Isolation, purification and characterization of an antifungal molecule produced by Bacillus licheniformis BC98, and its effect on phytopathogen Magnaporthe grisea.

AIMS: Isolation of bacterial antagonist for use in the biological control of phytopathogenic fungi like rice blast fungus, Magnaporthe grisea, and to further purify and characterize the antifungal molecule produced by the antagonist. METHODS AND RESULTS: Bacterial antagonist exhibiting highest antifungal activity against the rice blast fungus M. grisea was isolated from soil and identified as Bacillus licheniformis BC98. Besides M. grisea, the isolate also inhibited the growth of other phytopathogens such as Curvularia lunata and Rhizoctonia bataticola. Biologically active fractions were isolated from the culture filtrate and further fractionated by reverse-phase high-performance liquid chromatography (HPLC) enabling detailed structural characterization of a component of molecular mass 1035 Da. The active peptide was identified as surfactin after 500 MHz (1)H NMR analysis. Microscopic analysis of the effect of the antagonist on M. grisea revealed bulbous hyphae showing patchy and vacuolated cytoplasm when observed under the electron microscope. CONCLUSIONS: The antagonistic lipopeptide secreted by B. licheniformis BC98 and identified as surfactin, induced morphological changes in M. grisea, inhibiting its further growth, and thus exhibiting fungicidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The antagonist inhibits germination of M. grisea, a potent rice phytopathogen, and therefore appears to be a potential candidate for control of rice blast disease.

Purification and characterization of laccase from the rice blast fungus, Magnaporthe grisea.

A 70-kDa extracellular laccase was purified from the rice blast fungus Magnaporthe grisea using gel filtration and ion exchange chromatography The procedure provided 282-fold purification with a specific enzyme activity of 225.91 U mg(-1) and a yield of 11.92%. The enzyme oxidized a wide range of substrates. The highest level of oxidation was detected with syringaldazine as the substrate. Using syringaldazine as the substrate, the enzyme exhibited a pH optimum of 6 and temperature optimum of 30 degrees C, and its K(m) was 0.118 mM. The enzyme was strongly inhibited by Cu-chelating agents.

Chainia penicillin V acylase: strain characteristics, enzyme immobilization, and kinetic studies.

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5-8.0, and for the (NH4)2 SO4 fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55 degrees C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed Km value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively.

Production and secretion of biologically active human epidermal growth factor in Yarrowia lipolytica.

The gene encoding human epidermal growth factor (hEGF) was expressed as a fusion protein with the leader peptide and pro I region of alkaline extracellular protease in the yeast Yarrowia lipolytica. hEGF was purified from culture supernatant by reverse-phase chromatography and analysed by Western-blot hybridisations. The biologically active hEGF in the purified sample was assayed using the radioreceptor assay and estimated to be 100 microg/l. However, the level of expression was found to be substantially low compared to the levels of homologous protein, alkaline extracellular protease (AEP), possibly due to degradation by secreted acid protease(s). A novel and sensitive bioassay was developed to determine the biological activity of hEGF produced at low levels and is based on the effect produced by hEGF in the regenerating tails of the wall lizard. Intramuscular injections of culture supernatant from the recombinant yeast and the standard hEGF led to a drastic reduction in tail regeneration confirming the biological activity of the recombinant hEGF.

A counter-selectable marker for genetic transformation of the yeast Schwanniomyces alluvius.

We report here a counter-selectable marker system for genetic transformation of the yeast Schwanniomyces alluvius, based on the complementation of uracil auxotrophs defective in either orotidine-5'-phosphate decarboxylase (URA3) or orotidine-5'-pyrophosphate (URA5). Uracil auxotrophs of S. alluvius were obtained by ethyl methanesulphonate mutagenesis and complemented using the ura3 gene from S. cerevisiae. A transformation frequency of approximately 10(4)/micrograms DNA was obtained, which is tenfold higher than results described in either reports. Transformants were analysed by Southern blot hybridisation and were found to be mitotically stable. The extrachromosomal nature of the transforming DNA was confirmed by Southern hybridisation and plasmid rescue. The rescued plasmid DNA had a restriction pattern identical to that of the parent plasmid.

Organisation and molecular analysis of repeated DNA sequences in the rice blast fungus Magnaporthe grisea.

