Proliferating cell nuclear antigen transcription is repressed through an E2F consensus element and activated by geminivirus infection in mature leaves

Abstract

The geminivirus tomato golden mosaic virus (TGMV) amplifies its DNA genome in differentiated plant cells that lack detectable levels of DNA replication enzymes. Earlier studies showed that TGMV induces the accumulation of proliferating cell nuclear antigen (PCNA), the processivity factor for DNA polymerase delta, in mature cells of Nicotiana benthamiana. We sought to determine if PCNA protein accumulation reflects transcriptional activation of the host gene. RNA gel blot analysis detected an approximately 1200-nucleotide PCNA transcript in young leaves. The same RNA was found in mature leaves 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 of healthy 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 accumulation, PCNA promoter activity, and endogenous PCNA mRNA levels. Mutation of an E2F consensus element in the PCNA promoter had no effect on its activity in young leaves but increased transcription in healthy mature leaves. Unlike 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 by activating transcription of its gene in mature tissues, most likely by overcoming E2F-mediated repression.

Figure 1.

Mature Leaves Infected with TGMV Contain PCNA mRNA. Total RNA from young (lane 1; 15 μg), mock-inoculated mature (lane 2; 60 μg), and TGMV-infected mature (lane 3; 60 μg, 14 days after inoculation) leaves was resolved on agarose gels and transferred to nylon membranes. (A) The blots were hybridized with ³²P-labeled probe corresponding to N. benthamiana PCNA cDNA. The positions of nucleotide size markers are shown at right. (B) Ethidium bromide staining of the same blot. The stained bands correspond to rRNA.

Figure 2.

The N. benthamiana PCNA Promoter Is Active in Cycling Cells. (A) The sequence of the longest N. benthamiana (Nb) PCNA promoter fragment is shown. The transcription start site (▾) was determined by S1 nuclease protection (data not shown). The proximal sequences of the N. benthamiana and rice (Suzuka et al., 1991) PCNA promoters are compared. The TATA boxes and ATG start codons of the two genes are shown in bold lowercase letters. The G-box (white letters), the site II (dotted boxes) elements, and the transcription start site (▴) of the rice promoter (Kosugi et al., 1995) are marked. The regions of the N. benthamiana promoter that show limited homology with site II sequences are included in the dotted boxes. Two sequences that display strong homology with the E2F binding consensus and are conserved between the two promoters are shown in bold italic type. (B) A linear diagram of the N. benthamiana promoter indicating the site II–related elements, the E2F consensus elements, the TATA box, the transcription start site (+1), and the ATG. The end points of the 5′ deletions are marked. The relative reporter activities supported by the N. benthamiana promoter fragments in transiently transfected BY-2 cells are shown below. For each experiment, the activity of the enhanced Cauliflower mosaic virus 35S (E35S) promoter was set to 100 and the activities of the PCNA:uidA constructs were standardized against it. In these experiments, an average of 722 × 10³ light units/μg protein was detected for the E35S:uidA construct, whereas an average of 915 light units/μg protein was detected for a promoterless uidA cassette. Gus, β-glucuronidase. The error bars represent two standard errors.

Figure 3.

The N. benthamiana PCNA Promoter Is Regulated Developmentally. (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 a minimum of 10 plants. The ratio of reporter activity in young versus mature leaves (y/m) is shown at right for each line. (B) Same as in (A) except that the N. benthamiana lines were transformed with the −633 PCNA:luc construct and assayed for Luc activity. (C) Soluble protein extracts from individual leaves of plants representing four −633 PCNA:luc lines were assayed for Luc activity. The Luc specific activity was plotted against leaf number. The sizes of the corresponding leaves are indicated below. The error bars indicate two standard errors.

Figure 4.

The PCNA Promoter Is Activated in Mature Leaves of TGMV-Infected Plants. (A) Soluble protein extracts were isolated 14 days after inoculation from N. benthamiana lines (left) carrying the −633 PCNA:uidA construct and assayed for GUS (Gus) activity. The activity ratios for infected versus mock-inoculated plants are shown for young (gray bars) and mature (black bars) leaves. Each set of bars represents the mean ratios from three independent experiments. The ratio values for mature leaves are given at right. The dotted line corresponds to a ratio of 1. (B) Same as in (A) except that the N. benthamiana lines were transformed with the −633 PCNA:luc construct and ratios of Luc activity are shown. The error bars indicate two standard errors.

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 μg) was digested with BglII and analyzed on DNA gel blots using a TGMV B probe. The positions of double stranded (ds) and single stranded (ss) viral DNA are marked. Middle, total RNA (30 μg) was analyzed on RNA gel blots using an N. benthamiana PCNA probe. Bottom, the same RNA gel blots were 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 4 to obtain sufficient material for analysis. The DNA and RNA samples, which were isolated 14 days after inoculation, were from different plants because 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 protein extracts from individual leaves of infected (triangles and circles) and mock-inoculated (diamonds and squares) L-39 (left) and L-12 (right) plants carrying the −633 PCNA:luc transgene were assayed for Luc activity. Profiles from two plants are shown for each treatment. The average infected/mock-inoculated ratios for leaf 9 are given for each line above the corresponding peak.

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 mutant 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 relative to probe, as indicated by the triangles at the top of the gel. The shifted product corresponding to the specific E2F-2/protein complex is marked 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 are shown. The dotted line (mt) corresponds to the mean ratio for plants transformed with the mutant construct, whereas the solid line (wt) correlates with 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 and compared with lines carrying the wild-type −633 PCNA:luc construct shown in Figure 3B. The Luc-specific activity values from young (y) and mature (m) leaves of the mutant (mt) and wild-type (wt) constructs are compared in the scattergram. The eight independent lines given in (B) are shown 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 average of two plants. (Note the different scales relative to Figure 5C.) A gradual decrease in PCNA activity with leaf number and little effect of TGMV infection also were seen for line mt-29 (our unpublished data).