Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.     

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.     

Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus Giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea

The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea. In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T(2) homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea.     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.     

Prokaryotic expression of a constitutively expressed <Emphasis Type="Italic">Tephrosia villosa</Emphasis> defensin and its potent antifungal activity

Plant defensins are small, highly stable, cysteine-rich antimicrobial peptides produced by the plants for inhibiting a broad-spectrum of microbial pathogens. Some of the well-characterized plant defensins exhibit potent antifungal activity on certain pathogenic fungal species only. We characterized a defensin, TvD1 from a weedy leguminous herb, Tephrosia villosa. The open reading frame of the cDNA was 228 bp, which codes for a peptide with 75 amino acids. Expression analyses indicated that this defensin is expressed constitutively in T. villosa with leaf, stem, root, and seed showing almost similar levels of high expression. The recombinant peptide (rTvD1), expressed in the Escherichia coli expression system, exhibited potent in vitro antifungal activity against several filamentous soil-borne fungal pathogens. The purified peptide also showed significant inhibition of root elongation in Arabidopsis seedlings, subsequently affecting the extension of growing root hairs indicating that it has the potential to disturb the plant growth and development.     

Transgenic expression of gallerimycin, a novel antifungal insect defensin from the greater wax moth Galleria mellonella, confers resistance to pathogenic fungi in tobacco

A cDNA encoding gallerimycin, a novel antifungal peptide from the greater wax moth Galleria mellonella, was isolated from a cDNA library of genes expressed during innate immune response in the caterpillars. Upon ectopic expression of gallerimycin in tobacco, using Agrobacterium tumefaciens as a vector, gallerimycin conferred resistance to the fungal pathogens Erysiphe cichoracearum and Sclerotinia minor. Quantification of gallerimycin mRNA in transgenic tobacco by real-time PCR confirmed transgenic expression under control of the inducible mannopine synthase promoter. Leaf sap and intercellular washing fluid from transgenic tobacco inhibited in vitro germination and growth of the fungal pathogens, demonstrating that gallerimycin is secreted into intercellular spaces. The feasibility of the use of gallerimycin to counteract fungal diseases in crop plants is discussed.     

A protein from the mold Aspergillus giganteus is a potent inhibitor of fungal plant pathogens.

A purified preparation of antifungal protein (AFP) from Aspergillus giganteus exhibited potent antifungal activity against the phytopathogenic fungi Magnaporthe grisea and Fusarium moniliforme, as well as the oomycete pathogen Phytophthora infestans. Under conditions of total inhibition of fungal growth, no toxicity of AFP toward rice protoplasts was observed. Additionally, application of AFP on rice plants completely inhibited M. grisea growth. These results are discussed in relation to the potential of the afp gene to enhance crop protection against fungal pathogens in transgenic plants.     

Functional expression and subcellular localization of the Nectria haematococca Mak1 phytoalexin detoxification enzyme in transgenic tobacco

Medicarpin and maackiain are antifungal pterocarpan phytoalexins produced by many legumes, and are thought to be important components of the defense response of these legumes to certain fungal pathogens. The Mak1 gene from the fungal pathogen Nectria haematococca encodes an FAD-dependent mono-oxygenase, known to specifically hydroxylate the phytoalexins medicarpin and maackiain, converting them to less fungitoxic derivatives. Two binary vector constructs were made containing the coding regions from two fungal clones, a Mak1 cDNA (intronless) and a genomic (including three fungal introns) clone, regulated by an enhanced cauliflower mosaic virus 35S promoter. The constructs were introduced into tobacco to check for expression of active fungal enzyme in plant cells and for splicing of fungal introns. Leaves of tobacco plants transformed with the Mak1 cDNA construct readily metabolized infiltrated medicarpin to 1a-hydroxymedicarpin, indicating high levels of active enzyme. RT-PCR analysis of tobacco plants transformed with the Mak1 genomic construct indicated no processing of Mak1 introns, and no Mak1 activity was detected in these plants. When using plants containing the Mak1 cDNA construct, immunolocalization with a Mak1-specific antibody together with cellular fractionation indicated that Mak1 protein accumulated in the plant cytoplasm, associated with endoplasmic reticulum membranes; medicarpin biosynthetic enzymes have been localized to the same subcellular region. The Mak1 cDNA construct is therefore suitable for use in studies to selectively eliminate medicarpin accumulation to assess the relative importance of medicarpin in the antifungal defense mechanisms of alfalfa and other legumes.     

