Use of GRF-GIF chimeras and a ternary vector system to improve maize (Zea mays L.) transformation frequency.

Maize (Zea mays L.) is an important crop that has been widely studied for its agronomic and industrial applications and is one of the main classical model organisms for genetic research. Agrobacterium-mediated transformation of immature maize embryos is a commonly used method to introduce transgenes, but a low transformation frequency remains a bottleneck for many gene-editing applications. Previous approaches to enhance transformation included the improvement of tissue culture media and the use of morphogenic regulators such as BABY BOOM and WUSCHEL2. Here, we show that the frequency can be increased using a pVS1-VIR2 virulence helper plasmid to improve T-DNA delivery, and/or expressing a fusion protein between a GROWTH-REGULATING FACTOR (GRF) and GRF-INTERACTING FACTOR (GIF) protein to improve regeneration. Using hygromycin as a selection agent to avoid escapes, the transformation frequency in the maize inbred line B104 significantly improved from 2.3 to 8.1% when using the pVS1-VIR2 helper vector with no effect on event quality regarding T-DNA copy number. Combined with a novel fusion protein between ZmGRF1 and ZmGIF1, transformation frequencies further improved another 3.5- to 6.5-fold with no obvious impact on plant growth, while simultaneously allowing efficient CRISPR-/Cas9-mediated gene editing. Our results demonstrate how a GRF-GIF chimera in conjunction with a ternary vector system has the potential to further improve the efficiency of gene-editing applications and molecular biology studies in maize.

A myo-inositol dehydrogenase involved in aminocyclitol biosynthesis of hygromycin A.

Hygromycin A is a broad-spectrum antibiotic that contains a furanose, cinnamic acid, and aminocyclitol moieties. The biosynthesis of the aminocyclitol has been proposed to proceed through six enzymatic steps from glucose 6-phosphate through myo-inositol to the final methylenedioxy-containing aminocyclitol. Although there is some in vivo evidence for this proposed pathway, biochemical support for the individual enzyme activities is lacking. In this study, we verify the activity for one enzyme in this pathway. We show that Hyg17 is a myo-inositol dehydrogenase that has a unique substrate scope when compared to other myo-inositol dehydrogenases. Furthermore, we analyze sequences from the protein family containing Hyg17 and discuss genome mining strategies that target this protein family to identify biosynthetic clusters for natural product discovery.

Translation initiation factor eIF1A rescues hygromycin B sensitivity caused by deleting the carboxy-terminal tail in the GPN-loop GTPase Npa3.

The essential yeast protein GPN-loop GTPase 1 (Npa3) plays a critical role in RNA polymerase II (RNAPII) assembly and subsequent nuclear import. We previously identified a synthetic lethal interaction between a mutant lacking the carboxy-terminal 106-amino acid tail of Npa3 (npa3ΔC) and a bud27Δ mutant. As the prefoldin-like Bud27 protein participates in ribosome biogenesis and translation, we hypothesized that Npa3 may also regulate these biological processes. We investigated this proposal by using Saccharomyces cerevisiae strains episomally expressing either wild-type Npa3 or hypomorphic mutants (Npa3ΔC, Npa3K16R, and Npa3G70A). The Npa3ΔC mutant fully supports RNAPII nuclear localization and activity. However, the Npa3K16R and Npa3G70A mutants only partially mediate RNAPII nuclear targeting and exhibit a higher reduction in Npa3 function. Cell proliferation in these strains displayed an increased sensitivity to protein synthesis inhibitors hygromycin B and geneticin/G418 (npa3G70A > npa3K16R > npa3ΔC > NPA3 cells) but not to transcriptional elongation inhibitors 6-azauracil, mycophenolic acid or 1,10-phenanthroline. In all three mutant strains, the increase in sensitivity to both aminoglycoside antibiotics was totally rescued by expressing NPA3. Protein synthesis, visualized by quantifying puromycin incorporation into nascent-polypeptide chains, was markedly more sensitive to hygromycin B inhibition in npa3ΔC, npa3K16R, and npa3G70A than NPA3 cells. Notably, high-copy expression of the TIF11 gene, that encodes the eukaryotic translation initiation factor 1A (eIF1A) protein, completely suppressed both phenotypes (of reduced basal cell growth and increased sensitivity to hygromycin B) in npa3ΔC cells but not npa3K16R or npa3G70A cells. We conclude that Npa3 plays a critical RNAPII-independent and previously unrecognized role in translation initiation.

