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Plant macroevolutionary studies leverage the phylogenetic position of non-flowering model systems like the liverwort Marchantia polymorpha to investigate the origin and evolution of key plant processes. To date, most molecular genetic studies in Marchantia rely on hygromycin and/or chlorsulfuron herbicide resistance markers for the selection of stable transformants. Here, we used a sulfonamide-resistant dihydropteroate synthase (DHPS) gene to enable sulfadiazine-based transformation selection in M. polymorpha. We demonstrate the reliability of sulfadiazine selection on its own and in combination with existing hygromycin and chlorsulfuron selection schemes through transgene stacking experiments. The utility of this system is further demonstrated through confocal microscopy of a triple transgenic line carrying fluorescent proteins labelling the plasma membrane, cortical microtubules, and the nucleus. Collectively, our findings and resources broaden the capacity to genetically manipulate the increasingly popular model liverwort M. polymorpha., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20 bp, 40 bp, 60 bp and 80 bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20 bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Hornworts are a deeply diverged lineage of bryophytes and a sister lineage to mosses and liverworts. Hornworts have an array of unique features that can be leveraged to illuminate not only the early evolution of land plants, but also alternative paths for nitrogen and carbon assimilation via cyanobacterial symbiosis and a pyrenoid-based CO2-concentrating mechanism (CCM), respectively. Despite this, hornworts are one of the few plant lineages with limited available genetic tools. Here we report an efficient biolistics method for generating transient expression and stable transgenic lines in the model hornwort, Anthoceros agrestis. An average of 569 (±268) cells showed transient expression per bombardment, with green fluorescent protein expression observed within 48-72 h. A total of 81 stably transformed lines were recovered across three separate experiments, averaging six lines per bombardment. We followed the same method to transiently transform nine additional hornwort species, and obtained stable transformants from one. This method was further used to verify the localization of Rubisco and Rubisco activase in pyrenoids, which are central proteins for CCM function. Together, our biolistics approach offers key advantages over existing methods as it enables rapid transient expression and can be applied to widely diverse hornwort species., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Bacterial transformation is an important mode of horizontal gene transfer that helps spread genetic material across species boundaries. Yet, the factors that pose barriers to genome-wide cross-species gene transfer are poorly characterized. Here, we develop a replacement accumulation assay to study the effects of genomic distance on transfer dynamics. Using Bacillus subtilis as recipient and various species of the genus Bacillus as donors, we find that the rate of orthologous replacement decreases exponentially with the divergence of their core genomes. We reveal that at least 96% of the B. subtilis core genes are accessible to replacement by alleles from Bacillus spizizenii. For the more distantly related Bacillus atrophaeus, gene replacement events cluster at genomic locations with high sequence identity and preferentially replace ribosomal genes. Orthologous replacement also creates mosaic patterns between donor and recipient genomes, rearranges the genome architecture, and governs gain and loss of accessory genes. We conclude that cross-species gene transfer is dominated by orthologous replacement of core genes which occurs nearly unrestricted between closely related species. At a lower rate, the exchange of accessory genes gives rise to more complex genome dynamics., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error-prone repair pathways of nonhomologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize with respect to insertion site features, the genomic distribution of T-DNA incorporation, and the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Published by Oxford University Press on behalf of The Genetics Society of America 2023.)

Competing Interests: Conflict of interest statement: Although not involved in the highlighted study, the author is working in the same laboratory with M.C.K, H.A.P, and R.K.S.

Plastid engineering has several unique advantages such as high expression of transgenes due to high polyploidy of plastid genomes and environmental biosafety because of maternal inheritance of transgenes, and has become a promising tool for molecular farming, metabolic engineering, and genetic improvement. However, there are no standard vectors available for plastid transformation. Moreover, the construction of plastid transformation vectors containing long operons or genes encoding proteins that are toxic to Escherichia coli was tedious or difficult. Here, we developed a simple plastid transformation technology without the need for in vitro vector construction by using multiple linear DNA fragments which share homologous sequences (HSs) at their ends. The strategy is based on homologous recombination between HSs of DNA fragments via endogenous recombination machinery in plastids, which subsequently are integrated into the plastid genome. We found that HSs of 200 bp or longer were sufficient for mediating the integration into the plastid genome with at least similar efficiency to that of plasmid DNA-based plastid transformation. Furthermore, we successfully used this method to introduce a phage lysin-encoding gene and a long operon into a tobacco plastid genome. The establishment of this technology simplifies the plastid transformation procedure and provides a novel solution for expressing proteins, which are either toxic to the cloning host or large operons in plastids, without need of vector cloning., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium-mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T-DNA is integrated preferentially by homologous recombination and integration by non-homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T-DNA was mediated by homologous or non-homologous recombination. This correlates with an increase in double-strand DNA breaks, the putative entry sites for T-DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52-GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform., (© 2021 The Authors. Letters in Applied Microbiology published by John Wiley & Sons Ltd on behalf of Society for Applied Microbiology.)

