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We intend to investigate the drug-binding energy of each nucleotide inside the aminoglycoside hygromycin B (hygB) binding site of 30S ribosomal RNA (rRNA) subunit by using the molecular fractionation with conjugate caps (MFCC) strategy based on the density functional theory (DFT), considering the functional LDA/PWC, OBS, and the dielectric constant parametrization. Aminoglycosides are bactericidal antibiotics that have high affinity to the prokaryotic rRNA, inhibiting the synthesis of proteins by acting on the main stages of the translation mechanism, whereas binding to rRNA 16S, a component of the 30S ribosomal subunit in prokaryotes. The identification of the nucleotides presenting the most negative binding energies allows us to stabilize hygB in a suitable binding pocket of the 30S ribosomal subunit. In addition, it should be highlighted that mutations in these residues may probably lead to resistance to ribosome-targeting antibiotics. Quantum calculations of aminoglycoside hygromycin B-ribosome complex might contribute to further quantum studies with antibiotics like macrolides and other aminoglycosides.

Owing to the essential role in protection of the Aspergillus fumigatus cell against human defense reactions, its cell wall has long been taken as a promising antifungal target. The cell wall of A. fumigatus composed of chitin, glucan and galactomannan and mannoproteins. Although galactomannan has been used as a diagnostic target for a long time, its biosynthesis remains unknown in A. fumigatus. In this study, a putative α1,6-mannosyltransferase gene mnn9 was identified in A. fumigatus. Deletion of the mnn9 gene resulted in an increased sensitivity to calcofluor white, Congo red, or hygromycin B as well as in reduced cell wall components and abnormal polarity. Although there was no major effect on N-glycan synthesis, covalently-linked cell wall mannoprotein Mp1 was significantly reduced in the mutant. Based on our results, we propose that Mnn9p is a mannosyltransferase responsible for the formation of the α-mannan in cell wall mannoproteins, potentially via elongation of O-linked mannose chains.

A genetic transformation system was developed for the selective white rot basidiomycete Ceriporiopsis subvermispora using a modified protocol with polyethylene glycol and CaCl2 treatment of the protoplasts and plasmids harboring recombinant hygromycin phosphotransferase (hph) driven by a homologous promoter. During repeated transfer on fresh potato dextrose agar plates containing 100 µg/ml hygromycin B, most transformants lost drug resistance, while the remaining isolates showed stable resistance over five transfers. No drug-resistant colonies appeared in control experiments without DNA or using a promoter-less derivative of the plasmid, indicating that a transient expression of the recombinant hph was driven by the promoter sequence in these unstable drug-resistant transformants. Southern blot analysis of the stable transformants revealed random integration of the plasmid DNA fragment in the chromosome at different copy numbers. This transformation system yielding mostly transient transformants was successfully used for promoter assay experiments, and only a 141-bp fragment was found to be essential for the basic promoter function of glyceraldehyde dehydrogenase gene (gpd) in this fungus. Subsequent mutational analyses suggested that a TATAA sequence is important for the basic promoter function of gpd gene. The promoter assay system will enable the functional analysis of gene expression control sequences quickly and easily, mostly in the absence of undesirable effects from differences in copy number and chromosomal position of an integrated reporter gene among stable transformants.

The genetic-exchange process of some endophytic root fungi whose teleomorphs have not been discovered is unknown. To assess parasexuality in the root-colonizing fungus Glutinomyces brunneus, we investigated the transmission of genetic markers in this fungus. We generated strains resistant to benomyl and hygromycin B and performed co-cultivation; dual resistance was used as a selection marker for genetic exchange between hyphae. Co-cultivation of benomyl- and hygromycin B-resistant strains yielded significantly larger numbers of dual-resistant colonies than did co-cultivation of strains resistant to the same agent, which suggests genetic exchange between the two vegetative hyphae. This was not simply due to heterokaryosis because all dual-resistant strains were monokaryotic. Also, half of the analyzed dual-resistant strains were homozygous in β-tubulin gene which is associated with benomyl resistance; thus, their simple diploidization was rejected for these strains. During selective cultivation, temporal hyphal growth occurred from the point at which two hyphae touched, which stopped after 2 weeks; this was followed by growth of true dual-resistant hyphae. Transmission of resistance markers was more frequent during co-cultivation on 2% malt extract agar than on other media, which suggests a role of medium composition. Genetic exchange between resistant strains that did not share an ancestor was limited, which implies vegetative incompatibility in this fungus. Our findings suggest the partial presence of parasexual potential in G. brunneus.

