduatescholarships.

Received May 1, 2000; accepted June 1, 2000.

REFERENCES

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z.,Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: A new generation of protein database search programs.Nucleic Acids Res. 25, 3389–3402.

Barnes, W.M. (1990). Variable patterns of expression of luciferase intransgenic tobacco leaves. Proc. Natl. Acad. Sci. USA 87, 9183–9187.

Baumann, U., Frank, R., and Blöcker, H. (1989). Probing hairpinstructures of small DNAs by nondenaturing polyacrylamide gelelectrophoresis. Anal. Biochem. 183, 152–158.

Bazar, L., Meighen, D., Harris, V., Duncan, R., Levens, D., andAvignan, M. (1995). Targeted melting and binding of a DNA regu-latory element by a transactivator of c-myc. J. Biol. Chem. 270,8241–8248.

Breiteneder, H., Pettenburger, K., Bito, A., Valenta, R., Kraft, D.,Rumpold, H., Scheiner, O., and Breitenbach, M. (1989). Thegene coding for the major birch pollen allergen, Bet vI, is highlyhomologous to a pea disease resistance response gene. EMBO J.8, 1935–1938.

Bufe, A., Spangfort, M.D., Kahlert, H., Schlaak, M., and Becker,W.M. (1996). The major birch pollen allergen, Bet v1, shows ribo-nuclease activity. Planta 199, 413–415.

Constable, C.P., and Brisson, N. (1992). The defense-related STH-2gene product of potato shows race-specific accumulation afterinoculation with low concentrations of Phytophthora infestanszoospores. Planta 188, 289–295.

Dai, X., Greizerstein, M.B., Nadas-Chinni, K., and Rothman-Denes, L.B. (1997). Supercoil-induced extrusion of a regulatoryDNA hairpin. Proc. Natl. Acad. Sci. USA 94, 2174–2179.

Dayne, A., Malkhosyan, S., and Mirkin, S.M. (1992). Transcription-ally driven cruciform formation in vivo. Nucleic Acids. Res. 20,5991–5997.

Després, C., Subramaniam, R., Matton, D.P., and Brisson, N.(1995). The activation of the potato PR-10a gene requires the phos-phorylation of the nuclear factor PBF-1. Plant Cell 7, 589–598.

Dröge, P., and Nordheim, A. (1991). Transcription-induced confor-mational change in a topologically closed DNA domain. NucleicAcids Res. 19, 2941–2946.

Duncan, R., Bazar, L., Michelotti, G., Tomonaga, T., Krutzsch, H.,Avignan, M., and Levens, D. (1994). A sequence specific, single-strand binding protein activates the far upstream element of c-mycand defines a new DNA-binding motif. Genes Dev. 8, 465–480.

Euglem, T., Rushton, P.J., Schmelzer, E., Hahlbrock, K., andSomssich, I.E. (1999). Early nuclear events in plant defense sig-nalling: Rapid gene activation by WRKY transcription factors.EMBO J. 18, 4689–4699.

Foster, R., Izawa, T., and Chua, N.-H. (1994). Plant bZIP proteinsgather at ACGT elements. FASEB J. 8, 192–200.

Gerber, H.-P., Seipel, K., Georgiev, O., Höfferer, M., Hug, M.,Rusconi, S., and Schaffner, W. (1994). Transcriptional activationmodulated by homopolymeric glutamine and proline stretches.Science 263, 808–811.

Giaver, G.N., and Wang, J.C. (1988). Supercoiling of intracellularDNA can occur in eukaryotic cells. Cell 55, 849–856.

Haas, S., Gordon, J., and Khalili, K. (1993). A developmentally reg-ulated DNA-binding protein from mouse brain stimulates myelinbasic protein gene expression. Mol. Cell. Biol. 13, 3103–3112.

Haas, S., Steplewski, A., Siracusa, L., Amini, S., and Khalili, K.(1995). Identification of a sequence specific single-stranded DNAbinding protein that suppresses transcription of the mouse myelinbasic protein gene. J. Biol. Chem. 270, 12503–12510.

Horwitz, M.S.Z., and Loeb, L.A. (1988). An E. coli promoter thatregulates transcription by DNA superhelix-induced cruciformextrusion. Science 241, 703–705.

Israel, D.I. (1993). A PCR-based method for high-stringency screen-ing of DNA libraries. Nucleic Acids Res. 21, 2627–2631.

Izawa, T., Foster, R., and Chua, N.-H. (1993). Plant bZIP proteinDNA binding specificity. J. Mol. Biol. 230, 1131–1144.

Jupin, I., and Chua, N.-H. (1996). Activation of the CaMV as-1 cis-element by salicylic acid: Differential DNA-binding of a factorrelated to TGA1a. EMBO J. 15, 5679–5689.

Kahn, J.D., Yun, E., and Crothers, D.M. (1994). Detection of local-ized DNA flexibility. Nature 368, 163–166.

Kamada, S., and Miwa, T. (1992). A protein binding to CarG boxmotifs and to single-stranded DNA functions as a transcriptionalrepressor. Gene 119, 229–236.

