Raffaele Gallerani

PLANT-PIs: a database for plant protease inhibitors and their genes

PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs.

Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.

The mustard trypsin inhibitor 2 affects the fertility of Spodoptera littoralis larvae fed on transgenic plants

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco lines have been evaluated by feeding the lepidopteran Spodoptera littoralis throughout its larval life. Specific conditions were selected to study the long-term effects of feeding larvae on transgenic plants expressing the inhibitor at various levels. The data obtained led to the establishment of three relevant parameters to be considered during the experimentation: (i) the PI content of the plant lines to be used; (ii) the developmental stage of larvae sensitive to that PI content; (iii) the ratio of MTI-2/proteases sufficient to inhibit gut proteases. The experimental data obtained from feeding S. littoralis larvae using these conditions led to two main results. First, when L2 S. littoralis larvae were fed on high MTI-2 expressing tobacco plants, no effects on larval development were detected but there was a significantly reduced fertility. When the same larvae were fed on low expressing MTI-2 tobacco plants, only a less marked lowering of fertility was observed. Second, after the first generation, no differences in protease activity were observed in insects derived from larvae fed on high or low MTI-2 expressing tobacco lines, suggesting that genetic traits observed in previous studies were not inherited.

Genome walking in eukaryotes

Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T‐DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris.

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C‐terminal amino acids of the precursor form on the inhibitor activity, the C‐terminal precursor and the mature protein were both expressed. A third His‐tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40–160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine β‐trypsin compared to the precursor and His‐tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.

The gene coding for the mustard trypsin inhibitor-2 is discontinuous and wound-inducible

The gene coding for the mustard trypsin inhibitor‐2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5′ flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G‐☐ and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves.

Cystatins, Serpins and other Families of Protease Inhibitors in Plants

Plant protease inhibitors (PIs) are generally small proteins present in high concentrations in storage tissues (tubers and seeds), and to a lower level in leaves. Even if most of them are active against serine and cysteine proteases, PIs active against aspartic proteases and carboxypeptidases have also been identified. Inhibitors of serine proteases are further classifiable in several families on the basis of their structural features. They comprise the families known as Bowman-Birk, Kunitz, Potato I and Potato II, which are the subject of review articles included in this special issue. In the present article we aim to give an overview of other families of plant PIs, active either against serine proteases or other class of proteases, describing their distribution, activity and main structural characteristics.

Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction

Abstract. Transcription analysis of a mustard (Sinapis alba L.) serine proteinase inhibitor gene revealed identical 5′ termini of mRNAs synthesized during seed maturation and chemical or wounding induction. Polyadenylation of mRNAs on multiple or single sites differentiated gene expression, increasing the availability of stable mRNAs during seed maturation compared with chemical and wounding induction. Expression of the β-glucuronidase (GUS)-encoding region of the UidA reporter gene, detected under the control of deleted segments of the region flanking on the 5′ side the mti-2 gene, identified a stretch of about 520 bp essential for gene expression. The presence in this region of two ABRE motifs is relevant for plant response to gene induction. Expression of GUS was detectable under different induction stimuli in several organs such as seedlings and leaves and was active to varying extents in the vascular tissues and meristem.

DNA dependent RNA polymerase from rat liver mitochondria

The purification of mitochondrial DNA-dependent RNA polymerase from rat liver and other organisms appears to be particularly difficult since the enzyme is readily inactivated by a wide range of extraction procedures [ 1,2] . We recently succeeded in solubilizing the enzyme from rat liver mitochondria using the detergent deoxycholate [3]. Stabilization of the enzyme activity after extraction was achieved with large concentrations of dithiothreitol and glycerol [3]. Mitochondrial rat liver RNA polymerase solubilized either by sonication or by deoxycholate ap pears to belong to the bacterial class of RNA polymerases since is inhibited by rifampicin and insensitive to cw-amanitin [3,4] . A mitochondrial RNA polymerase has recently been purified from sonicates of Neurospora mitochondria by repeated glycerol gradient centrifugation [5]. Solubilization and partial purification of mitochondrial enzyme from wild type yeast has been reported by Tsai et al. [6] . In this paper we report the first attempt to purify a mitochondrial RNA polymerase from animal cells. Partial purification of the enzyme was achieved by a procedure including lysis at high ionic strength, DNase treatment, ammonium sulphate fractionation and DEAE-Sephadex A-25 column chromatography.

The structure of the cytochrome oxidase subunit II gene and its use as a new character in the construction of the phylogenetic tree of Angiospermae

Abstract With the aim of correlating the structure of the intron present in the mitochondrial gene coding for subunit II of the cytochrome oxidase complex ( coxII gene) with some aspects of Angiosperm evolution, we analysed the organization of the coxII gene from 30 plants belonging to 18 different orders. In all the monocotyledons tested, the gene contains an intron with a composite structure. Among dicot plants, 16 contain a split gene, 5 a continuous gene. The presence or the absence of intervening sequences in the genes of dicot plants does not seem to be a casual event, on the contrary it seems to be a feature of specific phylogenetic lines. Our results suggest that this approach, extended to a wider number of plant species, could make a contribution to the construction of the phylogenetic tree of Angiosperms.