Volvox carteri is an excellent model for investigating the evolution of multicellularity and cell differentiation, and the rate of future progress with this system will depend on improved molecular genetic tools. Several selectable markers for nuclear transformation of V. carteri have been developed, including the nitrate reductase (nitA) gene, but it would be useful to have additional markers to multiplex transgenes in this species. To further facilitate molecular genetic analyses of V. carteri, we developed two new selectable markers that provide rapid, easily selected, and stable resistance to the antibiotics hygromycin and blasticidin. We generated constructs with Volvox-specific regulatory sequences and codon-optimized hygromycin (VcHyg) and blasticidin (VcBlast) resistance genes from Coccidioides posadasii and Bacillus cereus, respectively. With these constructs, transformants were obtained via biolistic bombardment at rates of 0.5-13 per million target cells bombarded. Antibiotic-resistant survivors were readily isolated 7days post bombardment. VcHyg and VcBlast transgenes and transcripts were detected in transformants. Co-transformation rates using the VcHyg or VcBlast markers with unselected genes were comparable to those obtained with nitA. These results indicate that the pVcHyg and pVcBlast plasmids are highly efficient and convenient for transforming and co-transforming a broad range of V. carteri strains.

In yeast, the STB5 gene encodes a transcriptional factor belonging to binuclear cluster class (Zn2Cys6) of transcriptional regulators specific to ascomycetes. In this study, we prepared the Kluyveromyces lactis stb5Δ strain and assessed its responses to different stresses. We showed that KlSTB5 gene is able to complement the deficiencies of Saccharomyces cerevisiae stb5Δ mutant. The results of phenotypic analysis suggested that KlSTB5 gene deletion did not sensitize K. lactis cells to oxidative stress inducing compounds but led to Klstb5Δ resistance to 4-nitroquinoline-N-oxide and hygromycin B. Expression analysis indicated that the loss of KlSTB5 gene function induced the transcription of drug efflux pump encoding genes that might contribute to increased 4-nitroquinoline-N-oxide and hygromycin B tolerance. Our results show that KlStb5p functions as negative regulator of some ABC transporter genes in K. lactis.

An ability to synthesize extracellular enzymes degrading a wide spectrum of plant and algae polymeric substrates makes many fungi relevant for biotechnology. The terrestrial thermophilic and marine fungal isolates capable of plant and algae degradation have been tested for antibiotic resistance for their possible use in a new genetic transformation system. Plasmids encoding the hygromycin B phosphotransferase (hph) under the control of the cauliflower mosaic virus 35S promoter, the trpC gene promoter of Aspergillus nidulans, and the Aureobasidium pullulans TEF gene promoter were delivered into the fungal cells by electroporation. The effectiveness of different promoters was compared by transformation and growth of Thermothelomyces thermophila (formerly Myceliophthora thermophila) on the selective medium and by real-time PCR analysis. A highly efficient transformation was observed at an electric-pulse of 8.5 kV/cm by using 10 μg of DNA per 1 × 105 conidia. Although all promoters were capable of hph expression in the Th. thermophila cells, the trpC promoter provided the highest level of hygromycin resistance. We further successfully applied plant binary vector pPZP for co-transformation of hph gene and enhanced green fluorescent protein gene that confirmed this transformation system could be used as an appropriate tool for gene function studies and the expression of heterologous proteins in micromycetes.

Many antibiotics that bind to the ribosome inhibit translation by blocking the movement of tRNAs and mRNA or interfering with ribosome dynamics, which impairs the formation of essential translocation intermediates. Here we show how translocation inhibitors viomycin (Vio), neomycin (Neo), paromomycin (Par), kanamycin (Kan), spectinomycin (Spc), hygromycin B (HygB), and streptomycin (Str, an antibiotic that does not inhibit tRNA translocation), affect principal motions of the small ribosomal subunits (SSU) during EF-G-promoted translocation. Using ensemble kinetics, we studied the SSU body domain rotation and SSU head domain swiveling in real time. We show that although antibiotics binding to the ribosome can favor a particular ribosome conformation in the absence of EF-G, EF-G-induced transition to the rotated/swiveled state of the SSU is hardly affected. The major effect of the antibiotics is observed at the stage when the SSU body and the head domain move backward. Vio, Spc and high concentrations of Neo completely inhibit the backward movements of the SSU body and head domain. Kan, Par, HygB and low concentrations of Neo slow down both movements, but their sequence and coordination are retained. Finally, Str has very little effect on the backward rotation of the SSU body domain, but retards the SSU head movement. The data underscore the importance of ribosome dynamics for tRNA-mRNA translocation and provide new insights into the mechanism of antibiotic action.