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

ω3 Polyunsaturated fatty acids are currently obtained mainly from fisheries; thus, sustainable alternative sources such as oleaginous microorganisms are required. Here, we describe the isolation, characterization, and application of 3 novel ω3 desaturases with ω3 polyunsaturated fatty acid-producing activity at ordinary temperatures (28 °C). First, we selected Pythium sulcatum and Plectospira myriandra after screening for oomycetes with high eicosapentaenoic acid/arachidonic acid ratios and isolated the genes psulω3 and pmd17, respectively, which encode ω3 desaturases. Subsequent characterization showed that PSULω3 exhibited ω3 desaturase activity on both C18 and C20 ω6 polyunsaturated fatty acids while PMD17 exhibited ω3 desaturase activity exclusively on C20 ω6 polyunsaturated fatty acids. Expression of psulω3 and pmd17 in the arachidonic acid-producer Mortierella alpina resulted in transformants that produced eicosapentaenoic acid/total fatty acid values of 38% and 40%, respectively, at ordinary temperatures. These ω3 desaturases should facilitate the construction of sustainable ω3 polyunsaturated fatty acid sources., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Pseudomonas protegens CHA0, known as plant-growth-promoting rhizobacterium, showed positive chemotactic responses toward proteinaceous L-amino acids. Genomic analysis revealed that P. protegens CHA0 possesses four putative chemoreceptors for amino acids (designated CtaA, CtaB, CtaC, and CtaD, respectively). Pseudomonas aeruginosa PCT2, a mutant defective in chemotaxis to amino acids, harboring a plasmid containing each of ctaA, ctaB, ctaC , and ctaD showed chemotactic responses to 20, 4, 4, and 11 types of amino acids, respectively. To enhance chemotaxis toward amino acids, we introduced the plasmids containing ctaA, ctaB, ctaC , or ctaD into P. protegens CHA0. By overexpression of the genes, we succeeded in enhancing chemotaxis toward more than half of the tested ligands. However, unexpectedly, the P. protegens CHA0 transformants showed unchanged or decreased responses to some amino acids when compared to wild-type CHA0. We speculate that alternation of expression of a chemoreceptor may affect the abundance of other chemoreceptors., Abbreviations: cDNA: complementary DNA; LBD: ligand-binding domain; MCP: methyl-accepting chemotaxis protein; PDC: PhoQ/DcuS/CitA; PGPR: plant-growth-promoting rhizobacteria; qRT-PCR: quantitative reverse transcription PCR.

Genetic engineering of cis -regulatory elements in crop plants is a promising strategy to ensure food security. However, such engineering is currently hindered by our limited knowledge of plant cis -regulatory elements. Here, we adapted self-transcribing active regulatory region sequencing (STARR-seq)-a technology for the high-throughput identification of enhancers-for its use in transiently transformed tobacco ( Nicotiana benthamiana ) leaves. We demonstrate that the optimal placement in the reporter construct of enhancer sequences from a plant virus, pea ( Pisum sativum ) and wheat ( Triticum aestivum ), was just upstream of a minimal promoter and that none of these four known enhancers was active in the 3' untranslated region of the reporter gene. The optimized assay sensitively identified small DNA regions containing each of the four enhancers, including two whose activity was stimulated by light. Furthermore, we coupled the assay to saturation mutagenesis to pinpoint functional regions within an enhancer, which we recombined to create synthetic enhancers. Our results describe an approach to define enhancer properties that can be performed in potentially any plant species or tissue transformable by Agrobacterium and that can use regulatory DNA derived from any plant genome., (© 2020 American Society of Plant Biologists. All rights reserved.)

