Agrobacterium Tumefaciens-mediated Transformation Research Articles

Development of a Versatile Toolbox for Genetic Manipulation of Sporothrix brasiliensis.

Sporothrix brasiliensis has emerged as the most virulent species in the Sporothrix schenckii complex, accounting for sporotrichosis. Albeit the new insights into the understanding of host-pathogen interactions and comparative genomics of this fungi, the lack of genetic tools has hindered significant advances in this field of research. Here, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system to transform different strains of S. brasiliensis. We report parameters that account for a transformation efficiency of 3,179 ± 1,171 transformants/co-cultivation, which include the use of A. tumefaciens AGL-1 in a 2:1 ratio (bacteria:fungi) during 72 h at 26°C. Our data show that a single-copy transgene is transferred to S. brasiliensis that is mitotically stable in 99% of cells after 10 generations without selective pressure. In addition, we created a plasmid toolkit that allows the establishment of fusion proteins of any S. brasiliensis gene of interest with sGFP or mCherry under the control of the GAPDH or H2A endogenous promoters. These modules allow different levels of expression of the desired fusion. Moreover, we successfully targeted these fluorescent proteins to the nucleus and used fluorescence-tagged strains to assess phagocytosis. Overall, our data show that the ATMT system is an easy-to-use and efficient genetic toolbox for studies on recombinant expression and gene function in S. brasiliensis. IMPORTANCE Sporotrichosis is the most prevalent subcutaneous mycosis worldwide and has recently become a public health concern. Although immunocompetent hosts are also prone to sporotrichosis, immunodeficient hosts often develop a more severe and disseminated form of disease. To date, the Rio de Janeiro state in Brazil is the most significant feline zoonotic transmission epicenter in the world, with more than 4,000 human and feline diagnosed cases. Cats play an essential role in the S. brasiliensis infection due to their high susceptibility and transmissibility to other felines and humans. S. brasiliensis is the most virulent etiological agent of sporotrichosis, causing the most severe clinical manifestations. Despite the increasing incidence of sporotrichosis, the identification of virulence traits important for disease establishment, development, and severity has been lacking. In this work, we established an efficient genetic toolbox to manipulate S. brasiliensis that will guide future studies to define new virulence mechanisms and a better understanding of host-pathogen interactions from a molecular perspective.

Effect of VvLaeA on the growth and development of Beauveria bassiana

It is unclear how VvLaeA functions in regulating the growth and development of Volvariella volvacea (Bull. ex. Fr.) Sing.. Firstly, bioinformatics analysis of VvLaeA was carried out in this study. Subsequently, the Vvgpd promoter and the open reading frame (ORF) fragment of VvlaeA were amplified and fused by polymerase chain reaction (PCR). The fusion fragment was cloned into the pK2 (bar) plasmid. The recombinant construct pK2(bar)-OEVvlaeA was transfected into Beauveria bassiana by Agrobacterium tumefaciens-mediated transformation. Finally, the growth and development of the transformants were examined. The results showed that VvLaeA shared a low homology with similar proteins in other fungi. Compared with the wild type, the colony diameter of the transformant was significantly increased. However, the pigment deposition, conidial yields and germination rates were significantly decreased. The overexpression strains were more sensitive to stresses than that of the wild type. Further studies showed the conidial cell wall properties of the transformants were altered, and the expressions of genes related to the conidial development were significantly down-regulated. Collectively, VvLaeA increased the growth rate of B. bassiana strains and negatively regulated the pigmentation and conidial development, which shed a light for the functional identification of straw mushroom genes.

Reasons and riddance of Agrobacterium tumefaciens overgrowth in plant transformation.

Agrobacterium tumefaciens-mediated plant transformation has become routine work across the world to study gene function and the production of genetically modified plants. However, several issues hamper the transformation process in a profound way, both directly and indirectly. One of the major concerns is the overgrowth of Agrobacterium, which occasionally appears after the co-cultivation phase of the explant. This phenomenon is reported in several species and seems to spoil the whole transformation process. There are multiple approaches being employed to counter this unwanted growth of bacteria in a few plant species. In reality, once the overgrowth appears, it becomes nearly impossible to cure it. Hence, for the prevention of this phenomenon, numerous factors are regulated. These factors are: explant nature, A. tumefaciens strain, T-DNA vector, co-cultivation (time and condition), acetosyringone, washing medium, antibiotics (type, concentration, combination, incubation period), etc. In this article, we discuss these factors based on available reports. It can be of immense help in formulating viable strategies to control A. tumefaciens overgrowth.