Avocado (Persea americana) is a fruit crop of economic importance in Ecuador. Currently, a low incidence of Phytophthora cinnamomi has been reported, however, there are other soil pathogens that can affect this crop, even at the initial stages of plant multiplication (nursery), for this reason the use of rootstocks that tolerate these biotic adversities is recommended. In this research, the fungus Fusarium oxysporum was isolated from roots of nursery seedlings with symptoms of necrosis. In addition, an isolate of this pathogenic fungus modified with a strain of Agrobacterium tumefaciens was used to determine the infection of F. oxysporum in the roots of the Fuerte (commercial) and Criollo (local) cultivars. The results allowed to infer that the cultivar Criollo presented a greater tolerance to F. oxysporum than the cultivar Fuerte, which corroborates its use as a rootstock for commercial avocado varieties. Furthermore, to our knowledge, this is the first report of F. oxysporum affecting avocado nursery seedlings in Ecuador. El aguacate (Persea americana) es un frutal de importancia económica en el Ecuador. Actualmente se ha reportado una baja incidencia de Phytophthora cinnamomi, sin embargo, existen otros patógenos del suelo que afectan a este cultivo, incluso en etapas iniciales de multiplicación de la planta, por esta razón el uso de portainjertos que toleren estas adversidades bióticas es recomendado. En esta investigación, se aisló el hongo Fusarium oxysporum de plántulas de vivero de raíces con sintomatología de necrosis. Además, se utilizó un aislado de este hongo patógeno modificada con una cepa de Agrobacterium tumefaciens para determinar la infección de F. oxysporum en las raíces de los cultivares Fuerte y Criollo. Los resultados permiten inferir que el cultivar Criollo presentó una mayor tolerancia a F. oxysporum, con lo cual se corrobora su uso como portainjerto para variedades comerciales de aguacate. Además, de acuerdo a nuestro conocimiento, este es el primer reporte de F. oxysporum afectando a plántulas de aguacate. O abacate (Persea americana) é uma árvore frutífera de importância econômica no Equador. Atualmente, tem sido relatada uma baixa incidência de Phytophthora cinnamomi, entretanto, existem outros patógenos de solo que afetam esta cultura, mesmo nos estágios iniciais de multiplicação das plantas, por isso é recomendado o uso de porta-enxertos que toleram essas adversidades bióticas. Nesta pesquisa, o fungo Fusarium oxysporum foi isolado de mudas de viveiro de raízes com sintomas de necrose. Além disso, um isolado deste fungo patogênico modificado com uma cepa de Agrobacterium tumefaciens foi usado para determinar a infecção de F. oxysporum nas raízes dos cultivares Fuerte e Criollo. Os resultados permitem inferir que o cultivar Crioulo apresentou maior tolerância ao F. oxysporum, o que corrobora seu uso como porta-enxerto para variedades comerciais de abacate. Além disso, até onde sabemos, este é o primeiro relato de F. oxysporum afetando mudas de abacate.

Biofilm formation of Candida species is considered to be a pathogenic factor of host infection. Since biofilm formation of Candida glabrata has not been as well studied as that of Candida albicans, we performed genetic screening of C. glabrata, and three candidate genes associated with biofilm formation were identified. Candida glabrata SYN8 (CAGL0H06325g) was selected as the most induced gene in biofilm cells for further research. Our results indicated that the syn8Δ mutant was defective not only in biofilm metabolic activity but also in biofilm morphological structure and biomass. Deletion of SYN8 seemed to have no effect on extracellular matrix production, but it led to a notable decrease in adhesion ability during biofilm formation, which may be linked to the repression of two adhesin genes, EPA10 and EPA22. Furthermore, hypersensitivity to hygromycin B and various ions in addition to the abnormal vacuolar morphology in the syn8Δ mutant suggested that active vacuolar function is required for biofilm formation of C. glabrata. These findings enhance our understanding of biofilm formation in this fungus and provide information for the development of future clinical treatments.

Fungal attacks on stored fruit and vegetables are responsible for losses of products. There is an active research field to develop alternative strategies for postharvest disease management, and the use of biocontrol agents represents a promising approach. Understanding the molecular bases of the biocontrol activity of these agents is crucial to potentiate their effectiveness. The yeast Papiliotrema terrestris is a biocontrol agent against postharvest pathogens. Phenotypic studies suggest that it exerts its antagonistic activity through competition for nutrients and space, which relies on its resistance to oxidative and other cellular stresses. In this study, we developed tools for genetic manipulation in P. terrestris to perform targeted gene replacement and functional complementation of the transcription factors Yap1 and Rim101. In vitro phenotypic analyses revealed a conserved role of Yap1 and Rim101 in broad resistance to oxidative stress and alkaline pH sensing, respectively. In vivo analyses revealed that P. terrestris yap1Δ and rim101Δ mutants display decreased ability to colonize wounded fruit compared to that of the parental wild-type (WT) strain; the yap1Δ mutant also displays reduced biocontrol activity against the postharvest pathogens Penicillium expansum and Monilinia fructigena, indicating an important role for resistance to oxidative stress in timely wound colonization and biocontrol activity of P. terrestris. In conclusion, the availability of molecular tools developed in the present study provides a foundation to elucidate the genetic mechanisms underlying biocontrol activity of P. terrestris, with the goal of enhancing this activity for the practical use of P. terrestris in pest management programs based on biological and integrated control. IMPORTANCE The use of fungicides represents the most effective and widely used strategy for controlling postharvest diseases. However, their extensive use has raised several concerns, such as the emergence of plant pathogens’ resistance as well as the health risks associated with the persistence of chemical residues in fruit, in vegetables, and in the environment. These factors have brought attention to alternative methods for controlling postharvest diseases, such as the utilization of biocontrol agents. In the present study, we developed genetic resources to investigate at the molecular level the mechanisms involved in the biocontrol activity of Papiliotrema terrestris, a basidiomycete yeast that is an effective biocontrol agent against widespread fungal pathogens, including Penicillium expansum, the etiological agent of blue mold disease of pome fruits. A deeper understanding of how postharvest biocontrol agents operate is the basic requirement to promote the utilization of biological (and integrated) control for the reduction of chemical fungicides.