Four Agrobacterium tumefaciens strains AGL-1, C58C1, EHA105 and LBA4404 were tested for the effects of strain types on the transformation efficiency in Mortierella alpina. Results showed that AGL-1, C58C1 and EHA105 transformed M. alpina successfully. Among them, A. tumefaciens EHA105 was first proven successful transformation of M. alpina. AGL-1 and EHA105 had the highest transformation efficiency among the four strains, while LBA4404 failed to transform M. alpina. The reason leading to the transformation efficiency difference among the four strains was explored by determining transcription levels of the virulence (vir) gene in the induction medium. Results showed that the expressions of virD1, virD2, virD4 and virE1 genes were obviously induced by acetosyringone in all the strains, and their transcriptional levels as well as virA's of AGL-1, C58C1 and EHA105 were higher than that of LBA4404, suggesting high transcriptional levels of vir genes were important for successful transformation. The study selected A. tumefaciens with high transformation efficiency of M. alpina, and would accelerate the genetic management of M. alpina. SIGNIFICANCE AND IMPACT OF THE STUDY: Oleaginous filamentous fungus Mortierella alpina is a commercial strain for the production of arachidonic acid. Genetic manipulation of M. alpina requires highly efficient transformation method. In this study, we explore the effect of Agrobacterium tumefaciens strain types on the transformation efficiency of M. alpina and select A. tumefaciens with the highest transformation efficiency, which accelerates the genetic manipulation of M. alpina. Besides, high transcriptional levels of virulence genes in A. tumefaciens were proven to play an important role for successful transformation., (© 2020 The Society for Applied Microbiology.)

Maize ( Zea mays ) is one of the most important crops in the world. However, few agronomically important maize genes have been cloned and used for trait improvement, due to its complex genome and genetic architecture. Here, we integrated multiplexed CRISPR/Cas9-based high-throughput targeted mutagenesis with genetic mapping and genomic approaches to successfully target 743 candidate genes corresponding to traits relevant for agronomy and nutrition. After low-cost barcode-based deep sequencing, 412 edited sequences covering 118 genes were precisely identified from individuals showing clear phenotypic changes. The profiles of the associated gene-editing events were similar to those identified in human cell lines and consequently are predictable using an existing algorithm originally designed for human studies. We observed unexpected but frequent homology-directed repair through endogenous templates that was likely caused by spatial contact between distinct chromosomes. Based on the characterization and interpretation of gene function from several examples, we demonstrate that the integration of forward and reverse genetics via a targeted mutagenesis library promises rapid validation of important agronomic genes for crops with complex genomes. Beyond specific findings, this study also guides further optimization of high-throughput CRISPR experiments in plants., (© 2020 American Society of Plant Biologists. All rights reserved.)

The subfamily GH13_1 of alpha-amylases is typical of Fungi, but it is also found in some unicellular eukaryotes ( e.g. , Amoebozoa, choanoflagellates) and non-bilaterian Metazoa. Since a previous study in 2007, GH13_1 amylases were considered ancestral to the Unikonts, including animals, except Bilateria, such that it was thought to have been lost in the ancestor of this clade. The only alpha-amylases known to be present in Bilateria so far belong to the GH13_15 and 24 subfamilies (commonly called bilaterian alpha-amylases) and were likely acquired by horizontal transfer from a proteobacterium. The taxonomic scope of Eukaryota genomes in databases has been greatly increased ever since 2007. We have surveyed GH13_1 sequences in recent data from ca. 1600 bilaterian species, 60 non-bilaterian animals and also in unicellular eukaryotes. As expected, we found a number of those sequences in non-bilaterians: Anthozoa (Cnidaria) and in sponges, confirming the previous observations, but none in jellyfishes and in Ctenophora. Our main and unexpected finding is that such fungal (also called Dictyo-type) amylases were also consistently retrieved in several bilaterian phyla: hemichordates (deuterostomes), brachiopods and related phyla, some molluscs and some annelids (protostomes). We discuss evolutionary hypotheses possibly explaining the scattered distribution of GH13_1 across bilaterians, namely, the retention of the ancestral gene in those phyla only and/or horizontal transfers from non-bilaterian donors., (Copyright © 2020 Desiderato et al.)

Aspergillus flavus is a fungus that produces aflatoxin B1, one of the most carcinogenic secondary metabolites. Understanding the regulation mechanism of aflatoxin biosynthesis in this fungus requires precise methods for genomic integration of mutant alleles. To avoid the disadvantage of DNA integration into the genome by non-homologous or ectopic recombination, we developed a novel strategy for site-specific integration of foreign DNA by using a carboxin-resistant sdh2 R allele (His 249 Leu). Our results demonstrated that the transformants were generated with a high efficiency (>96%) of correct integration into the sdh2 -lcus of the genome of A. flavus NRRL 3357. The advantage of this method is that introduction of the eGFP expression cassette into the sdh2 -locus had little effect on fungal growth and virulence while also being rapid and efficient. This system will be a valuable tool for genetic manipulation in A. flavus To the best of our knowledge, this is the first report on the efficient site-specific integration at the sdh2 -locus in the genome of Aspergillus ., (Copyright © 2020 Tao et al.)

