For long time, studies on ectomycorrhiza (ECM) have been limited by inefficient expression of fluorescent proteins (FPs) in the fungal partner. To convert this situation, we have evaluated the basic requirements of FP expression in the model ECM homobasidiomycete Laccaria bicolor and established eGFP and mCherry as functional FP markers. Comparison of intron-containing and intronless FP-expression cassettes confirmed that intron-processing is indispensable for efficient FP expression in Laccaria. Nuclear FP localization was obtained via in-frame fusion of FPs between the intron-containing genomic gene sequences of Laccaria histone H2B, while cytosolic FP expression was produced by incorporating the intron-containing 5′ fragment of the glyceraldehyde-3-phosphate dehydrogenase encoding gene. In addition, we have characterized the consensus Kozak sequence of strongly expressed genes in Laccaria and demonstrated its boosting effect on transgene mRNA accumulation. Based on these results, an Agrobacterium-mediated transformation compatible plasmid set was designed for easy use of FPs in Laccaria. The four cloning plasmids presented here allow fast and highly flexible construction of C-terminal in-frame fusions between the sequences of interest and the two FPs, expressed either from the endogenous gene promoter, allowing thus evaluation of the native regulation modes of the gene under study, or alternatively, from the constitutive Agaricus bisporus gpdII promoter for enhanced cellular protein localization assays. The molecular tools described here for cell-biological studies in Laccaria can also be exploited in studies of other biotrophic or saprotrophic basidiomycete species susceptible to genetic transformation.

A novel endochitinase agar-plate assay has been developed and used to identify 11 full-length cDNAs encoding endochitinase I (ENC I) from aTrichoderma harzianum cDNA library by expression in yeast. The 1473-bpchil cDNA encodes a 424-residue precursor protein including both a signal sequence and a propeptide. The deduced ENC I amino-acid sequence is homologous to other fungal and bacterial chitinases, and the enzyme cross-reacts with a polyclonal antiserum raised against chitinase A1 fromBacillus circulans. TheT. harzianum endochitinase I was secreted into the culture medium by the yeastSaccharomyces cerevisiae in a functionally active form. The purified recombinant enzyme had a molecular mass of 44 kDa, an isoelectric point of 6.3, a pH optimum of 7.0 and a temperature optimum of 20 °C.

The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153–278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho^- or rho^o cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.

The mating type (MAT) locus of the lentil pathogen, Ascochyta lentis, was cloned and characterized using thermal asymmetric interlaced and inverse PCR with primers designed to the HMG-box of Ascochyta rabiei. A multiplex PCR assay for mating type was developed based on MAT idiomorph and flanking sequences. Primers were designed to specifically amplify MAT from several Ascochyta spp. including A. pisi, A. fabae and A. viciae-villosae in addition to A. lentis. Four hundred and fifty and 700 bp fragments were amplified from MAT1-1 and MAT1-2 isolates, respectively, and fragment size correlated perfectly with laboratory crosses using mating type tester strains. MAT-specific PCR allowed rapid scoring of mating type in crude DNA extracts from geographically diverse population samples of A. viciae-villosae from California and Washington State, USA. This co-dominant MAT-specific PCR assay will be a valuable tool for studying the population structure, biology and epidemiology of these fungi.

The budding yeast is currently one of the major model organisms for the study of a wide variety of biological processes. Genetic manipulation of yeast involves the extensive usage of selectable markers that can lead to undesired effects. Thus, marker-free genetic manipulation in yeast is highly desirable for gene/promoter replacement and various other applications. Here we combine the power of selectable markers followed by CRISPR/CAS9 genome editing for common genetic manipulations in yeast in a marker-free manner. We demonstrate our approach for whole gene and promoter replacements and for high-efficiency operator array integration. Our approach allows the utilization of many thousands of existing strains including library strains for the generation of significant genetic changes in yeast in a marker-free and cloning-free fashion.

