The Plant Cell, Vol. 13, 1437–1452, June 2001, www.plantcell.org © 2001 American Society of Plant Physiologists

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element and Activated by Geminivirus Infection in Mature Leaves

Erin M. Egelkrout,

a

Dominique Robertson,

b

and Linda Hanley-Bowdoin

a,1

a

Department of Biochemistry, North Carolina State University, Raleigh, North Carolina 27695-7622

b

Department of Botany, North Carolina State University, Raleigh, North Carolina 27695-7612

The geminivirus

tomato golden mosaic virus

(

TGMV

) amplifies its DNA genome in differentiated plant cells that lackdetectable levels of DNA replication enzymes. Earlier studies showed that

TGMV

induces the accumulation of prolifer-ating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase

�

, in mature cells of

Nicotiana benthami-ana

. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gelblot analysis detected an

�

1200-nucleotide PCNA transcript in young leaves. The same RNA was found in matureleaves of infected but not healthy plants. Reporter gene analysis showed that a 633-bp promoter fragment of the

N.benthamiana

PCNA gene supports high levels of expression in cultured cells and in young but not mature leaves ofhealthy transgenic plants. In contrast, PCNA promoter activity was detected in both young and mature leaves of

TGMV

-infected plants. Developmental studies established a strong relationship between symptom severity, viral DNA accu-mulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in thePCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Un-like the wild-type PCNA promoter, TGMV infection had no detectable effect on the activity of the mutant E2F promoter.Together, these results demonstrate that geminivirus infection induces the accumulation of a host replication factor byactivating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.

INTRODUCTION

Geminiviruses are DNA viruses that replicate their single-stranded genomes through double-stranded intermediatesin nuclei of mature plant cells (reviewed in Hanley-Bowdoinet al., 1999; Gutierrez, 2000). Because of their limited codingcapacity, geminiviruses supply only the factors required toinitiate rolling circle replication and use plant nuclear DNApolymerases to amplify their genomes. Many geminivirusesreplicate in differentiated cells that have exited the cell cycle(Coello et al., 1992; Nagar et al., 1995; Lucy et al., 1996). Asa consequence, these viruses must induce the synthesis ofthe requisite host enzymes before replication (Nagar et al.,1995) and thus are valuable tools for studying the mecha-nisms that mediate and regulate DNA replication and thecell cycle in plants.

Geminiviruses are a diverse family of plant-infecting vi-ruses that fall into three genera based on their genomestructure, insect vectors, and host range (Rybicki, 1994).

Tomato golden mosaic virus

(

TGMV

), a member of the bego-movirus genus, has a bipartite genome consisting of two2.6-kb DNA components designated A and B (Bisaro et al.,1982), which together encode seven proteins. Only the AL1protein (also designated C1 or Rep) is essential for replica-tion, whereas AL3 (or C3) enhances viral DNA accumulation(Elmer et al., 1988a; Sunter et al., 1990). AL1 is the originrecognition protein and catalyzes DNA cleavage and ligationto initiate and terminate rolling circle replication (Fontes etal., 1994; Laufs et al., 1995; Orozco and Hanley-Bowdoin,1996). The role of AL3 in viral replication is not well under-stood, and no catalytic functions have been assigned to thisviral protein.

DNA replication and the corresponding enzymes areconfined to meristematic and endoreduplicating tissues(Martinez et al., 1992; Staiger and Doonan, 1993). Somegeminiviruses are restricted to vascular tissue (Esau, 1977;Horns and Jeske, 1991; Sanderfoot and Lazarowitz, 1996;Morra and Petty, 2000) and may replicate in vascular paren-chyma cells that already contain some cell cycle compo-nents, such as the cyclin-dependent kinase cdc2a (Martinezet al., 1992; Hemerly et al., 1993). Other geminiviruses, suchas

TGMV

, are not limited to vascular tissue; instead, they

1

To whom correspondence should be addressed. E-mail linda@bchserver.bch.ncsu.edu; fax 919-515-2047.

1438 The Plant Cell

are found throughout terminally differentiated mesophylland epidermal cells of leaves (Rushing et al., 1987; Nagar etal., 1995; Lucy et al., 1996) and in various cell types in stemsand roots (S. Nagar, L. Hanley-Bowdoin, and D. Robertson,unpublished data). In animals, DNA tumor viruses, whichrely on host replicative enzymes, induce their synthesis byaltering cell cycle and transcriptional controls of quiescentcells (Chellappan et al., 1992; Jansen-Durr, 1996). There isevidence that

TGMV

also alters the cell cycle controls of itshost plant,

Nicotiana benthamiana

.

TGMV

-infected cells in-corporate high levels of bromodeoxyuridine into both viraland host DNA, indicative of progression into S phase andDNA replication (S. Nagar, L. Hanley-Bowdoin, and D.Robertson, unpublished data). A large fraction of

TGMV

-infected cells contain condensed chromatin, which is char-acteristic of early mitotic prophase (Bass et al., 2000). Inboth instances, uninfected cells immediately adjacent tocells positive for viral DNA showed no signs of reentry into thecell cycle, demonstrating that

TGMV

acts in a cell-autono-mous manner to reprogram its host.

Clues to how

TGMV

alters cell cycle controls come fromexperiments showing that both AL1 and AL3 bind to planthomologs of the cell cycle regulator retinoblastoma (pRB;designated in plants as pRBR [retinoblastoma related]) (Achet al., 1997; Settlage et al., 2000). By analogy with mamma-lian DNA viruses (Weinberg, 1995), these interactions maybypass a pRB phosphorylation requirement for cell cycleentry during geminivirus infection. Recent experimentsshowing that

TGMV

is confined to vascular cells when AL1/pRBR binding is impaired established the importance of thishost–pathogen interaction for geminivirus infection (Kong etal., 2000). Other geminiviruses also encode proteins thatbind to pRBR (Grafi et al., 1996; Xie et al., 1996; Horvath etal., 1998; Liu et al., 1999), providing general support for theimportance of this interaction during infection. In mam-malian cells, inactivation of pRB relieves E2F-mediatedtranscriptional repression of genes that encode proteinsnecessary for S phase and DNA replication (Herwig andStrauss, 1997). Recently, homologs of animal E2F transcrip-tion factors have been identified in plants (Ramirez-Parra etal., 1999; Sekine et al., 1999) and shown to regulate plantgene transcription in cultured cells (Albani et al., 2000;Chaboute et al., 2000). Like their mammalian counterparts,geminiviruses may alter host transcriptional controls throughpRBR and E2F.