Stalk lodging, which is generally determined by stalk strength, results in considerable yield loss and has become a primary threat to maize ( Zea mays ) yield under high-density planting. However, the molecular genetic basis of maize stalk strength remains unclear, and improvement methods remain inefficient. Here, we combined map-based cloning and association mapping and identified the gene stiff1 underlying a major quantitative trait locus for stalk strength in maize. A 27.2-kb transposable element insertion was present in the promoter of the stiff1 gene, which encodes an F-box domain protein. This transposable element insertion repressed the transcription of stiff1 , leading to the increased cellulose and lignin contents in the cell wall and consequently greater stalk strength. Furthermore, a precisely edited allele of stiff1 generated through the CRISPR/Cas9 system resulted in plants with a stronger stalk than the unedited control. Nucleotide diversity analysis revealed that the promoter of stiff1 was under strong selection in the maize stiff-stalk group. Our cloning of stiff1 reveals a case in which a transposable element played an important role in maize improvement. The identification of stiff1 and our edited stiff1 allele pave the way for efficient improvement of maize stalk strength., (© 2020 American Society of Plant Biologists. All rights reserved.)

High transformation efficiency is essential in genetic engineering for functional metabolic analysis and cell factory construction, in particular in construction of long biosynthetic pathways with multiple genes. Here, we found that free fatty acid (FFA)-overproducing strain showed higher transformation efficiency in Saccharomyces cerevisiae. We then verified that external supplementation of FFAs, to the culture media for competent cell preparation, improved yeast transformation efficiency significantly. Among all tested FFAs, 0.5 g/L C16:0 FFA worked best on promoting transformation of S. cerevisiae and Komagataella phaffii (previously named as Pichia pastoris). Furthermore, C16:0 FFA improved the assembly efficiency of multiple DNA fragments into large plasmids and genome by 100%, which will facilitate the construction and optimization of multigene-containing long pathways., (© FEMS 2019.)

Pearl millet is an important food crop in arid and semi-arid regions of South Asia and sub-Saharan Africa and is grown in Australia and the United States as a summer fodder crop. The d2 dwarf germplasm has been widely used in the last half-century to develop high-performing pearl millet hybrids. We previously mapped the d2 phenotype to a 1.6 cM region in linkage group (LG) 4 and identified the ABCB1 gene as a candidate underlying the trait. Here, we report the sequence, structure and expression of ABCB1 in tall ( D2D2 ) and d2 dwarf ( d2d2 ) germplasm. The ABCB1 allele in d2 dwarfs differs from that in tall inbreds by the presence of two different high copy transposable elements, one in the coding region and the second located 664 bp upstream of the ATG start codon. These transposons were present in all d2 dwarfs tested that were reported to be of independent origin and absent in the analyzed wild-type tall germplasm. We also compared the expression profile of this gene in different organs of multiple tall and d2 dwarf inbreds, including the near-isogenic inbreds at the d2 locus, Tift 23B ( D2D2 ) and Tift 23DB ( d2d2 ). Heterologous transformation of the tall ( Ca_ABCB1 ) and the d2 dwarf ( Ca_abcb1 ) pearl millet alleles in the Arabidopsis double mutant abcb1abcb19 showed that the pearl millet D2 but not the d2 allele complements the Arabidopsis abcb1 mutation. Our studies also show the importance of the COOH-terminal 22 amino acids of the ABCB1 protein in either protein function or stability., (Copyright © 2019 Parvathaneni et al.)

