Promoter analysis of
pyk
20, a gene from
Arabidopsis thaliana
Piotr S. Puzio, Jo¨rn Lausen, Petra Heinen, Florian M.W. Grundler *
Institut fu¨r Phytopathologie,Uni6ersita¨t Kiel,D-24098 Kiel, Germany
Received 15 November 1999; accepted 9 May 2000
Abstract
The gene pyk20 which has been isolated from Arabidopsis thaliana encodes a protein with a glutamine-rich domain in the C-terminal region. The transcription of this gene was shown to be induced in feeding sites of root-parasitic nematodes (Heterodera schachtii), in roots infected by a fungus-like organism (Plasmodiophora brassicae), by plant hormone treatment, and by wounding. In order to identify functional promoter regions seven different 5%and 3%pyk20 promoter (ppyk20) deletion fragments were fused to the uidA gene (gus) and transformed into A. thaliana plants. Histochemical analysis of plants containing the different ppyk20::uidAreporter constructs was performed during plant development in different plant tissues. Comparison of the promoter deletion constructs showed that the region between −277 and −1 bp is necessary to enhance the level of the GUS expression in nematode feeding sites and by plant hormone treatment. The region between −1912 and −278 is essential to provide specificity of GUS expression. Conserved regulatory elements were identified in the ppyk20 by sequence analysis. The activation pattern of ppyk20 makes it well suited to engineer resistance against nematodes and other pathogens. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:b-Glucuronidase; ppyk20; Promoter; Wounding; Plant hormone; Pathogen
www.elsevier.com/locate/plantsci
1. Introduction
The gene pyk20 was isolated from Arabidopsis
thaliana using a promoter tagging strategy. It
en-codes a protein of unidentified function with a glutamine-rich domain [1]. Although its function is still not known the gene is interesting because of the specific expression pattern. Analysis of induc-tion revealed that the pyk20 transcript rapidly accumulates in response to IAA- and kinetin-treat-ment. It is also strongly expressed in the feeding sites of sedentary nematodes [1] and root galls induced by the fungus-like organism Plasmodio
-phora brassicae [2].
Because of this expression pattern the transcrip-tional control of the gene was further analysed. As a first basis of the analysis the regulatory se-quences were used which were isolated in the promotor tagging approach. Subsequently, a cor-responding genomic clone was identified which contained the full-length regulatory sequence and the associated coding region [3,1]. Except for
in-tron sequences, the DNA sequence of the isolated cDNA clone pyk20 is identical with that of the genomic pyk20 clone, including the 5%- and 3% -un-translated regions [1].
Here, we present a detailed analysis of thepyk20 promoter (ppyk20) based on fusions of full-length and truncated promoter/reporter gene constructs (ppyk20::uidA) in transgenic A. thaliana plants. We were able to confirm the transcriptional activi-ation of the pyk20 gene by plant hormones and during the formation of nematode feeding sites. In addition we observed activation by wounding. In qualitative and quantitative reporter gene analyses
The nucleotide sequence data reported for ppyk20 is available in the EMBL, GenBank Database under the accession number AJ249204.
* Corresponding author. Tel.: +49-431-8804669; fax: + 49-431-8801583.
E-mail address: [email protected] (F.M.W. Grun-dler).
and RNA gel blots the complex expression pattern during plant development is documented. In this way several decisive promoter regions could be identified.
Due to its pathogen inducible activation pattern the promoter may be well suited to control expression of genes with anti-pathogenic pro-perties in transgenic crops.
2. Methods
2.1. Plasmid constructions
All DNA manipulations, including restriction digests, agarose gel electrophoresis, ligation and transformation toEschericha coli DH5a were car-ried out according to Sambrook et al. [4]. The genomic clone harbouring a 4027 bp fragment of
theA. thalianappyk20 [3,1] was used for
engineer-ing seven different promoter::uidA constructs (A – G). All ppyk20 fragments were produced by PCR using oligonucleotide primers including a Xho I site 3%(for the reverse primers) and aSma I site 5%
(for the forward primers) (Table 1). After PCR with the Pfu thermostable DNA polymerase (Stratagene GmbH, Heidelberg, Germany), the PCR products were restricted withXhoI and Sma
I (Promega, GmbH, Mannheim, Germany) and cloned into the binary pMOG819 vector [3] be-tween the uidA gene and the left border of the T-DNA at the corresponding sites.
