Laboratoire de Pathologie Comparée, UR 1231 INRA, 30380 Saint Christol les Alès, France
Correspondence
Miguel López Ferber
lopez{at}ensam.inra.fr
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ABSTRACT |
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Present address: Department of Biochemical Genetics, Institute of Molecular Biology and Genetics, Ukrainian National Academy of Sciences, Zabolotny St 150, 03143 Kiev, Ukraine.
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INTRODUCTION |
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The baculovirus cycle starts when larvae of susceptible species ingest viral occlusion bodies (OBs). OBs are protein matrixes containing a variable number of ODVs. The alkaline conditions of the midgut lumen dissolve OBs, liberating the ODVs. Then, ODVs enter the midgut cells by attaching and fusing viral envelopes with cell membranes (Horton & Burand, 1993; Granados & Lawyer, 1981
). Horton & Burand (1993)
showed that ODV entry into the midgut cells requires the interaction of ODV envelope proteins and cell surface proteins. Once the cell is infected, BVs are produced. BVs transmit infection to other host cells after budding out from the infected cell. In a later infection stage, ODVs are formed in the infected cell nucleus and are occluded within OBs. Once the larva dies, OBs are released in the immediate environment (Federici, 1997
).
The two types of virion differ mainly in the composition of their envelopes (Braunagel & Summers, 1994). Thus, the different behaviour and function of ODVs and BVs can be related to their specific proteins.
To date, only two ODV proteins have been demonstrated to be essential for ODV infection of midgut cells: P74 (Faulkner et al., 1997; Kuzio et al., 1989
) and PIF (for per os infectivity factor; Kikhno et al., 2002
). Both PIF and P74 have been detected only in the ODV envelope. Moreover, the pif and p74 genes are conserved within the sequenced baculovirus genomes. Recently, Pijlman et al. (2003)
have shown that pif-2, another conserved baculovirus gene, is essential for the primary infection, although its product has not yet been localized.
The pif and p74 genes share another interesting feature: their products are present in extremely low quantities during infection. Regulation of gene expression in baculoviruses appears to take place mainly at the transcriptional level (Lu & Miller, 1997) although examples of translation regulation have been described (Chang & Blissard, 1997
). Promoter recognition is considered one of the key transcription processes involved in baculovirus gene expression. So far, the promoters of four baculovirus late genes gp64, vp39, polh and p10 have been studied in detail (Garrity et al., 1997
; Thiem & Miller, 1990
; Ooi et al., 1989
; Weyer & Possee, 1988
, 1989
). These genes are expressed relatively well. In contrast, our knowledge on how weak gene expression is regulated in the baculoviruses is limited.
We are interested in understanding how PIF levels are kept low and the implications of these low levels for PIF function. In this study, we present a transcriptional analysis of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) pif gene region. In addition, we have analysed the SpliNPV pif gene promoter by studying the effect of deletions and mutations of the pif promoter region on oral infectivity. We show that pif gene transcription seems to be regulated by a surprisingly short promoter sequence. Moreover, a complex transcription unit in the pif region may also regulate SpliNPV pif expression.
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METHODS |
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Sequence signposting and plasmid construction.
Sequence positions are numbered in accordance with their location in the SpliNPV-M2 NotID fragment (accession no. AF527603; Fig. 1A). ORF names are given as in Kikhno et al. (2002)
.
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RNA isolation.
Total RNA was isolated from mock-infected and SpliNPV-M2-infected Sl52 cells at 2, 4, 8, 12, 24, 48 and 72 h post-infection (p.i.). The time defined as zero h p.i. corresponds to the moment the virus inoculum was added. Total RNA was also isolated from fat body tissue obtained at 120 h p.i. from mock-infected and SpliNPV-M2 orally infected S. littoralis larvae. Cells and tissue were homogenized in TRIzol (Invitrogen). Then, total RNA was isolated following the manufacturer's instructions. Messenger RNA (mRNA) was isolated from total RNA obtained from fat body using the PolyATract mRNA isolation system IV (Promega).
RT-PCR analysis and 3' RACE.