In a previous study, we showed that TGMV infection spe-cifically induces the accumulation of high levels of proliferat-ing cell nuclear antigen (PCNA), the processivity factor ofhost DNA polymerase

�

, in differentiated cells of infectedplants (Nagar et al., 1995). PCNA, albeit at lower levels, alsowas detected in differentiated cells of transgenic plants ex-pressing TGMV AL1, thereby establishing that the AL1 pro-tein alone is sufficient for PCNA accumulation. Theseexperiments did not determine if induction occurred throughtranscriptional activation of the

PCNA

gene or at a post-transcriptional step. PCNA protein and mRNA accumulate in

young but not mature tissues of rice, pea, rape, and maize(Kosugi et al., 1991; Citterio et al., 1992; Markley et al.,1993; Lopez et al., 1997). Analysis of the rice PCNA pro-moter showed that its activity is regulated developmentallyand identified

cis

elements that are necessary for high-levelactivity in young tissues (Kosugi et al., 1991, 1995; Ohashiet al., 1992). Although these studies were performed in trans-genic tobacco, they did not address how endogenous PCNAexpression is regulated in dicotyledonous plants. To betterunderstand how PCNA expression is controlled in dicots,we isolated the PCNA promoter region from

N. benthamiana

and characterized its activity during development and gemi-nivirus infection.

RESULTS

PCNA mRNA Accumulates in Mature Leaves of

TGMV

-Infected Plants

Increased PCNA protein levels in

TGMV

-infected plantsmost likely are due to de novo mRNA synthesis. To testthis hypothesis in the TGMV host

N. benthamiana

, we useda combination of reverse transcription–polymerase chainreaction (RT-PCR) and rapid amplification of cDNA ends toisolate cDNA clones corresponding to the endogenousPCNA mRNA. We analyzed one set of PCR products en-coding amino acids 64 to 237 of

N. benthamiana

PCNAand two independent sets of products corresponding tothe 5

�

and 3

�

ends of the cDNA. Sequence analysis re-vealed that these products represent a single mRNA (Gen-Bank accession number AF305075) that includes a 792-bpopen reading frame flanked by 110 and 170 bp of 5

�

and 3

�

sequences, respectively. Comparison of the

N. benthami-ana

sequence and other plant PCNA proteins revealed

�

95% homology.We used the

N. benthamiana

PCNA cDNA to probe RNAgel blots that compared mRNA levels in healthy versus in-fected plants. A single PCNA transcript of

�

1200 nucle-otides was detected in young leaves of healthy plants(Figure 1A, lane 1). A transcript of the same size was seen in

TGMV

-infected mature leaves (Figure 1A, lane 3) but not inequivalent mock-infected leaves (Figure 1A, lane 2). Thesedifferences were not due to loading because the expectedamounts of rRNA were seen by ethidium bromide staining inFigure 1B (one-fourth as much RNA was loaded in lane 1).Similar hybridization signals also were detected for matureleaves from mock-inoculated and infected plants using acDNA probe for the translation initiation factor eIF4a (seeFigure 5B, bottom panels). We typically observed 15- to25-fold less PCNA mRNA in infected mature leaves than inyoung leaves. Together, these data established that steadystate levels of

N. benthamiana

PCNA mRNA are regulatedby development and geminivirus infection.

Regulation of PCNA Transcription 1439

The PCNA Promoter Is Active in Cycling Cells and Young Leaves

To determine if the PCNA mRNA accumulation patterns re-flect changes in transcription, we developed a fully homolo-gous system to study the regulation of the

N. benthamiana

PCNA promoter. Two PCNA promoter fragments were am-plified independently from

N. benthamiana

chromosomalDNA. Both fragments contained 110 bp of leader sequenceand either 946 or 633 bp of 5

�

sequence. The overlappingsequences of the two promoter fragments were identical, in-dicating that they were derived from the same gene. Com-parison of the

N. benthamiana

PCNA promoter sequencewith the corresponding rice promoter (Kosugi et al., 1991)showed little conservation upstream of position

�

181 rela-tive to the transcription start site (data not shown) and lim-

ited homology downstream of this site (Figure 2A). Position

�

181 is located immediately 5

�

of an

N. benthamiana

se-quence that shows identity at seven of 10 nucleotides to thesite IIa element of the rice PCNA promoter. The

N.benthamiana

sequence displays less homology (five of 10nucleotides) with the downstream site IIb element of the ricepromoter. The site II elements have been shown to activatethe rice promoter in tobacco (Kosugi et al., 1995). A TATAconsensus is located 24 bp upstream of the

N. benthamiana

transcription start site. There also are short stretches ofconservation with variable spacing in the proximal promoterand leader regions of the

N. benthamiana

and rice se-quences. Two conserved stretches in the proximal promotershow strong homology with E2F consensus elements. TheE2F-1 sequence (TTTCCCGC), which is in inverse orienta-tion, is identical to the E2F consensus (TTTC/GC/GCGC)(Sidle et al., 1996; Herwig and Strauss, 1997). The E2F-2 se-quence (ATTCCCGCCAAT) contains an exact match of thecentral 10 bp of an inverted repeat E2F element (TTTC/GCC-GCC/GAAA) (Wade et al., 1995).

The

N. benthamiana

�

946 PCNA promoter fragment(designated by the position of its 5

�

end relative to the tran-scription start site marked in Figure 2A) was fused to the

�

-glucuronidase (GUS) reporter gene (

uidA

) and analyzed intransient assays using protoplasts from log phase BY-2cells. The

�

946 promoter supported high levels of GUS ex-pression and was as active as the

Cauliflower mosaic virus

35S promoter with a duplicated enhancer element (Figure2B). Transient analysis of a

�

633 PCNA promoter fragmentrevealed that the 5

�

truncation also supported high levels ofreporter activity similar to the

�

946 promoter. Further 5

�

de-letions to positions

�

357 and

�

165 resulted in progressive20 and 35% reductions in activity, whereas deletion to posi-tion

�

137 had no further effect. The abilities of the

�

165and

�

137 promoters to support

�

45% of full activity wereunexpected because these constructs lack one or both ofthe putative site II elements (Figure 2B). Although both puta-tive E2F elements were maintained in the

�

165 and

�

137promoter constructs, site-directed mutagenesis studiesfailed to uncover a role for either element during transcrip-tion in BY-2 cells (our unpublished results).

We next generated transgenic

N. benthamiana

carryingeither

uidA

or the luciferase gene (

luc

) fused to the

�

633PCNA promoter, the shortest fragment with full activity insuspension cells. Reporter activities were measured inyoung leaves (

�

2 cm in length) and fully expanded leaves(

�

10 cm). To ensure uniformity, young tissue was fromleaves 1 to 2 and mature tissue was from leaf 9. (Leaf 1measured

�

1 cm at the time of harvest, and all other leaveswere numbered consecutively.) In Figures 3A and 3B, all ofthe lines showed higher reporter activity in young than inmature leaves, with the absolute levels varying up to fivefoldbetween lines. The ratio of young/mature activity rangedfrom two- to 38-fold, with the

�

633 PCNA:

uidA

and

�

633PCNA:

luc

lines showing similar average ratios of 17- to19-fold, indicating that the choice of reporter had no effect

Figure 1. Mature Leaves Infected with TGMV Contain PCNA mRNA.

Total RNA from young (lane 1; 15 �g), mock-inoculated mature (lane2; 60 �g), and TGMV-infected mature (lane 3; 60 �g, 14 days afterinoculation) leaves was resolved on agarose gels and transferred tonylon membranes.(A) The blots were hybridized with 32P-labeled probe correspondingto N. benthamiana PCNA cDNA. The positions of nucleotide sizemarkers are shown at right.(B) Ethidium bromide staining of the same blot. The stained bandscorrespond to rRNA.

