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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 movement), 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, their kinetic effect on the EF-G-induced transition to the rotated/swiveled state of the SSU is moderate. The antibiotics mostly inhibit backward movements of the SSU body and/or the head domains. 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.

Dihydropyriculol is a major secondary metabolite of Pyricularia oryzae. However, the biological activity of dihydropyriculol has not been reported. Here, we showed that dihydropyriculol has inhibitory activity against Streptomyces griseus. Localization analysis of dihydropyriculol revealed that dihydropyriculol could reach to S. griseus under confrontation culture. These results suggest that dihydropyriculol can be used as a chemical weapon against S. griseus.

The growth of the polypeptide chain occurs due to the fast and coordinated work of the ribosome and protein elongation factors, EF-Tu and EF-G. However, the exact contribution of each of these components in the overall balance of translation kinetics remains not fully understood. We created an in vitro translation system Escherichia coli replacing either elongation factor with heterologous thermophilic protein from Thermus thermophilus. The rates of the A-site binding and decoding reactions decreased an order of magnitude in the presence of thermophilic EF-Tu, indicating that the kinetics of aminoacyl-tRNA delivery depends on the properties of the elongation factor. On the contrary, thermophilic EF-G demonstrated the same translocation kinetics as a mesophilic protein. Effects of translocation inhibitors (spectinomycin, hygromycin B, viomycin and streptomycin) were also similar for both proteins. Thus, the process of translocation largely relies on the interaction of tRNAs and the ribosome and can be efficiently catalysed by thermophilic EF-G even at suboptimal temperatures.

Insect Trichoplusia ni High Five™ (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this study, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e., iCherry, as an intracellular protein model) or two proteins (i.e., influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development., G3: Genes, Genomes, Genetics, 11 (8), ISSN:2160-1836

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.

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.

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.

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.

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.

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.

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.

Malassezia spp. are part of the normal human and animal mycobiota but are also associated with a variety of dermatological diseases. The absence of a transformation system hampered studies to reveal mechanisms underlying the switch from the non-pathogenic to pathogenic life style. Here we describe, a highly efficient Agrobacterium-mediated genetic transformation system for Malassezia furfur and M. pachydermatis. A binary T-DNA vector with the hygromycin B phosphotransferase (hpt) selection marker and the green fluorescent protein gene (gfp) was introduced in M. furfur and M. pachydermatis by combining the transformation protocols of Agaricus bisporus and Cryptococcus neoformans. Optimal temperature and co-cultivation time for transformation were 5 and 7 days at 19 °C and 24 °C, respectively. Transformation efficiency was 0.75–1.5% for M. furfur and 0.6–7.5% for M. pachydermatis. Integration of the hpt resistance cassette and gfp was verified using PCR and fluorescence microscopy, respectively. The T-DNA was mitotically stable in approximately 80% of the transformants after 10 times sub-culturing in the absence of hygromycin. Improving transformation protocols contribute to study the biology and pathophysiology of Malassezia.

An efficient Agrobacterium tumefaciens-mediated transformation (ATMT) approach was developed for the plant pathogenic fungus, Cercospora zeae-maydis, which is the causative agent of gray leaf spot in maize. The transformation was evaluated with five parameters to test the efficiencies of transformation. Results showed that spore germination time, co-cultivation temperature and time were the significant influencing factors in all parameters. Randomly selected transformants were confirmed and the transformants were found to be mitotically stable, with single-copy T-DNA integration in the genome. T-DNA flanking sequences were cloned by thermal asymmetric interlaced PCR. Thus, the ATMT approach is an efficient tool for insertional mutagenesis of C. zeae-maydis.

A plasmid pCAMBIA1301 containing Pleurotus eryngii cellulase gene (PEcbh), under the control of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (LEgpd) promoter, was constructed and used as an expression vector. The vector was transformed into the tissue of P. eryngii using Agrobacterium tumefaciens-mediated transformation (ATMT) method and 4 transformants (PET1-4) were obtained. The positive transformants were confirmed by cultivation in media containing hygromycin and by PCR amplification of hygromycin B resistance-LEgpd promoter gene fragment. Unpredicted, cellulase specific activities of the transformants were not higher than those of the wild type. Mushroom cultivation was performed in the laboratory and the results revealed that the average biological efficiency of PET4 was significantly 1.52 times higher than those of the wild type.

