Sensitive Yeast Strain Research Articles

Mn-CDF family genes enhance the manganese tolerance of the tea plants (Camellia sinensis) under acidic condition

The tea plants cultivated in acidic soils are vulnerable to excessive manganese (Mn), which increases the risk of Mn2+ toxicity to physiology and development. Mn-cation diffusion facilitator (CDF) family genes have been implicated in regulating Mn homeostasis and tolerance. However, the mechanism of Mn tolerance of tea plants in acidic environments is still unknown. In this study, we initially examined the phenotypic characteristics and Mn contents variability in different tissues of tea plants under various Mn concentration at pH 5 and 4. We observed that tea plants exhibited remarkably high Mn tolerance at pH 4, with Mn accumulation notably elevated in the aboveground tissues under pH 4 condition after 28-day treatment. We found the expression levels of Mn-CDF genes, had different subcellular localization, were tissue-specific and significantly induced by high Mn concentrations at pH 4 condition. Furthermore, the yeast complementation assays indicated that the heterologous expression of Mn-CDF genes restored the growth of a Mn2+ sensitive yeast strain, Δpmr1. Taken together, these results suggest that Mn-CDF family genes function as Mn transporters to participate in Mn tolerance in acidic environments. This study provides reference for further study on the mechanism of maintaining Mn homeostasis in tea plants under soil acidification.

Expression and mutagenesis studies in the Medicago truncatula iron transporter MtVTL8 confirm its role in symbiotic nitrogen fixation and reveal amino acids essential for transport.

The model legume Medicago truncatula establishes a symbiosis with soil bacteria (rhizobia) that carry out symbiotic nitrogen fixation (SNF) in plant root nodules. SNF requires the exchange of nutrients between the plant and rhizobia in the nodule that occurs across a plant-derived symbiosome membrane. One iron transporter, belonging to the Vacuolar iron Transporter-Like (VTL) family, MtVTL8, has been identified as essential for bacteria survival and therefore SNF. In this work we investigated the spatial expression of MtVTL8 in nodules and addressed whether it could be functionally interchangeable with a similar nodule-expressed iron transporter, MtVTL4. Using a structural model for MtVTL8 and the previously hypothesized mechanism for iron transport in a phylogenetically-related Vacuolar Iron Transporter (VIT), EgVIT1 with known crystal structure, we identified critical amino acids and obtained their mutants. Mutants were tested in planta for complementation of an SNF defective line and in an iron sensitive mutant yeast strain. An extended phylogenetic assessment of VTLs and VITs showed that amino acids critical for function are conserved differently in VTLs vs. VITs. Our studies showed that some amino acids are essential for iron transport leading us to suggest a model for MtVTL8 function, one that is different for other iron transporters (VITs) studied so far. This study extends the understanding of iron transport mechanisms in VTLs as well as those used in SNF.

A novel high-affinity potassium transporter SeHKT1;2 from halophyte Salicornia europaea shows strong selectivity for Na+ rather than K.

High-affinity K+ transporters (HKTs) are known as transmembrane cation transporters and are involved in Na+ or Na+-K+ transport in plants. In this study, a novel HKT gene SeHKT1;2 was isolated and characterized from the halophyte, Salicornia europaea. It belongs to subfamily I of HKT and shows high homology with other halophyte HKT proteins. Functional characterization of SeHKT1;2 indicated that it contributes to facilitating Na+ uptake in Na+-sensitive yeast strains G19, however, cannot rescue the K+ uptake-defective phenotype of yeast strain CY162, demonstrating SeHKT1;2 selectively transports Na+ rather than K+. The addition of K+ along with NaCl relieved the Na+ sensitivity. Furthermore, heterologous expression of SeHKT1;2 in sos1 mutant of Arabidopsis thaliana increased salt sensitivity and could not rescued the transgenic plants. This study will provide valuable gene resources for improving the salt tolerance in other crops by genetic engineering.

Novel auto-selection systems for transformation selection of Saccharomyces cerevisiae in rich complex media.

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry.

