Taha H. Al-Samarrai

A simple method for extraction of fungal genomic DNA

T. H. AL‐SAMARRAI and J. SCHMID.2000.We have developed a new, simple and effective method for extraction of fungal genomic DNA. The initial steps involved suspension of freeze‐dried mycelium in buffer containing sodium dodecyl sulphate, detachment of DNA from polysaccharides by mild shearing, NaCl precipitation of polysaccharides and protein, chloroform extraction and ethanol precipitation. The ethanol precipitate was then subjected to a second round of mild shearing, NaCl precipitation, chloroform extraction and ethanol precipitation. The procedure required approximately 1 h to perform. The method yielded 8–32 μg of high molecular weight DNA per 30 mg of freeze‐dried mycelium when tested on six fungal species: Aspergillus niger, A. flavus, Fusarium graminarum, Neotyphodium lolii, Penicillium citrinum and Rhizopus nigricanes. The DNA was digestible with EcoRI, HindIII, SalI and BamHI. For the slow‐growing N. lolii, a modification of the method was developed that removed the agar residue from colonies grown on agar plates by centrifugation at 13 000 rev min−1 in the presence of CsCl. The modified method yielded 1·5–2 μg of high molecular weight DNA per colony.

Analysis of a secreted aspartic peptidase disruption mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp112 and Asp297 were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a Km of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.

Heterologous expression, isotopic-labeling and immuno-characterization of Cin1, a novel protein secreted by the phytopathogenic fungus Venturia inaequalis.

The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel V. inaequalis gene, cin1, is strongly up-regulated in the early stages of infection. This gene codes for a 523 residue secreted protein, containing eight imperfect repeats of approximately 60 amino acids. Cin1 was expressed in the methanolytic yeast Pichia pastoris using the pPICZ vector system. A protein of 57 kDa was secreted by these transformants and peptide fingerprinting indicated that it was the Cin1 protein product. Multiple angle laser light scattering confirmed the predicted mass of Cin1, showing it was not glycosylated by Pichia and was monomeric in solution. Through measurements of the hydrodynamic properties of Cin1, the experimental Stokes radius of Cin1 was calculated and corresponded to the theoretical value for a natively folded globular protein of size 57 kDa. The mobility of recombinant Cin1 on native PAGE was also consistent with that of a folded protein. To simplify future structural analyses, a two-domain truncated version, Cin1-2D, consisting of domains one and two, was also expressed using the same vector system. Both proteins were purified to homogeneity. Conditions for maximal (>98%) incorporation of 13C and 15N were determined. A mouse polyclonal antibody and three monoclonal antibodies (MAbs) were raised against the full-length version of Cin1. Analysis of the three MAbs using surface plasmon resonance indicated binding to distinct epitopes on the Cin1 protein. Western blots confirmed the different specificities of each MAb.

The trans-Acting Protein Interacting with the DNA Motif Proximal to the Transcriptional Start Site of Plant L-Asparaginase Is Bacterial Sarcosine Oxidase

A trans-acting protein interacting with a specific sequence motif proximal to the transcriptional start site of the L-asparaginase promoter has been observed previously (E. Vincze, J. M. Reeves, E. Lamping, K. J. F. Farnden, and P. H. S. Reynolds, Plant Mol. Biol. 26:303-311, 1994). Gel retardation experiments in which protein extracts of Mesorhizobium loti and developing nodules were used suggested a bacterial origin for the repressor binding protein (rep2037). Nodulation tests were performed by using different Fix(-) Tn5 mutants of M. loti. Analyses of these mutants revealed a correlation between the presence of Mesorhizobium in the nodule-like structures and the ability of nodule protein extracts to bind the repressor binding domain (RBD). Through the use of mutated RBD sequences, the RBD sequence was identified as CTAAAAT. The repressor protein was isolated from M. loti NZP2037 by multiple chromatographic procedures and affinity separation by using concatemers of RBD attached to magnetic beads. Sequencing of the recovered protein resulted in identification of the repressor protein as the sarcosine oxidase alpha subunit. This was confirmed by expression of the gene encoding the M. loti alpha subunit of sarcosine oxidase in Escherichia coli. When the expressed peptide was bound to RBD, the gel retardation result was identical to the result obtained with rep2037 from M. loti strain NZP2037.

A Simple and Rapid Method for Selecting High Producers of Recombinant Proteins in Individual Clones of P. pastoris

The identification of clones expressing high levels of recombinant protein in Pichia pastoris is usually dependant upon SDS-PAGE, Western blotting, or bioactivity-based assays that are labour and time-consuming. We describe a rapid method that images green fluorescence protein (GFP) of individual P. pastoris clones transformed with vectors that express the proteins as GFP C- terminal fusion. In this report we have used the system to monitor expression of three proteins from Venturia inaequalis. Culture plates containing individual colonies were imaged on a Fuji LAS-3000 system and the intensity of fluorescence of GFP [Mean Gray Value (MGV)] of each colony recorded. Two common variables, the time course of expression and induction temperature were also optimised using this method. The results show that colonies with high levels of GFP fluorescence can be successfully used to identify, at an early stage, colonies expressing high levels of recombinant proteins. This correlation can be used to monitor the conditions for optimization of the expression and accumulation of extracellular recombinant protein in medium and to identify fractions containing GFP-tagged recombinant proteins during protein purification.

Characterization of Factors From Bacillus Amyloliquefaciens Strain PFR1 that Caused Adventitious Antheridia and Catenulate Vesicle Like Sac Formation of the Germinating Tubes of Venturia Inaequalis Conidiospores

Products, termed Antheridiogen Factor (AF), and vesicle like sac formation factor (VLS-F) were present in a culture of Bacillus amyloliquefaciens strain PFR-1 grown in either a defined or simple medium. The factors inhibited appressorium development and caused the formation of vesicle like sac formation of various sizes and adventitious antheridia at the end of the germinating tubes of Venturia inaequalis spores. The factors were separated on size; a fraction less that 500 Da contain the AFand stimulated the development of antheridia at the ends of germinated tubes. Another fraction between 3 and 5 KDa contained the VLS-F and caused development of catenulate vesicle like sac of the germinating tubes of conidiospores. Preliminary characterization showed both factors resistance to boiling. AF was sensitive to Trypsin and Proteinase-K, whereas VLS-F demonstrated a resistance, to Trypsin and proteinase-K treatment.