Chromoblastomycosis (CBM) is a chronic cutaneous or subcutaneous mycosis that is prevalent worldwide. Though CBM tends not to be fatal, it is difficult to treat and complications can include chronic, marked lesions, lymphatic damage, and neoplastic transformation. Fonsecaea monophora, as a new species segregated from Fonsecaea pedrosoi, is the predominant causative pathogen of CBM in southern China. However, research about F. monophora has been limited, which may be due to a lack of an effective genetic manipulation system for F. monophora. In this study, we successfully established a random insertional mutagenesis system by Agrobacterium tumefaciens-mediated transformation (ATMT) in F. monophora for the first time. In order to improve the efficiency of ATMT, various co-culture conditions were optimized, including: acetosyringone (AS) concentrations, co-culture duration, ratio of bacteria to conidia, and the A. tumefaciens strains. In addition, thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) was performed to identify the transferred DNA (T-DNA) flanking sequences of the F. monophora transformants. The valuable transformants obtained in this study will be used for research in the future.

Tilletia foetida causes wheat common smut disease with severe loss of yield production and seed quality. In this study, a low-cost, rapid, and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for T. foetida mutagenesis was constructed: Transformants were screened with hygromycin B at 100 μg/ml, cefotaxime sodium concentrations with 200 μg/ml, Acetosyringone (AS) concentration at 200 μmol/l, 1 × 106 T. foetida hypha cells/ml, co-cultivation at 22 °C with 24 h and culture was incubated at 16 °C up to day 7. Fourteen transformants were randomly selected and confirmed using the specific primers to amplify the fragment of hygromycin phosphotransferase gene. At the same time, PCR analysis was performed to detect Agrobacterium tumefaciens Vir gene to eliminate false positives. The transformants were cultivated up to 8 generations on hygromycine B-containing complete medium (CM) and confirmed by PCR. The results indicated that 80% of T. foetida transformants were hygromycine B resistant. In conclusion, our analyses identified an efficient T-DNA insertion system for T. foetida and the results will be useful for further understanding the pathogenic mechanism via generation of the insertional mutants.

Genome-editing techniques such as CRISPR/Cas9 have been widely used in crop functional genomics and improvement. To efficiently deliver the guide RNA and Cas9, most studies still rely on Agrobacterium-mediated transformation, which involves a selection marker gene. However, several limiting factors may impede the efficiency of screening transgene-free genome-edited plants, including the time needed to produce each life cycle, the response to selection reagents, and the labor costs of PCR-based genotyping. To overcome these disadvantages, we developed a simple and high-throughput method based on visual detection of antibiotics-derived H2O2 to verify transgene-free genome-edited plants. In transgenic rice containing hygromycin phosphotransferase (HPT), H2O2 content did not change in the presence of hygromycin B (HyB). In contrast, in transgenic-free rice plants with 10-h HyB treatment, levels of H2O2 and malondialdehyde, indicators of oxidative stress, were elevated. Detection of H2O2 by 3,3&prime, diaminobenzidine (DAB) staining suggested that H2O2 could be a marker to efficiently distinguish transgenic and non-transgenic plants. Analysis of 24 segregating progenies of an HPT-containing rice plant by RT-PCR and DAB staining verified that DAB staining is a feasible method for detecting transformants and non-transformants. Transgene-free genome-edited plants were faithfully validated by both PCR and the H2O2-based method. Moreover, HyB induced overproduction of H2O2 in leaves of Arabidopsis, maize, tobacco, and tomato, which suggests the potential application of the DAB method for detecting transgenic events containing HPT in a wide range of plant species. Thus, visual detection of DAB provides a simple, cheap, and reliable way to efficiently identify transgene-free genome-edited and HPT-containing transgenic rice.