The cloning of plasmids can be time-consuming or expensive. Yet, cloning is a prerequisite for many standard experiments for the functional analysis of genes, including the generation of deletion mutants and the localization of gene products. Here, we provide Golden Gate vectors for fast and easy cloning of gene fusion as well as gene deletion vectors applicable to diverse fungi. In Golden Gate cloning, restriction and ligation occur simultaneously in a one-pot reaction. Our vector set contains recognition sites for the commonly used type IIS restriction endonuclease BsaI. We generated plasmids for C- as well as N-terminal tagging with GFP, mRFP and 3xFLAG. For gene deletion, we provide five different donor vectors for selection marker cassettes. These include standard cassettes for hygromycin B, nourseothricin and phleomycin resistance genes as well as FLP/FRT-based marker recycling cassettes for hygromycin B and nourseothricin resistance genes. To make cloning most feasible, we provide robust protocols, namely (1) an overview of cloning procedures described in this paper, (2) specific Golden Gate reaction protocols and (3) standard primers for cloning and sequencing of plasmids and generation of deletion cassettes by PCR and split-marker PCR. We show that our vector set is applicable for the biotechnologically relevant Penicillium chrysogenum and the developmental model system Sordaria macrospora. We thus expect these vectors to be beneficial for other fungi as well. Finally, the vectors can easily be adapted to organisms beyond the kingdom fungi.

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.

Screening for a new yeast as an alternative host is expected to solve the limitations in the present yeast expression system. A yeast sample which was isolated from the traditional food starter &lsquo, ragi&rsquo, from Malaysia was identified to contain Meyerozyma guilliermondii strain SMB. This yeast-like fungus strain SMB was characterized to assess its suitability as an expression host. Lipase activity was absent in this host (when assayed at 30 &deg, C and 70 &deg, C) and Hygromycin B (50 &mu, g/mL) was found to be its best selection marker. Then, the hyg gene (Hygromycin B) was used to replace the sh ble gene (Zeocin) expression cassette in a Komagataella phaffii expression vector (designated as pFLDh&alpha, ). A gene encoding the mature thermostable lipase from Bacillus sp. L2 was cloned into pFLDh&alpha, followed by transformation into strain SMB. The optimal expression of L2 lipase was achieved using YPTM (Yeast Extract-Peptone-Tryptic-Methanol) medium after 48 h with 0.5% (v/v) methanol induction, which was 3 times faster than another K. phaffii expression system. In conclusion, a new host-vector system was established as a platform to express L2 lipase under the regulation of PFLD1. It could also be promising to express other recombinant proteins without inducers.

Red rot caused by Colletotrichum falcatum, is one of the economically important disease of sugarcane and breeding for resistant varieties is considered to be the major solution to manage the disease. However, breakdown of red rot resistance become usual phenomenon due to development of newer races by culture adaptation on newly released varieties. Hence it is needed to characterize the genes responsible for pathogen virulence in order to take care of host resistance or to manage the disease by other methods. The transcript studies gave foundation to characterize the huge number of pathogenicity determinants and their role in pathogenesis. Here we studied role of two important genes viz., Glucose Transporter (GT) and Sucrose Non-Fermenting1 (SNF1) during pathogenesis of C. falcatum, which said to be involved in carbon source metabolism. Sugar metabolism has a vital role in disease progression of C. falcatum by regulating their cell growth, metabolism and development of the pathogen during various stages of infection. The present study was aimed to find out the role of GT and SNF1 genes in response to pathogenicity by RNA silencing (RNAi) approach. Knock-down of the target pathogenicity gene homologs in standard C. falcatum isolate Cf671 was carried out by amplifying sense and antisense fragments of targets individually using pSilent-1 vector. The expression cassette was cloned into the binary vector pCAMBIA1300 followed by fungal transformation through Agarobacterium mediated transformation. Resulted mutants of both the genes showed less virulence compared to wild type isolate. Simultaneously, both the mutants did not produce spores. Moreover, the molecular confirmation of the mutants displayed the expression of hygromycin gene with reduced expression of the target gene during host-pathogen interaction. Knockdown of the pathogenicity related genes (GT and SNF1) by RNAi approach corroborate the possible role of the genes in causing the disease.