Efficient control of the fungal pathogens Colletotrichum gloeosporioides and Penicillium digitatum infecting citrus fruits by native soilborne Bacillus velezensis strains

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

Production of Bovine Rotavirus VP6 Subunit Vaccine in a Transgenic Fodder Crop, Egyptian Clover (Berseem, Trifolium alexandrinum) that Elicits Immune Responses in Rabbit.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock's health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

Engineering Agrobacterium tumefaciens with a Type III Secretion System to Express Type III Effectors.

Plants elicit defense responses when exposed to pathogens, which partly contribute to the resistance of plants to Agrobacterium tumefaciens-mediated transformation. Some pathogenic bacteria have sophisticated mechanisms to counteract these defense responses by injecting Type III effectors (T3Es) through the Type III secretion system (T3SS). By engineering A. tumefaciens to express T3SS to deliver T3Es, we suppressed plant defense and enhanced plant genetic transformation. Here, we describe the optimized protocols for mobilization of T3SS-expressing plasmid to engineer A. tumefaciens to deliver proteins through T3SS and fractionation of cultures to study proteins from pellet and supernatants to determine protein secretion from engineered A. tumefaciens.

Overexpression of ZxABCG11 from Zygophyllum xanthoxylum enhances tolerance to drought and heat in alfalfa by increasing cuticular wax deposition

Drought and heat stresses cause yield losses in alfalfa, a forage crop cultivated worldwide. Improving its drought and heat tolerance is desirable for maintaining alfalfa productivity in hot, arid regions. Cuticular wax forms a protective barrier on aerial surfaces of land plants against environmental stresses. ABCG11 encodes an ATP binding cassette (ABC) transporter that functions in the cuticular wax transport pathway. In this study, ZxABCG11 from the xerophyte Zygophyllum xanthoxylum was introduced into alfalfa by Agrobacterium tumefaciens-mediated transformation. Compared to the wild type (WT), transgenic alfalfa displayed faster growth, higher wax crystal density, and thicker cuticle on leaves under normal condition. Under either drought or heat treatment in greenhouse conditions, the plant height and shoot biomass of transgenic lines were significantly higher than those of the WT. Transgenic alfalfa showed excellent growth and 50% greater hay yield than WT under field conditions in a hot, arid region. Overexpression of ZxABCG11 up-regulated wax-related genes and resulted in more cuticular wax deposition, which contributed to reduction of cuticle permeability and thus increased water retention and photosynthesis capacity of transgenic alfalfa. Thus, overexpression of ZxABCG11 can simultaneously improve biomass yield, drought and heat tolerance in alfalfa by increasing cuticular wax deposition. Our study provides a promising avenue for developing novel forage cultivars suitable for planting in hot, arid, marginal lands.

Gene Editing Profiles in 94 CRISPR-Cas9 Expressing T0 Transgenic Tobacco Lines Reveal High Frequencies of Chimeric Editing of the Target Gene.

Chimeric editing is often reported in gene editing. To assess how the general chimeric editing is, we created a transgenic tobacco line carrying a marker, beta-glucuronidase gene (gusA), introduced a CRISPR-Cas9 editing vector into the transgenic tobacco line for knocking out gusA, and then investigated the gusA editing efficiencies in T0 and subsequent generations. The editing vector carried a Cas9 gene, which was driven by the cauliflower mosaic virus 35S promoter, and two guide RNAs, gRNA1 and gRNA2, which were driven by Arabidopsis U6 (AtU6) and U3 (AtU3) promoter, respectively. The two gRNAs were designed to knock out a 42-nucleotide fragment of the coding region of gusA. The editing vector was transformed into gusA-containing tobacco leaves using Agrobacterium tumefaciens-mediated transformation and hygromycin selection. Hygromycin-resistant, independent T0 transgenic lines were used to evaluate gusA-editing efficiencies through histochemical GUS assays, polymerase chain reactions (PCR), and next-generation sequencing of PCR amplicons. Profiles of targeted sequences of 94 T0 transgenic lines revealed that these lines were regenerated from non-edited cells where subsequent editing occurred and created chimeric-edited cells in these lines during or after regeneration. Two of them had the target fragment of 42 bp pairs of nucleotides removed. Detail analysis showed that on-target mutations at the AtU6-gRNA1 site and the AtU3-gRNA2 site were found in 4.3% and 77.7% of T0 transgenic lines, respectively. To overcome the issue of extremely low editing efficiencies in T0 lines, we conducted a second round of shoot induction from the chimeric line(s) to enhance the success of obtaining lines with all or most cells edited. The mutation profiles in T0 transgenic lines provide valuable information to understand gene editing in plant cells with constitutively expressed CRISPR-Cas9 and gRNAs.

