Bruno P. A. Cammue

The complexity of disease signaling in Arabidopsis

Not more than 10 years ago it was generally accepted that pathogen-inducible defense mechanisms in plants are triggered through a central signaling cascade that regulates a multicomponent defense response. Now we know that the plant defense system is regulated through a complex network of various signaling cascades.

Antimicrobial peptides from plants

Abstract Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell ...

Plant pathogenesis-related (PR) proteins: A focus on PR peptides

The novel classes of plant pathogenesis-related (PR) proteins identified during the last decade also include novel peptide families. This review specifically focuses on these pathogenesis-related peptides, including proteinase inhibitors (PR-6 family), plant defensins (PR-12 family), thionins (PR-13 family) and lipid transfer proteins (PR-14 family). For each family of PR peptides, the general features concerning occurrence, expression and possible functions of their members are described. Next, more specifically the occurrence of each PR peptide family in the model plant Arabidopsis thaliana is discussed. Single-gene studies performed on particular gene members of a PR peptide family are reported. In addition, expression data of yet undescribed gene members of that particular PR peptide family are presented by consultation of publicly available micro-array databases. Finally an update is provided on the potential role of these PR peptides in A. thaliana, with a focus on their possible involvement in plant defense.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

Pathogen-Responsive Expression of a Putative ATP-Binding Cassette Transporter Gene Conferring Resistance to the Diterpenoid Sclareol Is Regulated by Multiple Defense Signaling Pathways in Arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1 function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

A Novel Family of Small Cysteine-rich Antimicrobial Peptides from Seed of Impatiens balsamina Is Derived from a Single Precursor Protein

Four closely related peptides were isolated from seed of Impatiens balsamina and were shown to be inhibitory to the growth of a range of fungi and bacteria, while not being cytotoxic to cultured human cells. The peptides, designated Ib-AMP1, Ib-AMP2, Ib-AMP3, and Ib-AMP4, are 20 amino acids long and are the smallest plant-derived antimicrobial peptides isolated to date. The Ib-AMPs (I. balsamina antimicrobial peptides) are highly basic and contain four cysteine residues which form two intramolecular disulfide bonds. Searches of protein data bases have failed to identify any proteins with significant homology to the peptides described here. Characterization of isolated cDNAs reveals that all four peptides are encoded within a single transcript. The predicted Ib-AMP precursor protein consists of a prepeptide followed by 6 mature peptide domains, each flanked by propeptide domains ranging from 16 to 35 amino acids in length. Such a primary structure with repeated alternating basic mature peptide domains and acidic propeptide domains has, to date, not been reported in plants.

Genetic Transformation of Banana and Plantain (Musa spp.) via Particle Bombardment

We have developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the β-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

A set of modular plant transformation vectors allowing flexible insertion of up to six expression units

We have constructed a binary vector for Agrobacterium-mediated plant transformation, which has a multiple cloning site consisting of 13 hexanucleotide restriction sites, 6 octanucleotide restriction sites and 5 homing endonuclease sites. The homing endonuclease sites have the advantages to be extremely rare in natural sequences and to allow unidirectional cloning. We have also constructed a set of auxiliary vectors allowing the assembly of expression cassettes flanked by homing endonuclease sites. The expression cassettes assembled in these auxiliary vectors can be transferred into the binary vector with virtually no risk of cutting the vector within previously introduced sequences. This vector set is ideally suited for the construction of plant transformation vectors containing multiple expression cassettes and/or other elements such as matrix attachment regions. With this modular vector system, six different expression units were constructed in as many auxiliary vectors and assembled together in one plant transformation vector. The transgenic nature of Arabidopsis thaliana plants, transformed with this plant transformation vector, was assessed and the expression of each of the six genes was demonstrated.

Approaches to Minimize Variation of Transgene Expression in Plants

Genetic transformation of plants has become a widely used technology that serves multiple purposes in plant biology research. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct. This inter-transformant variation of transgene expression hampers proper evaluation of transgenes and might be most undesirable when high-throughput transgene screening is intended. The general plant transformation strategy today is to generate a sufficiently high number of transgenic plants to find some transformants with the desired level of expression. To reduce cost, labor and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to contribute to transgene expression variation including the transgene copy number, RNA silencing, transgene insertion site and the employment of certain regulatory sequences to drive transgene expression. This review provides an update on current methodologies to minimize inter-individual variation of transgene expression in nuclear transformed plants.

Interactions of antifungal plant defensins with fungal membrane components

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.