Characterization and Antifungal Properties of Wheat Nonspecific Lipid Transfer Proteins

• Published in: Molecular plant-microbe interactions Vol. 21; no. 3; pp. 346 - 360
• Main Authors: Sun, J.Y, Gaudet, D.A, Lu, Z.X, Frick, M, Puchalski, B, Laroche, A
• Format: Journal Article
• Language: English
• Published: St Paul, MN APS Press 01.03.2008; The American Phytopathological Society
• Subjects: aerial parts; amino acid sequences; Antifungal Agents - metabolism; Antifungal Agents - toxicity; antifungal properties; antimicrobial; Binding Sites; Biological and medical sciences; Carrier Proteins - genetics; Carrier Proteins - metabolism; Carrier Proteins - toxicity; Cell Membrane Permeability; Cloning, Molecular; complementary DNA; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; fungal pathogen; Fungal plant pathogens; genetic transformation; in vitro studies; lipids; molecular sequence data; multigene family; mycelium; nucleotide sequences; Phylogeny; Phytopathology. Animal pests. Plant and forest protection; Pichia pastoris; plant defense; plant pathogenic fungi; plant proteins; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Proteins - toxicity; species differences; spore germination; transport proteins; Triticum - metabolism; Triticum aestivum; wheat; antimicrobial; Monocotyledones; Toxicity; Lipids; Phylogeny; Pichia pastoris; Cereal crop; Fungi; Antifungal agent; fungal pathogen; Gene; Gramineae; Angiospermae; Immunoblotting assay; Vector; Ascomycota; Gene expression; Fatty acids; In vitro; Protein; Stress; plant defense; Complementary DNA; Sodium; Spermatophyta; multigene family; Triticum aestivum
• ISSN: 0894-0282; 1943-7706
• DOI: 10.1094/MPMI-21-3-0346

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZα and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 μM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.