Temporal analysis of pif, polh and egt transcriptions was done using RT-PCR. Total RNA from mock-infected and SpliNPV-M2 infected cells was digested with DNase (Promega) and tested for the presence of DNA using PCR. RT-PCR was performed using the Access RT-PCR System kit (Promega) with 1 µg DNA-free total RNA as template per time-point. Briefly, first cDNA synthesis from pif, polh and egt transcripts was carried out using AMV reverse transcriptase (Promega) and the Spli119_1, Pol1 and oligo(dT) primers (Table 1), respectively for each mRNA. The cDNAs obtained were amplified by PCR using the Spli119RT2, Pol2 and SpliegtRT primers (for pif, polh and egt, respectively, Table 1
).
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Northern blot analysis.
Total and messenger RNA samples were denatured in a formaldehyde/formamide buffer and then electrophoresed on a 1 % agarose, 2·2 M formaldehyde gel as described previously by Brown & Mackey (1997). Gels were stained with ethidium bromide before transfer to confirm equivalent sample quantities. RNA was transferred to a charged nylon membrane (Roche) and then fixed by UV irradiation.
DNA probes were prepared by labelling DNA fragments containing the ORF pif sequence with [-32P]dCTP and using the Megaprime DNA labelling system as specified by the manufacturer (Amersham).
Three riboprobes were synthesized using [-32P]CTP and T7 or SP6 polymerases (MAXIscript kit, Ambion; see Fig. 1A
for schema) to characterize transcription in the pif region. The pif riboprobe had 567 nt complementary to the 3' region of the pif gene (positions 5267 to 5834 bp). The ORF8 riboprobe was complementary to a sequence contained in ORF8 (206 nt, positions 7175 to 7381 bp). The ORF5-i riboprobe overlapped 264 nt of the 5' region of ORF5 sequence (positions 4271 to 4595 bp).
Hybridizations were carried out using ULTRAhyb buffer (Ambion) following the r manufacturer's instructions and then visualized with the Phosphoimager system (Amersham). Fragment sizes were determined by staining the molecular mass marker (Promega RNA marker) with methylene blue after transfer onto the membrane.
Primer extension and RNase protection analysis.
5' end mapping of the pif, ORF6 and polh messengers was carried out using primer extension. Fifty µg of total RNA from infected larvae was annealed to the corresponding 32P-labelled primer (see Table 1) and processed following the manufacturer's instructions (Primer Extension System-AMV, Promega). RNase protection for mapping the 5' extremity of the pif mRNA was performed using RPA III (Ambion) as stated by the manufacturer. The 32P-labelled 328 nt probe used overlaps 239 nt of the NotID sequence and contains 120 nt complementary to the 5' end of the pif gene. Primer extension and RNase protection products were electrophoresed and then visualized using the Phosphoimager system (Amersham). Sizes of primer extension products were determined by comparison with a sequence ladder obtained with the Spli119PE2 primer (Table 1
) and p282.1 (Fig. 2
A) following the manufacturer's instructions (T7 Sequencing Kit, Pharmacia).
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Six clones were built to study the effect of deletions on the pif promoter region. Deletions, which progressively removed nucleotides upstream from the first ATG of pif, were made in a NotID fragment containing a 315 bp promoter region and the ORF pif. The resulting fragments were cloned in pGEM-T Easy Vector (Promega). The obtained NotID-derived promoter regions had lengths of 315, 66, 28, 13, 8 and 0 bp (plasmids p282.1, p283.1, p281.1, p297.5, p298.22 and p260.1, respectively; Fig. 2A). All promoter regions except those from p298.22 and p260.1 contained a putative late promoter motif (TTAAG) situated from -13 to -8 bp from the initial ATG of the pif gene (Fig. 2B
).
The effect of single mutations on this TTAAG motif was studied. The TTAAG motif in p281.1 was mutated to obtain TCAAG and TTACG in p300.1 and p301.1 (Fig. 2B), respectively, using the Quick-Change Site-Directed Mutagenesis kit (Stratagene). The mutated sequences were verified by sequencing (Genome Express). Single mutations were again introduced in the mutated promoter motifs recovering the original motif sequence and obtaining plasmids p302.1 and p303.1.