2.2. A. thaliana transformation
The ppyk20::uidA constructs (A – G) in the bi-nary vector pMOG819 were mobilized from E.
coli DH5ainto theA. tumefaciensstrain MOG101 [5] by triparental mating, using the helper plasmid pRK2013 in E. coli DH5a. Roots of A. thaliana
(ecotype C-24) were transformed, regenerated and selected according to Valvekens et al. [6]. For each construct at least 25 independent transgenic A.
thaliana plants were regenerated.
2.3. Histochemical localisation of GUS acti6ity
GUS activity was histochemically detected by staining following the method as described [7] using a solution of 2 mM 5-bromo-4-chloro-3-in-dolyl-b-D-glucuronid acid (Biomol, Hamburg, Germany) in 0.1 M sodium phosphate pH 7.0, 0.1% Triton-X 100, 0.5 mM K3[Fe(CN)6], 0.5 mM
K4[Fe(CN)6], 10 mM Na2EDTA and incubated
overnight at 37°C. After staining, chlorophyll was extracted from photosynthetic tissues with 70% (v/v) ethanol. The GUS expression was detected microscopically by the distinct blue colour which results from the enzymatic cleavage of X-gluc. Of the 25 produced transgenic lines one representative was selected and analysed optically in detail.
2.4. Fluorometric determination of GUS acti6ity
Sixty individuals of the selected transgenic plants line were were grown in three Petri dishes and harvested as one sample. Proteins were ex-tracted by grinding material in extraction buffer (50 mM NaH2PO4 pH 7.0, 10 mM EDTA, 0.1%
Triton-X 100, 0.1% sarcosyl, 10 mM b -mercap-toethanol). The protein concentration was esti-mated by the Bradford method [8]. GUS enzyme activity was determined according to the proce-dure of Jefferson et al. [9] by measuring the fluorescence emitted by 4-methylumbelliferone, a GUS cleavage product from methyl-4-umbel-liferyl-b-D-glucuronide. The specific activity of GUS enzyme in the extracts was calculated as nanomoles of 4-methylumbelliferone produced per minute per mg total protein.
Table 1
Primers used for the creation of the ppyk20 constructs (A–G)
Primera Sequences (5%
3%)
2.5. Nematode and nematode infection assays
Second-stage juveniles (J2) of H. schachtii
cul-tures were harvested from in vitro stock culcul-tures on mustard (Sinapis alba) roots on 0.2 Knop medium [10]. Hatching was stimulated by soaking cysts on a 100mm nylon sieve in 3 mM ZnCl2. The
J2 juveniles were washed for four times in sterile
H20 and resuspend in 0.5% Gelrite before
inoculation.
Ten-day-old plant roots were inoculated under axenic conditions with a batch of 30 4-day-old hatched J2 juveniles of H. schachtii. The plants
were examined for the presence of GUS activity 2, 4, 7 and 12 days after inoculation.
2.6. Wounding
The leaves of the 21 days old transgenic A.
thalianaplants and wild type plants were wounded
by scissors and by pipette tips. Wounded trans-genic plants were harvested after 1 or 5 h and submitted to histochemical GUS staining as de-scribed above.
2.7. Plant hormone treatments
A. thaliana plants were grown under normal
growth and light conditions before hormonal treatment. For ABA and IAA, 20 in-vitro grown
A. thaliana plants were sprayed with 2 ml of 50
mM ABA (Sigma, Deisenhofen, Germany) or 50
mM IAA (Sigma) in water, respectively. As a control, A. thaliana plants were sprayed with 2 ml water. 8 h after initiation of these treatments, A.
thaliana plants were sampled, frozen in liquid
ni-trogen and stored at −80°C until further use.