Ceratocystis manginecans causes mango wilt with significant economic losses. In the infection court, cerato-platanin (CP) family proteins (CPPs) are believed to involve in pathogenesis but has not been determined in C. manginecans. To confirm this function, a CP protein (CmCP) of C. manginecans was characterized in this study. A protoplast of C. manginecans was prepared by treating its mycelia with driselase and lysing enzymes. The cmcp gene was edited using CRISPR/Cas-U6-1 expression vectors in 60% PEG and 50 μg/mL hygromycin B in the medium, resulting in mutants with cmcp deletion (Δcmcp). A complimented mutant (Δcmcp-C) was obtained by transforming cmcp to Δcmcp. Both Δcmcp and Δcmcp-C were characterized by comparing them with a wild-type strain on morphology, mycelial growth, conidial production and pathogenicity. Additionally, cmcp was transformed and expressed in Pichia pastoris, and the derived recombinant protein CmCP caused a severe necrosis on Nicotiana tabacum leaves. CmCP-treated plant leaves showed symptoms of hypersensitive response including electrolyte leakage, reactive oxygen species generation and overexpression of defense-related genes PR-1, PAD3, ERF1, HSR203J, and HIN1. All those results suggested that cmcp gene was required for the growth development of C. manginecans and functioned as a major pathogenicity factor in mango infection.

Secondary metabolites play important roles in microbial communities, but their natural functions are often unknown and may be more complex than appreciated. While compounds with antibiotic activity are often assumed to underlie microbial competition, they may alternatively act as signal molecules. In either scenario, microorganisms might evolve responses to sublethal concentrations of these metabolites, either to protect themselves from inhibition or to change certain behaviors in response to the local abundance of another species. Here, we report that violacein production by C. violaceum ATCC 31532 is induced in response to hygromycin A from Streptomyces sp. 2AW, and we show that this response is dependent on inhibition of translational polypeptide elongation and a previously uncharacterized two-component regulatory system. The breadth of the transcriptional response beyond violacein induction suggests a surprisingly complex metabolite-mediated microbe-microbe interaction and supports the hypothesis that antibiotics evolved as signal molecules. These novel insights will inform predictive models of soil community dynamics and the unintended effects of clinical antibiotic administration., Antibiotics produced by bacteria play important roles in microbial interactions and competition Antibiosis can induce resistance mechanisms in target organisms, and at sublethal doses, antibiotics have been shown to globally alter gene expression patterns. Here, we show that hygromycin A from Streptomyces sp. strain 2AW. induces Chromobacterium violaceum ATCC 31532 to produce the purple antibiotic violacein. Sublethal doses of other antibiotics that similarly target the polypeptide elongation step of translation likewise induced violacein production, unlike antibiotics with different targets. C. violaceum biofilm formation and virulence against Drosophila melanogaster were also induced by translation-inhibiting antibiotics, and we identified an antibiotic-induced response (air) two-component regulatory system that is required for these responses. Genetic analyses indicated a connection between the Air system, quorum-dependent signaling, and the negative regulator VioS, leading us to propose a model for induction of violacein production. This work suggests a novel mechanism of interspecies interaction in which a bacterium produces an antibiotic in response to inhibition by another bacterium and supports the role of antibiotics as signal molecules.

Metabolic engineering of Digitalis purpurea L. has emerged as a powerful biotechnological tool to enhance in vitro cardenolide biosynthesis. Thereto, Agrobacterium tumefaciens-mediated transformation protocols have been developed using nptII as selectable marker gene and uidA as reporter gene. In this work, the Arabidopsis thaliana L. VEP1 gene, encoding progesterone-5β-reductase, was expressed in cardenolide-producing D. purpurea plants. Resistance to hygromycin B was efficiently used as a selectable marker in callus induction and multiplication media. Successful transformation was confirmed in nine transgenic lines by PCR analyses using primers for the selectable marker gene (hpt) and the VEP1 gene. The number of copies of the transgene construct was estimated between one and three in different lines by quantitative PCR and inverse PCR. VEP1 expression was confirmed in eight transgenic lines by reverse transcription-quantitative PCR. Digitoxin and digoxin content were increased up to 3.8-fold (757 μg/gDW) and 2.2-fold (199 μg/gDW) respectively in transgenic plants cultivated in vitro. However, for plants grown in the greenhouse, digitoxin content was not significantly different from non-transgenic plants and digoxin production was enhanced up to 1.8-fold (87 μg/gDW). Genetic transformation thus allowed to enhance cardenolide production in vitro with higher efficiency than with other biotechnological strategies reported so far.

To visualize protein-protein interactions in Candida albicans with the bimolecular fluorescence complementation (BiFC) approach, we created a Tet-on system with the plasmids pWTN1 and pWTN2. Both plasmids bear a hygromycin B-resistant marker (CaHygB) that is compatible with the original Tet-on plasmid pNIM1, which carries a nourseothricin-resistant marker (CaSAT1). By using GFPmut2 and mCherry as reporters, we found that the two complementary Tet-on plasmids act synergistically in C. albicans with doxycycline in a dose-dependent manner and that expression of the fusion proteins, CaCdc11-GFPmut2 and mCherry-CaCdc10, derived from this system, is septum targeted. Furthermore, to allow detection of protein-protein interactions with the reassembly of a split fluorescent protein, we incorporated mCherry into our system. We generated pWTN1-RN and pNIM1-RC, which express the N-terminus (amino acids 1–159) and C-terminus (amino acids 160–237) of mCherry, respectively. To verify BiFC with mCherry, we created the pWTN1-CDC42-RN (or pWTN1-RN-CDC42) and pNIM1-RC-RDI1 plasmids. C. albicans cells containing these plasmids treated with doxycycline co-expressed the N- and C-terminal fragments of mCherry either N-terminally or C-terminally fused with CaCdc42 and CaRdi1, respectively, and the CaCdc42-CaRdi1 interaction reconstituted a functional form of mCherry. The establishment of this Tet-on-based BiFC system in C. albicans should facilitate the exploration of protein-protein interactions under a variety of conditions.