Malassezia encompasses a monophyletic group of basidiomycetous yeasts naturally found on the skin of humans and other animals. Malassezia species have lost genes for lipid biosynthesis, and are therefore lipid-dependent and difficult to manipulate under laboratory conditions. In this study, we applied a recently-developed Agrobacterium tumefaciens -mediated transformation protocol to perform transfer (T)-DNA random insertional mutagenesis in Malassezia furfur A total of 767 transformants were screened for sensitivity to 10 different stresses, and 19 mutants that exhibited a phenotype different from the wild type were further characterized. The majority of these strains had single T-DNA insertions, which were identified within open reading frames of genes, untranslated regions, and intergenic regions. Some T-DNA insertions generated chromosomal rearrangements while others could not be characterized. To validate the findings of our forward genetic screen, a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system was developed to generate targeted deletion mutants for two genes identified in the screen: CDC55 and PDR10 This system is based on cotransformation of M. furfur mediated by A. tumefaciens , to deliver both a CAS9 -gRNA construct that induces double-strand DNA breaks and a gene replacement allele that serves as a homology-directed repair template. Targeted deletion mutants for both CDC55 and PDR10 were readily generated with this method. This study demonstrates the feasibility and reliability of A. tumefaciens -mediated transformation to aid in the identification of gene functions in M. furfur , through both insertional mutagenesis and CRISPR/Cas9-mediated targeted gene deletion., (Copyright © 2019 by the Genetics Society of America.)

The red yeast Rhodosporidium toruloides is an excellent microbial host for production of carotenoids, neutral lipids and valuable enzymes. In recent years, genetic tools for gene expression and gene disruption have been developed for this red yeast. However, methods remain limited in terms of fine-tuning gene expression. In this study, we first demonstrated successful implementation of RNA interference (RNAi) in R. toruloides NP11, which was applied to down-regulate the expression of autophagy related gene 8 (ATG8), and fatty acid synthase genes (FAS1 and FAS2), respectively. Compared with the control strain, RNAi-engineered strains showed a silencing efficiency ranging from 11% to 92%. The RNAi approach described here ensures selective inhibition of the target gene expression, and should expand our capacity in the genetic manipulation of R. toruloides for both fundamental research and advanced cell factory development., (© FEMS 2019.)

Allelopathic plants exploit their chemical 'weapons' to prevail over the competition, suppress neighboring plants and consequently use the available resources more efficiently. However, the investigation of plant allelopathic interactions in rhizosphere is difficult to perform because of its high complexity due to interactions of biotic and abiotic factors. Thus, autonomous, aseptic root cultures of apple (Malus × domestica Borkh.) could facilitate allelopathic studies. We report on the successful genetic transformation of apple cultivars Melrose, Golden Delicious, Čadel and Gloster using Agrobacterium rhizogenes (Riker et al. 1930) Conn 1942 strain 15834 and for the first time the establishment of apple autonomous and permanent in vitro hairy root cultures that could be used as a new tool for apple allelopathic assays. Molecular characterization of transgenic hairy root lines was conducted to elucidate the possible relationship between expression of T-DNA genes and root growth characteristics that include branching. Similar content of phenolic acids (chlorogenic, caffeic, syringic, p-coumaric and ferulic), glycosilated flavonoids (rutin, quercitrin, isoquercitrin, kaempferol-3-glucoside) and flavonoid aglycones (quercetin and naringenin), and dihydrochalcone phloridzin, was detected in untransformed and transgenic apple root tissue by ultra high-performance liquid chromatography with mass spectrometry (UHPLC/(+/-)HESI-MS/MS) analyses, confirming that genetic transformation did not disturb secondary metabolite production in apple. Chlorogenic and caffeic acids and dihydrochalcones phloridzin and phloretin were detected as putative allelochemicals exuded into the growth medium in which transgenic roots were maintained for 4 weeks. Apple hairy root exudates significantly affected shoot and root development and growth of test plant Arabidopsis thaliana (L.) Heynh. seedlings after 5 or 10 days of treatment. Additionally, core cell-cycle genes CDKA1;1, CDKB2;1, CYCA3;1 and CYCB2;4 were down regulated in Arabidopsis shoots suggesting, in part, their role in inhibition of shoot growth. The present work highlighted an autonomous and permanent in vitro hairy root culture system as a valuable tool for studying allelopathic potential of apple, offering new perspective for allelopathy background elucidation in this important fruit species., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Fine-tuning of gene expression is crucial for protein expression and pathway construction, but it still faces formidable challenges due to the hierarchical gene regulation at multiple levels in a context-dependent manner. In this study, we defined the optimal targeting windows for CRISPRa and CRISPRi of the dCas9-α/ω system, and demonstrated that this system could act as a single master regulator to simultaneously activate and repress the expression of different genes by designing position-specific gRNAs. The application scope of dCas9-ω was further expanded by a newly developed CRISPR-assisted Oligonucleotide Annealing based Promoter Shuffling (OAPS) strategy, which could generate a high proportion of functional promoter mutants and facilitate the construction of effective promoter libraries in microorganisms with low transformation efficiency. Combing OAPS and dCas9-ω, the influences of promoter-based transcription, molecular chaperone-assisted protein folding and protease-mediated degradation on the expression of amylase BLA in Bacillus subtilis were systematically evaluated, and a 260-fold enhancement of BLA production was obtained. The success of the OAPS strategy and dCas9-ω for BLA production in this study thus demonstrated that it could serve as a powerful tool kit to regulate the expression of multiple genes multi-directionally and multi-dimensionally in bacteria., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cyclooxygenases are responsible for the production of prostaglandin H 2 (PGH 2 ) from arachidonic acid. PGH 2 can be converted into some bioactive prostaglandins, including prostaglandin F 2α (PGF 2α ), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF 2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF 2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF 2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF 2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