1440 The Plant Cell

on the outcome. The variation in absolute activity andyoung/mature ratios between the different lines may be in-dicative of copy number and/or position effects of the trans-gene. However, in all cases, the

N. benthamiana

PCNApromoter was more active in young leaves than in matureleaves.

Developmental regulation was characterized further bymeasuring reporter activity in individual leaves of four

�

633PCNA:

luc

lines (Figure 3C). In all four lines, Luc activity washighest in the upper leaves (leaves 1 and 2). Expanding mid-dle leaves (leaves 3 and 4) showed progressive loss of re-porter activity, whereas lower leaves (leaves 5 to 11) hadvery little activity. Analysis of endogenous PCNA mRNA re-vealed a similar expression pattern, with high levels in youngleaves, low levels in expanding leaves, and undetectablelevels in mature leaves (see Figure 5B). Together, these re-sults established that the

N. benthamiana

PCNA promoter isactive in cultured cells and is regulated developmentally inplants.

Geminivirus Infection Activates the PCNA Promoter in Mature Leaves

To assess the impact of TGMV infection on PCNA promoterfunction, we compared reporter activities in young and ma-ture leaves of infected and mock-inoculated plants. Plantscarrying the

�

633 PCNA:

uidA

or the

�

633 PCNA:

luc

fusionwere inoculated with

TGMV

by bombardment or agroinocu-lation, respectively. Bombarded plants typically developedsymptoms between 4 and 5 days after inoculation, whereasagroinoculated plants displayed symptoms by 5 to 6 days

Figure 2.

The

N. benthamiana

PCNA Promoter Is Active in CyclingCells.

(A)

The sequence of the longest

N. benthamiana

(Nb) PCNA pro-moter fragment is shown. The transcription start site (

�

) was deter-mined by S1 nuclease protection (data not shown). The proximalsequences of the

N. benthamiana

and rice (Suzuka et al., 1991)

PCNA promoters are compared. The TATA boxes and ATG startcodons of the two genes are shown in bold lowercase letters. TheG-box (white letters), the site II (dotted boxes) elements, and thetranscription start site (

�

) of the rice promoter (Kosugi et al., 1995)are marked. The regions of the

N. benthamiana

promoter that showlimited homology with site II sequences are included in the dottedboxes. Two sequences that display strong homology with the E2Fbinding consensus and are conserved between the two promotersare shown in bold italic type.

(B)

A linear diagram of the

N. benthamiana

promoter indicating thesite II–related elements, the E2F consensus elements, the TATA box,the transcription start site (

1), and the ATG. The end points of the5

�

deletions are marked. The relative reporter activities supported bythe

N. benthamiana

promoter fragments in transiently transfectedBY-2 cells are shown below. For each experiment, the activity of theenhanced Cauliflower mosaic virus 35S (E35S) promoter was set to100 and the activities of the PCNA:

uidA

constructs were standard-ized against it. In these experiments, an average of 722

10

3

lightunits/

�

g protein was detected for the E35S:

uidA construct, whereasan average of 915 light units/�g protein was detected for a promoter-less uidA cassette. Gus, �-glucuronidase. The error bars representtwo standard errors.

Regulation of PCNA Transcription 1441

after inoculation. Leaves 1 to 2 (young) and leaf 9 (mature)were harvested at 14 days after inoculation, and the ratios ofGUS (Figure 4A) or Luc (Figure 4B) activities for infected/mock-inoculated leaves were determined. The ratios foryoung leaves scattered around a value of 1 (Figure 4, dottedline), with some lines showing ratios �1 and others showingratios slightly �1. In contrast, the ratios for mature leavesranged from 1.4 to 7 and were always greater than thosedetected for young leaves of the same line. These datademonstrate that TGMV infection activates the N. benthami-ana PCNA promoter in mature leaves but has no detectableeffect in young leaves. However, in all cases, the absoluteactivity of the PCNA promoter was lower in mature than inyoung leaves (data not shown). This result is consistent withthe lower levels of steady state PCNA mRNA in infected ma-ture leaves than in young leaves (Figure 1).

To better characterize the relationship between geminivi-rus and developmental regulation of the N. benthamianaPCNA promoter, we monitored symptoms, TGMV DNA ac-cumulation, reporter activity, and endogenous PCNA mRNAlevels in individual leaves of �633 PCNA:luc plants (Figure5). For the plant depicted in Figure 5A, TGMV symptoms,which include chlorosis, leaf curling, and stunting, weremost severe for leaves 8 to 10. The oldest leaves, whichwere fully developed at the time of agroinoculation, showedmilder symptoms (leaf 11) or were free of symptoms (leaf12). Younger leaves (leaves 1 to 7) also displayed mildersymptoms. DNA gel blot analysis of TGMV B DNA showed asimilar pattern, with viral DNA highest in leaves 8 to 10 (Fig-ure 5B, top) and lower in older (leaves 11 and 12) andyounger (leaves 1 to 7) leaves.

PCNA promoter activity and endogenous PCNA mRNAlevels reflected the composite effect of TGMV infection anddevelopment. As shown for lines L-39 (Figure 5C, left) andL-12 (Figure 5C, right), Luc reporter activity was highest inyoung leaves (leaves 1 to 3) and decreased in expandingleaves (leaves 4 and 5) in both infected and mock-inocu-lated plants. A second, lower peak of Luc activity was de-tected in the most symptomatic leaves (Figure 5C, leaves 7to 9 or leaves 7 to 10) of the infected plants. In contrast, Lucactivity remained low in older leaves of the mock-inoculatedplants. A similar pattern was seen for endogenous PCNA

Figure 3. The N. benthamiana PCNA Promoter Is Regulated Devel-opmentally.

(A) Soluble protein extracts from young (y, gray bars) and mature(m, black bars) leaves of N. benthamiana lines (left) carrying the�633 PCNA:uidA construct were assayed for GUS (Gus) activity.Each set of bars corresponds to the mean activity/mg protein in aminimum of 10 plants. The ratio of reporter activity in young versusmature leaves (y/m) is shown at right for each line.

(B) Same as in (A) except that the N. benthamiana lines were trans-formed with the �633 PCNA:luc construct and assayed for Luc ac-tivity.(C) Soluble protein extracts from individual leaves of plants repre-senting four �633 PCNA:luc lines were assayed for Luc activity. TheLuc specific activity was plotted against leaf number. The sizes ofthe corresponding leaves are indicated below.The error bars indicate two standard errors.

1442 The Plant Cell

mRNA, with high levels in young leaves (leaves 1 to 4) anddecreasing levels in expanding leaves (leaves 5 and 6) ofboth infected and mock-inoculated plants (Figure 5B, mid-dle). In infected plants, PCNA mRNA steady state levels in-creased in lower leaves, with the highest amount in the mostsymptomatic leaves (Figure 5B, leaves 7 to 10), whereasPCNA mRNA could not be detected in mature leaves (leaves7 to 12) of mock-inoculated plants. Together, these datademonstrate a quantitative relationship between the severityof TGMV infection, the induction of the PCNA promoter, andthe accumulation of PCNA mRNA in mature leaves.

