Genome sequence of the plant pathogen Ralstonia solanacearum.

Ralstonia solanacearum is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. It is a model system for the dissection of molecular determinants governing pathogenicity. We present here the complete genome sequence and its analysis of strain GMI1000. The 5.8-megabase (Mb) genome is organized into two replicons: a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. Both replicons have a mosaic structure providing evidence for the acquisition of genes through horizontal gene transfer. Regions containing genetically mobile elements associated with the percentage of G+C bias may have an important function in genome evolution. The genome encodes many proteins potentially associated with a role in pathogenicity. In particular, many putative attachment factors were identified. The complete repertoire of type III secreted effector proteins can be studied. Over 40 candidates were identified. Comparison with other genomes suggests that bacterial plant pathogens and animal pathogens harbour distinct arrays of specialized type III-dependent effectors.

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

An ATM homologue from Arabidopsis thaliana: complete genomic organisation and expression analysis.

ATM is a gene mutated in the human disease ataxia telangiectasia with reported homologues in yeast, Drosophila, Xenopus and mouse. Whenever mutants are available they all indicate a role of this gene family in the cellular response to DNA damage. Here, we present the identification and molecular characterisation of the first plant homologue of ATM. The genomic locus of AtATM ( Arabidopsis thaliana homologue of ATM ) spans over 30 kb and is transcribed into a 12 kb mRNA resulting from the splicing of 79 exons. It is a single copy gene and maps to the long arm of chromosome 3. Transcription of AtATM is ubiquitous and not induced by ionising radiation. The putative protein encoded by AtATM is 3856 amino acids long and contains a phosphatidyl inositol-3 kinase-like (Pi3k-l) domain and a rad3 domain, features shared by other members of the ATM family. The AtAtm protein is highly similar to Atm, with 67 and 45% similarity in the Pi3k-l and rad3 domains respectively. Interestingly, the N-terminal portion of the protein harbours a PWWP domain, which is also present in other proteins involved in DNA metabolism such as human mismatch repair enzyme Msh6 and the mammalian de novo methyl transferases, Dnmt3a/b.

Evidence of the crucial role of sucrose synthase for sink strength using transgenic potato plants (Solanum tuberosum L.).

Sink strength of growing potato tubers is believed to be limited by sucrose metabolism and/or starch synthesis. Sucrose synthase (Susy) is most likely responsible for the entire sucrose cleavage in sink tubers, rather than invertases. To investigate the unique role of sucrose synthase with respect to sucrose metabolism and sink strength in growing potato tubers, transgenic potato plants were created expressing Susy antisense RNA corresponding to the T-type sucrose synthase isoform. Although the constitutive 35S CaMV promoter was used to drive the expression of the antisense RNA the inhibition of Susy activity was tuber-specific, indicating that independent Susy isoforms are responsible for Susy activity in different potato organs. The inhibition of Susy leads to no change in sucrose content, a strong accumulation of reducing sugars and an inhibition of starch accumulation in developing potato tubers. The increase in hexoses is paralleled by a 40-fold increase in invertase activities but no considerable changes in hexokinase activities. The reduction in starch accumulation is not due to an inhibition of the major starch biosynthetic enzymes. The changes in carbohydrate accumulation are accompanied by a decrease in total tuber dry weight and a reduction of soluble tuber proteins. The reduced protein accumulation is mainly due to a decrease in the major storage proteins patatin, the 22 kDa proteins and the proteinase inhibitors. The lowered accumulation of storage proteins is not a consequence of the availability of the free amino acid pool in potato tubers. Altogether these data are in agreement with the assumption that sucrose synthase is the major determinant of potato tuber sink strength. Contradictory to the hypothesis that the sink strength of growing potato tubers is inversely correlated with the tuber number per plant, no increase in tuber number per plant was found in Susy antisense plants.

Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs.

Sucrose synthase, an important enzyme in carbohydrate metabolism, catalyzes the reversible conversion of sucrose and UDP to UDP-glucose and fructose in vitro. To investigate the in vivo function of sucrose synthase, both the gene (Asus1) and a corresponding cDNA from roots of Arabidopsis were isolated. The Asus1 gene has homologies of 67-72% to sucrose synthase genes from other species. Histochemical GUS analysis of Arabidopsis and tobacco plants transformed with a 1.5 kb Asus1 promoter fragment transcriptionally fused to the beta-glucuronidase reporter gene showed that the Asus1 gene is expressed in the phloem of leaves, and in roots. Induction is found under conditions of limited ATP supply and increased demand for translocation of carbohydrates such as anaerobic or cold treatment. During anaerobiosis the increase in RNA level leads to increased sucrose synthase activity in roots. The expression pattern and regulation of the gene suggest that sucrose synthase is involved in the supply of energy for phloem loading in source tissues, and in metabolization of sucrose in sink tissues after unloading.

Analysis of the petunia nitrate reductase apoenzyme-encoding gene: a first step for sequence modification analysis.

In this paper, we describe the gene (nia) coding for the apoenzyme of the nitrate reductase (NR) of petunia. A full-size genomic clone was isolated from a genomic library, using the tobacco nia2 cDNA as a probe, and sequenced. The open reading frame is interrupted by three introns and encodes a protein of 909 amino acids which reveals between 92% and 68% identity to the NADH NR apoenzyme from other higher plants. Southern analyses indicated that the NR apoenzyme is encoded by a single-copy gene, although another region homologous to part of nia was also identified. The analysis of the steady-state level of nia mRNA showed that the petunia nia is regulated by the nitrogen source and is under the control of the circadian rhythm.

The steady-state level of potato sucrose synthase mRNA is dependent on wounding, anaerobiosis and sucrose concentration.

The steady-state level of potato sucrose synthase (SSase) mRNA was investigated in different plant organs and in response to certain stimuli. SSase mRNA is mainly present in developing tubers, but its presence is not restricted to this organ and the transcript is found at detectable levels in all the tissues analysed, except leaves. Wounding results in a decrease in SSase mRNA, but this can be overcome by incubation under anaerobic conditions. In leaves and petioles, an increase in sucrose concentration leads to an increase in SSase mRNA.

Molecular cloning and sequencing of sucrose synthase cDNA from potato (Solanum tuberosum L.): preliminary characterization of sucrose synthase mRNA distribution.

A cDNA library constructed from poly(A)+ RNA from potato tuber in lambda gt11 was screened for sucrose synthase using a maize sucrose synthase cDNA probe. The longest 2.7-kb insert was sequenced. An ATG located within the sequence CTGCAATGG starts an open reading frame of 805 codons. The nucleotide sequence, when compared to the maize sucrose synthase cDNA sequence, exhibits about 70% identity and the deduced amino acid sequence about 75%. Three amino acid regions are about 90% homologous and two of them could be important for the protein function. Expression studies show that transcription of the potato sucrose synthase gene is at least ten fold higher in tubers compared to photosynthetically active tissues.