2.8. RNA isolation and RNA gel blot analysis
Total RNA fromA.thaliana plants was isolated as described by Gurr and McPherson [11] with an additional chloroform extraction step. For North-ern hybridisation, 30 mg of total RNA was dena-tured, fractionated on a 1.5% agarose – formaldehyde gel, and blotted onto Qiabrane®
membrane (Qiagen GmbH, Hilden, Germany) ac-cording to the manufacturer’s instructions. The pyk20 cDNA clone [1] was used as a probe. To standardize the amounts of RNA loaded, 30mg of total RNA was short fractionated as described
above and then hybridized with 18s rDNA from sugar-beet [1]. The probes were labelled using ran-dom primers and [a-32P] dATP and [a-32P] dCTP (Amersham Buchler GmbH & Co. KG, Braun-schweig, Germany). Immobilized nucleic acids were prehybridized in solution containing 5% (v/v) Denhart’s solution, 5% (v/v) SSPE, 0.2% (w/v) SDS, 100 mg/ml denatured herring sperm DNA at 50°C for 6 h. The random-primed probe was added to the prehybridisation solution and incu-bated for 14 – 18 h at 50°C in a hybridisation oven. The membranes were washed once at 50°C for 20 min in 4×SSC, 0.1% SDS, then twice for 15 min in 1×SSC, 0.1% SDS, and exposed to Hyperfilm MP® (Amersham).
2.9. RT-PCR
Total RNA (ca. 2 mg) of wounded leaves were used in a RT reaction according to the manufac-turer’s instructions (Promega). 50 ng Oligo (dT)-Primer and 20 U M-MLV reverse transcriptase (Promega) in a total volume of 20 ml were incu-bated for 60 min at 42°C. Ten microliters were used in a PCR reaction containing 2 mM MgCl2,
10 mM Tris – HCl, pH 8.3, 33 mM KCl, 0.2 mM dNTPs, 50 nM of each primer and 2.5 U Taq – Polymerase (Promega). The following primers were used for the reaction: Pyk20-1: 5%– CACAG-CATGATCAGAGGA-3%; Pyk20-2: 5%
-TAC-CATTGGTGTAGGCAT-3%. The following PCR
conditions were employed: 35 times 45 s at 95°C, 45 s by 55°C, 1 min 74°C. Ten microliters of the PCR products were separated on a 1.5% agarose gel.
3. Results
3.1. Structure of the ppyk20
Using a promoter-tagging approach we previ-ously identified a nematode responsive gene called
pyk20 [1]. In order to characterise the correspond-ing promoter, ppyk20, 4027 bp 5%to the ATG start
codon sequence of thepyk20 gene were subdivided into six different regions (I – VI). Two of these promoter regions (I and II) were characterised earlier for its expression pattern [3].
regula-Fig. 1. Schematic map of ppyk20 with subcloned regions (I – VI) and putative promoter elements. TSP-transcription start point. CANNTG-box — nematode responsive box [23]. AS-1 — box of the 35SCaMV promoter [21]. Wun-box — wounding-respon-sive element [16,17]. ABA-box — ABA responwounding-respon-sive elements [18,20]. Poly-T — box a stress-responwounding-respon-sive element [13,14]. IAA-box — IAA responsive elements [12].
tory sequences homologous to that of known pro-moters (Fig. 1).The region with the highest homol-ogy to a TATA-box consensus sequence (5%-TATAA-3%) starts at the position 457 bp up-stream of the ATG and 169 bp upup-stream of the mapped transcription start (TSP) [1]. DNA se-quence analysis of the 170 bp, between the puta-tive TATA-box and the transcription start point comprised 80% pyrimidine nucleotides (CT). In addition, no obvious CAAT box was found to be located close to the TATA-box, even on the oppo-site strand. Twenty-nine sequence motifs with ho-mology to auxin-inducible elements (TCTC or TGTC) were located within the promoter [12]. A sequence with homology to the ‘poly-T box’, a stress-responsive element [13,14], was identified at position – 270 related to the TSP. Six elements similar to wounding responsive elements [15] were found in regions I, II and IV (Fig. 1). Moreover, in region V a sequence was found (5%
-TCATCTTCTT-3%) that is identical to the TCA
motif of wound- and pathogen-inducible pro-moters [16,17]. Ppyk20 also contains 2 domains (ACGTG) with similarities to the ABA responsive promoter domain of theOsemgene from rice [18]. The ACGT element was detected ten times in the ppyk20. This element was identified in many plant genes and is induced by diverse environmental and physiological factors like ABA treatment [19,20]. On the opposite strand, we found two domains
which are complementary to a TGACG domain. This motif is also included in the As1 domain of the CaMV 35S promoter and is responsible for the expression in seedlings and in the roots [21,22]. Twelve CANNTG motifs, which are known to bind proteins belonging to the superfamily of the helix-loop-helix (bHLH) transcriptional regulators [23,24] were found in promoter regions I, III and IV. The significance of all these boxes has to be established experimentally.