The PCNA Promoter Is Repressed through an E2F Element in Mature Leaves

Two E2F consensus sequences, which may play roles in de-velopmental regulation and geminivirus activation, are lo-cated in the proximal promoter region of the N. benthamianaPCNA promoter. Because the E2F-2 sequence is homolo-gous with an inverted repeat element that binds to E2F withvery high affinity in animals (Wade et al., 1995), our initialstudies have focused on this element. We used electro-phoretic mobility shift assays to determine if the E2F-2 se-quence is bound specifically by a nuclear protein(s) in vitro.A radiolabeled, double-stranded DNA (5�-gatcTCCATTCC-CGCCAATAGgatc-3�) containing 16 bp of PCNA promotersequence (uppercase letters) with the E2F-2 motif (under-lined) and 8 bp of unrelated flanking DNA (lowercase letters)was incubated with a nuclear protein extract from N.benthamiana suspension cells, and the resulting complexeswere resolved by PAGE. We observed a prominent band(Figure 6A, lane 2, arrow) that was not seen in the presenceof excess unlabeled E2F-2 competitor DNA (Figure 6A,lanes 3 to 5). The band was not competed by a DNA (5�-gatcTCCATTCttatCAATAGgatc-3�) altered in the centralcore of the E2F-2 element (Figure 6A, lanes 6 to 8) or by aDNA (5�-gatcATCCTCTCACGTACGACgatc-3�) containing arandomized sequence of equivalent nucleotide content (Fig-ure 6A, lanes 9 to 11). In contrast, the slower migratingbands seen with the E2F-2 probe and nuclear protein ex-tract (Figure 6A, lane 2) were competed by all three DNAs(Figure 6A, lanes 3 to 11). Together, these results demon-strated that the prominent band corresponds to a specificE2F-2 complex, whereas the slower migrating bands reflectnonspecific binding.

We next generated transgenic N. benthamiana carrying a�633 PCNA:luc construct with the 4-bp E2F-2 mutationthat prevented competition (Figure 6A, lanes 6 to 8). Figure6B shows Luc activity ratios in young versus mature leaves,which ranged from 1.2 to 5.2, for eight independent lines.The mutant promoter ratios were significantly lower thanthose calculated for plants carrying the wild-type �633PCNA:luc construct (Figure 3B), with mean values of 3 and19, respectively. These results established that mutation ofthe E2F-2 element alters developmental regulation of thePCNA promoter. Luc-specific activities in young and matureleaves of plants transformed with the wild-type and mutantpromoter constructs were compared to determine how pro-moter function is altered during development (Figures 6Cand 6D). The wild-type and mutant promoters displayedsimilar activities in young leaves, but the mutant promoterwas significantly more active than was the wild-type pro-moter in mature leaves. Because of the variability betweenlines, it was not possible to determine from the data pre-sented in Figures 6C and 6D whether mutation of the E2F-2sequence is sufficient to fully activate PCNA promoter ex-pression in mature leaves. To address this question, wemonitored Luc-specific activity in individual healthy leaves

Figure 4. The PCNA Promoter Is Activated in Mature Leaves ofTGMV-Infected Plants.

(A) Soluble protein extracts were isolated 14 days after inoculationfrom N. benthamiana lines (left) carrying the �633 PCNA:uidA con-struct and assayed for GUS (Gus) activity. The activity ratios for in-fected versus mock-inoculated plants are shown for young (graybars) and mature (black bars) leaves. Each set of bars represents themean ratios from three independent experiments. The ratio valuesfor mature leaves are given at right. The dotted line corresponds to aratio of 1. (B) Same as in (A) except that the N. benthamiana lines were trans-formed with the �633 PCNA:luc construct and ratios of Luc activityare shown.The error bars indicate two standard errors.

Regulation of PCNA Transcription 1443

Figure 5. TGMV Infection and Regulation of the PCNA Promoter.

(A) TGMV symptoms on an N. benthamiana plant 14 days after inoculation. Leaves are numbered consecutively from youngest to oldest.(B) Accumulation of TGMV DNA and PCNA mRNA in individual leaves of infected and mock-inoculated plants. Top, total DNA (10 �g) was di-gested with BglII and analyzed on DNA gel blots using a TGMV B probe. The positions of double stranded (ds) and single stranded (ss) viral DNAare marked. Middle, total RNA (30 �g) was analyzed on RNA gel blots using an N. benthamiana PCNA probe. Bottom, the same RNA gel blotswere reprobed with a tobacco eIF4a sequence. The position number of each leaf is given above the gels. It was necessary to pool leaves 1 to 4to obtain sufficient material for analysis. The DNA and RNA samples, which were isolated 14 days after inoculation, were from different plantsbecause of tissue limitations. The positions of the most symptomatic leaves ranged from positions 7 to 10 between individual plants.(C) Developmental regulation of the PCNA promoter in infected and mock-inoculated transgenic plants 14 days after inoculation. Soluble proteinextracts from individual leaves of infected (triangles and circles) and mock-inoculated (diamonds and squares) L-39 (left) and L-12 (right) plantscarrying the �633 PCNA:luc transgene were assayed for Luc activity. Profiles from two plants are shown for each treatment. The average in-fected/mock-inoculated ratios for leaf 9 are given for each line above the corresponding peak.

1444 The Plant Cell

Figure 6. Mutation of the E2F-2 Element Alters the Developmental Regulation and TGMV Activation of the N. benthamiana PCNA Promoter.

(A) Electrophoretic mobility shift assays with 32P-labeled E2F-2 DNA alone (lane 1) or incubated with N. benthamiana nuclear protein extract(lanes 2 to 11). Lane 2 had no added competitor DNA. Lanes 3 to 5 contained unlabeled E2F-2 DNA (wt). Lanes 6 to 8 contained unlabeled mu-tant E2F-2 DNA (mt). Lanes 9 to 11 contained a random control DNA (c). Competitor DNAs were used at 10-, 100-, or 250-fold molar excess rel-ative to probe, as indicated by the triangles at the top of the gel. The shifted product corresponding to the specific E2F-2/protein complex ismarked by the arrow.(B) The ratios of Luc activities in young versus mature leaves of N. benthamiana lines (left) carrying the mutant E2F-2 �633 PCNA:luc construct areshown. The dotted line (mt) corresponds to the mean ratio for plants transformed with the mutant construct, whereas the solid line (wt) correlateswith the mean ratio for plants transformed with the wild-type �633 PCNA:luc construct (Figure 3B). The error bars indicate two standard errors.(C) Soluble protein extracts from N. benthamiana lines carrying the mutant E2F-2 �633 PCNA:luc construct were assayed for Luc activity andcompared with lines carrying the wild-type �633 PCNA:luc construct shown in Figure 3B. The Luc-specific activity values from young (y) and

Regulation of PCNA Transcription 1445

of two lines, mt-16 and mt-35, both of which were trans-formed with the mutant E2F-2 �633:luc construct. Bothlines showed a gradual decrease in PCNA promoter activityduring development (Figure 6E, crosses), unlike the precipi-tous decrease seen in plants carrying the wild-type pro-moter (Figure 3C). Together, our data establish that the E2F-2sequence acts as a negative regulatory element during de-velopment but is not solely responsible for the decrease inPCNA promoter activity in mature tissues.

