Comparative analysis of Phytophthora infestans induced gene expression in potato cultivars with different levels of resistance.

Differential gene expression was analyzed after infection with Phytophthora infestans in six potato cultivars with different levels of resistance to late blight. To verify the infection of the potato leaflets, the amount of phytopathogen mRNA within the plant material was quantified by real-time quantitative PCR. The expression of 182 genes selected from two subtracted cDNA libraries was studied with cDNA array hybridization using RNA from non-infected and infected potato leaflets. Gene up- and down-regulation were clearly detectable in all cultivars 72 h post inoculation. Gene expression patterns in susceptible cultivars differed from those in potato varieties with a higher level of resistance. In general, a stronger gene induction was observed in the susceptible cultivars compared to the moderately to highly resistant potato varieties. Five genes with the highest homology to stress and/or defence-related genes were induced specifically in the susceptible cultivars. Four genes responded to pathogen attack independently of the level of resistance of the cultivar used, and three genes were repressed in infected tissue of most cultivars. Even in the absence of P. infestans infection, six genes showed higher expression levels in the somewhat resistant cultivars Bettina and Matilda. Possible reasons for the different levels of gene expression are discussed.

[Pericardial effusion in the hospital--diagnosis and therapy]. / Der Perikarderguss in der Klinik--Diagnose und Therapie.

A pericardial effusion is a relatively common disease confronting the clinician. The most frequent causes are neoplasias (lung, breast and ovarial carcinoma, leukemia and lymphoma) uremia or idiopathic. Infections (frequently virus and seldom bacteria), myocardial infarction and rheumatic disease are also common. We present the clinical picture, the differential diagnosis and the various investigations of the pericardial effusion.

Characterization and expression of the phytochrome gene family in the moss Ceratodon purpureus.

In the moss Ceratodon purpureus, phytochrome is encoded by two different genes, CpPHY1 and CpPHY2. CpPHY2 represents a conventional type phytochrome characterized by a C-terminus homologous to the catalytic domain of bacterial sensor histidine kinases, whereas CpPHY1 represents an unique phytochrome, which carries a C-terminus homologous to the catalytic domain of eukaryotic serine/threonine/tyrosine kinases. Southern blot analysis revealed that CpPHY1 is present in different Ceratodon cultivars which were collected in Germany and in Finland, implying that CpPHY1 represents a functional and active gene in Ceratodon, but CpPHY1 homologous genes could not be detected in another moss, Physcomitrella patens, or in Arabidopsis thaliana. cDNA analysis of CpPHY1 revealed the presence of a hitherto unnoticed intron within the 3' region. This results in a change of the sequence of the 11 C-terminal amino acids from KLSSHSYLTSK to FSSYQDSYPSTEELS. CpPHY1 and CpPHY2 mRNAs appear to accumulate in a light-independent manner, with CpPHY2 being much more strongly expressed than CpPHY1. Accordingly, in crude protein extracts, CpPHY2 is clearly detectable by Western blot analysis, whereas CpPHY1 is not. Light-dependent expression of CpPHY2 can be detected at the post-transcriptional level; during a 7-day period of dark adaptation, pronounced CpPHY2 accumulation occurs. Upon transfer to white light, dark-accumulated CpPHY2 is depleted within 24 h. That depletion can be completely inhibited by the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), implying that photosynthesis is strongly involved in the adjustment of phytochrome steady-state concentrations in Ceratodon. The presence of an ORF within the 5' UTR region of CpPHY2 (uORF) encoding peptide MKEFSSTSRSLMIVGIY suggests regulation at the translational level. The uORF resides on a short intron which is excised from the 5' leader in a light-dependent manner, resulting in the formation of an alternative uORF encoding peptide MEEEEDCVP.

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana.

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca(2+)-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.

Sequence similarities of phytochrome to protein kinases: implication for the structure, function and evolution of the phytochrome gene family.

