BEM46 shows eisosomal localization and association with tryptophan-derived auxin pathway in Neurospora crassa.

BEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein in Neurospora crassa is targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established a bem46 knockout strain of N. crassa. This Δbem46 mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterized bem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively spliced bem46 mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due to bem46 mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using the N. crassa ortholog of the eisosomal protein PILA from Aspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded by trp-1). This interaction was confirmed in vivo by a split-YFP (yellow fluorescent protein) approach. The Δtrp-1 mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the different bem46 transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced by bem46 overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

Guest, a transposable element belonging to the Tc1/mariner superfamily is an ancient invader of Neurospora genomes.

Guest is a transposable element of the Tc1/mariner superfamily with 30-40bp terminal inverted repeats and a TA dinucleotide target site duplication. Guest was originally discovered in the St. Lawrence 74A laboratory strain of the filamentous fungus Neurospora crassa. In this report, Guest iterations subcloned from a cosmid library of the Oakridge 74A strain were used to design PCR primers that permitted the detection of Guest in wild isolates of N. crassa. Guest is present in N. crassa as multiple copies ranging between 100bp and 2.4kb and is present in the mating type locus of several Neurospora species. Bioinformatic analysis of the entire N. crassa genome (Oakridge 74A strain) detected 48 Guest iterations. All iterations appeared to have been inactivated either by repeat-induced point mutation or sequence deletion, with the majority being remnants less than 400bp in length. The possible involvement of Guest in the evolution of the variable region that flanks the mating type idiomorphs in several Neurospora species is discussed.

Electroporation of isolated higher-plant mitochondria: transcripts of an introduced cox2 gene, but not an atp6 gene, are edited in organello.

To facilitate the analysis of RNA processing in plant mitochondria, a method was established for introducing foreign DNA into mitochondria isolated from maize and sorghum. This method permits the uptake of DNA of up to 11 kb into the mitochondrial matrix. In vitro incubation of maize mitochondria in a specific buffer system was found to permit splicing and editing of newly synthesized RNAs for a period of at least 7 h. This was shown both for transcripts of endogenous mitochondrial genes (atp6, cox2) and for transcripts derived from an introduced Arabidopsis thaliana cox2 gene. In contrast, when a Sorghum bicolor atp6 gene was introduced into isolated maize mitochondria, the gene was transcribed, but the RNA was not edited, although all the editing sites in maize and sorghum atp6 RNA are identical. This may indicate the presence of transcript-specific cis -acting regions in the up- or downstream untranslated sequences of the mRNA. The system described here should allow further dissection of the mechanism of RNA editing in plant mitochondria.

Hideaway, a repeated element from Ascobolus immersus, is rDNA-associated and may resemble a retrotransposon.

A lambda library of Ascobolus immersus stock 50 was screened for repeated DNA sequences by employing differential hybridization to identify transposable elements. Several lambda clones carrying repeated DNA sequences were obtained. One clone contains a repeated DNA sequence with unusual characteristics. This DNA element appears to be associated with rDNA sequences. Its methylation pattern is also reminiscent of rDNA. Sequence analysis indicates that all or at least many copies of the element have integrated in the external spacer sequence separating the rDNA repeats, possibly to avoid complete inactivation by methylation. The element has been named "Hideaway". It possesses structural characteristics of class I retrotransposons, most notably the presence of long direct repeats. The data presented here are discussed with respect to the ability of repeated DNA sequences to escape host defense mechanisms.

The hAT family: a versatile transposon group common to plants, fungi, animals, and man.

Transposons are ubiquitous mobile genetic elements found in all eu- and prokaryotic cells. The first transposon identified, the maize Activator element, belongs to the hAT family. hAT transposons have been identified in most eukaryotic lineages, including plants, fungi, animals and even man. The basic structural and functional features of this transposon family and its phylogenetic roots are discussed in detail, including a phylogenetic tree deduced from the amino acid sequence of the most conserved part of the transposon-encoded transposase. Emphasis is given to the use of hAT transposons as tools for gene tagging and insect transformation as well as to their biological function, i.e. are they selfish DNA, beneficial companions, or even both?

Methylation of the foreign transposon Restless in vegetative mycelia of Neurospora crassa.

Methylation of foreign and/or repeated sequences in the filamentous fungus Neurospora crassa is believed to be directed against invading transposable elements. To test this hypothesis, the fate of a transposon in N. crassa was investigated. Vectors were constructed which carried the transposon Restless, an active class-II element isolated from the fungus Tolypocladium inflatum. These vectors were introduced into N. crassa strains by protoplast transformation. Two strategies were employed: (1) ectopic multi-copy integration, and (2) site-specific single-copy integration at the his-3 locus. All ectopic transformants exhibited strong methylation as confirmed by Southern hybridization of genomic DNA digested with the methylation-sensitive endonuclease Sau3AI and the methylation-insensitive endonuclease NdeII. Single copies of Restless integrated at the his-3 locus were not methylated. These results are discussed with respect to non-RIP methylation and potential consequences for gene-tagging strategies based on the use of Restless.

