Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat.

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.

Development of a fumonisin-sensitive Saccharomyces cerevisiae indicator strain and utilization for activity testing of candidate detoxification genes.

IMPORTANCE: Fumonisins can cause diseases in animals and humans consuming Fusarium-contaminated food or feed. The search for microbes capable of fumonisin degradation, or for enzymes that can detoxify fumonisins, currently relies primarily on chemical detection methods. Our constructed fumonisin B1-sensitive yeast strain can be used to phenotypically detect detoxification activity and should be useful in screening for novel fumonisin resistance genes and to elucidate fumonisin metabolism and resistance mechanisms in fungi and plants, and thereby, in the long term, help to mitigate the threat of fumonisins in feed and food.

Chemical synthesis of culmorin metabolites and their biologic role in culmorin and acetyl-culmorin treated wheat cells.

The Fusarium metabolite culmorin (1) is receiving increased attention as an "emerging mycotoxin". It co-occurs with trichothecene mycotoxins and potentially influences their toxicity. Its ecological role and fate in plants is unknown. We synthesized sulfated and glucosylated culmorin conjugates as potential metabolites, which are expected to be formed in planta, and used them as reference compounds. An efficient procedure for the synthesis of culmorin sulfates was developed. Diastereo- and regioselective glucosylation of culmorin (1) was achieved by exploiting or preventing unexpected acyl transfer when using different glucosyl donors. The treatment of a wheat suspension culture with culmorin (1) revealed an in planta conversion of culmorin into culmorin-8-glucoside (6) and culmorin acetate, but no sulfates or culmorin-11-glucoside (7) was found. The treatment of wheat cells with the fungal metabolite 11-acetylculmorin (2) revealed its rapid deacetylation, but also showed the formation of 11-acetylculmorin-8-glucoside (8). These results show that plants are capable of extensively metabolizing culmorin.

A barley UDP-glucosyltransferase inactivates nivalenol and provides Fusarium Head Blight resistance in transgenic wheat.

Fusarium Head Blight is a disease of cereal crops that causes severe yield losses and mycotoxin contamination of grain. The main causal pathogen, Fusarium graminearum, produces the trichothecene toxins deoxynivalenol or nivalenol as virulence factors. Nivalenol-producing isolates are most prevalent in Asia but co-exist with deoxynivalenol producers in lower frequency in North America and Europe. Previous studies identified a barley UDP-glucosyltransferase, HvUGT13248, that efficiently detoxifies deoxynivalenol, and when expressed in transgenic wheat results in high levels of type II resistance against deoxynivalenol-producing F. graminearum. Here we show that HvUGT13248 is also capable of converting nivalenol into the non-toxic nivalenol-3-O-ß-d-glucoside. We describe the enzymatic preparation of a nivalenol-glucoside standard and its use in development of an analytical method to detect the nivalenol-glucoside conjugate. Recombinant Escherichia coli expressing HvUGT13248 glycosylates nivalenol more efficiently than deoxynivalenol. Overexpression in yeast, Arabidopsis thaliana, and wheat leads to increased nivalenol resistance. Increased ability to convert nivalenol to nivalenol-glucoside was observed in transgenic wheat, which also exhibits type II resistance to a nivalenol-producing F. graminearum strain. Our results demonstrate the HvUGT13248 can act to detoxify deoxynivalenol and nivalenol and provide resistance to deoxynivalenol- and nivalenol-producing Fusarium.

New tricks of an old enemy: isolates of Fusarium graminearum produce a type A trichothecene mycotoxin.