Ceratocystis paradoxa, the causal agent of stem-bleeding disease of the coconut palm, causes great losses to the global coconut industry. As the mechanism of pathogenicity of C. paradoxa has not been determined, an exogenous gene marker was introduced into the fungus. In this study, pCT74-sGFP, which contains the green fluorescent protein (GFP) gene, and the hygromycin B resistance gene as a selective marker, was used as an expression vector. Several protoplast release buffers were compared to optimize protoplast preparation. The plasmid pCT74-sGFP was successfully transformed into the genome of C. paradoxa, which was verified using polymerase chain reaction and green fluorescence detection. The transformants did not exhibit any obvious differences from the wild-type isolates in terms of growth and morphological characteristics. Pathogenicity tests showed that the transformation process did not alter the virulence of the X-3314 C. paradoxa strain. This is the first report on the polyethylene glycol-mediated transformation of C. paradoxa carrying a &lsquo, reporter&rsquo, gene GFP that was stably and efficiently expressed in the transformants. These findings provide a basis for future functional genomics studies of C. paradoxa and offer a novel opportunity to track the infection process of C. paradoxa.

To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples., This study was supported by grants AGL2014-55287-C2-1-R, BFU2015-66806-R, RTI2018-094263-B-I00, and AGL2017-84389-C2-2-R from the Ministry of Science, Innovation and Universities (Spain), Agencia Estatal de Investigación (AEI) and the Fondo Europeo de Desarrollo Regional (FEDER, EU). S. Rodríguez-Pires received a PhD fellowship from the Ministry of Science, Innovation and Universities (Spain).

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.

Hepatitis B virus (HBV) is considered as a global health concern and hepatitis B surface antigen (HBsAg) is the most immunogenic protein of HBV. The purpose of this study was to evaluate the expression of HBsAg on the cell surface of human embryonic kidney cell line (HEK293T). After transformation of expression vector pcDNA/HBsAg to E.coli TOP10F’, plasmid was extracted and digested with BglII. Afterwards, the linearized vector was transfected to cells and treated with hygromycin B for 5 weeks to expand the resulted clonies. The permanent expression of HBsAg followed by flow cytometry uptill now about one year. Genomic DNA was extracted from transfected cells and the existence of HBsAg gene was assessed by PCR. Real-time RT-PCR was utilized to measure the expression at the RNA level and flow cytometery was carried out to assess protein expression. Insertion of HBsAg cDNA in HEK293T genome was confirmed by PCR. The results of real-time RT-PCR illustrated that each cell expresses 2275 copies of mRNA molecule. Flow cytometry showed that compared with negative control cells, 99.9% of transfected cells express HBsAg on their surface. In conclusion, stable expression of hepatitis B surface antigen on the membrane of HEK293T provides an accurate post-translational modification, proper structure, and native folding in contrast with purified protein from prokaryotic expression systems. Therefore, these exposing HBsAg cells are practical in therapeutic, pharmaceutical, and biological sets of research.

Lovastatin is widely prescribed to reduce elevated levels of cholesterol and prevent heart-related diseases. Cultivation of Aspergillus terreus (ATCC 20542) with carbohydrates or low-value feedstocks such as glycerol produces lovastatin as a secondary metabolite and (+)-geodin as a by-product. An A. terreus mutant strain was developed (gedCΔ) with a disrupted (+)-geodin biosynthesis pathway. The gedCΔ mutant was created by inserting the antibiotic marker hygromycin B (hyg) within the gedC gene that encodes emodin anthrone polyketide synthase (PKS), a primary gene responsible for initiating (+)-geodin biosynthesis. The effects of emodin anthrone PKS gene disruption on (+)-geodin and lovastatin biosynthesis and the production of the precursors acetyl-CoA and malonyl-CoA were investigated with cultures based on glycerol alone and in combination with lactose. The gedCΔ strain showed improved lovastatin production, particularly when cultivated on the glycerol-lactose mixture, increasing lovastatin production by 80% (113 mg/L) while simultaneously inhibiting (+)-geodin biosynthesis compared to the wild-type strain. This study thus shows that suppression of the (+)-geodin pathway increases lovastatin yield and demonstrates a practical approach of manipulating carbon flux by modulating enzyme activity.

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions.The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD600 = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications.