Tagging of a nitrogen pathway-specific regulator gene in Tolypocladium inflatum by the transposon Restless.

Restless is an endogenous hAT transposon found in the cyclosporin-producing fungus Tolypocladium inflatum. This element is present in about 15 copies in a particular strain (ATCC34921) which was used for successful gene tagging. We have isolated a T. inflatum mutant with a defect in nitrogen metabolism. This mutant carries a copy of the Restless element in a gene encoding a C6 zinc-finger protein. The deduced amino acid sequence of the gene product shows a significant similarity to the NIT4 protein of Neurospora crassa, which is a regulator of nitrogen metabolism. The wild-type T. inflatum gene was shown to complement a nit-4 mutant of N. crassa. From these data, we conclude that the T. inflatum gene also encodes a regulator of nitrogen metabolism, which was named tnir1 (Tolypocladium nitrogen regulator 1). To the best of our knowledge, this is the first fungal gene to be identified by transposon-directed gene tagging. A general method for gene tagging using an endogenous fungal transposon is presented.

Nuclear genes from Tx CMS maintainer lines are unable to maintain atp6 RNA editing in any anther cell-type in the sorghum bicolor A3 cytoplasm.

RNA editing and cytoplasmic male sterility are two important phenomena associated with higher plant mitochondria. We recently have shown a potential function of RNA editing in CMS development. The frequency of atp6 RNA editing was specifically reduced in anthers of male-sterile Sorghum bicolor, which increased in frequency in partially restored progeny. Here we present data that show that the loss of RNA editing capability also occurs in a second nuclear background that allows the expression of male sterility. Loss of RNA editing thus appears to be associated with unique combinations of male-sterile cytoplasm and non-restoring nuclear backgrounds. In addition, the reduction of RNA editing affects both gametophytic and sporophytic anther cell-types but not other floral tissues. An analysis of F(2) plants exhibiting different levels of fertility indicates a co-segregation of fertility restoration and atp6 RNA editing. The atp6 transcript abundance is similar in seedlings and anthers of male-sterile, partially restored, and male-fertile lines and thus is not associated with loss of atp6 RNA editing in anthers. A model for RNA editing and male sterility based on the data available is presented. Functional correlations with other CMS systems are also discussed.

A unique two-gene gametophytic male sterility system in sorghum involving a possible role of RNA editing in fertility restoration.

The sorghum line IS1112C carries a male sterility-inducing cytoplasm when introduced into nuclear backgrounds that do not include fertility restoration genes. An mtDNA chimeric configuration resulting from recombination/duplication with atp9 resulted in the formation of orf107, a chimeric open reading frame. Transcription of orf107 is driven by three promoters, and abundant whole-length transcripts are detected in male-sterile lines. Fertility restoration is exacted through a unique two-gene gametophytic system requiring complementary action of genes designated Rf3 and Rf4. In male-sterile lines carrying Rf3, or lines restored to fertility, an enhanced nucleolytic transcript processing activity is targeted within orf107, cleaving 75% of whole-length transcripts. Rf3 thus confers or regulates the nucleolytic processing activity. A correlation between the frequency of RNA editing at two sites in orf107 and transcript processing suggests that processing may be dependent on templates edited at these sites. In addition, editing of atp6 transcripts is specifically reduced in anthers/pollen of male-sterile lines. Partially restored F1s and segregating F2s exhibit atp6 editing frequencies consistent with the possibility that Rf4 may confer the restitution of normal editing frequency. Thus RNA editing may be involved in features of fertility restoration in this unusual system.

Transposons in filamentous fungi--facts and perspectives.

Transposons are ubiquitous genetic elements discovered so far in all investigated prokaryotes and eukaryotes. In remarkable contrast to all other genes, transposable elements are able to move to new locations within their host genomes. Transposition of transposons into coding sequences and their initiation of chromosome rearrangements have tremendous impact on gene expression and genome evolution. While transposons have long been known in bacteria, plants, and animals, only in recent years has there been a significant increase in the number of transposable elements discovered in filamentous fungi. Like those of other eukaryotes, each fungal transposable element is either of class or of class II. While class I elements transpose by a RNA intermediate and employ reverse transcriptases, class II elements transpose directly at the DNA level. We present structural and functional features for such transposons that have been identified so far in filamentous fungi. Emphasis is given to specific advantages or unique features when fungal systems are used to study transposable elements, e.g., the evolutionary impact of transposons in coenocytic organisms and possible experimental approaches toward horizontal gene transfer. Finally, we focus on the potential of transposons for tagging and identifying fungal genes.

