A protein phosphatase was cloned that interacts with a serine-threonine receptor-like kinase, RLK5, from Arabidopsis thaliana. The phosphatase, designated KAPP (kinase-associated protein phosphatase), is composed of three domains: an amino-terminal signal anchor, a kinase interaction (KI) domain, and a type 2C protein phosphatase catalytic region. Association of RLK5 with the KI domain is dependent on phosphorylation of RLK5 and can be abolished by dephosphorylation. KAPP may function as a signaling component in a pathway involving RLK5.
Post-translational modification of nuclear proteins with poly(ADP-ribose) modulates chromatin structure and may be required for DNA processing events such as replication, repair and transcription. The polymer-catabolizing enzyme, poly(ADP-ribose) glycohydrolase, is crucial for the regulation of polymer metabolism and the reversibility of the protein modification. Previous reports have shown that glycohydrolase digests poly(ADP-ribose) via an exoglycosidic mechanism progressing from the protein-distal end of the polymer. Using two independent approaches, we investigated the possibility that poly(ADP-ribose) glycohydrolase also engages in endoglycosidic cleavage of polymers. First, partial glycohydrolase digestion of protein-bound poly(ADP-ribose) led to the production of protein-free oligomers of ADP-ribose. Second, partial glycohydrolase digestion of a fixed number of protein-free poly(ADP-ribose) polymers resulted in a transient increase in the absolute number of polymers while polymer size continuously decreased. Furthermore, endoglycosidic activity produced linear polymers from branched polymers although branch points themselves were not a preferential target of cleavage. From these data, we propose a mechanism whereby poly(ADPribose) glycohydrolase degrades polymers in three distinct phases ; (a) endoglycosidic cleavage, (b) endoglycosidic cleavage plus exoglycosidic, processive degradation, (c) exoglycosidic, distributive degradation.Poly(ADP-ribose) glycohydrolase is the only nuclear enzyme known to hydrolyze a-( 1"-2') glycosidic linkages. The natural substrate for this enzyme is poly(ADP-ribose), which is formed by the nuclear enzyme poly(ADP-ribose) polymerase and can contain branches. The polymers are covalently bound to various acceptor proteins, all of which bind to nucleic acids. Synthesis and degradation of these polymers is thought to regulate protein-DNA interactions during DNA processes involving single-strand and double-strand breaks (reviewed in [l -31). Polymer catabolism by poly(ADP-ribose) glycohydrolase is stimulated in mammalian cells exposed to increasing doses of DNA-damaging agents [4] and is reduced in a murine lymphoma cell line deficient in a specific repair function [5].The molecular mechanism by which poly(ADP-ribose) glycohydrolase degrades polymers has been investigated by several groups in the past. Most of these studies indicated that the glycohydrolase hydrolyzes ribose-ribose linkages from the protein-distal end of the polymer, i.e. exoglycosidically [6-lo]. The results from Ikejima and Gill, however, suggested the possibility of endoglycosidic attack [ 111. We present an in-depth study in which purified poly(ADP-ribose) glycohydrolase was incubated with protein-bound or protein-free ADP-ribose polymers, and the reaction products were directly analyzed on high-resolution DNA sequencing gels and by high-performance liquid chromatography. The results show that poly(ADP-ribose) glycohydrolase produces endoglycosidic reaction products in that (a) glycohydrolase activity releases...
In contrast to the well-defined tetrameric structure of animal and yeast casein kinase II (CKII), plant CKII is found in two forms: a monomeric form and an oligomeric form whose subunit composition is not well defined. The Arabidopsis homologs of the catalytic subunit alpha (CKA1) and the regulatory subunit beta (CKB1) of CKII were expressed in Escherichia coli to examine their ability to form complexes, the effect of CKB1 on the catalytic activity, and the relationship of the recombinant enzymes to those isolated from plant material. Both subunits were found mainly in the inclusion body fraction in the bacterial expression strains, and they were solubilized and renatured with the recovery of catalytic (CKA1) and stimulatory (CKB1) activities. The combination of purified CKA1 and CKB1 proteins resulted in up to 100-fold stimulation of casein kinase activity compared with the CKA1 activity alone, showing that CKB1 has biochemical properties similar to those of the beta subunit from animals. CKA1 and CKB1 spontaneously assembled into a tetrameric complex, CKA1(2)CKB12, which had properties very similar to those of the oligomeric CKII form isolated from broccoli. However, the properties of the catalytic subunit CKA1 alone differed from those of the broccoli monomeric form of CKII-like activity. Phosphorylation of transcription factor GBF1 with the reconstituted CKA1(2)CKB1(2) enzyme resulted in stimulation of its DNA binding activity and retardation of the protein-DNA complex; these results are identical to those obtained previously with isolated nuclear CKII from broccoli.
Summary Suppression Subtractive Hybridization (SSH) was applied in a search for genes induced during the compatible interaction between Phytophthora infestans and potato. Using potato leaves that had been treated with benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) as the control tissue, a low redundancy library with a relatively low frequency of the classic plant Pathogenesis-Related (PR) genes was generated. 288 of the clones were screened for induced sequences using Inverse Northern analysis (hybridizing the arrayed clones with radiolabelled cDNA populations). Of the 75 clones that were detectable by this method, 43 appeared to be induced. Eleven of these clones were then analysed by total RNA blot analysis, and elevation of transcript levels during P. infestans infection was confirmed for 10 of them. Some of the cDNAs analysed by RNA blot analysis have homology to genes already known to be induced during infection, e.g. to beta-1,3-glucanase. Another group of cDNAs have homology to enzymes involved in detoxification: gamma-glutamylcysteine synthetase, cytochrome P450, glutathione S-transferase and an MRP-type ABC transporter. Other infection induced cDNAs encode putative proteins that have not previously been reported to be induced by infection: e.g. the ER-located chaperone BiP, and a homologue of Aspergillus nidulans SudD, which was isolated as a suppressor of a mutation in chromosome disjunction. The differential library therefore presents the opportunity to analyse the metabolic changes occurring during infection, and the disease process itself in more detail.