Geminivirus Infection Does Not Alter the Activity of the Mutant E2F-2 Promoter

In animals, E2F acts as a transcriptional repressor by re-cruiting pRB and histone deacetylase to promoters (Luo etal., 1998). Mammalian DNA viruses can activate host geneexpression by binding to pRB and disrupting its interactionwith E2F (Weinberg, 1995). Because TGMV AL1 and AL3both bind to the plant pRB homolog pRBR (Ach et al., 1997;Settlage et al., 2000), we sought to determine if the activityof mutant E2F-2 PCNA promoter is influenced by geminivi-rus infection. In Figure 6E, the developmental profiles ofTGMV-infected (closed circles), mock-inoculated (open cir-cles), and untreated (crosses) plants are shown for lines mt-16 and mt-35. For both lines, there was no detectable differ-ence in promoter activity in mature leaves with the threetreatments. This result is consistent with the two- to fivefoldgreater activity detected for the mutant PCNA promoter inmature leaves than for the wild-type promoter in equivalentinfected leaves (cf. Figures 5C and 6E) and suggests thatTGMV does not further activate the mutant E2F-2 PCNApromoter during infection.

DISCUSSION

Eukaryotic viruses with small DNA genomes replicate in nu-clei using the replication machinery of their hosts. Becauseof their dependence on host enzymes, these viruses amplify

only in cells that contain the necessary DNA polymerasesand accessory factors, which generally are restricted to cy-cling cells. However, many small DNA viruses can overcomethis constraint by inducing the synthesis of replication en-zymes in terminally differentiated cells (Cheng et al., 1995).In animals, DNA tumor viruses alter replication competenceby modulating host gene transcription. They activate thetranscription of genes encoding cell cycle regulators andDNA replication enzymes, which in turn facilitate return tothe cell cycle and progression to S phase (Ohashi et al.,1992; Ogris et al., 1993; Chen et al., 1996; Tiainen et al.,1996). In a previous study, we demonstrated that TGMV in-duces PCNA in mature plant cells (Nagar et al., 1995). Here,we show that TGMV infection induces the accumulation ofPCNA mRNA by promoter activation. We also provide evi-dence that E2F transcription factors negatively regulateplant gene expression during development and that gemini-virus infection may overcome E2F-mediated repression toinduce PCNA expression in mature leaves. Thus, like theiranimal counterparts, geminiviruses alter developmental con-trols to activate the transcription of host genes whose prod-ucts are required for viral DNA replication.

To better understand how PCNA expression is controlledin healthy and geminivirus-infected plants, we developed afully homologous plant system for studying PCNA mRNAaccumulation and promoter function. For this system, weamplified and sequenced a series of PCNA cDNA and pro-moter clones from N. benthamiana. The 5� and 3� se-quences of the N. benthamiana cDNA as well as thepromoter region were represented by two independentclones of identical sequence. The sequence data, combinedwith the detection of only one PCNA transcript on RNA gelblots and by S1 nuclease protection assays, suggested thatN. benthamiana encodes a single PCNA gene. This conclu-sion was supported by genomic DNA hybridization experi-ments using probes corresponding to the 5� and 3� endsand the center of the N. benthamiana cDNA (data notshown). However, without intron sequence and position in-formation, we cannot unequivocally rule out the existence ofa second PCNA gene in N. benthamiana. Two PCNA geneshave been identified for carrot and maize (Hata et al., 1992;

mature (m) leaves of the mutant (mt) and wild-type (wt) constructs are compared in the scattergram. The eight independent lines given in (B) areshown for each treatment. The symbols indicate different lines.(D) P values of t test comparisons of the data shown in (C). Statistically significant values (P � 0.05) are shown in boldface type.(E) Developmental regulation of the mutant E2F-2 PCNA promoter in untreated, mock-inoculated, and infected plants (14 days after inoculation).Soluble protein extracts from individual leaves of untreated (crosses), mock-inoculated (open circles), and infected (closed circles) mt-16 (left)and mt-35 (right) plants carrying the mutant E2F-2 �633 PCNA:luc transgene were assayed for Luc activity. Each point represents the averageof two plants. (Note the different scales relative to Figure 5C.) A gradual decrease in PCNA activity with leaf number and little effect of TGMV in-fection also were seen for line mt-29 (our unpublished data).

Figure 6. (continued).

1446 The Plant Cell

Lopez et al., 1997) that display similar expression profiles(Lopez et al., 1997). As a consequence, if N. benthamianaencodes two PCNA genes, then they are likely to be regu-lated in parallel.

PCNA plays key roles in DNA replication, DNA repair, andcell cycle control (reviewed in Prosperi, 1997). In animals,PCNA expression is regulated tightly at the level of tran-scription during development and is altered by DNA virus in-fection (Cheng et al., 1995; Lee et al., 1995; Yamaguchi et al.,1995b). RNA gel blot analysis showed that, as in other plantspecies (Kosugi et al., 1991; Citterio et al., 1992; Markley etal., 1993; Lopez et al., 1997), PCNA mRNA levels in N.benthamiana are regulated developmentally. PCNA mRNAis abundant in young leaves, present in trace amounts in ex-panding leaves, and undetectable in fully expanded leaves.This pattern parallels the distribution of cells undergoing DNAreplication during leaf development and is consistent withstudies showing that PCNA mRNA is high in cycling plantcells (Kodama et al., 1991; Sekine et al., 1999). PCNA mRNAalso was detected in mature leaves of TGMV-infectedplants. PCNA mRNA levels were increased in leaves withthe most viral DNA, indicating a relationship between PCNAaccumulation—and presumably other components of thehost DNA replication apparatus—and efficient viral replica-tion in mature leaves. However, TGMV DNA levels were lowin young leaves, which have the most PCNA mRNA, demon-strating that other factors in addition to replication compe-tence influence geminivirus infection. This conclusion issupported by in situ results showing that TGMV does notaccumulate in meristematic cells even though they containplant DNA replication machinery (Nagar et al., 1995).

There is a strong correlation between PCNA mRNA levelsand PCNA promoter function in healthy and geminivirus-infected plants. Analysis of transgenic N. benthamianaplants carrying the homologous PCNA promoter fused to ei-ther the uidA or luc reporter gene established that PCNAmRNA accumulation is regulated primarily at the level oftranscription. Like PCNA mRNA, promoter activity is high inyoung leaves, decreases rapidly in expanding leaves, and isvery low in mature leaves of healthy plants. The PCNA pro-moter also is active in mature leaves of TGMV-infectedplants, with the most symptomatic leaves displaying thegreatest activity. Interestingly, PCNA promoter activity doesnot appear to be induced in expanding leaves or the oldestleaves of infected plants, both of which contain low but de-tectable levels of viral DNA. The limited viral replication inexpanding leaves may depend on residual replication ma-chinery produced earlier in development, whereas viral repli-cation in the oldest leaves may reflect the activity ofpreexisting DNA repair enzymes. Alternately, PCNA pro-moter induction may be below the limit of detection if fewercells are infected in these leaves, or, in the case of olderleaves, it may have occurred at an earlier time during the in-fection process. Earlier studies showed that �10% of thecells of a highly symptomatic leaf contain TGMV DNA, theviral replication proteins AL1 and AL3, and the PCNA pro-

tein (Nagar et al., 1995; Bass et al., 2000). This observationmay account for the 15- to 25-fold difference in activity be-tween young and infected mature leaves. However, PCNAexpression is regulated with respect to the cell cycle suchthat not all cells in asynchronous young tissue expressPCNA (Kodama et al., 1991; Sekine et al., 1999). Thus, it islikely that the difference in PCNA promoter activity in youngversus highly infected mature leaves reflects both the num-ber of cells actively transcribing the gene and the level oftranscription in a given cell. The same may be true for theless symptomatic flanking leaves.