Evidence for circular transposition derivatives from the fungal hAT-transposon Restless.

The structure and function of eukaryotic hAT-transposons has already been elucidated; however, their transposition mechanism is barely understood. We recently have discovered Restless, a fungal member of the hAT-transposon family, which shows transposition in its host Tolypocladium inflatum. Investigations of two strains from T. inflatum, carrying either about 15 copies or only a single copy of the Restless element, indicate the presence of circular transposition intermediates. Using PCR technology, amplicons were identified which carry the joined end of the Restless transposon fused at its inverted repeats. All of eight sequenced PCR fragments contained the intact transposon ends with a short insertion of 1-93 bp of genomic DNA. Remarkably, one of the discovered genomic sequences matches a previously described integration site. Our data are discussed with respect to the transposition mechanism and the horizontal transfer of hAT-transposons.

Interaction of mitochondrial RNA editing and nucleolytic processing in the restoration of male fertility in sorghum.

Nucleolytic processing of transcripts within mitochondrial orf107, associated with male sterility in sorghum, is regulated by the fertility restoration gene Rf3, conferring 75% cleavage of whole-length transcripts. Two transcript editing sites are 81% and 61% edited in rf3rf3 lines, while these sites are 41% and 10% edited in the remaining whole-length transcripts in an Rf3Rf3 line. RNA editing and processing efficiency in F1 progeny were similar to the Rf3Rf3 parent, and analyses of backcross progeny indicated that all rf3rf3 lines were characterized by high editing efficiency. We postulate that highly edited transcripts within the population are quickly processed in lines carrying Rf3, generating a residual population of poorly edited transcripts. Thus, action of Rf3 may have no direct affect on RNA editing, and may be dependent on a substrate of highly edited transcripts. These data indicate a potentially novel role of RNA editing in gene expression through an influence on the efficiency of transcript processing.

Distribution of the fungal transposon Restless: full-length and truncated copies in closely related strains.

The fungal transposon Restless of Tolypocladium inflatum ATCC 34921 is a member of the hAT family of mobile DNA elements. In order to study the distribution of this transposon we have looked at 13 fungal strains, most of which are taxonomically related to strain ATCC 34921. Three strains, which show identical banding patterns in a comparative RAPD analysis with strain ATCC 34921, similarly carry multiple copies of Restless. In addition, 1 T. inflatum strain and 2 Beauveria nivea strains contain only a few or even single copies of the transposon. Inverse PCR and DNA sequencing analysis revealed that 1 strain contains a nonmobile truncated version of the element, while the other one harbors a full-length transposon copy which was named Restless-2. The presence of a single transposon copy of Restless in a defined Beauveria strain indicates recent acquisition of this transposon, since class II transposons usually occur in several copies per haploid genome. Notably, the corresponding genomic location is not occupied by a transposon copy in strain ATCC 34921.

Mutations at specific atp6 codons which cause human mitochondrial diseases also lead to male sterility in a plant.

Defects in the human mitochondrial genetic system result in some diseases. These disorders are the result of rearrangements or point mutations in mitochondrial genes. In higher plants mutations and rearrangements in the mitochondrial DNA are believed to cause cytoplasmic male sterility (CMS), a mitochondrially inherited inability to produce viable pollen. In sorghum, formation of CMS is strongly correlated with anther-specific loss of mitochondrial atp6 RNA editing. Here we show that this loss of atp6 RNA editing mimics point mutations at codons that cause severe disorders in humans. We conclude that (i) loss of RNA editing in sorghum anthers probably causes CMS, (ii) similarities exist in the onset of mitochondrial dysfunction in plant and human tissues, and (iii) the evolutionary appearance of RNA editing provided a mechanism to compensate for otherwise lethal point mutations.

Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor.

RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of atp9, nad3, nad4, and rps12 transcripts in anthers was similar to etiolated shoots, mitochondrial atp6 RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of S. bicolor. atp6 transcripts of wheat and selected plastid transcripts in S. bicolor showed normal RNA editing, indicating that loss of atp6 RNA editing is specific for cytoplasmic male sterility S. bicolor mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of atp6 transcripts. Our data suggest that loss of atp6 RNA editing contributes to or causes cytoplasmic male sterility in S. bicolor. Further analysis of the mechanism of cell type-specific loss of atp6 RNA editing activity may advance our understanding of the mechanism of RNA editing.

restless, an active Ac-like transposon from the fungus Tolypocladium inflatum: structure, expression, and alternative RNA splicing.