Sl52 cells were co-transfected using DOTAP (Roche) with SpliNPV-4 DNA and one of the plasmids described above. The same quantities of plasmid and viral DNA were used in each co-transfection. SpliNPV
4 contains a 4·4 kb deletion that encompasses the pif gene. This virus replicates in cell culture but it is not infectious per os unless PIF is supplemented during OB formation (Kikhno et al., 2002
). Sl52 cells were also transfected with a quantity of SpliNPV-M2 DNA similar to that of SpliNPV-
4 DNA. The OBs formed in the co-transfected cells were collected and given to samples of 24 S. littoralis neonates using the droplet feeding technique (Hughes & Wood, 1981
). Mortality was recorded until all larvae had either died or pupated. All experiments were repeated at least three times.
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RESULTS |
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A single RT-PCR product at the expected size, approximately 1 kbp, was obtained using two internal primers to the pif gene sequence (Fig. 3A). This product was observed from 12 h p.i. The transcription analysis of polh showed a similar pattern to that found for pif transcription whereas that of egt showed amplification from 4 h p.i. (data not shown).
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Northern blot analysis of the pif region
The hybridization profile obtained from cell culture at 72 h p.i. with the pif riboprobe was similar to that obtained using total RNA (Fig. 4, profile pif) and mRNA (data not shown) isolated from infected larvae at 120 h p.i. Thus, the transcription pattern of the pif region was similar both in Sl52 cell culture and in S. littoralis larvae at late times in the infection.
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The 1·9 kb band obtained with the pif riboprobe (e in Fig. 4) had no corresponding band on the other two profiles. This polyadenylated 1·9 kb messenger is confined to the region between ORF8 and ORF5. In this region there are only two ORFs: pif and ORF6. Based on the transcript size and the location of the pif probe, it is likely that the 1·9 kb mRNA originated from transcription at the pif promoter.
The 0·5 kb transcript detected with the pif riboprobe (f in Fig. 4) had no corresponding transcript in the ORF8 and ORF5-i profiles. This transcript might originate from a spliced pif transcript. However, computer-assisted analysis did not detect any splicing motif in the pif region. An alternative explanation may be that this transcript resulted from ORF6 transcription. In this case, the 5' UTR of this putative ORF6 transcript should overlap the 3' region of the pif gene. In support of this idea, two late promoter motifs were found within the 3' region of the pif gene at positions 5307 and 5403 bp (Fig. 1
A). Hence, this 0·5 kb transcript could originate from ORF6 transcription.
The three profiles seem to have in common the three larger bands (a, b and c in Fig. 4). These three messengers probably start upstream from ORF8 and continue downstream from ORF5. They were not further characterized.
The profiles obtained with pif and ORF8 riboprobes had the 3·5 kb band in common (d in Fig. 4). The size and the presence of this band in both profiles suggest a late mRNA encompassing from ORF9-lef1 or ORF8, to ORF6. In the ORF8 promoter region, there is a late promoter motif at position 8382 bp (Fig. 1A
). The 3·5 kb transcript might start at this motif.
The relative intensity of bands a, b, c and d differed in the three hybridization profiles (Fig. 4) and this difference in intensity was again observed when the Northern blots were repeated using the same conditions (data not shown). The difference in intensity might be due to different hybridization efficiencies between the riboprobes.
The 1·6 kb band observed in the ORF8 profile (Fig. 4) had no equivalents in the pif or ORF5-i profiles. The size and situation of this transcript agree with those of a putative ORF8 messenger.
The presence of the 2·6 and 1·6 kb bands in the ORF8-probe profile (Fig. 4) is surprising. The 1·6 kb band was not present when mRNA was used as matrix (data not shown) and, thus, this band was considered an artefact. The size and localization of the 2·6 kb band could not be explained either by sequence analysis or by the Northern blot analysis. A transcript initiated within the ORF5 and encompassing the ORF4-egt might explain this band.