Although the N. benthamiana promoter shows weak ho-mology with the rice PCNA promoter, the only other plantPCNA promoter that has been examined to date, severalobservations suggest that the two promoters are regulateddifferently. First, the rice PCNA promoter contains a G-boxelement that is required for full activity of a truncated �262promoter (Kosugi et al., 1995). No G-box motif is found inthe N. benthamiana promoter. Second, the rice promoter in-cludes two sequences, sites IIa and IIb, that resemble auxinresponse elements (Kosugi et al., 1995). The N. benthami-ana promoter contains sequences that show weak homol-ogy with these elements but diverges at positions that areessential for rice promoter activity and binding to the basichelix-loop-helix transcription factors PCF1 and PCF2 (Kosugiet al., 1995; Kosugi and Ohashi, 1997). Third, deletion ormutation of both site II elements inactivates the rice PCNApromoter in transgenic tobacco (Kosugi et al., 1991, 1995).Our data do not eliminate the possibility that the site II–likesequences in the N. benthamiana PCNA promoter contrib-ute to its activity. However, the �137 PCNA:uidA reporter,which does not contain any site II–like sequences, retained45% of full-length promoter activity. Thus, unlike in rice,these putative elements are not required for N. benthamianaPCNA promoter function.

Unlike the G-box and site II elements, the E2F-1 and E2F-2sequences identified in Figure 2A are highly conserved in theN. benthamiana and rice PCNA promoters. An E2F-1–typeelement also is present in the proximal promoter region ofthe Arabidopsis PCNA gene (our unpublished observation),and E2F elements have been identified in Drosophila, hu-man, and murine PCNA genes (Yamaguchi et al., 1995a;Huang and Prystowsky, 1996; Tommasi and Pfeifer, 1999),suggesting that these elements may be general features ofeukaryotic PCNA genes. Analysis of transgenic plants carry-ing a mutation in the E2F-2 sequence of the N. benthamianaPCNA promoter demonstrated that it functions as a nega-tive regulatory element to repress PCNA transcription in ma-ture leaves. E2F also represses expression from theDrosophila PCNA promoter (Sawado et al., 1998). However,PCNA promoter function is complex, involving a variety oftranscription factors (Yamaguchi et al., 1996; Liu et al.,1998; Hayashi et al., 1999), and E2F can activate as well asrepress its activity (Sawado et al., 1998; Tommasi and Pfeifer,1999). This complexity may explain our observation that theE2F-2 sequence acts as a negative regulatory element dur-

Regulation of PCNA Transcription 1447

ing development but is not solely responsible for the de-crease in N. benthamiana PCNA promoter activity in maturetissues. One possibility is that the E2F-2 element does notact alone to repress PCNA promoter activity in mature tis-sues. A candidate for a second negative regulatory elementis the E2F-1 sequence, which is under analysis. Alternately,relevant transcriptional activators may decrease during de-velopment or be replaced by less effective activators, result-ing in a gradual decrease in PCNA promoter activity in theabsence of active repression.

The E2F transcription factor family modulates the activi-ties of many genes during cell division and development inanimals (Dyson, 1998; Lavia and Jansen-Durr, 1999). E2FcDNAs have been reported for wheat, tobacco, and carrot(Ramirez-Parra et al., 1999; Sekine et al., 1999; Albani et al.,2000). Genomic sequence data established that Arabidop-sis encodes six E2F homologs. Recently, DP1, the E2Fdimerization partner, was characterized from Arabidopsisand wheat (Magyar et al., 2000; Ramirez-Parra and Gutierrez,2000). However, little is known about the targets or mecha-nisms of E2F-mediated regulation of plant gene transcrip-tion. E2F has been shown to activate chimeric promoterscontaining E2F elements in cultured plant cells (Sekine etal., 1999; Albani et al., 2000). Recent studies of the tobaccoribonucleotide reductase small subunit gene (RNR2) indi-cate that its activity is regulated by E2F elements in BY-2cells (Chaboute et al., 2000). Like the N. benthamiana PCNApromoter, the tobacco RNR2 promoter contains multipleE2F elements, two of which activate transcription and onethat represses transcription outside of S phase. At present,there is no report of these elements also regulating RNR2transcription during plant development.

TGMV may modulate PCNA promoter function by alteringcell cycle controls and relieving E2F-mediated repression ofPCNA transcription in differentiated cells. Three lines of evi-dence support this hypothesis. First, TGMV infection in-creases the activity of the wild-type but not the mutant E2F-2promoter in mature leaves. Second, immunoblot analysisshowed that the E2F protein is ubiquitous in carrot (Albani etal., 2000). We also detected high levels of E2F protein inmature leaves of N. benthamiana plants (our unpublisheddata), consistent with its negative regulation of PCNA tran-scription. Third, TGMV AL1 binds to the host factor, pRBR,and this interaction influences the pattern of PCNA proteinaccumulation during infection (Ach et al., 1997; Kong et al.,2000). Although pRBR is not well characterized in plants (deJager and Murray, 1999), it is homologous with the animalpocket proteins pRB, p107, and p130, which repress thetranscription of genes required for S phase by binding to E2F(Herwig and Strauss, 1997). In animals, DNA tumor virusesinduce host gene transcription by binding to pocket proteinsand disrupting their interactions with E2F (Weinberg, 1995).By analogy, AL1 may disrupt pRBR/E2F complexes that re-press PCNA promoter function to induce transcription inmature leaves. Future experiments that assess the ability ofwild-type AL1 and pRBR binding mutants (Kong et al., 2000)

to activate the N. benthamiana PCNA promoter in matureleaves will provide insight into the roles of AL1/pRBR andpRBR/E2F interactions during geminivirus-mediated induc-tion of host transcription and into the mechanisms that con-trol gene expression during plant development.

METHODS

The Nicotiana benthamiana PCNA cDNA and Promoter

Total RNA was isolated from 4-week-old N. benthamiana seedlings(Hanley-Bowdoin et al., 1989) and used as a template in reverse tran-scription–polymerase chain reaction (RT-PCR) containing the degen-erate primers 5�-GGITTYGARCAYTAYMGITGYGA-3� and 5�-CCC-ATYTGNGCDATYTTRTAYTC-3�, which were based on carrot (Gen-Bank accession numbers Q00268 and Q00265), rice (AAA33913),and rape (AAB27811) proliferating cell nuclear antigen (PCNA) cDNAsequences. RT-PCR was performed using the Gene-Amp RNA-PCRkit (Perkin-Elmer, Foster City, CA) using seedling RNA as a template.The sequence of the amplified product was used to design primersfor subsequent rapid amplification of cDNA ends–PCR.