In vitro Antifungal Activity of Biopolymeric Foam Activated with Carvacrol

Background: Active packaging represents a defining strategy to improve food quality and safety of the packaged foods. This study aimed to evaluate the in vitro ability of commercial biopolymeric foams, namely Mater-Bi (MB), activated with 20% of carvacrol, to develop a completely biodegradable and compostable packaging to inhibit the growth of spoilage and pathogenic yeasts. Methods: MB foams, with and without carvacrol, were produced by melt mixing and the foaming process was performed in a laboratory press. The antifungal activity of foams containing carvacrol was tested applying the disk diffusion method. Statistical analysis was done using XLStat software version 7.5.2 for Excel. Results: Statistically significant differences (p<0.05) were observed between sensitivity of the tested yeasts. Candida zeylanoides 4G362 and Rhodotorula mucilaginosa ICE29 were found to be the most sensitive strains with a clear zone of 28.9±0.1 and 29.0±0.1 mm, respectively. However, Aureobasidium pullulans was the least sensitive yeast strain, showing clear zone of 20.4±0.3 mm. Conclusion: This study provided, for the first time, an in vitro analysis of the antifungal activity of MB foams activated with carvacrol against yeasts that commonly contaminate raw materials and processed foods. In conclusion, this biopolymer was highly effective against all the yeasts used as indicators strains.

Purification and characterization of Saccharomyces eubayanus killer toxin: Biocontrol effectiveness against wine spoilage yeasts

Microbiological contamination by spoilage yeasts species are frequent during winemaking, and biological control using antagonistic yeasts is considered a more beneficial alternative to conventional synthetic antimicrobials. Saccharomyces eubayanus killer toxin (SeKT) was produced and purified in a synthetic optimized medium. Purification procedure allowed the identification of SeKT as protein with an apparent molecular mass of 70 kDa and activity at physicochemical conditions suitable for winemaking process. Purified SeKT reduced the levels of volatile phenols produced by the spoilage yeasts Brettanomyces bruxellensis, Pichia membranifaciens, Meyerozyma guilliermondii and Pichia manshurica in wine-like medium. The putative mode of action of SeKT on sensitive yeast strains comprises cell wall disruption through β-glucanase and chitinase activities as well as necrotic and apoptotic death in a toxin dose dependent manner. Thus, SeKT appears to be a promising biocontrol agent against spoilage yeasts during wine aging and storing.

Metallomic and lipidomic analysis of S. cerevisiae response to cellulosic copper nanoparticles uncovers drivers of toxicity.

Nanotechnology is a promising new technology, of which antimicrobial metal nanocomposites are predicted to become valuable in medical and food packaging applications. Copper is a redox-active antimicrobial metal that can become increasingly toxic depending on the target biomolecule's donor atom selectivity and the chemical species of copper present. Mass is the traditional measurement of the intrinsic elemental chemistry, but this practice fails to reflect the morphology and surface area reactivity of nanotechnology. The carboxymethyl cellulose copper nanoparticles (CMC-Cu) investigated in this study have unique and undefined toxicity to Saccharomyces cerevisiae that is different from CuSO4. Cellular surface damage was found in scanning electron micrographs upon CMC-Cu exposure. Further investigation into the lipids revealed altered phosphatidylcholine and phosphatidylethanolamine membrane composition, as well as depleted triacylglycerols, suggesting an impact on the Kennedy lipid pathway. High levels of reactive oxygen species were measured which likely played a role in the lipid peroxidation detected with CMC-Cu treatment. Metal homeostasis was affected by CMC-Cu treatment. The copper sensitive yeast strain, YJM789, significantly decreased cellular zinc concentrations while the copper concentrations increased, suggesting a possible ionic mimicry relationship. In contrast to other compounds that generate ROS, no evidence of genotoxicity was found. As commonplace objects become more integrated with nanotechnology, humanity must look forward past traditional measurements of toxicity.

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.