Extremity mapping of the 1.9 and 0.5 kb transcripts
The 1·9 and 0·5 kb transcripts in the pif-probe profile (e and f in Fig. 4) may be pif and ORF6 messengers, respectively. To verify this hypothesis, the transcription initiation sites of these transcripts were determined by primer extension. In addition, primer extension was used to compare the amount of the 1·9 kb mRNA with that of the polh messenger.
Several primer extension products (Fig. 5A) were obtained with a primer complementary to the pif sequence (Spli119PE2, Table 1
). Only the 87 nt extension product was mapped to a consensus transcription initiation motif, TTAAG (Lu & Miller, 1997
). RNase protection was performed to interpret the primer-extension results. Two protected fragments of about 134 and 239 nt were obtained (Fig. 5B
) with a probe overlapping from 6724 to 6962 bp in the NotID fragment. The 134 nt fragment corresponded to the transcription initiation site of the 87 nt primer extension product. This transcription initiation site was mapped to the first T of the TTAAG motif (position 6853, Fig. 1A
). The 328 nt riboprobe used in the RNase protection assay overlaps 239 bp of the NotID sequence. Thus, the 239 nt fragment probably resulted from annealing of the riboprobe to the multiple messengers encompassing pif. These findings confirm the first T of the TTAAG motif situated 8 bp upstream from pif ORF as the transcription initiation site of the 1·9 kb transcript. The presence of primer extension products that are longer than 87 nt may be explained by incomplete extensions originated from the polycistronic messengers encompassing the pif gene. Secondary structures within these messengers may cause the premature termination of the extensions, resulting in the observed products.
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In the primer extension for the ORF6 transcript, a single extension product of approximately 196 nt was obtained with a primer complementary to the ORF6 sequence (Spli119.3_2, Table 1). This product corresponds to a transcription initiation site situated close upstream from a consensus late promoter motif (position 5403, Fig. 1A
) and within the 3' region of pif ORF.
A 3' RACE analysis was carried out to determine the 3' ends of the 1·9 and the 0·5 kb messengers. The Spli119RT primer (53615337 in NotID, Table 1) was designed to anneal to the cDNA obtained from the reverse transcription of both the 1·9 and 0·5 kb mRNAs. A single PCR product was obtained with the Spli119RT and the oligo(dT) primers. The 3' end of this product was located 15 bp downstream from a T-rich region and the combined poly(A) signal and stop triplet (AATAAA) of the ORF6. T-rich regions have been shown to be essential for baculovirus transcription termination (Jin & Guarino, 2000
). Therefore, the transcription termination of the 1·9 and 0·5 kb transcripts would take place close downstream from the ORF6 stop site. The 3' end of the 3·5 kb messenger detected in the Northern blot analysis (d in Fig. 4
) is likely to be situated in this region also. Taken together, the data from the extremity mapping are able to predict transcript lengths which are very similar to the 1·9 and 0·5 kb lengths obtained in the Northern blot analysis. Moreover, these data strongly support the interpretation given in Fig. 1(B)
.
Analysis of the pif gene promoter by functional complementation assays
A series of plasmids which contained the pif ORF under the control of modified promoter regions was constructed. The modifications consisted of progressive deletions of the pif promoter region or point mutations in the late promoter motif situated 8 bp upstream from the pif ORF. The effect of these modifications on the pif promoter activity was determined using co-transfections of each plasmid and SpliNPV-4 DNA, followed by bioassays using the OBs obtained from the co-transfected cells. Expression of the pif gene from the modified promoter would result in complementation of the pif-defective virus, SpliNPV
4, as indicated by death of larvae as a result of virus infection. When virus-induced mortality was observed in the complementation assays, it ranged from approximately 60 to 90 %. Fig. 2(A)
summarizes the results obtained for the deletion analysis. The mortality found with the OBs produced in the SpliNPV-M2 transfections was roughly similar to that observed in the complementation assays (80100 %).