3.2. Histochemical localisation of ppyk20
promoter acti6ity
Seven different 5%- and 3%-deletions of ppyk20 were made by PCR. The resulting constructs are shown in Fig. 2. Each of these constructs contains several regions of the ppyk20 fused to the uidA
reporter gene. For each construct more than 25 independent transformed lines were tested for GUS expression. The variability of gus activity between individual lines was evaluated optically and no obvious differences in the quality and quantity of GUS expression could be observed between the lines. Detailed analyses were per-formed with one selected line per construct. The GUS assay was performed at 2, 5, 14 and 21 days after germination (dag).
(Fig. 3A). In seedlings of the lines A, B, C, D and E the expression was detected in the entire hypocotyl, whereas in line F and G only the upper part of the hypocotyl (near the apical meristem) showed staining. In the hypocotyls (A – E) GUS was expressed in cortical and the vascular tissues but not in epidermal cells. GUS staining also appeared in the root tip in seedlings of line A, B, C and D (Fig. 3A).
At 5 dag, plants lines A, B, C, D, E and F showed GUS expression in the vascular tissue of root. Moreover, seedlings of lines A, B, C and D expressed GUS in the root tip (Fig. 3B). Line G showed no expression in the root. In all lines, staining was observed in the vascular tissue of leaves, in mesophyll cells of cotyledons and in apical meristem (Fig. 3C). In the hypocotyl, stain-ing was quite strong in the vascular tissue.
At 14 dag, all lines stained at different intensity in the mesophyll, vascular tissue and epidermal
cell layer of leaves. Lines D and G showed only very faint expression in tissues of younger shoots and leaf primordia. In addition, we observed GUS expression in young shoot buds in plant line A (Fig. 3D). GUS was also seen in hypocotyl of different intensities in all tested constructs. In roots of the A, B, C, D, E and F plants GUS staining was detected in vascular tissue and in root tips. In plants of line A the staining was seen also in lateral root formation tissue. Plants with con-struct G expressed no GUS in the roots.
GUS expression in flowers was examined at several developmental stages, from developing buds in which corolla was not yet visible, through several intermediate stages up to mature open flowers. However, only plants containing con-structs A, B, C and D exhibited a characteristic pattern of GUS staining in the stamina between two pollen sacks (Fig. 3E), in stigmatic tissue of gynoecium (Fig. 3F) and faint in the base and top
Fig. 3. Histochemical localisation of GUS activity in transgenicA.thaliana. (A) GUS expression in 2-day-old seedlings (construct C). Bar=4 mm. (B) GUS expression in vascular tissue and in root tip 5 days after germination (dag) (construct C). Bar=100
mm. (C) GUS expression in cotyledons 5 dag (construct C). Bar=1 mm. (D) GUS expression in young shoot buds of 14-day-old plants (construct A). Bar=250 mm. (E) GUS expression between two pollen sacks (construct C). Bar=10 mm. (F) GUS expression in flowers (construct C). Bar=250mm. (G) GUS expression in abscission zone of the fully elongated siliques (construct C). Bar=1 mm. (H) GUS expression in NFS, 7 days after inoculation with infective juveniles ofH. schachtii (construct C). Bar=100mm. (I) GUS expression in leaf 5 h after wounding (construct C). Bar=1 mm.
of sepals (Fig. 3F). In addition, GUS expression was also found in the abscission zone of the fully elongated siliques (Fig. 3G).