Elements of the hAT transposon family, such as the maize activator (Ac), have been discovered in a large number of eukaryotic species. This type of class II transposon, present in both plants and animals, has not been previously detected in any fungal organism. However, using a differential screening method to detect repetitive DNA, we have identified a hAT transposon in the hyphomycete Tolypocladium inflatum. The transposon, which we named restless, is 4,097 bp long, carries 20-bp inverted repeats and an 8-bp target site duplication, and encodes a long open reading frame which is interrupted by a single intronic sequence. The derived mRNA exhibits alternative splicing, resulting in the formation of two transcripts that may be translated into polypeptides of 157 or 803 amino acids. The predicted amino acid sequence of the larger polypeptide demonstrates significant homology with transposases from the hAT transposon family. A chromosomal analysis using pulsed-field gel electrophoresis showed that all seven chromosomal bands carry copies of the 4.1-kb transposon. This was confirmed in hybridization experiments with rare-cutting restriction endonucleases which indicate that about 15 copies are present in T. inflatum. The genomic distribution of restless and its transcriptional expression, alternative mRNA splicing, and genomic mobility all imply a potential role for this element in developing a transposon tagging system for use in filamentous fungi.

Mitochondrial RNA editing is sequence specific and independent of transcript abundance in Sorghum bicolor.

The DNA sequence which encodes the amino-terminal extension to the conserved core of the atp6-1 gene found in line IS1112C is absent from Tx398. Sequences further upstream are present in Tx398, but at a different genomic location. The atp6-2 genes are present in similar copy numbers in IS1112C and A3Tx398, a near-isogenic line carrying the IS1112C cytoplasm in a Tx398 background. However, transcript abundance of atp6-2 in these lines is about ten-times higher than that of atp6-1. RNA editing of atp6-1 transcripts is identical to that of atp6-2 and therefore sequence specific. A single non-silent editing site in the unique atp6-1 pre-piece sequence may indicate mitochondrial-guided RNA editing. While in Petunia the abundance and RNA editing of a transcript are correlated, we show here that RNA editing is independent of transcript abundance and is sequence specific in Sorghum.

The Use of Rare-Cutting Endonucleases in Electrophoretic Karyotyping of Fungal Genomes

A method for the fast and reliable restriction analysis of intact fungal chromosomes is presented. For this purpose rare-cutting restriction enzymes of pro- and eukaryotic origin were used. Restriction fragments were separated on pulsed-field gels. Complete inactivation of proteinases remaining from chromosome preparations was crucial in this attempt. Rare-cutting restriction enzymes were employed to demonstrate the applicability of this procedure to the analysis of complex fungal genomes. This includes the localization of genes on discrete chromosomal fragments,the identification of telomeric restriction fragments, and the analysis of fungal transformants.

A unique repeated DNA sequence in the cyclosporin-producing strain of Tolypocladium inflatum (ATCC 34921).

Recombinant lambda clones containing repeated DNA sequences were isolated from the cyclosporin A-producing fungus Tolypocladium inflatum (ATCC 34921) by differential hybridization with total fungal DNA and rDNA probes. From this survey 1% of the lambda clones appeared to contain repeated sequences. Subsequent analysis led to the identification of a dispersed repetitive DNA element. It was named CPA element (cyclosporin production associated) and appears to be strain specific, since it is absent from other related strains or fungi. Hybridization with chromosomal restriction fragments indicates an equal distribution of the CPA element in the genome. The copy number was estimated to be between 20 and 30 per haploid genome. Sequence analysis of a 0.9-kb XhoI fragment from three copies of the CPA element revealed strong conservation of this sequence among all copies. A 200-bp region exhibits similarities to a repeated sequence from Zea diploperennis. The use of this DNA sequence as a molecular marker for identification of this cyclosporin-producing strain ATCC 34921 is discussed as is the relevance of repeated DNA sequences for rearrangements of fungal karyotypes.

Horizontal transfer of a mitochondrial plasmid.

Direct evidence for horizontal transfer of a mitochondrial plasmid from the discomycete Ascobolus immersus to the pyrenomycete Podospora anserina is presented. Southern blot hybridisation analysis, polymerase chain reaction (PCR) amplification, and DNA sequencing demonstrate transmission of a linear plasmid upon hyphal contact. DNA extraction from isolated organelles indicates a mitochondrial localisation for the plasmid in P. anserina. This is the first report of horizontal gene transfer among unrelated fungi. These results have important evolutionary implications for plasmid propagation in fungi.