A promoter region of 13 bp was sufficient to trigger virus-induced mortality. A late promoter motif, TTAAG, is situated at the 5' end of this promoter region. No mortality was detected when this motif was deleted. TAAG motifs are essential elements of late baculovirus promoters (Lu & Miller, 1997). The TTAAG motif encompassed by the 28 bp promoter region of p281.1 was modified to give TCAAG and TTACG in the plasmids p300.1 and p301.1 (Fig. 2B
), respectively. No mortality was detected when p300.1 and p301.1 were used in the complementation assays. On the other hand, p302.1 and p303.1, which had reconstructed promoter motifs, gave mortality levels similar to those obtained with p281.1. These results confirm the mutations in the TTAAG motif as the reason for the absence of mortality observed in the complementation assays with p300.1 and p301. Taken together, these findings demonstrated that the TTAAG motif was essential for pif promoter activity in the assay conditions. Furthermore, a 13 bp region containing the TTAAG motif in its 5' end was sufficient to allow production of orally infectious ODVs in the assay.
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DISCUSSION |
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RT-PCR analysis detected pif transcripts from 12 h p.i. in infected Sl52 cells. Comparison with the temporal patterns obtained for egt and polh transcription suggests that pif is a late gene. This temporal classification was further supported by Northern blot analysis. Moreover, late expression is in accordance with a gene encoding an ODV structural protein (Funk et al., 1997).
Transcription analysis of the pif region revealed a complex transcription unit during the late phase of infection. In this transcription unit, six messengers encompassed the pif coding region most of them entirely (Fig. 1B). Three of these mRNAs (transcripts d, e and f in Fig. 1B
) probably share their 3' termini, using the transcription-termination sequence located close downstream from the ORF6. The extremities of the other mRNAs encompassing pif (transcripts a, b and c in Fig. 1B
) have not been rigorously mapped, but there is evidence that their 5' ends contain ORFs other than pif.
From the set of messengers overlapping pif, a 1·9 kb mRNA was found to originate from the pif promoter. Taking the data of the transcription and the promoter analysis together, the 1·9 kb mRNA was considered the genuine pif transcript. This mRNA was not the most abundant in the set. The amount of the 1·9 kb mRNA synthesized was extremely low, about 300 times less than the amount of polh mRNA. The low expression of the 1·9 kb mRNA correlates well with the low amount of the PIF protein observed by Kikhno et al. (2002). PIF translation from other polycistronic mRNAs cannot be ruled out. Nevertheless, the intercistronic distance considered necessary for efficient translation reinitiation ranges from approximately 50 to 150 nt (Kozak, 1987
) whereas the intercistronic distance between pif ORF and the upstream ORF is much longer (244 nt).
The 1·9 kb mRNA initiated 13 nt upstream from the initial ATG of pif and completely encoded ORF6. No messenger was found to exclusively encode pif. Other baculovirus genes are encoded by polycistronic messengers (Carstens et al., 1993; Blissard & Rohrmann, 1989
). Okano et al. (2001)
detected pif messengers in Bombyx mori NPV-infected B. mori cells by EST analysis. Our analysis of these ESTs showed that the pif transcripts detected also overlapped an ORF6 homologue, suggesting that pif may be transcribed as a bicistronic mRNA in other baculoviruses.
In this work the SpliNPV ORF6 has been found to be encoded by a late 0·5 kb transcript which has been characterized. No consistent homology with proteins of known function was detected for the SpliNPV ORF6. ORF6 homologues were observed in the same position and orientation with respect to the pif gene in most of the sequenced lepidopteran NPVs (see Fig. 6). In addition, SpliNPV-fgf homologues were also associated to the pif+ORF6 cluster in the group II NPVs. The gene arrangement found in the region downstream from the pif gene is surprising as gene order is poorly conserved among the baculoviruses (Herniou et al., 2003
). Two hypotheses have been suggested for the conservation of gene clusters in the baculoviruses: a regulation of transcription in the cluster region or a role of the cluster sequences in virus replication. Since we have not observed a need for the presence of the ORF6 and fgf sequences during replication of the virus (Kikhno et al., 2002
), this cluster might have a role in transcription regulation.