The fluorometric GUS assay on protein extracts from whole 21 days old transgenic A. thaliana
plants is shown in Fig. 4A. The highest level of
GUS activity was observed in line C. However, no significant differences in the GUS activity could be seen in lines A – D, whereas expression levels in lines E – G were significantly reduced.
To confirm these results the transcription of the
moni-tored by Northern blot analysis. Equal amounts of total-RNA from silique, flower, stem, root, rosette, and leaf of the wild type A. thaliana were probed with thepyk20 cDNA clone. High levels of
pyk20 mRNA were found in the rosette and stem tissues (Fig. 5).
3.3. Histochemical localisation of ppyk20 acti6ity
after nematode infection
The number of GUS expressing NFS (nematode
feeding structures) was scored 2, 4, 7 and 12 days after inoculation (dai) with J2 juveniles of H.
schachtii. The highest number of GUS expressing
NFS was observed 7 dag in all lines (Table 2) (Fig. 3H). Cross sections of the 7-day-old NFS showed strong GUS staining inside the NFS. From all tested lines the highest percentage of GUS positive NFS was detected in plants harbouring construct C (Table 2).
After nematode infection, no changes in the GUS expression was observed in the histological
Fig. 4. Fluorometric GUS assay of the 21-day-oldA.thalianaplants containing the seven different ppyk20::uidAconstructs A – G. (A) Control plants after treatment with 2 ml water. (B) After treatment with 2 ml of 50mM ABA solution. (C) After treatment with 2 ml of 50mM IAA.
Fig. 5. Comparison of the expression of thepyk20 gene in different organs ofA.thalianawildtype C-24. Total RNA (5mg) from silique (lane 1), flowers (lane 2), stem (lane 3), roots (lane 4), rosette (lane 5) and leaves (lane 6) was examined by gel blot analysis. The gel blot was probed with the32P-labelled cDNA clone of thepyk20 gene. Control hybridisation to standardize the amounts
Table 2
Number of NFS showing GUS expression driven by the ppyk20 constructs
No. of GUS (−) Total number of NFS
aNumber of NFS expressed GUS.
bNumber of NFS without GUS expression.
and the fluorometric assay in whole plants. Al-though we did not quantify GUS expression in NFS, in line G the GUS staining was obviously lower than in all other lines.
3.4. Effect of wounding, IAA and ABA on ppyk20
acti6ity
In order to determine the effect of mechanical wounding lines A – G were wounded with scissors and pipette tips. Only in lines A, B and C blue staining was observed around the wounded leaf tissue 1 and 5 h after wounding (Fig. 3I). In plants containing other promoter::uidAconstructs no re-sponse was observed. In order to confirm these results, we also tested induction of pyk20 gene by RT-PCR and Northern blots. Pyk20 transcripts were detected 1 and 5 h after wounding using RT-PCR (Fig. 6) and Northern blot analysis.
We demonstrated previously that transcription of pyk20 gene is specifically up-regulated by IAA and down-regulated by ABA treatment [1]. In order to determine the ppyk20 regions responsible for IAA- and ABA-responsiveness, 21-day-old plants of lines A – G were sprayed with 50mM ABA or 50 mM IAA solutions. After 8 h in the case of ABA treatment lines A – D showed down-regula-tion of the GUS expression (Fig. 4B). Lines A – C responded to IAA treatment with a clear up-regula-tion of GUS expression, while lines D – G did not alter GUS levels compared to the control (Fig. 4C).
4. Discussion
The genepyk20 has previously been shown to be
activated in feeding sites of H. schachtii and to respond to IAA and ABA treatment [1]. Fragments of its promoter, ppyk20, (from −963 to −4027, relative to the ATG of pyk20) were shown to be able to drive GUS expression in NFS of H.
schachtii [3]. In the present study the activity of
the entire promoter was analysed during plant development and in response to nematode in-fection, wounding, and plant hormone treat-ments in order to identify specifically responses to stimuli.
4.1. Putati6e promoter regions in ppyk20
The region of 4027 bp 5%to ATG of ppyk20 may
be long compared to other A. thaliana promoters, on the other hand sequence analysis revealed no long open reading frame of a preceding gene.