Establishment of an efficient regeneration and Agrobacterium transformation system in mature embryos of calla lily (Zantedeschia spp.).

Calla lily (Zantedeschia spp.) have great aesthetic value due to their spathe-like appearance and richness of coloration. However, embryonic callus regeneration is absent from its current regeneration mechanism. As a result, constructing an adequate and stable genetic transformation system is hampered, severely hindering breeding efforts. In this research, the callus induction effectiveness of calla lily seed embryos of various maturities was evaluated. The findings indicated that mature seed embryos were more suitable for in vitro regeneration. Using orthogonal design experiments, the primary elements influencing in vitro regeneration, such as plant growth regulators, genotypes, and nanoscale materials, which was emergent uses for in vitro regeneration, were investigated. The findings indicated that MS supplemented with 6-BA 2mg/L and NAA 0.1mg/L was the optimal medium for callus induction (CIM); the germination medium (GM) was MS supplemented with 6-BA 2mg/L NAA 0.2mg/L and 1mg/L CNTs, and the rooting medium (RM) was MS supplemented with 6-BA 2mg/L NAA 0.7mg/L and 2mg/L CNTs. This allowed us to verify, in principle, that the Agrobacterium tumefaciens-mediated genetic transformation system operates under optimal circumstances using the GUS reporter gene. Here, we developed a seed embryo-based genetic transformation regeneration system, which set the stage for future attempts to create new calla lily varieties.

Hypothetical protein FoDbp40 influences the growth and virulence of Fusarium oxysporum by regulating the expression of isocitrate lyase.

Fungal growth is closely related to virulence. Finding the key genes and pathways that regulate growth can help elucidate the regulatory mechanisms of fungal growth and virulence in efforts to locate new drug targets. Fusarium oxysporum is an important plant pathogen and human opportunistic pathogen that has research value in agricultural and medicinal fields. A mutant of F. oxysporum with reduced growth was obtained by Agrobacterium tumefaciens-mediated transformation, the transferred DNA (T-DNA) interrupted gene in this mutant coded a hypothetical protein that we named FoDbp40. FoDbp40 has an unknown function, but we chose to explore its possible functions as it may play a role in fungal growth regulatory mechanisms. Results showed that F. oxysporum growth and virulence decreased after FoDbp40 deletion. FOXG_05529 (NCBI Gene ID, isocitrate lyase, ICL) was identified as a key gene that involved in the reduced growth of this mutant. Deletion of FoDbp40 results in a decrease of more than 80% in ICL expression and activity, succinate level, and energy level, plus a decrease in phosphorylated mammalian target of rapamycin level and an increase in phosphorylated 5'-adenosine monophosphate activated protein kinase level. In summary, our study found that the FoDbp40 regulates the expression of ICL at a transcriptional level and affects energy levels and downstream related pathways, thereby regulating the growth and virulence of F. oxysporum.

Generating a construct harboring the rpb1 promoter for enhancing recombinant protein expression in the medicinal mushroom Cordyceps militaris

Cordyceps militaris is a valuable medicinal mushroom that contains numerous bioactive ingredients such as cordycepin, adenosine, pentostatin, polysaccharides, and carotenoids. However, the contents of these substances in wild-type strains of C. militaris are relatively low. Recently, the genome of C. militaris has been fully sequenced, and genes involved in the biosynthesis of beneficial constituents have been identified. Therefore, research on enhancing the biosynthesis of the bioactive compounds in this fungus by recombinant DNA technology has advantages. In the present study, the authors have successfully generated a binary vector with a T-DNA region carrying a construct regulated by the native rpb1 promoter that can enhance gene expression in C. militaris. The T-DNA structure from the binary vector was then transferred into the C. militaris genome by Agrobacterium tumefaciens-mediated transformation. Analysis of some fungal transformants by PCR using the specific primer pairs indicated that the T-DNA structure was successfully integrated into the fungal genome. Observation of the transformants under fluorescence microscopy confirmed the overexpression of the DsRed fluorescent protein under the regulation of the rpb1promoter. The binary vector carrying the rpb1 promoter constructed in this study can be employed to promote the expression of target genes in the medicinal mushroom C. militaris.