Deciphering the in vivo redox behavior of human peroxiredoxins I and II by expressing in budding yeast

Peroxiredoxins (Prxs), scavenge cellular peroxides by forming recyclable disulfides but under high oxidative stress, hyperoxidation of their active-site Cys residue results in loss of their peroxidase activity. Saccharomyces cerevisiae deficient in human Prx (hPrx) orthologue TSA1 show growth defects under oxidative stress. They can be complemented with hPRXI but not by hPRXII, but it is not clear how the disulfide and hyperoxidation states of the hPrx vary in yeast under oxidative stress. To understand this, we used oxidative-stress sensitive tsa1tsa2Δ yeast strain to express hPRXI or hPRXII. We found that hPrxI in yeast exists as a mixture of disulfide-linked dimer and reduced monomer but becomes hyperoxidized upon elevated oxidative stress as analyzed under denaturing conditions (SDS-PAGE). In contrast, hPrxII was present predominantly as the disulfide in unstressed cells and readily converted to its hyperoxidized, peroxidase-inactive form even with mild oxidative stress. Interestingly, we found that plant extracts containing polyphenol antioxidants provided further protection against the growth defects of the tsa1tsa2Δ strain expressing hPrx and preserved the peroxidase-active forms of the Prxs. The extracts also helped to protect against hyperoxidation of hPrxs in HeLa cells. Based on these findings we can conclude that resistance to oxidative stress of yeast cells expressing individual hPrxs requires the hPrx to be maintained in a redox state that permits redox cycling and peroxidase activity. Peroxidase activity decreases as the hPrx becomes hyperoxidized and the limited protection by hPrxII compared with hPrxI can be explained by its greater sensitivity to hyperoxidation.

The Protective Effect of DAS and DADS Against Doxorubicin Induced Toxicity

PurposeCancer survival rates are increasing but the mechanisms involved in the adverse effects of cancer therapy such as doxorubicin are still being investigated along with treatment for these effects. Doxorubicin, an established chemotherapeutic agent used for various cancers, has limited use due to cardiotoxicity. The objective of this study, is to determine how garlic constituents, Diallyl sulfide (DAS) and Diallyl disulfide (DADS) may decrease the adverse effects of doxorubicin exposure.With a S. cerevisiae deletion library, a genome wide screen was conducted to detect mutants sensitive to doxorubicin exposure. A mutant deleted for the gene enconding for Mac1, a copper sensing transcription factor, was found to be highly sensitive. In S. cerevisiae the MAC1 gene is activated upon copper deficiency, resulting in the induction of copper transporters. Other doxorubicin sensitive strains included the sod1 mutant and its copper chaperone ccs1 mutant, both of which are associated with MAC1. The sensitivity of the mutant strains to doxorubicin, was in part due to the drug's capacity to generate reactive oxygen species (ROS). To test if garlic constituents, DAS and DADS could reduce the sensitivity of the mutants to ROS, the viability of the sensitive yeast strains were determined by exposing them to doxorubicin in the presence or absence of DAS and DADS.MethodsYeast survival assays were completed to determine the sensitivity of wt, mac1, ccs1, and sod1 strains to doxorubicin, menadione and etoposide, co‐treated with DAS and DADS. Statistical significance was set at p<0.05.Resultsmac1 shows sensitivity to doxorubicin and etoposide but not menadione. Menadione contains a quinone ring like doxorubicin, and acts through the induction of reactive oxygen species. Etoposide, a chemotherapeutic agent acts only through inhibition of DNA topoisomerase II. The sensitivity of mac1 to both etoposide and doxorubicin, shows that doxorubicin's sensitivity to mac1 is through its action on topoisomerase II. Unlike DAS, DADS was able to decrease the sensitivity of mac1 to doxorubicin and etoposide. Menadione has greater toxicity to ccs1 and sod1 than doxorubicin and etoposide, with DAS and DADS both increasing the survival of all chemicals to ccs1 and sod1.ConclusionGarlic constituents, das and dads may decrease the adverse effects of doxorubicin exposure, improving the quality of life of patients expose to doxorubicin.Support or Funding InformationResearch supported by National Institutes of Health (NIH) under Award Number G12MD007582This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris

BackgroundVirus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.ResultsIn this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.ConclusionSuccessful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus.