The distribution of a previously described repeated DNA sequence present as a 1.3-kb PstI fragment in the genome of the rice blast fungus Magnaporthe grisea was analysed by carrying out DNA fingerprint analysis of 36 isolates including rice, non-rice and laboratory strains. The analysis of various higher-molecular-weight PstI fragments with homology to the 1.3-kb repeat revealed that these may arise predominantly from transposon insertions or point mutations. Analysis of a 5.1-kb derivative revealed both a point mutation at a PstI site and an insertion of a putative transposable element which caused an increase in molecular weight from 1.3 to 5.1 kb. Another repeat element of 1.4 kb was identified and found to exist in association with the 1.3-kb repeat. Both 1.3- and 1.4-kb elements were found to be parts of MGR583 (Hamer et al. 1989), a LINE-like element. These elements were present in a high copy number in all the rice and a majority of non-rice pathogens indicating that MGR583 is not a host-specific sequence as reported earlier. Our results suggest that repeated DNA elements in M. grisea have amplified independently of one another and further indicate that different isolates of M. grisea may have evolved from several distinct lines of origin.

Analysis of host-induced response in the rice blast fungus Magnaporthe grisea using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional (2-D) polyacrylamide gel electrophoresis of proteins was used to study the response of the rice blast fungus to extracts prepared from resistant and susceptible rice cultivars. A protein of molecular mass 31 kDa was induced by a susceptible host extract, while the fungus exposed to extract from the resistant cultivar and the untreated samples did not show the presence of this protein. Levels of this 31 kDa protein increased 30-fold, 72 h after treatment with plant extracts, with the concomitant appearance of at least sixteen other novel proteins. Fungus treated with extracts of resistant host or the untreated samples did not show any of these proteins while the proteins specific to different growth stages appeared as expected. Analysis of the extracellular samples showed induction of a 17 kDa protein after 72 h in the culture treated with susceptible host extract. Since the resistant host extract does not cause induction of any protein it is likely that the proteins induced in response to the susceptible host are expressed during the disease process and/or its establishment. Our study demonstrates usefulness of 2-D analysis in understanding host-pathogen interactions.

Development of a sequence characterized amplified region (SCAR) based indirect selection method for a dominant blast-resistance gene in rice.

Two randomly amplified polymorphic DNA (RAPD) markers, OPF6(2700) and OPH18(2400), tightly linked to Pi-10, a dominant blast-resistance gene conferring complete resistance to isolate 106 (international race IB46) of the blast fungus were identified. To derive sequence characterized amplified regions (SCARs) from OPF6(2700) and OPH18(2400), these amplified RAPD products were cloned and sequenced. Nucleotide sequence information, obtained for each end of the two linked RAPD markers, was used to design 24-mer oligonucleotide primers for PCR amplification of the respective SCARs. Polymorphisms appearing as differences in the length of the SCAR's alternate alleles were considered for the indirect selection of Pi-10. Such polymorphisms converted the linked dominant RAPD loci into codominant SCAR markers and also facilitated the indirect scoring of the blast-resistant and blast-susceptible genotypes. The development of length variant codominant SCAR markers linked to a major gene for blast resistance in rice is described. The codominant SCARs will facilitate marker-assisted selection of the Pi-10 locus in rice breeding programs and will also be useful as genetic markers for high resolution mapping of the Pi-10 region.

Mg-SINE: a short interspersed nuclear element from the rice blast fungus, Magnaporthe grisea.

A short interspersed nuclear element, Mg-SINE, was isolated and characterized from the genome of the rice blast fungus, Magnaporthe grisea. Mg-SINE was isolated as an insertion element within Pot2, an inverted-repeat transposon from M. grisea and shows typical features of a mammalian SINE. Mg-SINE is present as a 0.47-kb interspersed sequence at approximately 100 copies per haploid genome in both rice and non-rice isolates of M. grisea, indicating a common evolutionary origin. Secondary structure analysis of Mg-SINE revealed a tRNA-related region at the 5' end which folds into a cloverleaf structure. Genomic fusions resulting in chimeric Mg-SINEs (Ch-SINEs) composed of a sequence homologous to Mg-SINE at the 3' end and an unrelated sequence at its 5' end were also isolated, indicating that this and other DNA rearrangements mediated by these elements may have a major effect on the genomic architecture of this fungus.

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea.

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 bp in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fot1, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tc1. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.

Cloning and growth-regulated expression of the gene encoding the hepatitis B virus middle surface antigen in Yarrowia lipolytica.

Expression of the gene encoding the hepatitis B virus middle surface antigen (pre-HBsAg) in the yeast Yarrowia lipolytica has been studied. The preS2-HBsAg gene was expressed from the alkaline extracellular protease-encoding gene (XPR2) promoter. In the fusion construct, the membrane-spanning 'a' domain of preS2-HBsAg has been replaced by the leader peptide and the proI region of the alkaline protease, thus eliminating the epitope responsible for the immune escape mechanism. Expression has been found to be growth-stage dependent with the highest protein accumulation during the stationary phase, accounting for around 2.35% of the total soluble intracellular proteins. The produced protein was assembled into Dane particles and was immunogenic in mice. The expression vector was found to be stable for at least 100 generations under non-selective conditions.