RNA-based detection of genetically modified plants via current-voltage characteristic measurement.

The widespread adoption of genetically modified (GM) crops has escalated concerns about their safety and ethical implications, underscoring the need for efficient GM crop detection methods. Conventional detection methods, such as polymerase chain reaction, can be costly, lab-bound, and time-consuming. To overcome these challenges, we have developed RapiSense, a cost-effective, portable, and sensitive biosensor platform. This sensor generates a measurable voltage shift (0.1-1 V) in the system's current-voltage characteristics, triggered by an increase in membrane's negative charge upon hybridization of DNA/RNA targets with a specific DNA probe. Probes designed to identify the herbicide resistance gene hygromycin phosphotransferase show a detection range from ∼1 nM to ∼10 µM and can discriminate between complementary, non-specific, and mismatched nucleotide targets. The incorporation of a small membrane sensor to detect fragmented RNA samples substantially improve the platform's sensitivity. In this study, RapiSense has been effectively used to detect specific DNA and fragmented RNA in transgenic variants of Arabidopsis, sweet potato, and rice, showcasing its potential for rapid, on-site GM crop screening.

Taxonomic characterization of Pseudomonas hygromyciniae sp. nov., a novel species discovered from a commercially purchased antibiotic.

In an attempt to identify novel bacterial species, microbiologists have examined a wide range of environmental niches. We describe the serendipitous discovery of a novel gram-negative bacterial species from a different type of extreme niche: a purchased vial of antibiotic. The vial of antibiotic hygromycin B was found to be factory contaminated with a bacterial species, which we designate Pseudomonas hygromyciniae sp. nov. The proposed novel species belongs to the P. fluorescens complex and is most closely related to P. brenneri, P. proteolytica, and P. fluorescens. The type strain Pseudomonas hygromyciniae sp. nov. strain SDM007T (SDM007T) harbors a novel 250 kb megaplasmid which confers resistance to hygromycin B and contains numerous other genes predicted to encode replication and conjugation machinery. SDM007T grows in hygromycin concentrations of up to 5 mg/mL but does not use the antibiotic as a carbon or nitrogen source. While unable to grow at 37°C ruling out its ability to infect humans, it grows and survives at temperatures between 4 and 30°C. SDM007T can infect plants, as demonstrated by the lettuce leaf model, and is highly virulent in the Galleria mellonella infection model but is unable to infect mammalian A549 cells. These findings indicate that commercially manufactured antibiotics represent another extreme environment that may support the growth of novel bacterial species. IMPORTANCE Physical and biological stresses in extreme environments may select for bacteria not found in conventional environments providing researchers with the opportunity to not only discover novel species but to uncover new enzymes, biomolecules, and biochemical pathways. This strategy has been successful in harsh niches such as hot springs, deep ocean trenches, and hypersaline brine pools. Bacteria belonging to the Pseudomonas species are often found to survive in these unusual environments, making them relevant to healthcare, food, and manufacturing industries. Their ability to survive in a variety of environments is mainly due to the high genotypic and phenotypic diversity displayed by this genus. In this study, we discovered a novel Pseudomonas sp. from a desiccated environment of a sealed antibiotic bottle that was considered sterile. A close genetic relationship with its phylogenetic neighbors reiterated the need to use not just DNA-based tools but also biochemical characteristics to accurately classify this organism.

Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae.

Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

Antimicrobial mechanisms and secondary metabolite profiles of Streptomyces hygroscopicus subsp. hygroscopicus 5-4 against banana fusarium wilt disease using metabolomics.

Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungus Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identified Streptomyces hygroscopicus subsp. hygroscopicus 5-4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5-4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5-4 cultivated at different times were characterized by a liquid chromatography-mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5-4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5-4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50 concentration was 7.4 µg/mL. These results showed that strain 5-4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite. Streptomyces hygroscopicus subsp. hygroscopicus 5-4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent.