BackgroundStable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step.ResultsAdding a combination of terbinafine (1 µM) and timentin (200 mg/L) to solid media delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from ten different wild-type and mutant Arabidopsis thaliana accessions. The method was also compatible with Nicotiana tabacum germination. Seedlings sown in non-sterile conditions could be maintained on antimicrobial media for up to a week without observable contamination. The antimicrobial cocktail was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as four days after stratification and transferred to soil before the onset of visible microbial contamination.ConclusionThe antimicrobial cocktail presented here delays microbial growth for long enough to permit germination of non-sterile Arabidopsis thaliana seedlings on solid media and it is compatible with rapid screening methods. We were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.

Some members of the Sporothrix genus can cause sporotrichosis, a worldwide distributed mycosis that affects several mammalian species, including human beings. Sporothrix schenckii and Sporothrix brasiliensis are the fungal species frequently associated with this disease, and the latter has gained significant interest because of the increased number of cases associated with transmission by cats. Despite the relevance of these organisms in the medical field, limited strategies for their genetic manipulation have been explored. Thus far, gene silencing using the hygromycin B resistance cassette is the sole strategy currently available to study these organisms. Here, we report the generation of a cassette that confers resistance to nourseothricin, which was successfully transferred from Agrobacterium tumefaciens to Sporothrix cells. Therefore, this can be used as a second selective marker to manipulate the genome of these organisms.

Lack of an efficient transformation system has hampered the molecular breeding of Agaricus bisporus . Here, we describe a highly efficient Agrobacterium-mediated transformation system for A. bisporus using foxtail millet (Setaria italica L. Beauv) grains as cultivation and infection media. Mycelium-millet complexes were prepared for co-culture and treated with ultrasonication for 10 s to improve infection efficiency. After a 72-h culture period, the newly grown mycelium-surrounded millet grain was transferred to selection medium supplemented with 200 μg/mL cefotaxime and 15 μg/mL hygromycin B (hyg) to screen positive transformants. Putative transformants were analyzed for the presence of the hyg gene by polymerase chain reaction and Southern blotting. Expression of eGFP in A. bisporus transformants was detected by fluorescence imaging, and the β-glucuronidase (GUS) protein was detected by histochemical staining. Our protocol resulted in an average 53.85% transformation frequency, and over 85% of the transformants tested remained mitotically stable, even after five successive rounds of subculturing. A feasible method for A. bisporus mushroom transformation using foxtail millet as an innovative culture medium was developed, which will benefit future functional genetic studies of this mushroom.

Transformation of rice was done through Agrobacterium mediated, utilizing a binary vector pBS, harboring a fungal gene endo α-1, 3-glucanase from Trichoderma harzianum under rice constitutive promoter actin2 and Agrobacterium nopaline synthase (nos) transcriptional terminator in a T-DNA. Likewise, a selectable marker gene hygromycin phosphotransferese (hpt) resistant to Hygromycin B was cloned in the middle of actin2 and nos terminator in a similar T-DNA. The expression of endo α-1, 3-glucanase was affirmed by cloning gfp gene after the fungal gene in the transformation DNA cassette. In the first generation of transgenic rice lines, out of 912 just 209 plants were false positive affirmed through PCR based screening for the transgene. The positive transgenic lines were tried with fungal infection by leaf cut test in vitro and foliar leaf shower technique in vivo. They demonstrated an exceptional protection against sheath blight disease. Further, seeds of all positive transgenic plants of the first generation were developed and screened in next generation. Just 62 plants were false positive out of 873 transgenic lines in this generation. In the comparative way, they were tried against the fungal disease and they demonstrated the exceptional protection once more. In this way, in this investigation, a fungal gene endo α-1, 3-glucanase was transformed into rice (IR 64) effectively which demonstrated protection against fungus Rhizoctonia solani.