Construction of nucleus-directed fluorescent reporter systems and its application to verification of heterokaryon formation in Morchella importuna.

Morchella importuna (M. importuna) is a rare fungus with high nutrition value and distinct flavor. Despite the successful artificial cultivation, its genetic characteristics and biological processes such as life cycle, reproductive system, and trophic mode remain poorly understood. Considering this, we constructed pEH2B and pMH2B vectors by fusing M. importuna endogenous histone protein H2B with fluorescent proteins eGFP or mCherry, respectively. Based on the constructed pEH2B and pMH2B vectors, nuclear fluorescence localization was performed via Agrobacterium tumefaciens-mediated transformation (ATMT). These two vectors were both driven by two endogenous promoters glyceraldehyde 3-phosphate dehydrogenase (GPD) and ubiquitin (UBI). The vector-based reporter systems were tested by the paired culture of two genetically modified strains pEH2B-labeled M04M24 (24e, MAT1-1-1) and pMH2B-abeled M04M26 (26m, MAT1-2-1). The fluorescence observation and molecular identification results indicated the successful hyphal fusion and heterokaryon formation. We found that the expression of the reporter genes was stable, and it did not interfere with the growth of the fungus. Our constructed nucleus-directed fluorescent systems in M. importuna can be used for monitoring the dynamic development and reproductive processes in living cells and also for monitoring the interaction between morels and plant roots. Therefore, morels exhibit the potential to be a candidate organism used for the research on basic biology and genetics of ascomycetes.

Biocontrol action of Trichothecium roseum against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

Trichothecium roseum is known to be a mycoparasite and inhibit phytopathogenic fungi. However, so far, only scarce information is available on the impacts of T. roseum on powdery mildews. Based on the morphological and molecular analysis, we identified T. roseum as a mycoparasite on colonies of the wheat powdery mildew fungus (Blumeria graminis f. sp. tritici, Bgt, recently clarified as B. graminis s. str.) and then showed that T. roseum was capable of efficiently impairing colony formation and conidial distribution of Bgt. After inoculation of T. roseum conidia on Bgt colonies, the biomasses of Bgt significantly decreased 1.46, 1.64, 7.55, and 10.49 times at 2, 4, 6, and 8 dpi, respectively. Thus, T. roseum, acting as a potential biological agent, impeded the developments of Bgt, making it a viable alternative for wheat powdery mildew control. Utilizing the Agrobacterium tumefaciens-mediated transformation (ATMT) system, a T. roseum strain that constitutively expressed green fluorescent protein was produced to improve the visualization of the T. roseum-Bgt interaction and showed direct hyphae interaction of T. roseum with Bgt structures during parasitic processes. These findings indicate that ATMT is a potent and efficient method for transforming T. roseum. Nevertheless, our results suggest that T. roseum is an antagonistic parasite of the wheat powdery mildew fungus, and hence, can be considered for phytopathogen management.

Multiplex CRISPR/Cas9-mediated knockout of the phytoene desaturase gene in Coffea canephora

Coffea canephora (2n = 2x = 22 chromosomes) is a species with extensive genetic diversity and desirable agronomic traits for coffee breeding programs. However, obtaining a new coffee cultivar through conventional breeding techniques may require more than 30 years of crossing cycles and selection, which hampers the effort of keeping up with market demands and rapidly proposing more resilient to climate change varieties. Although, the application of modern biotechnology tools such as precision genetic engineering technologies may enable a faster cultivar development process. Therefore, we aimed to validate the CRISPR/Cas9 system to generate mutations on a selected genotype of C. canephora, the clone 14. Embryogenic calli and a multiplex binary vector containing two sgRNAs targeting different exons of the CcPDS gene were used. The sgRNAs were under the C. canephora U6 promoter regulation. The target gene encodes phytoene desaturase, an enzyme essential for photosynthesis involved in β-carotene biosynthesis. Somatic seedlings and embryos with albino, variegated and green phenotypes regenerated after Agrobacterium tumefaciens-mediated genetic transformation were analyzed by verifying the insertion of the Cas9 gene and later by sequencing the sgRNAs target regions in the genome of Robusta modified seedlings. Among them, 77% had the expected mutations, and of which, 50% of them had at least one target with a homozygous mutation. The genotype, temperature of co-cultivation with the bacteria, and light intensity used for subsequent embryo regeneration appeared to strongly influence the successful regeneration of plants with a mutated CcPDS gene in the Coffea genus.