A new expression cassette (EC0) consisting of the fused 5′ and 3′ intergenic regions (IGRs) of the Eremothecium cymbalariae translational elongation factor 1α (EcTEF1) gene was evaluated through expression of the bacterial hygromycin B phosphotransferase (hph) resistance gene in the common baker’s yeast Saccharomyces cerevisiae. Progressively shorter versions of the hph-containing EC cassette (hphEC1 though hphEC6) with trimmed 5′ and 3′ EcTEF1 IGRs were tested for their ability to confer resistance to hygromycin B in S. cerevisiae. Hygromycin B resistance was retained in all six generated hphEC variants up to a concentration of 400 mg/L. The hphEC6 cassette was the shortest cassette to be assayed in this study with 366 and 155 bp of the EcTEF1 5′ and 3′ IGRs, respectively. When tested for deletion of the S. cerevisiae proline oxidase gene PUT1, the hphEC6 cassette was shown to successfully act as a selection marker on hygromycin B-containing medium. The hphEC6 cassette could be placed immediately adjacent to a kanMX4 G418 disulfate resistance marker without any discernable effect on the ability of the yeast to grow in the presence of both hygromycin B and G418 disulfate. Co-cultivation experiments under non-selective conditions demonstrated that a PUT1 deletion strain carrying the hphEC6 cassette displayed equivalent fitness to an otherwise isogenic PUT1 deletion strain carrying the kanMX4 cassette.

Morchella importuna is a worldwide distributed edible mushroom with high ecological and economic values, but the molecular and genetic research about this mushroom has been hindered due to lack of an efficient transformation method. Here, we report for the first time the successful transformation of M. importuna by using a hypervirulent Agrobacterium tumefaciens strain bearing the constructed binary plasmid p1391-U-GUS. The selectable markers used were the genes for hygromycin resistance under the control of the polyubquitin promoter from M. importuna. The reporter genes were those for enhanced green fluorescent protein (EGFP) and the β-Glucuronidase (GUS) under the control of glyceraldehyde-3-phosphate dehydrogenase promoter and polyubquitin promoter respectively. The presence of the reporter gene EGFP in the transformants was confirmed by the fluorescence and confocal microscope and molecular analysis and that of the reporter gene GUS was verified by enzyme activity and molecular analysis. The analysis results of both reporter genes indicated that Agrobacterium-mediated transformation was successfully performed in M. importuna.

Agrobacterium tumefaciens-mediated transformation (ATMT) has been widely used in filamentous fungi. In this study, an efficient Agrobacterium tumefaciens-mediated transformation approach was developed for the plant pathogenic fungus, Kabatiella zeae, the causative pathogen of eyespot in maize. Five parameters were selected to optimize efficiencies of transformation. A. tumefaciens concentration, conidia concentration of K. zeae and mixing ratio of A. tumefaciens and K. zeae were found to exert a significant influence on all parameters. Transformants emitted green fluorescence under fluorescence microscopy. The presence of mitotically stable hygromycin resistance gene (hph) integration in the genome was confirmed by PCR. Up to 148 transformants per 107 conidia could be obtained under optimal conditions. In this way, ATMT approach is an efficient tool for insertional mutagenesis of K. zeae.

The increase in multi-drug resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P- and E-site tRNAs bound, and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A-site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates, and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

Streptomyces sp. Tü 6176 produces the cytotoxic benzoxazole nataxazole. Bioinformatic analysis of the genome of this organism predicts the presence of 38 putative secondary-metabolite biosynthesis gene clusters, including those involved in the biosynthesis of AJI9561 and its derivative nataxazole, the antibiotic hygromycin B, and ionophores enterobactin and coelibactin. The nataxazole biosynthesis gene cluster was identified and characterized: it lacks the O-methyltransferase gene required to convert AJI9561 into nataxazole. This O-methyltransferase activity might act as a resistance mechanism, as AJI9561 shows antibiotic activity whereas nataxazole is inactive. Moreover, heterologous expression of the nataxazole biosynthesis gene cluster in S. lividans JT46 resulted in the production of AJI9561. Nataxazole biosynthesis requires the shikimate pathway to generate 3-hydroxyanthranilate and an iterative type I PKS to generate 6-methylsalicylate. Production of nataxazole was improved up to fourfold by disrupting one regulatory gene in the cluster. An additional benzoxazole, 5-hydroxynataxazole is produced by Streptomyces sp. Tü 6176. 5-Hydroxynataxazole derives from nataxazole by the activity of an as yet unidentified oxygenase; this implies cross-talk between the nataxazole biosynthesis pathway and an unknown pathway.

Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription., Inositol pyrophosphates are phosphate-rich metabolic messengers that regulate many cellular processes. We observed that RNA polymerase I is pyrophosphorylated by inositol pyrophosphates, and its transcription elongation activity was reduced in budding yeast strains devoid of inositol pyrophosphates.