The ubiquitous filamentous fungus Fusarium graminearum causes the important disease Fusarium head blight on various species of cereals, leading to contamination of grains with mycotoxins. In a survey of F. graminearum (sensu stricto) on wheat in North America several novel strains were isolated, which produced none of the known trichothecene mycotoxins despite causing normal disease symptoms. In rice cultures, a new trichothecene mycotoxin (named NX-2) was characterized by liquid chromatography-tandem mass spectrometry. Nuclear magnetic resonance measurements identified NX-2 as 3α-acetoxy-7α,15-dihydroxy-12,13-epoxytrichothec-9-ene. Compared with the well-known 3-acetyl-deoxynivalenol (3-ADON), it lacks the keto group at C-8 and hence is a type A trichothecene. Wheat ears inoculated with the isolated strains revealed a 10-fold higher contamination with its deacetylated form, named NX-3, (up to 540 mg kg(-1) ) compared with NX-2. The toxicities of the novel mycotoxins were evaluated utilizing two in vitro translation assays and the alga Chlamydomonas reinhardtii. NX-3 inhibits protein biosynthesis to almost the same extent as the prominent mycotoxin deoxynivalenol, while NX-2 is far less toxic, similar to 3-ADON. Genetic analysis revealed a different TRI1 allele in the N-isolates, which was verified to be responsible for the difference in hydroxylation at C-8.

Deoxynivalenol-sulfates: identification and quantification of novel conjugated (masked) mycotoxins in wheat.

We report the identification of deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate as two novel metabolites of the trichothecene mycotoxin deoxynivalenol in wheat. Wheat ears which were either artificially infected with Fusarium graminearum or directly treated with the major Fusarium toxin deoxynivalenol (DON) were sampled 96 h after treatment. Reference standards, which have been chemically synthesized and confirmed by NMR, were used to establish a liquid chromatography-electrospray ionization (LC-ESI)-MS/MS-based "dilute and shoot" method for the detection, unambiguous identification, and quantification of both sulfate conjugates in wheat extracts. Using this approach, detection limits of 0.003 mg/kg for deoxynivalenol-3-sulfate and 0.002 mg/kg for deoxynivalenol-15-sulfate were achieved. Matrix-matched calibration was used for the quantification of DON-sulfates in the investigated samples. In DON-treated samples, DON-3-sulfate was detected in the range of 0.29-1.4 mg/kg fresh weight while DON-15-sulfate concentrations were significantly lower (range 0.015-0.061 mg/kg fresh weight). In Fusarium-infected wheat samples, DON-3-sulfate was the only detected sulfate conjugate (range 0.022-0.059 mg/kg fresh weight). These results clearly demonstrate the potential of wheat to form sulfate conjugates of DON. In order to test whether sulfation is a detoxification reaction in planta, we determined the ability of the sulfated DON derivatives to inhibit in vitro protein synthesis of wheat ribosomes. The results demonstrate that both DON-sulfates can be regarded as detoxification products. DON-15-sulfate was about 44× less inhibitory than the native toxin, and no toxicity was observed for DON-3-sulfate in the tested range.

Methylthiodeoxynivalenol (MTD): insight into the chemistry, structure and toxicity of thia-Michael adducts of trichothecenes.

Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.

Isolation and structure elucidation of pentahydroxyscirpene, a trichothecene Fusarium mycotoxin.

Pentahydroxyscirpene, a novel trichothecene-type compound, was isolated from Fusarium-inoculated rice. The structure of pentahydroxyscirpene was elucidated by 1D and 2D NMR spectroscopy and X-ray single-crystal diffraction. The conformation in solution was determined by NOESY experiments supported by quantum chemical calculations. In vitro toxicity tests showed that pentahydroxyscirpene inhibits protein synthesis as do other trichothecenes.

The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe-S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe-S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

Mechanism of Fumonisin Self-Resistance: Fusarium verticillioides Contains Four Fumonisin B1-Insensitive-Ceramide Synthases.

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker's yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides "housekeeping" ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

Functional characterization of two clusters of Brachypodium distachyon UDP-glycosyltransferases encoding putative deoxynivalenol detoxification genes.

Plant small-molecule UDP-glycosyltransferases (UGT) glycosylate a vast number of endogenous substances but also act in detoxification of metabolites produced by plant-pathogenic microorganisms. The ability to inactivate the Fusarium graminearum mycotoxin deoxynivalenol (DON) into DON-3-O-glucoside is crucial for resistance of cereals. We analyzed the UGT gene family of the monocot model species Brachypodium distachyon and functionally characterized two gene clusters containing putative orthologs of previously identified DON-detoxification genes from Arabidopsis thaliana and barley. Analysis of transcription showed that UGT encoded in both clusters are highly inducible by DON and expressed at much higher levels upon infection with a wild-type DON-producing F. graminearum strain compared with infection with a mutant deficient in DON production. Expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae revealed that only two B. distachyon UGT encoded by members of a cluster of six genes homologous to the DON-inactivating barley HvUGT13248 were able to convert DON into DON-3-O-glucoside. Also, a single copy gene from Sorghum bicolor orthologous to this cluster and one of three putative orthologs of rice exhibit this ability. Seemingly, the UGT genes undergo rapid evolution and changes in copy number, making it difficult to identify orthologs with conserved substrate specificity.

Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum.

Chromatin modifications and heterochromatic marks have been shown to be involved in the regulation of secondary metabolism gene clusters in the fungal model system Aspergillus nidulans. We examine here the role of HEP1, the heterochromatin protein homolog of Fusarium graminearum, for the production of secondary metabolites. Deletion of Hep1 in a PH-1 background strongly influences expression of genes required for the production of aurofusarin and the main tricothecene metabolite DON. In the Hep1 deletion strains AUR genes are highly up-regulated and aurofusarin production is greatly enhanced suggesting a repressive role for heterochromatin on gene expression of this cluster. Unexpectedly, gene expression and metabolites are lower for the trichothecene cluster suggesting a positive function of Hep1 for DON biosynthesis. However, analysis of histone modifications in chromatin of AUR and DON gene promoters reveals that in both gene clusters the H3K9me3 heterochromatic mark is strongly reduced in the Hep1 deletion strain. This, and the finding that a DON-cluster flanking gene is up-regulated, suggests that the DON biosynthetic cluster is repressed by HEP1 directly and indirectly. Results from this study point to a conserved mode of secondary metabolite (SM) biosynthesis regulation in fungi by chromatin modifications and the formation of facultative heterochromatin.

Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Gramiketides, Novel Polyketide Derivatives of Fusarium graminearum, Are Produced during the Infection of Wheat.

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane.

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe(2+) homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe(2+) utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe(2+) which was driven by the external Fe(2+) concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Delta failed to show this rapid Fe(2+) uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe(2+) uptake rates. Cu(2+) was transported at similar rates as Fe(2+), while other divalent cations, such as Zn(2+) and Cd(2+) apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe(2+) across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose.

Sal1p, a novel Ca2+-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K(m) of 0.20 +/- 0.03 mM and 0.28 +/- 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca2+ dependent. Ca2+ increases the V(max) with a S(0.5) of 15 muM, and no changes in the K(m) for ATP-Mg. Glucose sensing in yeast generates Ca2+ transients involving Ca2+ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H+-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca2+-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.

Biochemical Characterization of the Fusarium graminearum Candidate ACC-Deaminases and Virulence Testing of Knockout Mutant Strains.

Fusarium graminearum is a plant pathogenic fungus which is able to infect wheat and other economically important cereal crop species. The role of ethylene in the interaction with host plants is unclear and controversial. We have analyzed the inventory of genes with a putative function in ethylene production or degradation of the ethylene precursor 1-aminocyclopropane carboxylic acid (ACC). F. graminearum, in contrast to other species, does not contain a candidate gene encoding ethylene-forming enzyme. Three genes with similarity to ACC synthases exist; heterologous expression of these did not reveal enzymatic activity. The F. graminearum genome contains in addition two ACC deaminase candidate genes. We have expressed both genes in E. coli and characterized the enzymatic properties of the affinity-purified products. One of the proteins had indeed ACC deaminase activity, with kinetic properties similar to ethylene-stress reducing enzymes of plant growth promoting bacteria. The other candidate was inactive with ACC but turned out to be a d-cysteine desulfhydrase. Since it had been reported that ethylene insensitivity in transgenic wheat increased Fusarium resistance and reduced the content of the mycotoxin deoxynivalenol (DON) in infected wheat, we generated single and double knockout mutants of both genes in the F. graminearum strain PH-1. No statistically significant effect of the gene disruptions on fungal spread or mycotoxin content was detected, indicating that the ability of the fungus to manipulate the production of the gaseous plant hormones ethylene and H2S is dispensable for full virulence.

Untargeted LC-MS based 13C labelling provides a full mass balance of deoxynivalenol and its degradation products formed during baking of crackers, biscuits and bread.