Paracoccidioides lutzii Formamidase Contributes to Fungal Survival in Macrophages.

Nitrogen is a crucial nutrient for microorganisms that compose essential biomolecules. However, hosts limit this nutrient as a strategy to counter infections, therefore, pathogens use adaptive mechanisms to uptake nitrogen from alternative sources. In fungi, nitrogen catabolite repression (NCR) activates transcription factors to acquire nitrogen from alternative sources when preferential sources are absent. Formamidase has been related to nitrogen depletion in Aspergillus nidulans through formamide degradation to use the released ammonia as a nitrogen source. In Paracoccidioides spp., formamidase is highly expressed in transcriptomic and proteomic analyses. Here, we aim to investigate the importance of formamidase to Paracoccidioides lutzii. Thereby, we developed a P. lutzii silenced strain of fmd gene (AsFmd) by antisense RNA technology using Agrobacterium tumefaciens-mediated transformation (ATMT). The AsFmd strain led to increased urease expression, an enzyme related to nitrogen assimilation in other fungi, suggesting that P. lutzii might explore urease as an alternative route for ammonia metabolism as a nitrogen source. Moreover, formamidase was important for fungal survival inside macrophages, as fungal recovery after macrophage infection was lower in AsFmd compared to wild-type (WT) strain. Our findings suggest potential alternatives of nitrogen acquisition regulation in P. lutzii, evidencing formamidase influence in fungal virulence.

Production of marker-free transgenic plants from mature tissues of navel orange using a Cre/loxP site-recombination system

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1–0.5 (1–2 weeks), > 0.5 cm (3–4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange (Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase (IPT) and an anti-bacterial peptide (PR1aCB). Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained. Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants (deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange (Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

In vitro</em></strong><strong> direct regeneration and <em>Agrobacterium tumefaciens</em> mediated <em>in planta</em> transformation of <em>Ocimum sanctum L.

An in vitro regeneration system for propagation has been successfully developed for a valuable medicinal and aromatic plant ‘ Ocimum sanctum L’. In the present study, petiole explants, from in-vitro grown cultures of O. sanctum, was used for direct regeneration. The developed protocol employed 98% of regeneration frequency in addition to 9.6 shoots per explant when cultured on Murashige and Skoog (MS) medium fortified with 3 mg/L benzylamino purine (BAP) and 1 mg/L Naphthalene acetic acid (NAA). Furthermore, Agrobacterium tumefaciens Mediated genetic Transformation (ATMT) protocol (transient and stable) was also developed using LBA4404 strain harboring pBI121 with uid-A and neomycin phosphotransferase genes. The regenerated transformants were shifted on MS with kanamycin (50 mg/L) and afterwards placed on the half-strength MS medium. The validation was done through polymerase chain reaction (PCR) with neomycin phosphotransferase-II ( npt-II) & β-glucoronidase ( uid-A) gene primers. The maximum stable transformation frequency of 70% ± 0.35 was achieved. Hence, it is apparent that the established protocols i.e. in vitro direct regeneration and ATMT are appropriate for integrating novel enzymes/genes through high throughput techniques such as gene tagging, and targeted gene replacement to modulate the primary as well as secondary metabolic flux towards desired agronomic product or trait in planta.

Production of benzylglucosinolate in genetically engineered carrot suspension cultures.