Yarrowia lipolytica is an industrial host organism with incredible potential for metabolic engineering. However, the genetic tools and capacities in this host lag behind those of conventional counterparts. In this study, we sought to increase the transformation efficiency of Y. lipolytica by creating a simple protocol using electroporation. Efficiency was increased by optimizing wash buffers, pre-culture growth time, OD600 of competent cells, voltage, competent cell volume, DNA concentration, and recovery time. The outcome of these optimizations led to a simple protocol with maximum linear fragment transformation efficiency of 1.6 × 104 transformants per μg DNA and 2.8 × 104 transformants per μg DNA for episomal plasmid transformation. The protocol presented here is superior to other Y. lipolytica transformation protocols as it requires no lengthy pretreatment and no required carrier DNA to achieve efficiencies on par with, or exceeding, previously reported methods.

Brettanomyces bruxellensis is usually considered a spoilage microorganism, responsible for significant economic losses during the production of fermented beverages such as wine, beer and cider, though for some styles of beer its influence is essential. In recent years, the competitiveness of this yeast in bioethanol production processes has brought to attention its broader biotechnological potential. Furthermore, the species has evolved key fermentation traits in parallel with Saccharomyces cerevisiae. Attempts to better understand B. bruxellensis physiology through genomics-driven research have been hampered by a lack of functional genomics tools. Genetic transformation for B. bruxellensis has only been developed recently and with limited efficiency. Here we describe gene transformation cassettes tailored for B. bruxellensis, which provide multiple drug-resistant markers and the ability to tag B. bruxellensis with different fluorescent proteins. All marker cassettes resulted in increased transformation efficiency compared to the maximum reported in literature, with one cassette, TDH1p natMX, showing five times greater efficiency. Transformation cassettes encoding fluorescent proteins enabled discrimination between subpopulations of transformed B. bruxellensis cells by flow cytometry and fluorescent microscopy. Thus, the genetic transformation toolkit described here unlocks several molecular applications such as strain tagging, insertional mutagenesis and potentially targeted gene deletion.

Aims: The aim of this study was to optimize the potential of Beauveria bassiana through cell electrofusion technique with emphasis on improving its thermotolerance and virulence against western flower thrips, Frankliniella occidentalis (Thysanoptera: Thripidae) (WFT)., Methods and Results: Nine B. bassiana isolates were tested against western flower thrips, F. occidentalis (Thysanoptera: Thripidae) (WFT) to estimate their pathogenicity and speed of infection under laboratory conditions. The isolates were also exposed to 25, 36, 38 and 40°C, for 24, 48, 72 and 120 h. Two isolates with the highest virulence against WFT and highest thermotolerance were selected and paired using a cell electrofusion technique to obtain a single strain that was both highly virulent and thermotolerant. To determine the hybrid conidia among the fusion cells, each selected isolate was loaded with a different nuclei-staining fluorescent dye before the fusion process. Hybridized fungal strains were acquired by a series of sequential dilutions and were tested for thermotolerance and virulence against WFT. Those that demonstrated both characteristics (high thermotolerance and virulence) similar to or greater than the parental isolates were considered hybrids., Conclusions: The results demonstrated that genetic transformation using cell electrofusion can be used to obtain hybrid isolates of fungal pathogens that contain the desired genes from selected isolates. Further DNA-based analysis is required to determine the genetic variation between hybrids and the parental isolates., Significance and Impact of the Study: This is the first study to demonstrate genetic transformation in B. bassiana by use of cell electrofusion and it could be developed to create superior hybrid isolates with various desired properties for integrated pest management and industrial uses in the future., (© 2018 The Society for Applied Microbiology.)