We present a versatile vector toolkit for nuclear transgene expression in the model green microalga Chlamydomonas reinhardtii. The vector was designed in a modular fashion which allows quick replacement of regulatory elements and genes of interest. The current toolkit comprises two antibiotic resistance markers (paromomycin and hygromycin B), five codon-optimized light emission reporters, including the Gaussia princeps luciferase, as well as bright cyan, green, yellow, and red fluorescent protein variants. The system has demonstrated robust functional flexibility with signal options to target the protein of interest to the cytoplasm, the nucleus, cellular microbodies, the chloroplast, mitochondria, or via the endoplasmic reticulum-Golgi apparatus secretory pathway into the culture medium. Successful fluorescent reporter protein fusion to C. reinhardtii Rubisco small subunit 1 was accomplished with this system. Localization of the fluorescently tagged protein was observed in the chloroplast pyrenoid via live cell fluorescence microscopy, the first report of heterologous protein localization to this cellular structure. The functionalities of the vector toolkit, the individual modular elements, as well as several combinations thereof are demonstrated in this manuscript. Due to its strategic design, this vector system can quickly be adapted to individual tasks and should therefore be of great use to address specific scientific questions requiring nuclear recombinant protein expression in C. reinhardtii.

The abundant molecular chaperone Hsp90 is essential for the folding and stabilization of hundreds of distinct client proteins. Hsp90 is assisted by multiple cochaperones that modulate Hsp90's ATPase activity and/or promote client interaction, but the in vivo functions of many of these cochaperones are largely unknown. We found that Cpr6, Cpr7, and Cns1 interact with the intact ribosome and that Saccharomyces cerevisiae lacking CPR7 or containing mutations in CNS1 exhibited sensitivity to the translation inhibitor hygromycin. Cpr6 contains a peptidyl-prolyl isomerase (PPIase) domain and a tetratricopeptide repeat (TPR) domain flanked by charged regions. Truncation or alteration of basic residues near the carboxy terminus of Cpr6 disrupted ribosome interaction. Cns1 contains an amino-terminal TPR domain and a poorly characterized carboxy-terminal domain. The isolated carboxy-terminal domain was able to interact with the ribosome. Although loss of CPR6 does not cause noticeable growth defects, overexpression of CPR6 results in enhanced growth defects in cells expressing the temperature-sensitive cns1-G90D mutation (the G-to-D change at position 90 encoded by cns1 ). Cpr6 mutants that exhibit reduced ribosome interaction failed to cause growth defects, indicating that ribosome interaction is required for in vivo functions of Cpr6. Together, these results represent a novel link between the Hsp90 molecular-chaperone machine and protein synthesis.

Choosing a potent selection antibiotic (SA), is a crucial success factor when creating stably transfected cell lines using an antibiotic selection marker. The selection capacity of this antibiotic is defined as its ability to kill sensitive, untransfected parental cells, while leaving resistant, transfected cells unharmed. Currently, no procedure has been described to determine this selection capacity. Therefore, a protocol to obtain a numerical value, called the "selectivity factor" (SF), that defines the selection capacity of SAs is developed. The SF is determined by using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-diphenyltetrazolium bromide) assay for both sensitive and resistant cells, and applies to commonly used cell lines. To prove the concept, the SF of the SA G418 and hygromycin B (HmB) on several cell lines is determined. The SF of G418 on BHK-21 cells is very high, indicating that G418 is an ideal SA for transfected BHK-21 cells. For HeLa cells, the SF of G418 is very low suggesting G418 is not an optimal SA for selecting transfected HeLa cells. For these cells, HmB would be a better choice. These conclusions are confirmed by an independent cell death assay. The SF identifies the most optimal SA for a certain cell line, reduces the risk of selecting spontaneously resistant cell clones, and streamlines the process of generating stable cell lines. Most importantly, the method is especially time saving when obtaining stable cell lines expressing toxic genes, and reduces culture times for generating large numbers of cell lines from the same parental cell line.

Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.