The eukaryotic origin recognition complex (ORC) is made up of six subunits and functions in nuclear DNA replication, chromatin structure, and gene silencing in both fungi and metazoans. We demonstrate that disruption of a plant ORC subunit homolog, AtORC2 of Arabidopsis (Arabidopsis thaliana), causes a zygotic lethal mutant phenotype (orc2). Seeds of orc2 abort early, typically producing embryos with up to eight cells. Nuclear division in the endosperm is arrested at an earlier developmental stage: only approximately four nuclei are detected in orc2 endosperm. The endosperm nuclei in orc2 are dramatically enlarged, a phenotype that is most similar to class B titan mutants, which include mutants in structural maintenance of chromosomes (SMC) cohesins. The highest levels of ORC2 gene expression were found in preglobular embryos, coinciding with the stage at which homozygous orc2 mutant seeds arrest. The homologs of the other five Arabidopsis ORC subunits are also expressed at this developmental stage. The orc2 mutant phenotype is partly suppressed by a mutation in the Polycomb group gene MEDEA. In double mutants between orc2 and medea (mea), orc2 homozygotes arrest later with a phenotype intermediate between those of mea and orc2 single mutants. Either alterations in chromatin structure or the release of cell cycle checkpoints by the mea mutation may allow more cell and nuclear divisions to occur in orc2 homozygous seeds.
To find out more about the interaction between potato and Phytophthora infestans at the molecular level, we screened for genes induced early after infection using mRNA differential display. Among the twenty cDNA clones recovered in the screen, two were found to represent plant genes whose transcript levels increased during infection of intact plants. These two genes differed strikingly in their response to wounding. Stprx2, a putative peroxidase, responded slowly and transiently to wounding, and its expression pattern was similar to that of gst1, a well-described pathogen-induced gene of potato. The second gene, StNAC, was induced rapidly and strongly after wounding but not systemically. Transcript levels reached a maximum after around 1 h and returned to basal levels after ca. 24 h. StNAC has strong similarity to the ATAF subfamily of NAC domain proteins, a large family of putative transcriptional activators. Arabidopsis ATAF1 and ATAF2 were also shown to be induced by wounding. This implies that the ATAF genes are not merely structurally similar but also share a conserved role in stress responses.
Casein kinase II is thought to play an essential role in the control of cell division and differentiation in all eukaryotes. Through complementation of a defective casein kinase II catalytic subunit gene from Saccharomyces cerevisiae, we isolated an Arabidopsis thaliana casein kinase II regulatory subunit homologue, CKB1. A second regulatory subunit was identified by low-stringency hybridization with CKB1. Casein kinase II from S. cerevisiae is composed of two catalytic (alpha) and two regulatory (beta) subunits. Simultaneous disruption of the genes for the alpha and alpha' subunits, CKA1 and CKA2, respectively, is lethal. Strain YDH8 has disruptions of CKA1 and CKA2; its viability depends on a temperature-sensitive allele of CKA2, cka2-8, carried on a centromeric plasmid. We screened an A. thaliana cDNA library, whose inserts are under the control of the galactose-inducible GAL10 promoter, for cDNAs which enabled YDH8 cells to grow at the restrictive temperature. One cDNA, CKB1, was isolated by this screen which had homology to cDNAs of casein kinase II beta subunits. A second cDNA, CKB2, was isolated by hybridization and was also able to suppress the YDH8 mutant phenotype. The proteins encoded by CKB1 and CKB2 are 80% identical. The carboxy-terminal two thirds of both proteins is ca. 54% identical to the regulatory beta subunits of casein kinase II from other species. The amino termini are unrelated to any other known proteins. CKB1 and CKB2 lack the conserved autophosphorylation site characteristic of animal beta subunits, but have potential casein kinase II phosphorylation sites in the same region. Suppression of the cka1 delta cka2-8 mutant phenotype occurs by interaction of CKB1 with the defective, cka2-8-encoded, catalytic subunit. Cells with disruptions in CKA1 and CKA2 are not rescued by expression of CKB1.
SUMMARY Suppression Subtractive Hybridization (SSH) was used to search for genes of Phytophthora infestans that are induced during the infection of potato. To avoid having to distinguish the genes of the pathogen from the plant genes involved in defence responses and to isolate the genes involved in the early stages of interaction, mycelium of P. infestans was induced by contact with the host plant and then separated from the plant tissue. A differential cDNA library was generated by SSH that compared such induced mycelium with mycelium incubated in water. The expression of about 100 cDNA fragments from this differential cDNA library was analysed by hybridization of the arrayed PCR products with mRNA from control and induced mycelium. Twenty per cent of them showed increased transcript levels in mycelium within the first 24 h after exposure to a potato leaf. For six of these cDNA clones the elevated expression in response to the potato leaf could be proven by RNA gel blot analysis. Five of these cDNA clones have predicted amino acid sequence homologies to entries in the databases, including an amino acid transporter, a sucrose transporter, a spliceosome-associated factor, an ABC transporter, and a cell division control protein. We showed that the genes corresponding to these six cDNA clones are differentially regulated during their life. Reliable gene expression analysis of Phytophthora in infected leaf tissue is not possible until c. 48 h post-infection, but for two of the genes we identified, induction during in planta growth was detectable by RNA gel blot analysis. Therefore the SSH library that we have created provides a basis for the identification of P. infestans genes that are up-regulated during the interaction with the plant, which could be important for pathogenicity.
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