Ppyk20 was subdivided into six regions (I – VI) located 5%to the ATG of the pyk20 gene. Four of these six regions (I – IV) are located 5% to the
mapped transcription start point (TSP) [1], while two regions (V and VI) are located in the 3%
direction to the mapped TSP.
The highest number of GUS expressing NFS was observed in lines containing construct C, which include regions III – VI. Lines which in addi-tion contain regions I and II (constructs A and B) showed similar number GUS expressing NFS. These findings indicate that regions I and II are not essential in enhancing the number of GUS expressing NFS. Lack of region VI led to a radical reduction of GUS positive NFS (59.4; 55.7%). This phenomenon could partially compensated by the presence of region II (73.5%), whereas the GUS level in 21-day-old plants remained low also in line E. (Fig. 4A). Regions V and VI contain in the 5%-untranslated leader (5%-UTR) of the pyk20
mRNA. Several experiments provided evidence that the secondary structure of the 5%-UTR can be involved in the regulation of gene expression [25 – 27]. In maize and tobacco the presence of the 5%
UTR increased expression of the hsp gene more than 10fold [28].
The importance of region III to drive the ex-pression of GUS in the NFS was shown in an earlier study [3]. However, only the combination of regions III, IV, V and VI gave maximum activ-ity in NFS.
Wounding and IAA treatment was shown to induce the GUS expression in plant lines A – C. Similar to nematode infection studies, in lines missing regions VI (construct F) or regions V and VI (construct G), did not respond to wounding or IAA treatment. This indicated that the 5%-UTR of
the pyk20 mRNA is involved in the regulation of gene expression. The importance of this 5%-UTR
became obvious in ABA treatment.
In conclusion, it can be stated that construct C including regions III, IV, V, and VI comprises all elements important for the observed expression patterns, while regions III and IV seem to deter-mine the specificty. The 5% UTR consisting of regions V and VI apparently is decisive of the level of transcription.
4.2. Putati6e cis-elements in ppyk20
The major question arising from our experi-ments is, what sequences within the regions III and IV may serve as a binding sites for transcrip-tion factors that regulate the observed gene
ex-pression? Comparison of both regions with the regulatory regions of other genes reveals several similarities in the occurrence of certain elements. Whether these elements plays a crucial role during the activation of pyk20 gene in NFS has to be analysed experimentally.
The sequence motif CANNTG as a core se-quence of specific boxes is often found in animal and plant organisms [29]. Recently, Escobar [23] found the motif in the Lemmi9 promoter, which is strongly induced in feeding sites of plant parasitic root-knot nematodes (Meloidogyne incognita). They were able to prove binding of the motif with nuclear proteins from these root galls in tomato. Several stress-induced promoters in dicots have a particular primary structure characterised by the presence of a poly T-box, which is separated from the TATA-box by a pyrimidine-rich region [13]. This structure is also present in the ppyk20 core promoter.
Pyk20 gene expression has been shown to be responsive to auxin treatment [1]. Considering this finding and the increasing level of auxin in NFS [16,30,31], one can expect the presence of auxin-re-sponsive elements in the ppyk20. In fact, several TGTCTC-like boxes, which are known to be re-quired for the auxin inducibility in several plant genes could be found [12].
In region V a cis-element was found (5% -TCATCTTCTT-3%), which (the same or very
closely related motifs) are also present in the pro-moter and non-coding regions of over 30 different stress-inducible plant genes [32]. These similarities in the structure indicate an involvement in re-sponse to stress stimuli.
In the regions I and IV we found a CGTCA motif, which is complementary to the as-1 box of the 35S CaMV promoter [21]. This box binds to the ASF-1 transcription factor and is important for root specific gene expression [21], However, several promoters active in feeding sites of root nematodes lack the as-1 box [33,34]. In addition, Sijmons [34] showed that after replacement or mutation of the as-1 box from the 35S CaMV promoter::uidA construct in A. thaliana the fre-quency of GUS expressing NFS increased from 5 to 20%. Therefore, the role of this box in ppyk20 has to be clarified.
binding studies will show, whether these motifs serve as binding sites.
Acknowledgements
The authors would like to thank Stephan Ohl for critical reading the manuscript. P.H. is sup-ported by ZENECA-MOGEN.
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