Mariateresa Volpicella

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.

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.

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.

Genome Walking by Next Generation Sequencing Approaches

Genome Walking (GW) comprises a number of PCR-based methods for the identification of nucleotide sequences flanking known regions. The different methods have been used for several purposes: from de novo sequencing, useful for the identification of unknown regions, to the characterization of insertion sites for viruses and transposons. In the latter cases Genome Walking methods have been recently boosted by coupling to Next Generation Sequencing technologies. This review will focus on the development of several protocols for the application of Next Generation Sequencing (NGS) technologies to GW, which have been developed in the course of analysis of insertional libraries. These analyses find broad application in protocols for functional genomics and gene therapy. Thanks to the application of NGS technologies, the original vision of GW as a procedure for walking along an unknown genome is now changing into the possibility of observing the parallel marching of hundreds of thousands of primers across the borders of inserted DNA molecules in host genomes.

Rhodobacter sphaeroides adaptation to high concentrations of cobalt ions requires energetic metabolism changes

Rhodobacter sphaeroides has for a long time been investigated for its adaptive capacities to different environmental and nutritional conditions, including presence of heavy metals, which make it a valuable model organism for understanding bacterial adaptation to metal stress conditions and future environmental applications, such as bioremediation of polluted sites. To further characterize the capability of R. sphaeroides to cope with high cobalt ion concentrations, we combined the selection of adaptive defective mutants, carried out by negative selection of transposon insertional libraries on 5 mM Co(2+) -enriched solid medium, with the analysis of growing capacities and transcriptome profiling of a selected mutant (R95). A comparative analysis of results from the mutant and wild-type strains clearly indicated that the adaptive ability of R. sphaeroides strongly relies on its ability to exploit any available energy-supplying metabolisms, being able to behave as photo- or chemotrophic microorganism. The selected R95 mutant, indeed, exhibits a severe down-expression of an ABC sugar transporter, which results nonpermissive for its growth in cobalt-enriched media under aerobic conditions. Interestingly, the defective expression of the transporter does not have dramatic effects on the growth ability of the mutant when cultivated under photosynthetic conditions.

One of the three proteinase inhibitor genes newly identified in the Brassica napus genome codes for an inhibitor of glutamyl endopeptidase

In germinating seeds, only the transcript of one gene, coding for a trypsin inhibitor, is detectable by Northern analysis. The other two genes are transcribed at basal levels detectable only by reverse transcription PCR. One of the other two genes (rti‐2) encodes a polypeptide with a glutamic residue in the P1 position, characteristic of glutamyl proteinase inhibitors. The recombinant RTI‐2 protein strongly inhibits (K i = 44 nM) a glutamyl proteinase from Streptomyces griseus.

PlantPIs – An Interactive Web Resource on Plant Protease Inhibitors

PlantPIs is a web querying system for a database collection of plant protease inhibitors data. Protease inhibitors in plants are naturally occurring proteins that inhibit the function of endogenous and exogenous proteases. In this paper the design and development of a web framework providing a clear and very flexible way of querying plant protease inhibitors data is reported. The web resource is based on a relational database, containing data of plants protease inhibitors publicly accessible, and a graphical user interface providing all the necessary browsing tools, including a data exporting function. PlantPIs contains information extracted principally from MEROPS database, filtered, annotated and compared with data stored in other protein and gene public databases, using both automated techniques and domain expert evaluations. The data are organized to allow a flexible and easy way to access stored information. The database is accessible at http://www.plantpis.ba.itb.cnr.it/.

Application of a genome walking method for the study of the spinach Lhcb1 multigene family.

We describe the application of a novel genome walking (GW) strategy for the one-shot identification of members of multigene families. The method was used to study the spinach Lhcb1 family (encoding the light harvesting complex protein Lhcb1), for which three cDNAs were known. Two additional genes and regulatory regions of the five members of the family were identified. For one of the newly detected genes, sequencing of full-length cDNA and analysis of regulatory regions by gel-shift are also reported. To our best knowledge, this is the first report on the use of a GW approach for the study of a multigene family.

Identification and characterization of protease inhibitors in Diplotaxis species

PCR analysis of the genomes of two wild Brassicaceae plants, Diplotaxis muralis and Diplotaxis tenuifolia, demonstrated the presence of several genes coding for potential protease inhibitors, classifiable within the mustard inhibitor family (MSI). This is a small family of plant protease inhibitors named after the mustard trypsin inhibitor MTI-2, the first protease inhibitor characterized in Brassicaceae. From identified sequences two recombinant inhibitors were expressed in Pichia pastoris. In comparison with MTI-2, they show a reduced activity against bovine trypsin. However, when tested against trypsin-like proteases present in the guts of Helicoverpa zea larvae, the Diplotaxis inhibitors and MTI-2 show similar activities, indicating that the usually adopted procedure of reporting activity of plant protease inhibitors against bovine trypsin may lead to wrong estimation of their effect on insect proteases. This issue is of particular relevance when planning the use of PI genes for developing insect resistant plants.