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na(+)/H(+) antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4% identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na(+) and accumulate more K(+). Expression of HtSOS1 decreased Na(+) content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.

Construction of a genetic analysis-available multidrug sensitive yeast strain by disruption of the drug efflux system and conditional repression of the membrane barrier system

Construction of a genetic analysis-available multidrug sensitive yeast strain by disruption of the drug efflux system and conditional repression of the membrane barrier system

Purification, characterization and gene cloning of the killer toxin produced by the marine-derived yeast Williopsis saturnus WC91-2

As the killer toxin produced by Williopsis saturnus WC91-2 could kill many sensitive yeast strains, including the pathogenic ones, the extracellular killer toxin in the supernatant of cell culture of the marine yeast strain was purified and characterized. The molecular mass of the purified killer toxin was estimated to be 11.0kDa according to the data from SDS-PAGE. The purified killer toxin had killing activity, but could not hydrolyze laminarin. The optimal conditions for action of the purified killer toxin against the pathogenic yeast Metschnikowia bicuspidate WCY were the assay medium with 10% NaCl, pH 3–3.5 and temperature 16°C. The gene encoding the killer toxin from the marine killer yeast WC91-2 was cloned and the ORF of the gene was 378bp. The deduced protein from the cloned gene encoding the killer toxin had 125 amino acids with calculated molecular weight of 11.6kDa. It was also found that the N-terminal amino acid sequence of the purified killer toxin had the same corresponding sequence deduced from the cloned killer toxin gene in this marine yeast, confirming that the purified killer toxin was indeed encoded by the cloned gene.

Ectopic expression of a LEA protein gene TsLEA1 from Thellungiella salsuginea confers salt-tolerance in yeast and Arabidopsis

Thellungiella salsuginea is a valuable halophytic genetic model plant in the Brassicaceae family. Based on previous construction of a salt treated Thellungiella cDNA library carried by pGAD-GH shuttle vector which could directly express in Saccharomyces cerevisiae, a putative salt-tolerance gene TsLEA1 was identified by large-scale stress-tolerance screen in salt sensitive yeast strain G19. The longest 483bp ORF of TsLEA1 cDNA coding a 160 amino acids protein with a predicted conserved pfam domain shared an 89% amino acid sequence similarity to Arabidopsis LEA group 4 proteins. The transcription level of TsLEA1 gene in T. salsuginea seedlings increased upon salt treatment and its transcript accumulated more in roots than in aerial parts. The ability of the TsLEA1 to facilitate salinity tolerance was analyzed in yeast and transgenic Arabidopsis. It was confirmed that TsLEA1 exhibits conserved salt tolerance in plant as well as in yeast. The results suggested that the TsLEA1 may participate in response to stresses in over expressed circumstance, protecting yeast and plant cells under stress conditions.

Cloning and heterologous expression of candidate DON-inactivating UDP-glucosyltranferases from rice and wheat in yeast

Fusarium graminearum and related species causing Fusarium head blight of cereals and ear rot of maize produce the trichothecene toxin and virulence factor deoxynivalenol (DON). Plants can detoxify DON to a variable extent into deoxynivalenol-3-O-glucoside (D3G). We have previously reported the DON inactivat- ing glucosyltransferase (UGT) AtUGT73C5 from Arabidopsis thaliana (Poppenberger et al, 2003). Our goal was to identify UGT genes from monocotyledonous crop plants with this enzymatic activity. The two selected rice candidate genes with the highest sequence similarity with AtUGT73C5 were expressed in a toxin sensitive yeast strain but failed to protect against DON. A full length cDNA clone corresponding to a transcript derived fragment (TDF108) from wheat, which was reported to be specifically expressed in wheat genotypes contain- ing the quantitative trait locus Qfhs.ndsu-3BS for Fusarium spreading resistance (Steiner et al, 2009) was reconstructed. Only cDNAs with a few sequence deviations from TF108 could be cloned. However, toxin sensitive yeast strains expressing this wheat UGT cDNA did not show a resistant phenotype. The main diffi- culty in generating full length cDNAs for functional validation by heterologous expression in yeast is the enormous number of the UGT superfamily members in plants, with 107 UGT genes plus some pseudogenes in Arabidopsis thaliana and about 150 putative UGT genes in grasses. We conclude that neither sequence simi- larity nor inducibility are good predictors of substrate specificity.