Phytochrome, the best characterised plant photoreceptor, is encoded by a small multigene family within the plant kingdom. The different phytochrome types are composed of a conserved light-sensing chromophore domain of about 80 kDa and a less-conserved C-terminal domain of about 50 kDa. The C-terminus of phytochrome of the moss Ceratodon purpureus is homologous to the catalytic domain of eukaryotic serine/threonine or tyrosine protein kinases; in contrast, for all other phytochromes (conventional phytochromes) sequence similarities within the C-terminal domain to the catalytic domain of bacterial histidine kinases have been reported. We performed careful sequence comparisons of the putative catalytic domains of phytochrome with each other, with authentic serine/threonine, tyrosine and with histidine kinases. We report that conventional phytochromes exhibit structural elements of the catalytic domains of both histidine and, to a lesser extent, of serine/threonine and tyrosine kinases. The significance of these observations is discussed in the framework of the structure, function and evolution of phytochrome.

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture.

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly to moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immunoprecipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.

Biochemical evidence that phytochrome of the moss Ceratodon purpureus is a light-regulated protein kinase.

The phytochrome gene of the moss Ceratodon purpureus (phyCer) codes for a novel phytochrome polypeptide with a predicted molecular mass of 145 kDa that has a COOH-terminal domain which is homologous to the catalytic domain of eukaryotic protein kinases. In this paper we report the first biochemical evidence that in fact, as predicted from the gene sequence, PhyCer represents an active, light-regulated protein kinase. In vitro phosphorylation experiments with protonemata extracts revealed the existence of a 140 kDa protein, phosphorylated in a red/far-red light dependent manner. The binding of a polyclonal antibody directed to the protein kinase catalytic domain of PhyCer enhanced the phosphorylation of a 140 kDa band when assayed in a renaturation-auto-phosphorylation experiment with nitrocellulose bound protein. These findings strongly implicate that the phyCer gene product has protein kinase activity and is capable of auto-phosphorylation. The results of the renaturation-phosphorylation experiments were essentially the same, no matter whether protein extracts from light grown or dark adapted moss protonemata were used. Thus, phyCer expression most likely is not light regulated.

Molecular cloning of a novel phytochrome gene of the moss Ceratodon purpureus which encodes a putative light-regulated protein kinase.

The phytochrome gene (phyCer) of the moss Ceratodon purpureus was isolated and characterized. phyCer is composed of three coding exons: exon I of 2035 bp, exon II of 300 bp and exon III of 1574 bp. The deduced polypeptide encoded by exon I and II exhibits substantial sequence homology to the conserved NH2-terminal chromophore domain of known phytochromes. In contrast, the COOH-terminal polypeptide encoded by exon III shows no sequence homology to any phytochrome molecule. phyCer most likely represents a single-copy gene and is expressed in a light-independent manner. From the DNA sequence analysis it can be deduced that the PhyCer polypeptide is composed of 1303 amino acids (including the starting Met) which predicts a molecular mass for PhyCer of 145 kDa. The polypeptide encoded in exon III exhibits striking homology within the 300 carboxy-terminal amino acids to the catalytic domain of protein kinases. The carboxy terminus of PhyCer was found to be most homologous to protein-tyrosine kinases of Dictyostelium discoideum and to the products of retroviral oncogenes which belong to the Raf-Mos serine/threonine kinase family. From the hydropathy profile PhyCer appears to be a soluble protein. The predicted structure suggests that PhyCer represents a soluble light-sensor protein kinase which is linked with a cellular phosphorylating cascade.

Expression of oat phyA cDNA in the moss Ceratodon purpureus.

The possibility of transforming Ceratodon purpureus protoplasts by PEG-mediated direct DNA uptake was tested. Transformation with a plasmid carrying a kanamycin-resistance gene resulted in kanamycin-resistant colonies of C. purpureus protonemata. A full-length cDNA clone coding for oat phyA phytochrome was isolated. The clone HM4.1 which is 3.7-kb long exhibits about 99% nucleotide sequence identity to the known phytochrome clone AP3. The expression of HM4.1 in C. purpureus protonemata was tested. A construct with the 35S-promotor and the structural gene of HM4.1 was cotransformed with the plasmid containing the kanamycin-resistance. Kanamycin-resistant colonies were tested for the presence of HM4.1 sequences in a genomic Southern experiment. Two out of 19 kanamycin-resistant colonies reacted positively with a HM4.1 specific probe. The expression of phyA in the positive colonies was examined with monoclonal antibodies specific for oat phytochrome. The Western blot experiment with protein extracts of the two positive colonies grown in the dark revealed clear signals at 124-kDa which were not detected in control plants. These data demonstrate the possibility of expressing oat phyA-apoprotein in C. purpureus protonemata. The transgenic moss protonemata did not show phenotypical alterations in response to the foreign phytochrome polypeptide; it is not known at the moment if the tetrapyrole chromophore is attached to the oat polypeptide in the protonemata or not.

