Vectors for construction of translational fusions to beta-galactosidase

The use of lacZ as a reporter gene requires appropriate plasmid constructions to produce fusions of fungal promoter and translations initiation sequences to the E. coli lacZ gene. I have constructed three plasmids which may facilitate construction of lacZ fusions

A rapid and simple method for isolation of Neurospora crassa homokaryons using microconidia

Current approaches for obtaining homokaryons of asexually growing N. crassa rely on serial passages of macroconidia, which are usually multinuclear (Davis and de Serres 1970 Methods Enzymol. 17A:79- 143). This method is time consuming and labor intensive. We have devised a simple method for purifying viable uninucleate microconidia from conidiating strains of N. crassa grown on Westergaard and Mitchell synthetic crossing medium (SC) supplemented with iodoacetate (IAA). Rossier, Oulevey and Turian (1973 Arch. Mikrobiol. 91:345-353) showed that standing liquid cultures containing 1x SC and 1 mM IAA produced microconidia. We have modified their method to reliably obtain microconidia from 150 mm slants of solid agar medium (0.1 x SC/0.5% sucrose/2% agar/1 mM IAA). We have purified these microconidia free of macroconidia and mycelia using Millipore Durapore Millex 5 µm filters. We are using this technique for the one step purification of homokaryons following DNA-mediated transformation. These methods may be generally useful for studies with heterokaryons

The use of lacZ gene fusions in Neurospora crassa

Systematic analyses of gene expression in diverse organisms have relied on genetic fusions in which the regulated expression of the product of the Escherichia coli lacZ gene, ß-galactosidase, is used to assay gene activity. Because there are low but readily detectable levels of ß-galactosidase in Neurospora crassa (e.g. Landman, Arch. Biochem. Biophys. 52:93-109, 1954), the use of E. coli lacZ as a reporter gene in this organism has not been extensively investigated. Here we report that lacZ fusion proteins can be used to analyze the regulation of two N. crassa genes, arg-2 and con-10. The levels of ß-galactosidase produced by strains carrying the fused genes indicate that they are developmentally regulated in a manner similar to the intact genes (Davis, Microbiol. Rev. 50: 280-313, 1986; Orbach, Sachs, and Yanofsky, J. Biol. Chem. in press 1990; Roberts, Berlin, Hager and Yanofsky, Mol. Cell. Biol. 8:2411-2418, 1988; Sachs and Yanofsky, in preparation)

Mutants of Neurospora crassa that alter gene expression and conidia development.

Several genes have been identified that are highly expressed during conidiation. Inactivation of these genes has no observable phenotypic effect. Transcripts of two such genes, con-6 and con-10, are normally absent from vegetative mycelia. To identify regulatory genes that affect con-6 and/or con-10 expression, strains were prepared in which the regulatory regions for these genes were fused to a gene conferring hygromycin resistance. Mutants were then selected that were resistant to the drug during mycelial growth. Mutations in several of the isolates had trans effects; they activated transcription of the corresponding intact gene and, in most isolates, one or more of the other con genes. Most interestingly, resistant mutants were obtained that were defective at different stages of conidiation. One mutant conidiated under conditions that do not permit conidiation in wild type

Identification and functional analysis of secreted proteins from Magnaporthe grisea.

Secreted proteins are the most likely candidates for being elicitors of resistance gene products in rice and other host plants. To identify the candidate secreted protein genes in M. grisea, the 5' ends of genes in the M. grisea EST databases, and genes from genomic sequences were analyzed using the SIGNALP-2.0 program

Novos aspectos da patogenicidade de Magnaporthe grisea.

O principal objetivo deste estudo foi identificar e analisar o papel que o gene PTH1 exerce na formação e maturação do apressório em M. grisea. Isolados de M. grisea mutantes (denominado pth1) para o gene PTH1 são incapazes de perfurar o tecido do hospedeiro para dar início à colonização e assim estabelecer uma interação patógeno-hospedeiro