Functional characterization of pathogen-responsive protein AtDabb1 with an antifungal activity from Arabidopsis thaliana

A plant antifungal protein was purified from Arabidopsis thaliana leaves by using a typical procedure consisting of anion exchange chromatography and high-performance liquid chromatography. We determined the amino acid sequence of the purified protein using MALDI-TOF/MS analysis, and found that the sequence matched that of a hypothetical Arabidopsis protein in GenBank (accession number NP_175547). We designated the protein as AtDabb1. After the cDNA encoding the AtDabb1 gene was cloned from an Arabidopsis leaf cDNA library, the recombinant protein was expressed in Escherichia coli and found to significantly inhibit cell growth of various pathogenic fungal strains. mRNA expression of the AtDabb1 gene was induced by pathogen-related signaling molecules including salicylic acid and jasmonic acid. These results suggest that AtDabb1 may contribute to the induced plant defense mechanism against diverse pathogenic fungi.     

Molecular Cloning of the cDNA Encoding an Antifungal Protein in Gastrodia elata Bl.

Antifungal protein is the main inhibitor of fungal infection in the secondary corm of Gastrodia elata B1. was isolated and purified antifungal protein (GAFP) from the plant. Its molecular weight was about 14 kD. Polyclonal antibody against GAFP was produced. In vitro test, this antifungal protein inhibited the growth of some fungi in some crop including Gibberella zeae. cDNA was synthesized from poly (A) mRNA purified from G. elata. The cDNA was ligated into phage vector λgtll DNA and packaged in vitro and the phages were propagated on E. coli Y1090 and a λgtll expression library was constructed. A cDNA clone encoding for antifungal protein was screened out by immunoscreening of the library using the protein as a probe. The λDNA containing insert was digested by Eco RI after isolated and purified recombinants λDNA, the insert was obtained. The cDNA was 300 bp in length. The authors had isolated the cDNA clone encoding antifungal protein from G. elata.     

Antifungal activity and enhancement of plant growth by Bacillus cereus grown on shellfish chitin wastes

Bacillus cereus QQ308 produced antifungal hydrolytic enzymes, comprising chitinase, chitosanase and protease, when grown in a medium containing shrimp and crab shell powder (SCSP) produced from marine waste. The growth of the plant-pathogenic fungi Fusarium oxysporum, Fusarium solani, and Pythium ultimum were considerably affected by the presence of the QQ308 culture supernatant. The supernatant inhibited spore germination and germ tube elongation of F. oxysporum, F. solani, and P. ultimum. The increase in the growth time of the fungal culture was associated with a gradual decrease in inhibition. Besides antifungal activity, QQ308 enhanced growth of Chinese cabbage. These characteristics were unique among known strains of B. cereus. To our knowledge, this is the first report on the antifungal and Chinese cabbage growth enhancing compounds produced by B. cereus.     

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins

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 pPICZalpha 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 microM. 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.     

Piperazinyl quinolines as chemosensitizers to increase fluconazole susceptibility of Candida albicans clinical isolates

The effectiveness of the potent antifungal drug fluconazole is being compromised by the rise of drug-resistant fungal pathogens. While inhibition of Hsp90 or calcineurin can reverse drug resistance in Candida, such inhibitors also impair the homologous human host protein and fungal-selective chemosensitizers remain rare. The MLPCN library was screened to identify compounds that selectively reverse fluconazole resistance in a Candida albicans clinical isolate, while having no antifungal activity when administered as a single agent. A piperazinyl quinoline was identified as a new small-molecule probe (ML189) satisfying these criteria.     

