Plant Science 157 2000 245 – 255
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 pyk
20
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 pyk
20
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::uidA reporter 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.comlocateplantsci
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 pyk
20
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 pyk
20
is identical with that of the genomic pyk
20
clone, including the 5- and 3-un- translated regions [1].
Here, we present a detailed analysis of the pyk
20
promoter ppyk20 based on fusions of full-length and truncated promoterreporter gene constructs
ppyk20::uidA in transgenic A. thaliana plants. We were able to confirm the transcriptional activi-
ation of the pyk
20
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
:
fgrundlerphytomed.uni-kiel.de F.M.W. Grun- dler.
0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 0 0 0 0 2 8 7 - 9
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