The examinations of conditions for establishing a variety independent Agrobacterium-mediated transformation procedure for wheat are preferable since many of cultivars and breeding lines remain recalcitrant for biotechnological manipulation, mainly due to low efficiency of plant regeneration in vitro, which is highly genotype specific. This paper describes and discusses an improved protocol for enhanced and low-genotype dependent Agrobacterium-mediated transformation using a super-binary vector LBA4404/pTOK233 carrying reporter gus-intron gene and hygromycin (hpt) and kanamicyn (nptII) selectable marker genes. The protocol was optimized on highly responsive common wheat cv. Vesna. Transient expression monitored by the gus-intron on explants after 3, 6 and 25 days of co-cultivation, followed by GUS expression and hygromycin resistance in whole plants indicated the protocol including a co-cultivation of freshly isolated immature embryos in the presence of ascorbic acid, and acetosyringone added only in the bacteria-containing infection medium combined with a delayed and stepwise increasing hygromycin B selection procedure significantly enhanced the transformation efficiency in cv. Vesna that exceed 7\% of treated explants from previously 0.41\%. Explant pre-cultivation did not additionally improve transformation efficiency. The optimized protocol was successful in evoking satisfactory transformation efficiencies from 3.6\% to 10.8\% in 5 less-responsive wheat genotypes. All 57 T0 hygromycin-resistant and GUS-positive lines were phenotypically normal and fertile. Therefore, the conditions employed in this study may serve as a base to facilitate the transformation in other, particularly recalcitrant wheat cultivars. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}ON173015]

Valsa mali var. mali, the causal agent of valsa canker of apple, causes great loss of apple production in apple producing regions. The pathogenic mechanism of the pathogen has not been studied extensively, thus a suitable gene marker for pathogenic invasion analysis and a random insertion of T-DNA for mutants are desirable. In this paper, we reported the construction of a binary vector pKO1-HPH containing a positive selective gene hygromycin phosphotransferase (hph), a reporter gene gfp conferring green fluorescent protein, and an efficient protocol for V. mali var. mali transformation mediated by Agrobacterium tumefaciens. A transformation efficiency up to about 75 transformants per 10(5) conidia was achieved when co-cultivation of V. mali var. mali and A. tumefaciens for 48 h in A. tumefaciens inductive medium agar plates. The insertions of hph gene and gfp gene into V. mali var. mali genome verified by polymerase chain reaction and southern blot analysis showed that 10 randomly-selected transformants exhibited a single, unique hybridization pattern. This is the first report of A. tumefaciens-mediated transformation of V. mali var mali carrying a 'reporter' gfp gene that stably and efficiently expressed in the transformed V. mali var. mali species.

Lecanicillium lecanii has been used in the biological control of several insects in agricultural practice. Since the gene manipulation tools for this entomopathogenic fungus have not been sufficiently developed, Agrobacterium tumefaciens-mediated transformation (ATMT) in L. lecanii was investigated in this study, using the wild-type isolate FZ9906 as a progenitor strain and the hygromycin B resistance (hph) gene as a selection marker. Furthermore, a field carbendazim-resistant (mrt) gene from Botrytis cinerea was expressed in L. lecanii FZ9906 via the ATMT system. The results revealed that the frequency of transformation surpassed 25transformants/10(6) conidia, most of the putative transformants contained a single copy of T-DNA, and the T-DNA inserts were stably inherited after five generations. All putative transformants had indistinguishable biological characteristics relative to the wild-type strain, excepting two transformants with altered growth habits or virulence. Moreover, the resistance of the putative transformants to carbendazim (MBC) was improved, and the highest one was 380-fold higher than the wild-type strain. In conclusion, ATMT is an effective and suitable system for L. lecanii transformation, and will be a useful tool for the basic and application research of gene functions and gene modifications of this strain.

We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD600) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.

An upstream region of cellobiohydrolase I gene promoter was modified from Trichoderma reesei to enhance heterologous gene expression. The base pairs from −677 to −724 were deleted indicating glucose repressor binding sites. Trichoderma reesei is considered an important workplace for homologous/heterologous genes expressions under the modified cbh1 promoter. However, constitutive expression is desirable in some cases over inducible expression that leads to production of undesirable cellulase components as byproduct. Here we report that the reporter β-glucosidase gene (bgl1) from Aspergillus niger BE-2 was cloned and recombined with cbh1 promoter followed by terminator trpC cloned from fungal expression vector, pUR5750. The expression cassette was ligated to binary vector and transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT) using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformant Bgl-3 increased by 39% and 47.1% under repression condition respectively. The expression level of Bgl-3 was found 7.9 folds increase over host strain EU7-22. Our results indicate that cbh1 is a strong promoter and has successfully driven over-expression of bgl1 gene in EU7-22 under repression condition and has surprisingly improved the efficiency of heterologous expression of cellulase genes in EU7-22.