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn(2+) nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn(2+) and Cd(2+) chelator with capacity to bind 10 Zn(2+) ions per C terminus. When AtHMA4 is expressed in a Zn(2+)-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn(2+) and Cd(2+) tolerance and lowered Zn(2+) and Cd(2+) content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn(2+) and Cd(2+) chelator (sensor) and as a regulator of the efficiency of Zn(2+) and Cd(2+) export. The identification of a post-translational handle on Zn(2+) and Cd(2+) transport efficiency opens new perspectives for regulation of Zn(2+) nutrition and tolerance in eukaryotes.

Optimization of a Small-scale Fermentation Test to Predict the Premature Yeast Flocculation Potential of Malts

Rapid and early detection of malts likely to cause premature yeast flocculation (PYF) is of major importance both to the brewer and the maltster. To date, the most reliable and widely applied diagnostic tests for predicting the PYF status of malts involve conducting a small scale fermentation test, which usually takes several days to be completed. This study investigated the impact of selected factors on the ability of such a test to distinguish between PYF positive and PYF negative malts and the fermentation time after which the test results, and therefore the PYF status of the malts, could be confirmed. The results obtained suggest that the PYF potential of malts could be predicted 40 hours post pitching by adding 6 mg.L−1 linoleic acid and using a highly flocculent PYF sensitive yeast strain.

Occurrence of killer yeast strains in industrial and clinical yeast isolates.

The secretion of proteinaceous toxins is a widespread characteristic in environmental and laboratory yeast isolates, a phenomenon called "killer system". The killer phenotype (K+) can be encoded by extrachromosomal genetic elements (EGEs) as double stranded DNA or RNA molecules (dsDNA, dsRNA) or in nuclear genes. The spectrum of action and the activity of killer toxins are influenced by temperature, salinity and pH of media. In the present work we determined the existence of K+ in a collection of S. cerevisiae and P. anomala yeasts isolated from environmental, industrial and clinical sources. The assays were performed in strains belonging to three yeast genera used as sensitive cells and under a wide range of pH and temperatures. Approximately 51 % of isolates tested showed toxicity against at least one sensitive yeast strain under the conditions tested. The K+ P. anomala isolates showed a wide spectrum of action and two of them had toxic activity against strains of the three yeast genera assayed, including C. albicans strains. In all S. cerevisiae K+ isolates an extrachromosomal dsRNA molecule (4.2 Kb) was observed, contrary to P. anomala K+ isolates, which do not possess any EGEs. The K+ phenotype is produced by an exported protein factor and the kinetics of killer activity production was similar in all isolates with high activity in the log phase of growth, decaying in the stationary phase.

Ubiquitin and fusarium resistance: Lessons from wheat cDNAS conferring deoxynivalenol resistance in yeast

The production of the trichothecene mycotoxin deoxynivalenol (DON) is an important virulence factor of the plant pathogenic fungus Fusarium graminearum on wheat. We have engineered a DON sensitive yeast strain and constructed a cDNA library from DON treated wheat suspension culture cells in a yeast expression vector. The library was used to select DON resistance conferring clones. Besides ORFs of unknown function, we found 3 classes of cDNAs that in addition to DON resistance conferred hypersensitivity to hygromycin and canavanine. The predicted functions of several of the wheat cDNAs (putative E3 ligase, ubiquitin specific protease, proteasome subunit) suggested a role for ubiquitin-proteasome mediated protein degradation in DON resistance. Results with a coupled wheat germ in vitro translation system and a GUS-luciferase fusion gene showed that DON is a powerful translation elongation inhibitor. The truncated proteins formed in the presence of DON most likely lead to ubiquitin depletion and consequently growth inhibition in yeast. Ubiquitin is essential for many processes in plants, including plant defense. Our results warrant the re-evaluation of the relevance of proteasome system components found to be differentially regulated during Fusarium infection.