Kawata, T., Imada, T., Shiraishi, H., Okada, K., Shimura, Y., andIwabuchi, M. (1992). A cDNA encoding HBP-1b homologue inArabidopsis thaliana. Nucleic Acids Res. 20, 1141.

Keen, N.T. (1992). The molecular biology of disease resistance.Plant Mol. Biol. 19, 109–122.

Korfhage, U., Trezzini, G.F., Meier, I., Hahlbrock, K., and Somssich,I.E. (1994). Plant homeodomain protein involved in transcriptional reg-ulation of a pathogen defense-related gene. Plant Cell 6, 695–708.

Kowalski, D., Natale, D.A., and Eddy, M.J. (1988). Stable DNAunwinding, not “breathing,” accounts for single-strand–specificnuclease hypersensitivity of specific A1T-rich sequences. Proc.Natl. Acad. Sci. USA 85, 9464–9468.

Lamb, C.J., Lawton, M.A., Dron, M., and Dixon, R.A. (1989). Sig-nals and transduction mechanisms for activation of plantdefenses against microbial attack. Cell 56, 863–867.

Lamb, P., and McKnight, S.L. (1991). Diversity and specificity intranscriptional regulation: The benefits of heterotypic dimeriza-tion. Trends Biochem. Sci. 16, 417–422.

Lebel, E., Heifetz, P., Thorne, L., Uknes, S., Ryals, J., and Ward,E. (1998). Functional analysis of regulatory sequences controllingPR-1 gene expression in Arabidopsis. Plant J. 16, 223–233.

Linthorst, H.J.M. (1991). Pathogenesis-related proteins of plants.Crit. Rev. Plant Sci. 10, 123–150.

Liu, L.F., and Wang, J.C. (1987). Supercoiling of the DNA templateduring transcription. Proc. Natl. Acad. Sci. USA 84, 7024–7027.

Magnien, E., Dalschaert, X., and Devreux, M. (1980). Differentradiosensitivities of Nicotiana plumbaginifolia leaves and regener-ating protoplasts. Plant Sci. Lett. 19, 231–241.

ssDNA Binding Plant Nuclear Factor 1489

Marineau, C., Matton, D.P., and Brisson, N. (1987). Differential accu-mulation of potato mRNAs during the hypersensitive responseinduced by arachidonic acid elicitor. Plant Mol. Biol. 9, 335–342.

Matton, D.P., and Brisson, N. (1989). Cloning, expression, andsequence conservation of pathogenesis-related gene transcriptsof potato. Mol. Plant Microbe Interact. 2, 325–331.

Matton, D.P., Prescott, G., Bertrand, C., Camirand, A., and Brisson,N. (1993). Identification of cis-acting elements involved in the reg-ulation of the pathogenesis-related gene STH-2 in potato. PlantMol. Biol. 22, 279–291.

Michelotti, G., Tomonaga, T., Krutzsh, H., and Levens, D. (1995).Cellular nucleic acid binding protein regulates the CT element ofthe human c-myc protooncogene. J. Biol. Chem. 270, 9494–9499.

Michelotti, G., Michelotti, E.F., Pullner, A., Duncan, R.C., Eick, D.,and Levens, D. (1996). Multiple single-stranded cis elements areassociated with activated chromatin of the human c-myc gene invivo. Mol. Cell. Biol. 16, 2656–2669.

Moiseyev, G.P., Beintema, J.J., Fedoreyeva, L.I., and Yakovlev,G.I. (1994). High sequence similarity between a ribonuclease fromginseng calluses and fungus-elicited proteins from parsley indi-cates that intracellular pathogenesis-related proteins are ribonu-cleases. Planta 193, 470–472.

Moons, A., Prinsen, E., Bauw, G., and Van Montagu, M. (1997).Antagonistic effects of abscisic acid and jasmonates on salt stress–inducible transcripts in rice shoots. Plant Cell 9, 2243–2259.

Muralidharan, V., Tretiakova, A., Steplewski, A., Haas, S., Amini,S., Johnson, E., and Khalili, K. (1997). Evidence for inhibition ofMyEF-2 binding to MBP promoter by MEF-1/Pur a. J. Cell. Bio-chem. 66, 524–531.

Niderman, T., Genetet, I., Bruyere, T., Gees, R., Stinzi, A., Legrand,M., Fritig, B., and Mosinger, E. (1995). Pathogenesis-relatedPR-1 proteins are antifungal: Isolation and characterization ofthree 14-kilodalton proteins of tomato and of a basic PR-1 oftobacco with inhibitory activity against Phytophthora infestans.Plant Physiol. 108, 17–27.

Oakley, B.R., Kirsch, D.R., and Morris, N.R. (1980). A simplifiedultrasensitive silver stain for detecting proteins in polyacrylamidegels. Anal. Biochem. 105, 361–363.

