Fungal Glucosylceramide-Specific Camelid Single Domain Antibodies Are Characterized by Broad Spectrum Antifungal Activity.

Chemical crop protection is widely used to control plant diseases. However, the adverse effects of pesticide use on human health and environment, resistance development and the impact of regulatory requirements on the crop protection market urges the agrochemical industry to explore innovative and alternative approaches. In that context, we demonstrate here the potential of camelid single domain antibodies (VHHs) generated against fungal glucosylceramides (fGlcCer), important pathogenicity factors. To this end, llamas were immunized with purified fGlcCer and a mixture of mycelium and spores of the fungus Botrytis cinerea, one of the most important plant pathogenic fungi. The llama immune repertoire was subsequently cloned in a phage display vector to generate a library with a diversity of at least 108 different clones. This library was incubated with fGlcCer to identify phages that bind to fGlcCer, and VHHs that specifically bound fGlcCer but not mammalian or plant-derived GlcCer were selected. They were shown to inhibit the growth of B. cinerea in vitro, with VHH 41D01 having the highest antifungal activity. Moreover, VHH 41D01 could reduce disease symptoms induced by B. cinerea when sprayed on tomato leaves. Based on all these data, anti-fGlcCer VHHs show the potential to be used as an alternative approach to combat fungal plant diseases.

Arabidopsis thaliana plant defensin AtPDF1.1 is involved in the plant response to biotic stress.

*Previously, it was shown that the Arabidopsis thaliana plant defensins AtPDF1.1 (At1g75830) and AtPDF1.2a (At5g44420) exert in vitro antimicrobial properties and that their corresponding genes are expressed in seeds and induced in leaves upon pathogen attack, respectively. *In this study, the expression profile of both AtPDF1.1 and AtPDF1.2a is analysed in wild-type plants upon different stress-related treatments and the effect of modulation of their expression in transgenic plants is examined in both host and nonhost resistance. *AtPDF1.1, which was originally considered to be seed-specific, is demonstrated to be locally induced in leaves upon fungal attack and exhibits an expression profile distinct from that of AtPDF1.2a, a gene frequently used as marker for the ethylene/jasmonate-mediated signaling pathway. Transgenic plants with modulated AtPDF1.1 or AtPDF1.2a gene expression show no altered phenotype upon Botrytis cinerea inoculation. However, constitutive overexpression of AtPDF1.1 in A. thaliana leads to a reduction in symptoms caused by the nonhost Cercospora beticola causing non-spreading spots on A. thaliana leaves. *These results indicate that AtPDF1.1 and AtPDF1.2a clearly differ regarding their expression profile and functionality in planta. It emphasizes the additional level of complexity and fine-tuning within the highly redundant plant defensin genes in A. thaliana.

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.

Use of a PTGS-MAR expression system for efficient in planta production of bioactive Arabidopsis thaliana plant defensins.

Plant defensins, exhibiting various levels of inhibitory activity against fungal pathogens, are potent candidates for pharmaceutical or agricultural antimycotics. Study of the plant defensins from the model plant Arabidopsis thaliana requires the purification of these peptides. However, heterologous production of defensins for large-scale in vitro bioactivity assays is often experienced as a major problem. In this study we describe the transgenic expression of a previously identified seed-specific and a so far uncharacterized plant defensin gene in their host A. thaliana using a formerly developed plant expression system. Therefore, both genes were cloned in a matrix attachment region (MAR) based plant transformation vector and expressed in post-transcriptional gene silencing (PTGS) impaired A. thaliana plants. The peptides were purified to homogeneity and were correctly processed, as confirmed by mass spectrometry analysis. Finally, they were assessed for their in vitro antifungal activity and mode of antifungal action. Our results indicate that the PTGS-MAR expression system can be applied to obtain significant amounts of bioactive, rightly processed plant peptides from leaves of first generation transgenic plants.

The influence of matrix attachment regions on transgene expression in Arabidopsis thaliana wild type and gene silencing mutants.

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a beta-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chi-MARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chi-MARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.

The role of ethylene in host-pathogen interactions.

The phytohormone ethylene is a principal modulator in many aspects of plant life, including various mechanisms by which plants react to pathogen attack. Induced ethylene biosynthesis and subsequent intracellular signaling through a single conserved pathway have been well characterized. This leads to a cascade of transcription factors consisting of primary EIN3-like regulators and downstream ERF-like transcription factors. The latter control the expression of various effector genes involved in various aspects of systemic induced defense responses. Moreover, at this level significant cross-talk occurs with other defense response pathways controlled by salicylic acid and jasmonate, eventually resulting in a differentiated disease response.

Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions.

Basic and applied research involving transgenic plants often requires consistent high-level expression of transgenes. However, high inter-transformant variability of transgene expression caused by various phenomena, including gene silencing, is frequently observed. Here, we show that stable, high-level transgene expression is obtained using Arabidopsis thaliana post-transcriptional gene silencing (PTGS) sgs2 and sgs3 mutants. In populations of first generation (T1) A. thaliana plants transformed with a beta-glucuronidase (GUS) gene (uidA) driven by the 35S cauliflower mosaic virus promoter (p35S), the incidence of highly expressing transformants shifted from 20% in wild type background to 100% in sgs2 and sgs3 backgrounds. Likewise, when sgs2 mutants were transformed with a cyclin-dependent kinase inhibitor 6 gene under control of p35S, all transformants showed a clear phenotype typified by serrated leaves, whereas such phenotype was only observed in about one of five wild type transformants. p35S-driven uidA expression remained high and steady in T2 sgs2 and sgs3 transformants, in marked contrast to the variable expression patterns observed in wild type T2 populations. We further show that T-DNA constructs flanked by matrix attachment regions of the chicken lysozyme gene (chiMARs) cause a boost in GUS activity by fivefold in sgs2 and 12-fold in sgs3 plants, reaching up to 10% of the total soluble proteins, whereas no such boost is observed in the wild type background. MAR-based plant transformation vectors used in a PTGS mutant background might be of high value for efficient high-throughput screening of transgene-based phenotypes as well as for obtaining extremely high transgene expression in plants.

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.

Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins.

We developed a method for expression in Arabidopsis of a transgene encoding a cleavable chimeric polyprotein. The polyprotein precursor consists of a leader peptide and two different antimicrobial proteins (AMPs), DmAMP1 originating from Dahlia merckii seeds and RsAFP2 originating from Raphanus sativus seeds, which are linked by an intervening sequence ("linker peptide") originating from a natural polyprotein occurring in seed of Impatiens balsamina. The chimeric polyprotein was found to be cleaved in transgenic Arabidopsis plants and the individual AMPs were secreted into the extracellular space. Both AMPs were found to exert antifungal activity in vitro. It is surprising that the amount of AMPs produced in plants transformed with some of the polyprotein transgene constructs was significantly higher compared with the amount in plants transformed with a transgene encoding a single AMP, indicating that the polyprotein expression strategy may be a way to boost expression levels of small proteins.