Deoxynivalenol (DON) is considered to be one of the most important contaminants in cereals and food commodities produced thereof. So far it is not clear i) to which extent DON is degraded during baking and ii) if a degradation results in reduced toxicity. We have elucidated the fate of DON during baking of crackers, biscuits and bread, which were produced from fortified dough and processed under pilot plant conditions. Untargeted stable isotope assisted liquid chromatography (LC) high resolution mass spectrometry was used to determine all extractable degradation products. Targeted LC - tandem mass spectrometry based quantification revealed that DON was partially degraded to isoDON (1.3-3.9%), norDON B (0.2-0.9%) and norDON C (0.3-1.2%). A DON degradation of 6% (crackers), 5% (biscuits) and 2% (bread), respectively, was observed. In vitro translation experiments indicate that isoDON is less toxic than DON.

Zearalenone and ß-Zearalenol But Not Their Glucosides Inhibit Heat Shock Protein 90 ATPase Activity.

The mycotoxin zearalenone (ZEN) is produced by many plant pathogenic Fusarium species. It is well known for its estrogenic activity in humans and animals, but whether ZEN has a role in plant-pathogen interaction and which process it is targeting in planta was so far unclear. We found that treatment of Arabidopsis thaliana seedlings with ZEN induced transcription of the AtHSP90.1 gene. This heat shock protein (HSP) plays an important role in plant-pathogen interaction, assisting in stability and functionality of various disease resistance gene products. Inhibition of HSP90 ATPase activity impairs functionality. Because HSP90 inhibitors are known to induce HSP90 gene expression and due to the structural similarity with the known HSP90 inhibitor radicicol (RAD), we tested whether ZEN and its phase I metabolites α- and ß-zearalenol are also HSP90 ATPase inhibitors. Indeed, AtHSP90.1 and wheat TaHSP90-2 were inhibited by ZEN and ß-zearalenol, while α-zearalenol was almost inactive. Plants can efficiently glycosylate ZEN and α/ß-zearalenol. We therefore tested whether glucosylation has an effect on the inhibitory activity of these metabolites. Expression of the A. thaliana glucosyltransferase UGT73C6 conferred RAD resistance to a sensitive yeast strain. Glucosylation of RAD, ZEN, and α/ß-zearalenol abolished the in vitro inhibitory activity with recombinant HSP90 purified from Escherichia coli. In conclusion, the mycotoxin ZEN has a very prominent target in plants, HSP90, but it can be inactivated by glycosylation. This may explain why there is little evidence for a virulence function of ZEN in host plants.

RNA degradation in fission yeast mitochondria is stimulated by a member of a new family of proteins that are conserved in lower eukaryotes.

We report here on the role of open reading frame (ORF) SPCC1183.04c of Schizosaccharomyces pombe in mitochondrial RNA metabolism. A mutant deleted for this ORF on chromosome III accumulates mitochondrial transcripts with the exception of the cob mRNA. A detailed Northern blot analysis showed that the effect results from a decrease in RNA degradation but not from RNA processing deficiencies. Overexpression of the SPCC1183.04c gene in a S. pombe wild-type strain is characterized by slow growth at 37 degrees C on non-fermentable carbon sources and a significant reduction of steady-state levels of mitochondrial transcripts. A NCBI BLASTP search with the amino acid sequence deduced from the S. pombe gene identified significant similarity to a number of proteins in fungi (e.g. Ascomycota, Basidiomycota) and in some non-fungal eukaryotes (e.g. ciliate, slime mold, red algae). By heterologous expression of SPCC1183.04c in a Saccharomyces cerevisiae pet127Delta strain, we demonstrate that the fission yeast protein and Pet127p from S. cerevisiae function similarly: The fission yeast gene complemented the respiratory defect associated with the pet127Delta allele and partially restored the RNA processing phenotype. Although it lacks any recognizable targeting signal, the S. pombe protein is imported into S. cerevisiae mitochondria in vivo. We conclude from our results that the fission yeast SPCC1183.04c gene is a member of a new protein family that functions to stimulate mitochondrial RNA degradation, a function that is conserved within the mitochondria of lower eukaryotes but seems to have been replaced by alternative pathways in metazoans and higher plants.