Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae
Introduction
Plant infectious diseases caused by phytopathogenic fungi produce an important reduction of plant yield across many crop species, and require the continued use of fungicides for control. However, there are several problems associated to their use, such as the limited spectrum of action, the emergence of resistant fungal isolates and the negative impact on human health and the environment [1]. To substitute for or complement current fungicides, more pathogen-specific compounds with lower unspecific toxicity could be identified by screening for molecules which specifically block infection-related processes rather than inhibit vegetative hyphal growth. The identification and application of these target-oriented molecules has been proposed as an environmentally friendly alternative to avoid some drawbacks of current fungicides [1], [2].
Natural and synthetic antimicrobial peptides (AMPs) have emerged as promising compounds to use in plant protection as alternative to fungicides [3], [4], [5]. The diversity available through combinatorial chemistry provides a powerful tool to identify novel peptides targeted to specific crop protection problems. “Non-defined” synthetic combinatorial libraries (SCL) enable the high-throughput (HTP) assay of millions of compounds without sequence restrictions in relatively short time [6]. In most cases, the primary peptide screens are cell-based assays of in vitro growth inhibition, which enables the HTP formats required for the analysis of compound libraries [7], [8]. Indeed, the library used in this work was previously assayed against the phytopathogen Penicillium digitatum, identifying several peptides called PAFs [7]. However, few examples of peptide screens targeted to specific pathogen functions have been applied to plant pathogens [9], [10], [11].
This work focuses on the screening of a peptide library to identify inhibitors of an essential infection process of the rice blast fungus Magnaporthe oryzae, a devastating rice pathogen [12]. M. oryzae passes through a set of well-defined developmental stages to successfully invade and infect host plants: adhesion to plant surface and germination, appressorium differentiation and maturation, penetration and invasive growth. Multiple external signals and several transduction pathways, including Pmk1 and Mps1 mitogen-activated protein kinases (MAPK) and cAMP-dependent signaling pathways, regulate this developmental process [12], [13], [14], [15], [16].
We have previously characterized the heptapeptidePAF104 as an inhibitor of appressorium formation in M. oryzae that affects the expression of genes coding for components of the Pmk1 pathway [17]. PAF104 was identified as a derivative of PAF26, and both peptides resulted from combinatorial screens to find inhibitors of fungal growth [7], [18]. The purpose of this study was the improvement of the properties of PAF104 through combinatorial chemistry by screening of a “non-defined” hexapeptide library specifically against appressorium formation. As a result of the screening, the improved peptides MgAPI24, MgAPI40 and MgAPI47 have been identified.
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Microbial strains and growth conditions
M. oryzae isolate PR9 (CIRAD collection, Montpellier, France) was used for the in vitro appressorium inhibition assays. The fungus was grown on complete medium at 25 °C under 16 h/8 h (light/dark) photoperiod. The composition of complete medium (CM) is minimal medium (MM, 6 g/l NaNO3, 0.52 g/l KCl, 0.52 g/l MgSO4·7H2O, 1.52 g of KH2PO4, 0.001% thiamine, 0.1% trace elements supplemented with 10 g/l d-glucose) supplemented with 2 g/l peptone, 1 g/l yeast extract, and 1 g/l casamino acids. Conidia were
Screening of a PS-SCL for inhibition of appressorium formation in M. oryzae
The PS-SCL represent 47 millions of peptides and consisted of six sub-libraries as previously described [7], [20]. Each sub-library is composed of 19 complex mixtures, each one specifying one of the natural amino acids (except cysteine) at each one of the peptide position. The assay of the peptide mixtures provides information about the most important amino acid residue at each position of the peptide. In this work, the PS-SCL was tested for the in vitro inhibition of appressorium formation in
Acknowledgments
We thank Dr. M. Coca and Dr. B. San Segundo for their helpful comments during the course of this investigation. M. oryzae PR9 isolate was kindly provided by Dr. Tharreau from CIRAD, Montpellier, France. This work was supported by grant AGL2010-16847 and BIO2012-34381 from the Ministerio de Ciencia e Innovación (MICINN, Spain).
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