The 5� end of the N. benthamiana PCNA cDNA was amplified usinga 5� rapid amplification of cDNA ends system from Gibco BRL(Grand Island, NY). The nested PCNA-specific primers were primer 1(5�-AATCAGCAATCTTGTCTTGGGTGGGGC-3�) and primer 2 (5�-CCATGTTACCAAGGTTCATCCCCATTG-3�). Reverse transcriptionwas performed with SuperScriptII reverse transcriptase (200 units;Gibco BRL) using primer 1 and 1 �g of N. benthamiana seedlingRNA. The resulting cDNA was treated with RNase, isolated using aGlassMax spin cartridge (Gibco BRL), and extended with dCTP usingterminal deoxynucleotidyl transferase. PCR was performed using theabridged universal anchor primer (Gibco BRL) and 400 nM of PCNAprimer 2, and the resulting PCR product was purified and reampli-fied. The 3� end of the N. benthamiana PCNA cDNA was isolatedby the same procedure using the adapter primer 5�-GACTCGAGT-CGACATCGATTTTTTTTTTTTTTTTT-3� (Genosys, The Woodlands,TX) and PCNA-specific primer 3 (5�-CAGTAATAGAGATGAATGAGC-CAGTAT-3�).

The N. benthamiana PCNA promoter was amplified using the Uni-versal Genome Walker Kit (Clontech, Palo Alto, CA) and nestedPCNA-specific primers 4 (5�-CTTCCCTGAACAAGCCGTAATTCC-AAC-3�) and 5 (5�-CTATGAAAAAGGGGTTTGAGAAGGTC-3�) de-rived from the sequence of the 5� rapid amplification of cDNA endsproduct. Genomic DNA (Dellaporta et al., 1983) from young leaf tis-sue of 5-week-old N. benthamiana seedlings was dissolved in 1 MNaCl, purified using a Tip 500 column (Qiagen, Valencia, CA), and di-gested separately with EcoRV and ScaI. The fragments were purifiedand ligated to Clontech Universal Genome Walker Kit adapter DNA.The resulting products were amplified according to the manufac-turer’s instructions first using the kit AP1 primer and PCNA primer 4and second using the kit AP2 primer and PCNA primer 5.

The various PCR products were treated with Klenow, cloned intothe SmaI site of pUC119, and sequenced. �-Glucuronidase (GUS)reporter constructs containing the PCNA promoter were generatedusing pMON8677, which contains a Cauliflower mosaic virusE35S:uidA:rbcS E9 cassette (Coruzzi et al., 1984; Jefferson et al.,1987; Kay et al., 1987). The PCNA promoter fragments were isolatedwith BamHI and trimmed SacI ends and cloned in place of the E35S

1448 The Plant Cell

promoter of pMON8677 (BglII and trimmed PstI ends) to give �633PCNA:uidA (pNSB713) and �946 PCNA:uidA (pNSB716). pNSB716was digested with AccI and BglII, treated with Klenow, and religatedto give pNSB717, the �357 PCNA:uidA construct. pNSB716 alsowas digested with SacII and religated to give pNSB748, the �165PCNA:uidA construct. The �137 PCNA promoter fragment was gen-erated by PCR using pNSB716 as a template and the primers 5�-CGTTGACTGCCTCTTCGCTG-3� and 5�-CAGCCCAAAATAGAG-GCGGG-3�. The product was treated with Klenow, digested withBstBI, and cloned into pMON8677 with BstBI and trimmed PstI endsto give pNSB1021, the �137 PCNA:uidA construct. A luciferase (Luc)reporter construct was generated by cloning the �633 promoter frag-ment with BamHI and trimmed SacI ends into pMON8796 (Eagle et al.,1994) with BglII and trimmed PstI ends to give pNSB931.

The E2F-2 sequence in the �633 PCNA promoter fragment wasmutated by a two-step overlap extension PCR process using the uni-versal and reverse primers (New England Biolabs, Beverly, MA) andtwo promoter-specific oligonucleotides, E2F-2 1 (5�-GGCTCGCTA-TTGataaGAATGGAAATGAAC-3�) and E2F-2 2 (5�-GTTCATTTCCAT-TCttatCAATAGCGAGCC-3�). The mutation is represented by lower-case letters. The first set of reactions used pNSB712 as a templateand either E2F-2 1 and the universal primer or E2F-2 2 and the re-verse primer. The resulting products were gel purified, mixed, andreamplified using the universal and reverse primers. The final productwas gel purified, repaired with Klenow, cloned into pUC119 to givepNSB926, and sequenced. The mutated promoter was fused to theLuc reporter gene in pMON8796 as described above for the wild-type promoter to produce pNSB942.

Transgenic Plants

Plant transformation vectors were made by cloning NotI fragmentscontaining the expression cassettes of pNSB713, pNSB931, andpNSB942 into pMON721 (Lanahan et al., 1994) digested with NotI.The resulting vectors contained the �633 PCNA:uidA (pNSB714),the �633 PCNA:luc (pNSB935), and the mutant E2F-2 �633PCNA:luc (pNSB937) cassettes, respectively. pNSB714, pNSB935,and pNSB937 were introduced by direct DNA transformation(Holsters et al., 1978) into Agrobacterium tumefaciens ABI, whichcontains a chromosomal copy of pMP90RK (Zannis-Hadjopoulos etal., 1988). N. benthamiana seedlings grown in vitro were cocultivatedwith the Agrobacterium strains, and independent transformants wereselected on kanamycin (300 �g/mL) and regenerated using standardprocedures (Horsch et al., 1985).

Geminivirus Infection

Wild-type or transgenic N. benthamiana plants at the six-leaf stagewere infected by either bombardment or agroinoculation. For bom-bardment, 5 �g each of tomato golden mosaic virus (TGMV) A(pMON1565) and B (pTG1.4B) DNAs cloned as partial tandem copiesinto pUC (Fontes et al., 1994; Orozco and Hanley-Bowdoin, 1996)were precipitated onto gold particles and inoculated as describedpreviously (Nagar et al., 1995). For agroinoculation, overnight cul-tures of Agrobacterium carrying partial tandem copies of TGMV A(pMON337) or B (pMON393) in their T-DNAs (Elmer et al., 1988b)were mixed 1:1 and syringe inoculated onto the stem immediatelybelow the apex. Infected and mock-inoculated tissues were har-vested �2 weeks after infection using leaves 1 to 2 as young tissue

and leaf 9 as mature tissue. For viral DNA analysis, total DNA wasisolated from the indicated leaves at 14 days after agroinoculation(Dellaporta et al., 1983), digested with BglII, and analyzed on DNA gelblots using a TGMV B–specific probe (Fontes et al., 1994).