Evaluation of Antibiotic-Based Selection Methods for Camelina sativa Stable Transformants.

Camelina sativa (Camelina) is an oilseed crop that in recent years has gained importance due to its closeness to the plant model organism Arabidopsis thaliana (Arabidopsis), its low agronomical requirements, and the ability to grow under temperate conditions. To explore all the agronomical and biotechnological possibilities of this crop, it is important to evaluate the usability of the molecular procedures currently available for plants. One of the main tools for plant genetic modification and genetic studies is stable plant transformation. In the case of Arabidopsis, as well as Camelina, floral dipping is the easiest and most used method, which is followed by a selection for stable transformants. Commonly used selection methods for Camelina involve Discosoma sp. red protein (DsRed) fluorescence screening. However, many widely used plant transformation vector systems, for example those used in Arabidopsis and grasses, rely on antibiotic resistance selection. In this study, we evaluated the usability of different antibiotics including kanamycin (Kan), hygromycin (Hyg) and BASTA, and propose optimised protocols for selecting T1 and subsequent generation Camelina transformants, as well as crossing of Camelina lines expressing different transgenes. Finally, we also showed that overexpression of genes encoding enzymes from the seco-iridoid pathway of Catharanthus roseus using Hyg or BASTA-based expression constructs could be successfully achieved in Camelina, demonstrating the potential of these methods for metabolic engineering. Overall, in this study we show an efficient way to sterilize seeds, handle and perform selection of Camelina for use with transformation vectors designed for Arabidopsis thaliana. We also demonstrate a successful method to cross Camelina sativa and provide qRT-PCR results to prove its effectiveness.

Degron tagging of BleoR and other antibiotic-resistance genes selects for higher expression of linked transgenes and improved exosome engineering.

Five antibiotic resistance (AR) genes have been used to select for transgenic eukaryotic cell lines, with the BleoR, PuroR, HygR, NeoR, and BsdR cassettes conferring resistance to zeocin, puromycin, hygromycin, geneticin/G418, and blasticidin, respectively. We recently demonstrated that each AR gene establishes a distinct threshold of transgene expression below which no cell can survive, with BleoR selecting for the highest level of transgene expression, nearly ∼10-fold higher than in cells selected using the NeoR or BsdR markers. Here, we tested the hypothesis that there may be an inverse proportionality between AR protein function and the expression of linked, transgene-encoded, recombinant proteins. Specifically, we fused each AR protein to proteasome-targeting degron tags, used these to select for antibiotic-resistant cell lines, and then measured the expression of the linked, recombinant protein, mCherry, as a proxy marker of transgene expression. In each case, degron-tagged AR proteins selected for higher mCherry expression than their cognate WT AR proteins. ER50BleoR selected for the highest level of mCherry expression, greater than twofold higher than BleoR or any other AR gene. Interestingly, use of ER50BleoR as the selectable marker translated to an even higher, 3.5-fold increase in the exosomal loading of the exosomal cargo protein, CD63/Y235A. Although a putative CD63-binding peptide, CP05, has been used to decorate exosome membranes in a technology known as "exosome painting," we show here that CP05 binds equally well to CD63-/- cells, WT 293F cells, and CD63-overexpressing cells, indicating that CP05 may bind membranes nonspecifically. These results are of high significance for cell engineering and especially for exosome engineering.

Covalent molecularly imprinted electrochemical sensor modulated by borate ester bonds for hygromycin B detection based on the synergistic signal amplification of Cu-MOF and MXene.

A molecularly imprinted electrochemical sensor (MIES) modified with Cu-MOF and Ti3C2Tx was successfully prepared for the detection of hygromycin B in food. According to the pH sensitive property of reversible borate ester bonds, a molecularly imprinted polymers (MIP) were constructed on the modified gold electrode (GE) surface by electropolymerization to realize the controllable elution and adsorption of template molecules. Cu-MOF with large specific surface area and Ti3C2Tx with excellent electrical conductivity cooperate to improve the overall analytical performance of MIES. The morphology and electrochemical performance of the sensor were evaluated. Under the optimal experimental conditions, the method showed superior selectivity to hygromycin B with a linear range of 5 × 10-9-5 × 10-6 M and a detection limit of 1.92 × 10-9 M (S/N = 3). The detection results of this method are in good agreement with HPLC-MS/MS, which proves that the method has reliable practicability and accuracy.