Osmark, P., Boyle, B., and Brisson, B. (1998). Sequential and struc-tural homology between intracellular pathogenesis-related proteinsand a group of latex proteins. Plant Mol. Biol. 38, 1243–1246.

Ponstein, A.S., Bres-Vloemans, S.A., Sela-Buurlage, M.B., vanden Elzen, P.J., Melchers, L.S., and Cornelissen, B.J. (1994). Anovel pathogen- and wound-inducible tobacco (Nicotiana tabacum)protein with antifungal activity. Plant Physiol. 104, 109–118.

Rushton, P.J., Torres, J.T., Parniske, M., Wernert, P., Hahlbrock,K., and Sommsich, I.E. (1996). Interaction of elicitor-inducedDNA binding proteins with elicitor response elements in the pro-moters of parsley PR1 genes. EMBO J. 15, 5690–5700.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989). MolecularCloning: A Laboratory Manual. (Cold Spring Harbor, NY: ColdSpring Harbor Laboratory Press).

Schlumbaum, A., Mauch, F., Voegeli, U., and Boller, T. (1986).

Plant chitinases are potent inhibitors of fungal growth. Nature324, 365–367.

Schwechheimer, C., Smith, C., and Bevan, M.W. (1998). Theactivities of acidic and glutamine-rich transcriptional activationdomains in plant cells: Design of modular transcription factors forhigh-level expression. Plant Mol. Biol. 36, 195–204.

Seipel, K., Georgiev, O., and Schaffner, W. (1992). Different activa-tion domains stimulate transcription from remote (‘enhancer’) andproximal (‘promoter’) positions. EMBO J. 11, 4961–4968.

Smidt, M.P., Russchen, B., Snippe, L., Wijnholds, J., and Ab, G.(1995). Cloning and characterization of a nuclear, site specificssDNA binding protein. Nucleic Acids Res. 23, 2389–2395.

Somssich, I.E., Schmelzer, E., Kawalleck, P., and Hahlbrock, K.(1988). Gene structure and in situ transcript localization of patho-genesis-related protein 1 in parsley. Mol. Gen. Genet. 213, 93–98.

Subramaniam, R., Després, C., and Brisson, N. (1997). A func-tional homolog of mammalian protein kinase C participates in theelicitor-induced defense response in potato. Plant Cell 9, 653–664.

Swoboda, I., Hoffmannsommergruber, K., Oriordain, G., Scheiner,O., Heberlebors, E., and Vicente, O. (1996). BET v1 proteins, themajor birch pollen allergens and members of a family of con-served pathogenesis-related proteins, show ribonuclease activityin vitro. Physiol. Plant. 96, 433–438.

Takimoto, M.T., Tomonaga, T., Matunis, M., Avignan, M.,Krutzsch, H., Dreyfuss, G., and Levens, D. (1993). Specific bindingof heterogeneous ribonucleoprotein particle K to the human c-mycpromoter, in vitro. J. Biol. Chem. 268, 18249–18258.

Van Loon, L.C., Pierpoint, W.S., Boller, T., and Conejero, V.(1994). Recommendations for naming plant pathogenesis-relatedproteins. Plant Mol. Biol. Rep. 12, 245–264.

Vigers, A.J., Roberts, W.K., and Selitrennikoff, C.P. (1991). A newfamily of plant antifungal proteins. Mol. Plant-Microbe Interact. 4,315–323.

Walter, M.H., Liu, J.-W., Grand, C., Lamb, C., and Hess, D. (1990).Bean pathogenesis-related (PR) proteins deduced from elicitor-induced transcripts are members of a ubiquitous new class ofconserved PR proteins including pollen allergens. Mol. Gen.Genet. 222, 353–360.

Warner, S.A.J., Scott, R., and Draper, J. (1992). Characterizationof a wound-induced transcript from the monocot asparagus thatshares similarity with a class of pathogenesis-related (PR) pro-teins. Plant Mol. Biol. 19, 555–561.

Wilkins, R.C., and Lis, J.T. (1999). DNA distortion and multimeriza-tion: Novel functions of the glutamine-rich domain of GAGA fac-tor. J. Mol. Biol. 285, 515–525.

Xiang, C., Miao, Z., and Lam, E. (1997). DNA-binding proteins,genomic organization and expression pattern of TGA6, a newmember of the TGA family of bZIP transcription factors in Arabi-dopsis thaliana. Plant Mol. Biol. 34, 403–415.

Zhang, Y., Fan, W., Kinkema, M., Li, X., and Dong, X. (1999). Inter-action of NPR1 with basic leucine zipper protein transcription fac-tors that bind sequences required for salicylic acid induction ofthe PR-1 gene. Proc. Natl. Acad. Sci. USA 96, 6523–6528.

DOI 10.1105/tpc.12.8.1477 2000;12;1477-1489Plant Cell

Darrell Desveaux, Charles Després, Alexandre Joyeux, Rajagopal Subramaniam and Normand BrissonPotato

Gene Activation inPR-10aPBF-2 Is a Novel Single-Stranded DNA Binding Factor Implicated in

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