RNA Analysis

Total RNA was isolated from leaf tissue, resolved on 1% agarose-formaldehyde-Tris-borate/EDTA gels containing 0.4 �g/mL ethidiumbromide, and transferred to Genescreen nylon membranes (DuPont–New England Nuclear, Boston, MA), as described previously (Hanley-Bowdoin et al., 1988). Blots were hybridized in 50% formamide, 5 Denhardt’s solution (1 Denhardt’s solution is 0.02% Ficoll, 0.02%polyvinylpyrrolidone, and 0.02% BSA), 1% SDS, 5 SSPE (1

SSPE is 0.115 M NaCl, 10 mM sodium phosphate, and 1 mM EDTA,pH 7.4), 100 �g/mL tRNA (Sigma, St. Louis, MO), and 50 �g/mLpoly(A) RNA (Sigma). A 523-bp fragment from the middle of the N.benthamiana PCNA cDNA was colabeled with �-32P-dATP and�-32P-dCTP using a random-primed labeling kit (New England Biolabs)and hybridized at 106 to 3 106 cpm/mL. After hybridization overnightat 42�C, the membranes were washed at room temperature using thefollowing sequence: two 30-min washes and one 60-min wash in 1 SSPE and 0.1% SDS followed by one 30-min wash and one 60-minwash in 0.3 SSPE and 0.1% SDS. The blots were reprobed witha 1372-bp fragment of the translation factor eIF4a from tobacco(Mandel et al., 1995) as a control for loading and transfer. Ethidiumbromide staining also was used to monitor loading and transfer. RNAhybridization signals were quantified by phosphorimager analysis.

Reporter Assays

For transient assays, BY-2 protoplasts (5 106) were electroporatedin the presence of 10 �g of expression cassette, as described previ-ously (Eagle et al., 1994). Total soluble protein extracts were isolated�40 hr after transfection, and reporter activity was measured using aGUS-Light Kit (Tropix, Bedford, MA). The assays were standardizedagainst the activity resulting from pMON8677, which contains a GUSexpression cassette under the control of the Cauliflower mosaic virusE35S promoter with a duplicated enhancer. The activity of eachPCNA promoter:uidA cassette was measured in triplicate in at leastthree independent experiments.

For plant assays, tissue (250 mg) was harvested from the indicatedleaves, ground in liquid nitrogen, and mixed with 250 �L of GUS-Light Kit lysis buffer (Tropix). Extracts were centrifuged at 16,000g for5 min, and the supernatants were recovered and monitored for re-porter activity using a Monolight 3010 luminometer (Pharmingen,San Diego, CA). Reporter assays were standardized for total protein,which was measured by Bradford assays (Bio-Rad, Hercules, CA).Leaf tissue was harvested similarly for luciferase assays, which wereperformed as described previously (Eagle et al., 1994). Reporter ac-tivity in healthy, mock-inoculated, and infected leaves was assessedfor a minimum of 10 plants for each line.

Nuclear Protein Extracts and Electrophoretic MobilityShift Assays

Protoplasts were prepared from N. benthamiana suspension cells asdescribed previously (Eagle et al., 1994), except that digestion was

Regulation of PCNA Transcription 1449

for 130 min. Nuclear protein extracts were prepared using a modifiedprotocol based on that of Albani et al. (2000). The protoplasts (�5 107) were suspended in 15 mL of resuspension buffer (0.4 M sucrose,25 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 0.3% Triton X-100, 5 mM�-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride[PMSF]) and disrupted by 12 strokes with a Dounce homogenizer.The homogenate was filtered through two layers of nylon mesh andcentrifuged at 3000g for 5 min. The pellet was suspended in 5 mL ofresuspension buffer, centrifuged at 3000g for 15 min, and sus-pended in 5 mL of wash buffer (0.4 M sucrose, 50 mM Tris-HCl, pH7.6, 5 mM MgCl2, 20% glycerol, 5 mM �-mercaptoethanol, and 0.5mM PMSF). The nuclei were then centrifuged at 3000g for 20 minand suspended in 1 mL of lysis buffer (25 mM Hepes, pH 7.6, 40 mMKCl, 0.5 mM EDTA, 5 mM MgCl2, 20% glycerol, 2 mM DTT, 1 mg/mLantipain, and 1 mg/mL leupeptin). After addition of 100 �L of satu-rated ammonium sulfate, the mixture was rocked for 30 min at 4�C.The extract was centrifuged at 200,000g in a Sorvall (Newtown, CT)70.1 Ti rotor for 1 hr. The supernatant was recovered, precipitated bythe addition of 600 �L of saturated ammonium sulfate and rockingfor 1 hr at 4�C, and recentrifuged for 1 hr at 200,000g . The pellet wassuspended in 800 �L of dialysis buffer (25 mM Hepes, pH 7.6, 40 mMKCl, 0.5 mM EDTA, 1 mM MgCl2, 20% glycerol, 2 mM DTT, and 0.5mM PMSF) and dialyzed for 3 hr against 1 liter of the same buffer.The extract was divided into aliquots, frozen in liquid N2, and storedat �80�C until use.

Oligonucleotides corresponding to the wild-type E2F-2 sequence(top, 5�-gatcTCCATTCCCGCCAATAG-3�; bottom, 5�-gatcCTATT-GGCGGGAATGGA-3�), a mutated E2F-2 sequence (top, 5�-gatcTC-CATTCttatCAATAG-3�; bottom, 5�-gatcCTATTGataaGAATGGA-3�),and an unrelated control sequence (top, 5�-gatcATCCTCTCACG-TACGAC-3�; bottom, 5�-gatcGTCGTACGTGAGAGGAT-3�) weresynthesized commercially. The lowercase letters represent 5�

overhangs or mutations in the E2F consensus. Probe for electro-phoretic mobility shift assays was prepared by mixing 0.5 pmol ofthe wild-type E2F-2 oligonucleotides, which were then annealedand repaired using Klenow in the presence of 100 �Ci of �-32P-dATP.For binding assays, 5 �L of nuclear protein extract (1.9 �g) was in-cubated with probe DNA (105 cpm, corresponding to �7 fmol) in a20-�L reaction containing 100 ng of poly(dI-dC) and 4 �L of 5 buffer (125 mM Hepes, pH 7.5, 0.5 M KCl, 5 mM EDTA, 5 mMMgCl2, 25% glycerol, and 50 mM DTT). The reactions were incu-bated at room temperature for 30 min and resolved on 4% poly-acrylamide gels in 0.5 TBE (1 TBE is 0.9 M Tris-borate and2 mM EDTA) for 45 min at 15 mA. For competition experiments, 10-,100-, or 250-fold molar excess of annealed, unlabeled oligonucle-otides was incubated with the reactions for 30 min on ice beforeprobe addition.

ACKNOWLEDGMENTS

We thank Ling-Jie Kong and Dr. Cindy Hemenway for critical readingof the manuscript. This research was supported by National Re-search Initiative Competitive Grants Program Grants 96-0052 and98-01392 to L.H.-B. and D.R. from the United States Department ofAgriculture.

Received January 4, 2001; accepted April 6, 2001.

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DOI 10.1105/TPC.010004 2001;13;1437-1452Plant Cell

Erin M. Egelkrout, Dominique Robertson and Linda Hanley-Bowdoinand Activated by Geminivirus Infection in Mature Leaves

Proliferating Cell Nuclear Antigen Transcription Is Repressed through an E2F Consensus Element

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