Development of an Agrobacterium tumefaciens-mediated transformation system for Tilletia controversa Kühn.

Dwarf bunt of wheat caused by Tilletia controversa Kühn has been identified an international quarantine disease, which replace the grain material into millions of teliospores. Agrobacterium tumefaciens-mediated transformation (ATMT) system is a powerful tool for fungi transformation with significant advantages of simple operation, high efficiency, and genetic stability of transformants. In this study, we constructed ATMT system for T. controversa. All the transformants were tested using Acetosyringone (AS) concentration at 150 µmol/l, hygromycin B at 25 µg/ml, 1 × 106 T. controversa hypha cells/ml, A. tumefaciens with OD600 of 0.5 co-cultivation at 16 °C for 48 h and culture was incubated at 16 °C for 20 days. Using the ATMT method, we cultivated 8 generations of transformants on complete medium (CM) containing hygromycin B antibiotic and validated by PCR, which indicate that T-DNA had been successfully inserted into each of T. controversa transformants. In addition, thermal asymmetric interlaced PCR (TAIL-PCR) evaluated the Ti element inserts were at random sites in the fungal genome. Thus, ATMT approach is an efficient tool for insertional mutagenesis of T. controversa.

Collaborative Study of Thresholds for Mutagens: Hormetic Responses in Cell Proliferation Tests Using Human and Murine Lymphoid Cells.

BACKGROUND: We previously showed that hormetic responses can be established in cell activity tests using human and murine adherent cells. This time, we examined whether hormetic responses can be established in cell proliferation tests using suspended human and murine lymphoid cells. METHODS: Human lymphoblastoid cells (TK6) and mouse lymphoma cells (L5178Y) were cultured in multi-well culture plates and treated with mitomycin C, ethyl methansulfonate, hygromycin B, aclarubicin or colchicine at various dose levels and the number of cells was measured at varied times using a flow cytometer. RESULTS: When the ratio of the number of cells treated with a test chemical to those in the negative control was plotted, the dose-response relationship typically showed a reverse U-shaped curve, indicating the occurrence of hormesis and existence of thresholds in cell toxicity. The hormetic responses depended largely on the test chemical, dose level and exposure time. When examining responses over the course of time, a J-shaped or fallen S-shaped curve was also observed. CONCLUSIONS: The dose-response relationship showed a reverse U-shaped curve, a hallmark of hormesis, at least some time points for all chemicals tested here, indicating that chemical hormesis can be established in in vitro cell proliferation tests.

Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation.

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

SCRaMbLE: A Study of Its Robustness and Challenges through Enhancement of Hygromycin B Resistance in a Semi-Synthetic Yeast.

Recent advances in synthetic genomics launched the ambitious goal of generating the first synthetic designer eukaryote, based on the model organism Saccharomyces cerevisiae (Sc2.0). Excitingly, the Sc2.0 project is now nearing its completion and SCRaMbLE, an accelerated evolution tool implemented by the integration of symmetrical loxP sites (loxPSym) downstream of almost every non-essential gene, is arguably the most applicable synthetic genome-wide alteration to date. The SCRaMbLE system offers the capability to perform rapid genome diversification, providing huge potential for targeted strain improvement. Here we describe how SCRaMbLE can evolve a semi-synthetic yeast strain housing the synthetic chromosome II (synII) to generate hygromycin B resistant genotypes. Exploiting long-read nanopore sequencing, we show that all structural variations are due to recombination between loxP sites, with no off-target effects. We also highlight a phenomenon imposed on SCRaMbLE termed "essential raft", where a fragment flanked by a pair of loxPSym sites can move within the genome but cannot be removed due to essentiality restrictions. Despite this, SCRaMbLE was able to explore the genomic space and produce alternative structural compositions that resulted in an increased hygromycin B resistance in the synII strain. We show that among the rearrangements generated via SCRaMbLE, deletions of YBR219C and YBR220C contribute to hygromycin B resistance phenotypes. However, the hygromycin B resistance provided by SCRaMbLEd genomes showed significant improvement when compared to corresponding single deletions, demonstrating the importance of the complex structural variations generated by SCRaMbLE to improve hygromycin B resistance. We anticipate that SCRaMbLE and its successors will be an invaluable tool to predict and evaluate the emergence of antibiotic resistance in yeast.

Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana.

The green oleaginous microalgae, Chlorella sorokiniana, is a highly productive Chlorella species and a potential host for the production of biofuel, nutraceuticals, and recombinant therapeutic proteins. The lack of a stable and efficient genetic transformation system is the major bottleneck in improving this species. We report an efficient and stable Agrobacterium tumefaciens-mediated transformation system for the first time in C. sorokiniana. Cocultivation of C. sorokiniana cells (optical density at λ 680 = 1.0) with Agrobacterium at a cell density of OD600 = 0.6, on BG11 agar medium (pH 5.6) supplemented with 100 µM of acetosyringone, for three days at 25 ± 2 °C in the dark, resulted in significantly higher transformation efficiency (220 ± 5 hygromycin-resistant colonies per 106 cells). Transformed cells primarily selected on BG11 liquid medium with 30 mg/L hygromycin followed by selecting homogenous transformants on BG11 agar medium with 75 mg/L hygromycin. PCR analysis confirmed the presence of hptII, and the absence of virG amplification ruled out the Agrobacterium contamination in transformed microalgal cells. Southern hybridization confirmed the integration of the hptII gene into the genome of C. sorokiniana. The qRT-PCR and Western blot analyses confirmed hptII and GUS gene expression in the transgenic cell lines. The specific growth rate, biomass doubling time, PSII activity, and fatty-acid profile of transformed cells were found similar to wild-type untransformed cells, clearly indicating the growth and basic metabolic processes not compromised by transgene expression. This protocol can facilitate opportunities for future production of biofuel, carotenoids, nutraceuticals, and therapeutic proteins. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02750-7.

Efficient Agrobacterium tumefaciens-mediated transformation system of Diaporthe caulivora.

This is the first report describing the genetic transformation of Diaporthe caulivora, the soybean stem canker fungus. A simple and 100% efficient protocol of Agrobacterium tumefaciens-mediated transformation used mycelium as starting material and the hygromycin B resistance and green fluorescent protein (GFP) as a selection and reporter agents, respectively. All transgenic isolates were mitotically stable in two independent experiments and polymerase chain reaction with hygromycin B resistance primers confirmed successful T-DNA integration into the fungal genome. Plant-fungus interaction studies, including pathogenicity, latency, and endophytism, as well as further studies of random and targeted mutagenesis will be possible with GFP-expressing isolates of D. caulivora and other species in the Diaporthe / Phomopsis complex.

Resistance Marker- and Gene Gun-Mediated Transformation of Trichoderma reesei.

Transformation enables the transfer of DNA into fungal cells for subsequent integration into the genome. Due to its versatility in industrial application, transformation is of utmost importance in Trichoderma reesei and hence continuously optimized. As one of the most crucial obstacles in fungal transformation efforts, removal of the cell wall is required to efficiently target genome modification cassettes to the genome. Here we describe resistance marker-mediated gene gun (biolistic) transformation of fungal spores of T. reesei as an alternative to protoplast transformation.

Enhanced resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B induced by sea salt.

OBJECTIVES: To determine the effect of sea salt on the resistance of Trichoderma harzianum LZDX-32-08 to hygromycin B and speculate the possible mechanisms involved via transcriptome analysis. RESULTS: Sea salt addition in media to simulate marine environment significantly increased the tolerance of marine-derived fungus Trichoderma harzianum LZDX-32-08 to hygromycin B from 40 to 500 µg/ml. Meanwhile, sea salt addition also elicited the hygromycin B resistance of 5 other marine or terrestrial fungi. Transcriptomic analyses of T. harzianum cultivated on PDA, PDA supplemented with sea salt and PDA with both sea salt and hygromycin B revealed that genes coding for P-type ATPases, multidrug resistance related transporters and acetyltransferases were up-regulated, while genes coding for Ca2+/H+ antiporter and 1,3-glucosidase were down-regulated, indicating probable increased efflux and inactivation of hygromycin B as well as enhanced biofilm formation, which could jointly contribute to the drug resistance. CONCLUSIONS: Marine environment or high ion concentration in the environment could be an importance inducer for antifungal resistance. Possible mechanisms and related key genes were proposed for understanding the molecular basis and overcoming this resistance.