Role of media constituents and proline in callus growth, somatic embryogenesis and regeneration of Oryza sativa cv Indica.

Conditions were optimised for efficient callus induction from seeds of four local indica rice cultivars, GR-3, GR-102, Jaya and Te-Tep. Addition of 2,4-D to MS medium at 2.5 mg/l resulted in 100% callus induction. N6 medium was superior to MS medium for callus growth, formation of embryogenic callus as well as regeneration. Cultivar Te-Tep showed highest callus growth while GR-102 the least. Addition of casein hydrolysate enhanced growth of callus but did not yield more embryogenic calli. Supplementation of MS or N6 media with proline, not only increased callus growth but also showed an increase in embryogenic callus formation. GR-102 callus was most embryogenic followed by Te-Tep, GR-3 and Jaya. Histological observation of embryogenic calli revealed the presence of pro-embryo like structures. It was also observed that calli induced on N6 medium supplemented with proline could maintain regeneration potential for a longer period as compared to other media. Cytokinins like BAP or kinetin alone could not initiate shoot formation. Regeneration frequency, and the number of shoots formed per callus increased significantly. Cultivar Te-Tep gave the best response for regeneration followed by Jaya, GR-3 and GR-102.

Selection of lys2 Mutants of the Yeast SACCHAROMYCES CEREVISIAE by the Utilization of alpha-AMINOADIPATE.

Normal strains of Saccharomyces cerevisiae do not use alpha-aminoadipate as a principal nitrogen source. However, alpha-aminoadipate is utilized as a nitrogen source by lys2 and lys5 strains having complete or partial deficiencies of alpha-aminoadipate reductase and, to a limited extent, by heterozygous lys2/+ strains. Lys2 mutants were conveniently selected on media containing alpha-aminoadipate as a nitrogen source, lysine, and other supplements to furnish other possible auxotrophic requirements. The lys2 mutations were obtained in a variety of laboratory strains containing other markers, including other lysine mutations. In addition to the predominant class of lys2 mutants, low frequencies of lys5 mutants and mutants not having any obvious lysine requirement were recovered on alpha-aminoadipate medium. The mutants not requiring lysine appeared to have mutations at the lys2 locus that caused partial deficiencies of alpha-aminoadipate reductase. Such partial deficiencies are believed to be sufficiently permissive to allow lysine biosynthesis, but sufficiently restrictive to allow for the utilization of alpha-aminoadipate. Although it is unknown why partial or complete deficiencies of alpha-aminoadipate reductase cause utilization of alpha-aminoadipate as a principal nitrogen source, the use of alpha-aminoadipate medium has considerable utility as a selective medium for lys2 and lys5 mutants.

Patterns of Genetic and Phenotypic Suppression of lys2 Mutations in the Yeast SACCHAROMYCES CEREVISIAE.

A total of 358 lys2 mutants of Saccharomyces cerevisiae have been characterized for suppressibility by the following suppressors: UAA and UAG suppressors that insert tyrosine, serine or leucine; a putative UGA suppressor; an omnipotent suppressor SUP46; and a frameshift suppressor SUF1-1. In addition, the lys2 mutants were examined for phenotypic suppression by the aminoglycoside antibiotic paromomycin, for osmotic remediability and for temperature sensitivity. The mutants exhibited over 50 different patterns of suppression and most of the nonsense mutants appeared similar to nonsense mutants previously described. A total of 24% were suppressible by one or more of the UAA suppressors, 4% were suppressible by one or more of the UAG suppressors, while only one was suppressible by the UGA suppressor and only one was weakly suppressible by the frameshift suppressor. One mutant responded to both UAA and UAG suppressors, indicating that UAA or UAG mutations at certain rare sites can be exceptions to the specific action of UAA and UAG suppressors. Some of the mutants appeared to require certain types of amino acid replacements at the mutant sites in order to produce a functional gene product, while others appeared to require suppressors that were expressed at high levels. Many of the mutants suppressible by SUP46 and paromomycin were not suppressible by any of the UAA, UAG or UGA suppressors, indicating that omnipotent suppression and phenotypic suppression need not be restricted to nonsense mutations. All of the mutants suppressible by SUP46 were also suppressible by paromomycin, suggesting a common mode of action of omnipotent suppression and phenotypic misreading.