Here, two methods are described for efficient genetic modification of Saccharomyces cerevisiae using CRISPR/Cas9. The first method enables the modification of a single genetic locus using in vivo assembly of a guide RNA (gRNA) expression plasmid without the need for prior cloning. A second method using in vitro assembled plasmids that could contain up to two gRNAs was used to simultaneously introduce up to six genetic modifications (e.g. six gene deletions) in a single transformation step by transforming up to three gRNA expression plasmids simultaneously. The method is not only suitable for gene deletion but is also applicable for in vivo site-directed mutagenesis and integration of multiple DNA fragments in a single locus. In all cases, the strain transformed with the gRNA expression plasmids was equipped with a genomic integration of Spcas9, leading to strong and constitutive expression of SpCas9. The protocols detailed here have been streamlined to be executed by virtually any yeast molecular geneticist.

The establishment of the mitochondrion is seen as a transformational step in the origin of eukaryotes. With the mitochondrion came bioenergetic freedom to explore novel evolutionary space leading to the eukaryotic radiation known today. The tight integration of the bacterial endosymbiont with its archaeal host was accompanied by a massive endosymbiotic gene transfer resulting in a small mitochondrial genome which is just a ghost of the original incoming bacterial genome. This endosymbiotic gene transfer resulted in the loss of many genes, both from the bacterial symbiont as well the archaeal host. Loss of genes encoding redundant functions resulted in a replacement of the bulk of the host's metabolism for those originating from the endosymbiont. Glycolysis is one such metabolic pathway in which the original archaeal enzymes have been replaced by bacterial enzymes from the endosymbiont. Glycolysis is a major catabolic pathway that provides cellular energy from the breakdown of glucose. The glycolytic pathway of eukaryotes appears to be bacterial in origin, and in well-studied model eukaryotes it takes place in the cytosol. In contrast, here we demonstrate that the latter stages of glycolysis take place in the mitochondria of stramenopiles, a diverse and ecologically important lineage of eukaryotes. Although our work is based on a limited sample of stramenopiles, it leaves open the possibility that the mitochondrial targeting of glycolytic enzymes in stramenopiles might represent the ancestral state for eukaryotes.

Late embryogenesis abundant (LEA) proteins play key roles in plant drought tolerance. In this study, 157, 85 and 89 candidate LEA2 proteins were identified in G. hirsutum , G. arboreum and G. raimondii respectively. LEA2 genes were classified into 6 groups, designated as group 1 to 6. Phylogenetic tree analysis revealed orthologous gene pairs within the cotton genome. The cotton specific LEA2 motifs identified were E, R and D in addition to Y, K and S motifs. The genes were distributed on all chromosomes. LEA2s were found to be highly enriched in non-polar, aliphatic amino acid residues, with leucine being the highest, 9.1% in proportion. The miRNA, ghr-miR827a/b/c/d and ghr-miR164 targeted many genes are known to be drought stress responsive. Various stress-responsive regulatory elements, ABA-responsive element (ABRE), Drought-responsive Element (DRE/CRT), MYBS and low-temperature-responsive element (LTRE) were detected. Most genes were highly expressed in leaves and roots, being the primary organs greatly affected by water deficit. The expression levels were much higher in G. tomentosum as opposed to G. hirsutum The tolerant genotype had higher capacity to induce more of LEA2 genes. Over expression of the transformed gene Cot_AD24498 showed that the LEA2 genes are involved in promoting root growth and in turn confers drought stress tolerance. We therefore infer that Cot_AD24498 , CotAD_20020 , CotAD_21924 and CotAD_59405 could be the candidate genes with profound functions under drought stress in upland cotton among the LEA2 genes. The transformed Arabidopsis plants showed higher tolerance levels to drought stress compared to the wild types. There was significant increase in antioxidants, catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) accumulation, increased root length and significant reduction in oxidants, Hydrogen peroxide (H 2 O 2 ) and malondialdehyde (MDA) concentrations in the leaves of transformed lines under drought stress condition. This study provides comprehensive analysis of LEA2 proteins in cotton thus forms primary foundation for breeders to utilize these genes in developing drought tolerant genotypes., (Copyright © 2018 Magwang et al.)

Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer.