Agrobacterium tumefaciens-Mediated Genetic Transformation of the Ect-endomycorrhizal Fungus Terfezia boudieri.

Mycorrhizal desert truffles such as Terfezia boudieri, Tirmania nivea, and Terfezia claveryi, form mycorrhizal associations with plants of the Cistaceae family. These valued truffles are still collected from the wild and not cultivated under intensive farming due to the lack of basic knowledge about their biology at all levels. Recently, several genomes of desert truffles have been decoded, enabling researchers to attempt genetic manipulations to enable cultivation. To execute such manipulations, the development of molecular tools for genes transformation into truffles is needed. We developed an Agrobacterium tumefaciens-mediated genetic transformation system in T. boudieri. This system was optimized for the developmental stage of the mycelia explants, bacterial optical density, infection and co-cultivation durations, and concentrations of the selection antibiotics. The pFPL-Rh plasmid harboring hph gene conferring hygromycin resistance as a selection marker and the red fluorescent protein gene were used as visual reporters. The optimal conditions were incubation with 200 µM of acetosyringone, attaining a bacterial optical density of 0.3 OD600; transfer time of 45 min; and co-cultivation for 3 days. This is the first report on a transformation system for T. boudieri, and the proposed protocol can be adapted for the transformation of other important desert truffles as well as ectomycorrhizal species.

Selection markers for transformation of the sequenced reference monokaryon Okayama 7/#130 and homokaryon AmutBmut of Coprinopsis cinerea.

BACKGROUND: Two reference strains have been sequenced from the mushroom Coprinopsis cinerea, monokaryon Okayama 7/#130 (OK130) and the self-compatible homokaryon AmutBmut. An adenine-auxotrophy in OK130 (ade8-1) and a para-aminobenzoic acid (PABA)-auxotrophy in AmutBmut (pab1-1) offer selection markers for transformations. Of these two strains, homokaryon AmutBmut had been transformed before to PABA-prototrophy and with the bacterial hygromycin resistance marker hph, respectively. RESULTS: Gene ade8 encodes a bifunctional enzyme with an N-terminal glycinamide ribonucleotide synthase (GARS) and a C-terminal aminoimidazole ribonucleotide synthase (AIRS) domain required for steps 2 and 5 in the de novo biosynthesis of purines, respectively. In OK130, a missense mutation in ade8-1 rendered residue N231 for ribose recognition by the A loop of the GARS domain into D231. The new ade8 + vector pCcAde8 complements the auxotrophy of OK130 in transformations. Transformation rates with pCcAde8 in single-vector and co-transformations with ade8 +-selection were similarly high, unlike for trp1 + plasmids which exhibit suicidal feedback-effects in single-vector transformations with complementation of tryptophan synthase defects. As various other plasmids, unselected pCcAde8 helped in co-transformations of trp1 strains with a trp1 +-selection vector to overcome suicidal effects by transferred trp1 +. Co-transformation rates of pCcAde8 in OK130 under adenine selection with nuclear integration of unselected DNA were as high as 80% of clones. Co-transformation rates of expressed genes reached 26-42% for various laccase genes and up to 67% with lcc9 silencing vectors. The bacterial gene hph can also be used as another, albeit less efficient, selection marker for OK130 transformants, but with similarly high co-transformation rates. We further show that the pab1-1 defect in AmutBmut is due to a missense mutation which changed the conserved PIKGT motif for chorismate binding in the C-terminal PabB domain to PIEGT in the mutated 4-amino-4-deoxychorismate synthase. CONCLUSIONS: ade8-1 and pab1-1 auxotrophic defects in C. cinerea reference strains OK130 and AmutBmut for complementation in transformation are described. pCcAde8 is a new transformation vector useful for selection in single and co-transformations of the sequenced monokaryon OK130 which was transformed for the first time. The bacterial gene hph can also be used as an additional selection marker in OK130, making in combination with ade8 + successive rounds of transformation possible.