Antagonistic fungi are well-known as viable alternatives to chemical control of root-knot nematodes. In this paper, serine protease Ac1 as an important pathogenicity factor was used to enhance the antagonistic activity of Arthrobotrys conoides and A. oligospora against Meloidogyne javanica J2 (second stage juveniles). Ac1 gene was extracted from A. conoides and cloned in pCAMBIA1304 vector. The recombinant plasmid was then transferred to these fungi using two strains of Agrobacterium tumefaciens (LBA4404 and AGL1). Transgenic isolates were confirmed by PCR amplification of Hygromycin B resistance gene (hph) as selectable marker, protease assay using casein substrate and in vitro bioassay. Transferring of Ac1 gene by homologous recombination to A. conoides, increased protease activity. Our results showed that production of protease in both transgenic species was increased compared to wild type. Bioassay results indicated that pathogenicity rates of transformants, in both the number of traps formed and the number of trapped J2, increased compared to the wild type. The results showed that inhibition of root-knot nematode, M. javanica, was markedly increased by transgenic isolates of Arthrobotrys spp. compared to the wild types.

The mesophilic fungus Eupenicillium parvum 4-14 is an important producer of thermotolerant hemicellulolytic and cellulolytic enzymes. The aim of this study was to establish a method for genetic manipulation of the fungus by Agrobacterium tumefaciens. The promotor PgpdA of a glyceraldehyde-3-phosphate dehydrogenase gene was isolated from E. parvum 4-14. To transform the fungus, an expression plasmid containing a superfolder green fluorescent protein (sfGFP) gene under the control of PgpdA promotor was constructed using the plasmid pAg1-H3 as a parental plasmid. Using the fungal ascospores as receptor and hygromycin B resistance as a selection marker, the recombinant plasmid was successfully introduced into the fungal cells by A. tumefaciens-mediated transformation (ATMT) method. Acetosyringone (AS) was essential to the successful transformation. The transformation frequency was significantly affected by the co-culture temperature and time, the quantity of fungal spores and the AS concentration. The highest transformation frequency was up to 373 transformants per 105 fungal spores, which was higher than those of other fungal species. The fungal transformants were genetically stable after five subcultures in the absence of antibiotic. GFP protein was strongly expressed in the hypha of fungal transformants. In conclusion, the ATMT is a highly efficient method for genetic manipulation of E. parvum 4-14, and will improve the molecular researches on the fungus.

Fonsecaea pedrosoi, a melanized fungal pathogen that causes Chromoblastomycosis, a human disease with a worldwide distribution. Biolistic is a widely used technique for direct delivery of genetic material into intact cells by particles bombardment. Another well-established transformation method is Agrobacterium-mediated transformation (ATMT), which involves the transfer of a T-DNA from the bacterium to the target cells. In F. pedrosoi there are no reports of established protocols for genetic transformation, which require optimization of physical and biological parameters. In this work, intact conidia of F. pedrosoi were particle bombarded and subjected to ATMT. In addition, we proposed hygromycin B, nourseothricin and neomycin as dominant selective markers for F. pedrosoi and vectors were constructed. We tested two parameters for biolistic: the distance of the particles to the target cells and time of cells recovery in nonselective medium. The biolistic efficiency was 37 transformants/μg of pFpHYG, and 45 transformants/μg of pAN7.1. Transformants expressing GFP were successfully obtained by biolistic. A co-culture ratio of 10: 1 (bacterium: conidia) and co-incubation time of 72 h yielded the largest number of transformants after ATMT. Southern blot analysis showed the number of foreign DNA insertion into the genome is dependent upon the plasmid used to generate the mutants. This work describes for the first time two efficient methods for genetic modification of Fonsecaea and these results open new avenues to better understand the biology and pathogenicity of the main causal agent of this neglected disease.

Soybean transformation is limited by the lack of multiple efficient selectable marker systems. Biolistic transformation of somatic proliferative embryogenic cultures, one of the commonly used soybean transformation methods, relies largely on hygromycin phosphotransferase II (hptII) selection. The purpose of the present study was to establish another efficient selectable marker system to facilitate multiple gene transformations of soybean. We tested neomycin phosphotransferase II (nptII) that has been used successfully in cotyledonary node transformation, but with limited success in transformation of embryogenic cultures. Transgenic events were obtained using nptII with improved G418 selection without generating escapes. G418 selection required longer recovery and selection periods, and resulted in a lower efficiency of initial transformants compared to hygromycin selection. Six independent fertile transgenic plants were recovered using nptII and G418, a frequency similar to that obtained with hygromycin selection. Soybean embryogenic cultures co-transformed with the hptII and nptII markers showed resistance to both hygromycin B and G418, while regeneration and plant fertility were not adversely affected. The nptII will be useful as a second selectable marker for multiple gene transformations in basic and applied soybean research.

Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controlling this pathogen. In this study, we have characterized a virulent strain of P. digitatum isolated in Vietnam and established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for this fungal strain with two newly constructed binary vectors. These binary vectors harbor dominant selectable markers for hygromycin or nourseothricin resistance, and expression cassettes for the red fluorescent protein (DsRed) or the green fluorescent protein (GFP), respectively. Using the established ATMT system, the transformation efficiency of the Vietnamese strain could reach a very high yield of 1240±165 transformants per 106 spores. Interestingly, we found that GFP is much better than DsRed for in situ visualization of citrus fruit colonization by the fungus. Additionally, we showed that the transformation system can also be used to generate T-DNA insertion mutants for screening non-pathogenic or less virulent strains. Our work provides a new platform including a virulent tropical strain of P. digitatum, an optimized ATMT method and two newly constructed binary vectors for investigation of the postharvest pathogen. This platform will help develop strategies to dissect molecular mechanisms of host-pathogen interactions in more detail as well as to identify potential genes of pathogenicity by either insertional mutagenesis or gene disruption in this important pathogenic fungus.