Phytochrome in lower plants. Detection and partial sequence of a phytochrome gene in the moss Ceratodon purpureus using the polymerase chain reaction.

The polymerase chain reaction was carried out with primers hybridizing to conserved regions of the phytochrome genes. With DNA from the moss Ceratodon purpureus 5 overlapping fragments were obtained resulting in a continuous nucleotide sequence of 1474 bp. The deduced amino acid sequence showed homology of around 60% with all known phytochrome sequences. The sequences contained a conserved chromophore attachment site. In light-grown Ceratodon protonemata the phytochrome mRNA with the size of about 4.5 kb was detected.

Nucleotide sequence of cDNA clones encoding the entire precursor polypeptides for subunits IV and V of the photosystem I reaction center from spinach.

Using lambda gt11 expression cloning and immunoscreening, cDNA-containing recombinant phages for subunits IV and V of the photosystem I reaction center were isolated, sequenced and used to probe Northern blots of polyadenylated RNA prepared from spinach seedlings. The mRNA sizes for both components are approximately 1000 and 850 nucleotides, respectively. The 968 nucleotide cDNA sequence and derived amino acid sequence for subunit IV predict a single open reading frame of 231 amino acid residues (25.4 kDa). Comparison with a 13-residue N-terminal amino acid sequence determined for subunit IV suggests a mature protein of 17.3 kDa (154 residues) and a transit sequence of 77 amino acids (8.1 kDa). The corresponding data for subunit V are 677 bp (cDNA), 167 residues for the precursor protein (18.2 kDa), 98 residues for the mature polypeptide (10.8 kDa) and 69 residues for the transit peptide (7.4 kDa). Secondary structure predictions indicate that both proteins possess greatly different transit sequences and that none is membrane-spanning.

Nodulin-100 of soybean is the subunit of sucrose synthase regulated by the availability of free heme in nodules.

A cDNA sequence encoding a nodule-specific protein, nodulin-100, was identified among the abundant transcripts of poly(A)+ RNA from soybean nodules. Purification of nodulin-100 from the soluble fraction of nodule extract yielded an abundant protein with a subunit of approximately 90 kDa having properties of sucrose synthase (UDP-glucose:D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13). Nodule sucrose synthase of soybean is a tetrameric enzyme. Antibodies raised against this protein cross-reacted with the hybrid-released translation product of nodulin-100 cDNA, suggesting that nodulin-100 is the subunit of this enzyme. This was confirmed by partial DNA sequence analysis which showed 73% sequence homology at the amino acid level with maize sucrose synthase. Nodule sucrose synthase was found to dissociate rapidly into monomers in the presence of heme, suggesting that the availability of free heme may regulate the activity of this enzyme. Sucrose synthase activity increases rapidly during nodule development and declines during senescence. A model is presented which suggests that this enzyme plays a key role in maintaining the carbon economy of the nodules and the free heme may be involved in the flow of carbon to the bacteroids. As the degradation of leghemoglobin occurs during senescence, a concomitant decrease in sucrose synthase activity is observed.

Chromophore structure of the physiologically active form (P(fr)) of phytochrome.

Chromopeptides were prepared by proteolytic digestion of phytochrome (far-red absorbing form, P(fr)) and of phycocyanin. The phycocyanobilin peptide, the chromophore of which is Z,Z,Z-configurated, was modified to the Z,Z,E isomeric chromophore. It has been demonstrated earlier that the P(fr) chromopeptide and the Z,Z,E-configurated phycocyanin chromopeptide behave similarly with regard to spectral and chromatographic properties and reactivity. We present evidence here, obtained by high-resolution (1)H NMR spectroscopy, that both the modified phycocyanobilin chromophore and the phytochrome chromophore obtained directly from P(fr) are 15E-configurated.