Nicolai Strizhov

An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics.

The GABI-Kat population of T-DNA mutagenized Arabidopsis thaliana lines with sequence-characterized insertion sites is used extensively for efficient progress in plant functional genomics. Here we provide details about the establishment of the material, demonstrate the populations functionality and discuss results from quality control studies. T-DNA insertion mutants of the accession Columbia (Col-0) were created by Agrobacterium tumefaciens-mediated transformation. To allow selection of transformed plants under greenhouse conditions, a sulfadiazine resistance marker was employed. DNA from leaves of T1 plants was extracted and used as a template for PCR-based amplification of DNA fragments spanning insertion site borders. After sequencing, the data were placed in a flanking sequence tag (FST) database describing which mutant allele was present in which line. Analysis of the distribution of T-DNA insertions revealed a clear bias towards intergenic regions. Insertion sites appeared more frequent in regions in front of the ATG and after STOP codons of predicted genes. Segregation analysis for sulfadiazine resistance showed that 62% of the transformants contain an insertion at only one genetic locus. In quality control studies with gene-specific primers in combination with T-DNA primers, 76% of insertions could be confirmed. Finally, the functionality of the GABI-Kat population was demonstrated by exemplary confirmation of several new transparent testa alleles, as well as a number of other mutants, which were identified on the basis of the FST data.

Differential expression of two P5CS genes controlling proline accumulation during salt‐stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis

Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of delta 1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2-78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3-101.3 contributes 20-40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt-induction. Mutations abi1 and axr2, affecting ABA-perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt-stress, whereas ABA-signalling functions defined by the abi2 and abi3 mutations have no effect on salt-induction of the AtP5CS genes.

Digestion of δ-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC

The present study describes the correlation between gut protease activity of lepidopteran larvae of different instars, the inactivation of Bacillus thuringiensis delta-endotoxins in crystalline and noncrystalline forms, and the reduced susceptibility of advanced larval instars of Spodoptera littoralis to the toxin. The original assembly of delta-endotoxins in a crystal structure is essential for causing efficient larval mortality. Denaturation and renaturation (D/R) of delta-endotoxin crystals increased the vulnerability of the toxin molecules to proteolysis, reduced their capability to kill neonate larvae of S. littoralis, but sustained most of their larval growth-inhibition activity. E. coli-produced CryIC delta-endotoxin applied as a fraction of inclusion bodies exerted a growth inhibition effect, similar to the molecules released from the crystals by denaturation and subsequent renaturation. Incubation of CryIC with gut juice of 1st or 2nd instar larvae, left part of the CryIC toxin intact, while the toxin was completely degraded when incubated with gut juice of 5th instar larvae. The degradation rate was consistent with the increase of protease specific activity of the gut juice during larval development. This increase in toxin degradation may account for the loss of sensitivity of 5th instar larvae to CryIC. Specific protease inhibitors such as PMSF and Leupeptin were shown to inhibit gut proteases activity in all instar larvae, while, 1,10 phenanthroline, TLCK and TPCK were effective only in young instar larvae. The differential effect of protease inhibitors on proteases obtained from different larval instars indicated that gut juice protease profiles change with larval age. The observed quantitative and qualitative differences in degradation of delta-endotoxin by larval gut proteases that occur during larval maturation may account for the difference in susceptibility to the delta-endotoxin. This finding should be taken into consideration when designing strategies for the development of transgenic crops expressing delta-endotoxins as potent insecticidal proteins.

T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants.

Oncogenes carried by the transferred DNA (T‐DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants. Other T‐DNA genes regulate the tumorous growth in ways that are not yet understood. To determine the function of T‐DNA gene 5, its coding region was expressed in Escherichia coli. Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28‐fold increase in conversion of tryptophan to indole‐3‐lactate (ILA), an auxin analogue. Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin. Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells. cis‐regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax‐1. ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole‐3‐acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins. It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

Isolation and characterization of two different cDNAs of Δ1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress

Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding Δ1-pyrroline-5-carboxylate synthase (P5CS), the first enzyme of the proline biosynthetic pathway, were isolated from a λZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

GABI-Kat SimpleSearch: a flanking sequence tag (FST) database for the identification of T-DNA insertion mutants in Arabidopsis thaliana

SUMMARY GABI-Kat SimpleSearch is a database of flanking sequence tags (FSTs) of T-DNA mutagenized Arabidopsis thaliana lines that were generated by the GABI-Kat project. Sequences flanking the T-DNA insertion sites were aligned to the A.thaliana genome sequence, annotated with information about the FST, the insertion site and the line from which the FST was derived. A web interface permits text-based as well as sequence-based searches for relevant insertions. GABI-Kat SimpleSearch aims to help biologists to quickly find T-DNA insertion mutants for their research. AVAILABILITY http://www.mpiz-koeln.mpg.de/GABI-Kat/

Mapping of the entomocidal fragment of Spodoptera -specific Bacillus thuringiensis toxin CryIC

Abstract Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI poteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.

Structure, Function and Regulation of ATP5CS Genes in Arabidopsis

ABSTRACTProline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of Δ1-pyrroline-5-carboxylate synthase (P5CS) controlling the rate-limiting step of glutamate-derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2–78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3–101.3, and is responsible for the synthesis of abundant P5CS mRNA in dividing cells. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt-treated seedlings involves an immediate early transcriptional response regulated by ABA signaling. Inhibition of protein synthesis by cycloheximide affects the induction of AtP5CS mRNA accumulation. Mutat...