Since the genetic transformation of the apple is strongly genotype-dependent and generally inefficient, the evaluation of factors affecting shoot regeneration are crucial for the establishment of a successful transformation process. In this report, we evaluated the effects of the β-lactam antibiotics meropenem and timentin on in vitro regeneration via de novo shoot organogenesis from leaf explants of apple cv. Golden Delicious, as well as on the growth of the Agrobacterium tumefaciens strain EHA 105, and compared them with the commonly used β-lactam cefotaxime. Also, we report for the first time the effect of hygromycin B as a selective agent in the domesticated apple, as regards shoot regeneration and shoot multiplication efficiency. We observed that cefotaxime and timentin at concentrations higher than 100 mg L -1 were sufficient to prevent Agrobacterium growth during a two-week period, while meropenem exhibited an inhibitory effect on bacterial growth at all tested concentrations (25-150 mg L -1 ). Cefotaxime at a concentration of 300 mg L -1 increased the number of regenerated shoots per explant (9.39) in comparison with the control (7.67). In contrast to cefotaxime, meropenem and timentin caused a decrease in shoot regeneration efficiency, but larger and more developed shoots were obtained on meropenem (25-125 mg L -1 ) after the same period of cultivation. Hygromycin B at a concentration of 5 mg L -1 or higher completely inhibited shoot regeneration and induced explant tissue necrosis. Therefore, the selection procedure with a final concentration of 4 mg L -1 throughout organogenesis and 10 mg L -1 for further shoot growth and multiplication is recommended for an efficient transformation process in apple cv. Golden Delicious. https://doi.org/10.2298/ABS170731037S Received: July 31, 2017; Revised: September 23, 2017; Accepted: September 25, 2017; Published online: October 11, 2017 How to cite this article: Stanisic M, Ninkovic S, Savic J, Cosic T, Mitic N. The effects of β-lactam antibiotics and hygromycin B on de novo shoot organogenesis in apple cv. Golden Delicious. Arch Biol Sci. 2018;70(1):179-90.

Currently ectomycorrhizal research suffers from a lack of molecular tools specifically adapted to study gene expression in fungal symbionts. Considering that, we designed pReNuK, a cloning vector for transcriptional promoter studies in the ectomycorrhizal basidiomycete Laccaria bicolor. The pReNuK vector offers the use of a nuclear localizing and chromatin incorporating histone H2B-mCherry fluorescent reporter protein and it is specifically optimized for efficient transgene expression in Laccaria. Moreover, pReNuK is designed to work in concert with Agrobacterium-mediated transformation under hygromycin B resistance selection. The functionality of the pReNuK reporter system was tested with the constitutive Laccaria glyceraldehyde 3-phosphate dehydrogenase gene promoter and further validated with the nitrogen source regulated nitrate reductase gene promoter. The expression of the nucleus-directed H2B-mCherry reporter is highly stable in time. Moreover, the transformation of Laccaria with pReNuK and the expression of the reporter do not have negative effects on the growth of the fungus. The pReNuK offers a novel tool for studying in vivo gene expression regulation in Laccaria, the leading fungal model for ectomycorrhizal research.

Dwarf bunt of wheat caused by Tilletia controversa Kuhn 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.

Background/Purpose: Candida albicans is a common fungal pathogen in humans. In healthy individuals, C. albicans represents a harmless commensal organism, but infections can be life threatening in immunocompromised patients. The complete genome sequence of C. albicans is extremely useful for identifying genes that may be potential drug targets and important for pathogenic virulence. However, there are still many uncharacterized genes in the Candida genome database. In this study, we investigated C. albicans Hom6, the functions of which remain undetermined experimentally. Methods: HOM6-deleted and HOM6-reintegrated mutant strains were constructed. The mutant strains were compared with wild-type in their growth in various media and enzyme activity. Effects of HOM6 deletion on translation were further investigated by cell susceptibility to hygromycin B or cycloheximide, as well as by polysome profiling, and cell adhesion to polystyrene was also determined. Results: C. albicans Hom6 exhibits homoserine dehydrogenase activity and is involved in the biosynthesis of methionine and threonine. HOM6 deletion caused translational arrest in cells grown under amino acid starvation conditions. Additionally, Hom6 protein was found in both cytosolic and cell-wall fractions of cultured cells. Furthermore, HOM6 deletion reduced C. albicans cell adhesion to polystyrene, which is a common plastic used in many medical devices. Conclusion: Given that there is no Hom6 homologue in mammalian cells, our results provided an important foundation for future development of new antifungal drugs. Keywords: Candida albicans, cell adhesion, Hom6, homoserine dehydrogenase, protein synthesis