Psc-AFP, an antifungal protein with trypsin inhibitor activity from Psoralea corylifolia seeds

An antifungal protein designated as Psc-AFP, with an apparent molecular mass of 18kDa, was isolated from a traditional Chinese herb, malaytea scurfpea (Psoralea corylifolia L.). The isolation procedure entailed extraction, cation exchange chromatography on CM FF, gel filtration chromatography on Superdex 75 and reversed-phase high performance liquid chromatography on SOURCE 5RPC column. Automated Edman degradation determined the partial N-terminal sequence of Psc-AFP to be NH2-EWEPVQNGGSSYYMVPRIWA, which displayed homology with plant trypsin inhibitors. The protease inhibitor activity of Psc-AFP was then confirmed by the inhibition on trypsin. Psc-AFP at 10 microM inhibited the mycelial growth of Alternari brassicae, Aspergillus niger, Fusarium oxysporum and Rhizoctonia cerealis, suggesting that Psc-AFP has a role in the defense against pathogens.     

A potent antifungal protein from Helianthus annuus flowersis a trypsin inhibitor

A 16-kDa protein was isolated from Helianthus annuus flowers by its ability to inhibit the germination of fungal spores. This protein, SAP16, displays an associated activity of trypsin inhibitor and was further purified to apparent homogeneity by affinity chromatography on trypsin-agarose. SAP16 causes the complete inhibition of Sclerotinia sclerotiorum ascospores germination at a concentration of 5 μg·mL^–1 (0.31 μM) and a clear reduction of mycelial growth at lower concentrations, indicating a strong antifungal potency against this natural pathogen of sunflower. Our data suggest that the antifungal ability of SAP16 would not be the result of the inhibition of a fungal protease. This study contributes to the characterization of the emerging family of antifungal proteins with an associated activity of trypsin inhibition and emphasizes their role in plant resistance against fungal attack.     

Bacisubin, an antifungal protein with ribonuclease and hemagglutinating activities from Bacillus subtilis strain B-916

An antifungal protein, with a molecular mass of 41.9 kDa, and designated as bacisubin, was isolated from a culture of Bacillus subtilis strain B-916. The isolation procedure consisted of ion exchange chromatography on DEAE-Sepharose Fast Flow, and fast protein liquid chromatography on Phenyl Sepharose 6 Fast Flow and hydroxyapatite columns. The protein was adsorbed on all three chromatographic media. Bacisubin exhibited inhibitory activity on mycelial growth in Magnaporthe grisease, Sclerotinia sclerotiorum, Rhizoctonia solani, Alternaria oleracea, A. brassicae and Botrytis cinerea. The IC50 values of its antifungal activity toward the last four fungal species were 4.01 microM, 0.087 microM, 0.055 microM and 2.74 microM, respectively. Bacisubin demonstrated neither protease activity, nor protease inhibitory activity. However, it manifested ribonuclease and hemagglutinating activities.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

Characterization and biocontrol ability of fusion chitinase in Escherichia coli carrying chitinase cDNA from Trichothecium roseum

The antifungal mechanism of mycoparasitic fungi involves fungal cell wall degrading enzymes such as chitinases. Trichothecium roseum is an important mycoparasitic fungus with significant antifungal ability, but studies on chitinases of T. roseum were poor. Here, we report a novel chitinase cDNA isolated from T. roseum by PCR amplification based on conserved chitinase sequences. Southern blot analysis suggested that a single copy of the gene exists in the genome of T. roseum. The deduced open reading frame of 1,143 nucleotides encodes a protein of 380 amino acids with a calculated molecular weight of 41.6 kDa. The fusion chitinase expressed in Escherichia coli has been purified by single-step chromatography. It has a pI of pH 5.4 and expresses a thermal stability, but is insensitive to pH in a broad pH range. According to expectation, E. coli efficiently yielded a high amount of active chitinase. Remarkably, the fusion chitinase offered high antifungal activity.