CRISPR/Cas9 is a transformative tool for making targeted genetic alterations. In plants, high mutation efficiencies have been reported in primary transformants. However, many of the mutations analyzed were somatic and therefore not heritable. To provide more insights into the efficiency of creating stable homozygous mutants using CRISPR/Cas9, we targeted LsNCED4 ( 9-cis-EPOXYCAROTENOID DIOXYGENASE4) , a gene conditioning thermoinhibition of seed germination in lettuce. Three constructs, each capable of expressing Cas9 and a single gRNA targeting different sites in LsNCED4 , were stably transformed into lettuce (Lactuca sativa) cvs. Salinas and Cobham Green. Analysis of 47 primary transformants (T 1 ) and 368 T 2 plants by deep amplicon sequencing revealed that 57% of T 1 plants contained events at the target site: 28% of plants had germline mutations in one allele indicative of an early editing event (mono-allelic), 8% of plants had germline mutations in both alleles indicative of two early editing events (bi-allelic), and the remaining 21% of plants had multiple low frequency mutations indicative of late events (chimeric plants). Editing efficiency was similar in both genotypes, while the different gRNAs varied in efficiency. Amplicon sequencing of 20 T 1 and more than 100 T 2 plants for each of the three gRNAs showed that repair outcomes were not random, but reproducible and characteristic for each gRNA. Knockouts of NCED4 resulted in large increases in the maximum temperature for seed germination, with seeds of both cultivars capable of germinating >70% at 37°. Knockouts of NCED4 provide a whole-plant selectable phenotype that has minimal pleiotropic consequences. Targeting NCED4 in a co-editing strategy could therefore be used to enrich for germline-edited events simply by germinating seeds at high temperature., (Copyright © 2018 Bertier et al.)

Resistance to antifungals is a leading concern in the treatment of human mycoses. We demonstrate that the salA gene, encoding salicylate 1-monooxygenase, is involved in resistance of the dermatophyte Trichophyton rubrum to terbinafine, one of the most effective antifungal drugs against dermatophytes. A strain with multiple copies of salA was constructed and exhibited elevated expression of salA and increased terbinafine resistance. This reflects a mechanism not yet reported in a pathogenic fungus.

Cryptococcus neoformans is a fungal pathogen that claims hundreds of thousands of lives annually. Targeted genetic manipulation through biolistic transformation in C. neoformans drove the investigation of this clinically important pathogen at the molecular level. Although costly and inefficient, biolistic transformation remains the major method for editing the Cryptococcus genome as foreign DNAs introduced by other methods such as electroporation are predominantly not integrated into the genome. Although the majority of DNAs introduced by biolistic transformation are stably inherited, the transformation efficiency and the homologous integration rate (∼1-10%) are low. Here, we developed a Tra nsient C RISPR (clustered regularly interspaced short palindromic repeat)-Cas9 coupled with E lectroporation (TRACE) system for targeted genetic manipulations in the C. neoformans species complex. This method took advantages of efficient genome integration due to double-strand breaks created at specific sites by the transient CRISPR-Cas9 system and the high transformation efficiency of electroporation. We demonstrated that TRACE can efficiently generate precise single-gene deletion mutants using the ADE2 locus as an example. This system can also effectively delete multiple genes in a single transformation, as evident by the successful generation of quadruple mf α 1 Δ 2 Δ 3 Δ 4 Δ mutants. In addition to generating gene deletion mutants, we complemented the ade2 Δ mutant by integrating a wild-type ADE2 allele at the "safe haven" region ( SH2 ) via homologous recombination using TRACE. Interestingly, introduced DNAs can be inserted at a designated genetic site without any homologous sequences, opening up numerous other applications. We expect that TRACE, an efficient, versatile, and cost-effective gene editing approach, will greatly accelerate research in this field., (Copyright © 2018 by the Genetics Society of America.)

Received wisdom in the field of fungal biology holds that the process of editing a genome by transformation and homologous recombination is inherently mutagenic. However, that belief is based on circumstantial evidence. We provide the first direct measurement of the effects of transformation on a fungal genome by sequencing the genomes of 29 transformants and 30 untransformed controls with high coverage. Contrary to the received wisdom, our results show that transformation of DNA segments flanked by long targeting sequences, followed by homologous recombination and selection for a drug marker, is extremely safe. If a transformation deletes a gene, that may create selective pressure for a few compensatory mutations, but even when we deleted a gene, we found fewer than two point mutations per deletion strain, on average. We also tested these strains for changes in gene expression and found only a few genes that were consistently differentially expressed between the wild type and strains modified by genomic insertion of a drug resistance marker. As part of our report, we provide the assembled genome sequence of the commonly used laboratory strain Cryptococcus neoformans var. grubii strain KN99α., (Copyright © 2018 Friedman et al.)