A high concentration of glucose in the medium could greatly inhibit the expression of cellulase in filamentous fungi. The aspartic protease from fungus Hypocrea orientalis EU7-22 could efficiently express under both induction condition and glucose repression condition. Based on the sequence of structure gene of aspartic protease, the upstream sequence harboring the putative promoter proA for driving the expression of aspartic protease was obtained by genome walking. The upstream sequence contained the typical promoter motifs “TATA” and “CAAT”. The β-glucosidase gene (Bgl1) from H. orientalis was cloned and recombined with promoter proA and terminator trpC. The expression cassette was ligated to the binary vector to form pUR5750-Bgl1, and then transferred into the host strain EU7-22 via Agrobacterium tumefaciens mediated transformation (ATMT), using hygromycin B resistance gene as the screening marker. Four transformants Bgl-1, Bgl-2, Bgl-3 and Bgl-4 were screened. Compared with the host strain EU7-22, the enzyme activities of filter paper (FPA) and β-glucosidase (BG) of transformant Bgl-2 increased by 10.6% and 19.1% under induction condition, respectively. The FPA and BG activities were enhanced by 22.2% and 700% under 2% glucose repression condition, respectively, compared with the host strain. The results showed that the putative promoter proA has successfully driven the over-expression of Bgl1 gene in H. orientalis under glucose repression condition.

An efficient Agrobacterium tumefaciens-mediated genetic transformation method was successfully established for a newly isolated Taxol-producing fungus, Ozonium sp EFY21. A specific hygromycin B resistance expression vector, pCAMBIA1304'AN7-1, was constructed for fungal transformation. Key factors affecting transformation efficiency were thoroughly investigated and optimized. PCR amplification and Southern hybridization were used to verify the transformation events. This study should pave the way for future genetic modification studies of Ozonium sp EFY21.

An Agrobacterium-mediated genetic transformation system for the rice false smut fungus Ustilaginoidea virens was developed using conidia as recipients. A binary vector, pCAMBIA1301-PgpdA-GUS-TtrpC, was constructed. The gpdA promoter (PgpdA) from Aspergillus nidulans was used to drive the expression of the β-glucuronidase (GUS) gene which enabled GUS activity visualization. The conidia transformation efficiency reached approximately 110 to 250 transformants per 1×105 conidia. Based on the analysis made on five successive generations of subcultures and PCR, the pCAMBIA1301-GUS cassette had integrated into the genomes of all transformants and clearly showed mitotic stability. The novel reporter vector constructed will promote the functional characterization of genes and the construction of genetically engineered strains of this important fungus.

Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Fusarium proliferatum is an important pathogen that is associated with plant diseases and primarily affects aerial plant parts by producing different mycotoxins, which are toxic to humans and animals. Within the last decade, this fungus has also been described as one of the causes of red root rot or sudden death syndrome in soybean, which causes extensive damage to this crop. This study describes the Agrobacterium tumefaciens-mediated transformation of F. proliferatum as a tool for the disruption of pathogenicity genes. The genetic transformation was performed using two binary vectors (pCAMDsRed and pFAT-GFP) containing the hph (hygromycin B resistance) gene as a selection marker and red and green fluorescence, respectively. The presence of acetosyringone and the use of filter paper or nitrocellulose membrane were evaluated for their effect on the transformation efficiency. A mean processing rate of 94% was obtained with 96 h of co-cultivation only in the presence of acetosyringone and the use of filter paper or nitrocellulose membrane did not affect the transformation process. Hygromycin B resistance and the presence of the hph gene were confirmed by PCR, and fluorescence due to the expression of GFP and DsRed protein was monitored in the transformants. A high rate of mitotic stability (95%) was observed. The efficiency of Agrobacterium-mediated transformation of F. proliferatum allows the technique to be used for random insertional mutagenesis studies and to analyze fungal genes involved in the infection process.