During meiotic chromosome pairing, a loop of unpaired DNA induces the silencing of all paired and unpaired homologous DNA via meiotic silencing, an RNA-mediated post-transcriptional gene-silencing mechanism. To test the effect of unpaired DNA on adjacent genes, we constructed strains containing the DNA of a transformation marker integrated immediately downstream of the Ascospore maturation-1 (Asm-1) gene and tested whether this unpaired DNA silences asm-1⁺. We conclude that unpaired downstream DNA has no effect on Asm-1 expression during meiosis or ascospore development, which suggests that the silencing signal produced by unpaired DNA does not propagate onto adjacent paired regions.

Abstract.

Genome sequencing of a large number of organisms has provided a wealth of previously uncharacterized genes. Rapid functional analysis of these genes relies on efficient methods for targeted gene disruption. Gene replacement requires homologous recombination at the target locus. The efficiency of homologous recombination largely depends on the size of the flanking homology regions provided with the disruption cassette. Therefore, the ratio of targeted versus random integration into the genome governs the choice of tools applicable in any organism. PCR-based methods for gene disruption were first reported in Saccharomyces cerevisiae. Over the past years, additional tools have been developed for epitope- or green fluorescent protein-tagging of genes and for promoter exchanges. The attractiveness of these tools led to the generation of PCR modules for use in a wide variety of bacterial and fungal species. The high capacity of in vivo recombination of Sac. cerevisiae and Escherichia coli may also be used for heterologous DNA manipulations. This facilitates the generation of disruption cassettes for organisms that cannot be transformed with very short flanks of target homology regions. Furthermore, laborious cloning procedures, e.g. the generation of point mutations or the deletion of internal domains of genes, can be simplified by using these organisms as workhorses which will advance the general genetic toolkit.

We have cloned a pyr4 gene encoding orotidine-5′-monophosphate decarboxylase of the filamentous fungus Rhizopus niveus. The pyr4 gene of R. nivens has an open reading frame composed of 265 amino-acid residues and has two putative introns. We have also isolated a pyr4 mutant of Rhizopus delemar from 5-fluoroorotic acid-resistant mutants and transformed it with the pyr4 gene of R. niveus as a selectable marker. Introduced DNA was integrated into the chromosome in a multiple tandem array. The mitotic stability of the introduced DNA was increased by a repeated sporulation process. The expression of the Escherichia coli β-glucuronidase gene in R. delemar was successfully obtained under the control of the pgk2 gene promoter of R. niveus by co-transformation with the pyr4 gene.

Molecular genetic analyses in Schizosaccharomyces pombe rely on selectable markers that are used in cloning vectors or to mark targeted gene deletions and other integrated constructs. In this study, we used genetic mapping data and genomic sequence information to predict the identity of the S. pombelys2⁺ gene, which is homologous to Saccharomyces cerevisiaeLYS4⁺. We confirmed this prediction, showing that the cloned SPAC343.16 gene can complement a lys2-97 mutant allele, and constructed the lys2⁺-based cloning vector pRH3. In addition, we deleted the S. pombe his7⁺ gene with a lys2⁺-marked polymerase chain reaction (PCR) product and the S. pombe lys2⁺ gene with a his7⁺-marked PCR product. Strains carrying these deletions of lys2⁺ or his7⁺ serve as relatively efficient hosts for the deletion of the ade6⁺ gene by lys2⁺- or his7⁺-marked PCR products when compared with hosts carrying lys2 or his7 point mutations. Therefore, these studies provide plasmids and strains allowing the use of lys2⁺ as a selectable marker, along with improved strains for the use of his7⁺ to mark gene deletions.

Plasmids containing the AMA1 replicon are capable of autonomous maintenance in Aspergillus nidulans. It has been reported previously that these plasmids can form concatenates by recombination in a transformed mycelium, and up to 10% of molecules are involved in such events. The present study demonstrates that plasmid recombination, although frequent during transformation, rarely occurs during vegetative growth. As a result, the structure and phenotypic stability of AMA1 plasmids generally remains unaltered for many asexual (conidial) generations. It is also evident that plasmid replication does not require specific recombination events in the AMA1 palindrome. However, during sexual reproduction, autonomous plasmids exhibit increased recombination, which results in both plasmid concatenation and integration into the chromosome.