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Another regulatory mechanism proposed for the sets of overlapping transcripts is interference with gene expression by antisense transcription (Friesen & Miller, 1987; Ooi & Miller, 1990
). In the present study, antisense transcripts to the pif mRNA have not been detected, ruling out this mode of regulation.
The SpliNPV pif promoter is likely to be a weak promoter as indicated by the transcriptional analysis. To date, no baculovirus weak promoter has been studied in detail. Here, we present an analysis of the SpliNPV pif promoter using a functional complementation assay. This assay allowed testing for PIF expression levels with a biological significance; in other words, PIF levels giving orally infectious ODVs. However, this assay did not allow quantitative interpretation of the results.
The 13 bp sequence situated upstream of the pif coding region was required and sufficient for the production of orally infectious virions in the assay conditions. The 5' end of this sequence starts with a consensus late promoter motif (TTAAG). Previous work by Morris & Miller (1994) demonstrated that the 8 bp situated upstream from the TAAG motif have a significant influence on Autographa californica MNPV (AcMNPV) vp39 promoter activity. Similar results were obtained for the AcMNPV polh promoter (Rankin et al., 1988
). In the case of the SpliNPV pif promoter, the region upstream from the TTAAG was not necessary for mortality of larva. Thus, the promoter core of SpliNPV pif gene seems to be shorter than those of the studied late genes. Recently, Pijlman et al. (2003)
suggested that an 18 bp promoter region of an S. exigua MNPV (SeMNPV) pif homologue might be sufficient for the production of orally infectious virions. Hence, a short promoter core may be conserved in other pif homologues. Such a short promoter core may be explained by the quantity of PIF required by the virus. If the virus requires only small amounts of PIF to produce orally infectious ODVs, then, the pif promoter may be reduced to a minimum to obtain just a basal expression. Alternatively, high PIF levels might somehow interfere in the virus cycle and, thus, PIF expression would have to be down-regulated.
Our results show that the TTAAG motif situated 8 bp upstream from the pif initial ATG is essential to obtain PIF expression sufficient for detection by bioassay. These findings are in accordance with an essential role of the TAAG motif in late promoter activity as demonstrated for other baculovirus late genes (Garrity et al., 1997; Morris & Miller, 1994
; Ooi et al., 1989
; Rankin et al., 1988
; Thiem & Miller, 1990
; Todd et al., 1995
). TAAG motifs are present in the pif promoter regions of all the sequenced baculoviruses. Curiously, we have observed unconventional late promoter motifs (CTAAG) in the pif promoter regions of SeMNPV and Helicoverpa armigera NPV-G4. It would be interesting to determine if such motifs are active as late promoter motifs. No other appreciable similarity was observed among the different pif promoter regions. Thus, the TAAG motif seems to be the only element which is conserved in the pif promoter region as is the case in other conserved late genes of baculoviruses (Theilmann et al., 1996
). In fact, Garrity et al. (1997)
have shown that Orgyia pseudotsugata MNPV gp64 promoters provided only basal expression levels in AcMNPV-infected cells. These authors suggested that sequence recognition by late transcription factors is virus-specific.
pif gene expression seems to be regulated mainly at the transcriptional level, as it is thought to be the case for the majority of the baculovirus genes (Lu & Miller, 1997). However, regulation of pif gene expression at other levels cannot be excluded. Recent work (López-Ferber et al., 2003
) has shown that a significant proportion of genotypes (19·5 %) within a wild S. frugiperda NPV population do not contain the pif gene, among others. However, the p74 gene is present in all these genotypes. It is tempting to speculate that pif expression is also regulated at the population level by reducing the number of pif gene copies within a population.
To summarize, transcription of the SpliNPV pif gene seems to be regulated by a very short promoter. In addition, pif gene transcription may also be regulated by a set of multiple overlapping messengers. Our results suggest that tight regulation of pif expression takes place at the transcriptional level. Further research on the precise role of PIF is required to understand the reasons for this regulation.
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ACKNOWLEDGEMENTS |
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Received 2 September 2003;
accepted 22 October 2003.