Glucosinolates, a group of sulfur-containing specialized metabolites of the Brassicales, have attracted a lot of interest in nutrition, medicine and agriculture due to their positive health effects and their involvement in plant defense. Their biological activities and the extensive knowledge of their biosynthesis have inspired research into development of crops with enhanced glucosinolate contents as well as their biotechnological production in homologous and heterologous systems. Here, we provide proof-of-concept for transgenic suspension cultures of carrot (Daucus carota, Apiacae) as a scalable production platform for plant specialized metabolites using benzylglucosinolate as a model. Two T-DNAs carrying in total six genes of the benzylglucosinolate biosynthesis pathway from Arabidopsis thaliana as well as NPTII and BAR as selectable markers were transferred to carrot cells by Agrobacterium tumefaciens-mediated transformation. Putative transformants selected based on their kanamycin and BASTA resistances were subjected to HPLC-MS analysis. Of 79 putative transformants, 17 produced benzylglucosinolate. T-DNA-integration was confirmed for the five best producers. Callus from these transformants was used to establish suspension cultures for quantitative analysis. When grown in 60-ml-cultures, the best transformants produced roughly 2.5 nmol (g fw)-1 benzylglucosinolate, together with up to 10 nmol (g fw)-1 desulfobenzylglucosinolate. Only one transformant produced more benzylglucosinolate than desulfobenzylglucosinolate. The concentration of sulfate in the medium was not a major limiting factor. High production seemed to be associated with poor growth and vice versa. Therefore, future research should try to optimize medium and cultivation process and to separate growth and production phase by using an inducible promoter.

Interference Expression of StMSD Inhibited the Deposition of Suberin and Lignin at Wounds of Potato Tubers by Reducing the Production of H2O2.

Superoxide dismutase (SOD) actively participates in the wound stress of plants. However, whether StMSD mediates the generation of H2O2 and the deposition of suberin polyphenolic and lignin at potato tuber wounds is elusive. In this study, we developed the StMSD interference expression of potato plants and tubers by Agrobacterium tumefaciens-mediated transformation. The StSOD expression showed a marked downregulation in StMSD-interference tubers, especially StCSD2 and StCSD3. The content of O2•− exhibited a noticeable increase together with the inhibition in H2O2 accumulation. Moreover, the gene expression levels of StPAL (phenylalanine ammonia-lyase) and StC4H (cinnamate-4-hydroxylase) were downregulated in StMSD-interference tubers, and less suberin polyphenolic and lignin depositions at the wounds were observed. Taken together, the interference expression of StMSD can result in less suberin polyphenolic and lignin deposition by inhibiting the disproportionation of O2•− to H2O2 and restraining phenylpropanoid metabolism in tubers.

Development of Genetic Tools in Glucoamylase-Hyperproducing Industrial Aspergillus niger Strains.

Simple SummaryGlucoamylase is one of the most needed industrial enzymes in the food and biofuel industries. Aspergillus niger is a commonly used cell factory for the production of commercial glucoamylase. For decades, genetic manipulation has promoted significant progress in industrial fungi for strain engineering and in obtaining deep insights into their genetic features. However, genetic engineering is more laborious in the glucoamylase-producing industrial strains A. niger N1 and O1 because their fungal features of having few conidia (N1) or of being aconidial (O1) make them difficult to perform transformation on. In this study, we targeted A. niger N1 and O1 and successfully developed high-efficiency transformation tools. We also constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 editing marker-free system using an autonomously replicating plasmid to express Cas9 protein and to guide RNA and the selectable marker. By using the genetic tools developed here, we generated nine albino deletion mutants. After three rounds of sub-culturing under nonselective conditions, the albino deletions lost the autonomously replicating plasmid. Together, the tools and optimization process above provided a good reference to manipulate the tough working industrial strain, not only for the further engineering these two glucoamylase-hyperproducing strains, but also for other industrial strains.The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes, particularly glucoamylase. Although a variety of genetic techniques have been successfully used in wild-type A. niger, the transformation of industrially used strains with few conidia (e.g., A. niger N1) or that are even aconidial (e.g., A. niger O1) remains laborious. Herein, we developed genetic tools, including the protoplast-mediated transformation and Agrobacterium tumefaciens-mediated transformation of the A. niger strains N1 and O1 using green fluorescent protein as a reporter marker. Following the optimization of various factors for protoplast release from mycelium, the protoplast-mediated transformation efficiency reached 89.3% (25/28) for N1 and 82.1% (32/39) for O1. The A. tumefaciens-mediated transformation efficiency was 98.2% (55/56) for N1 and 43.8% (28/64) for O1. We also developed a marker-free CRISPR/Cas9 genome editing system using an AMA1-based plasmid to express the Cas9 protein and sgRNA. Out of 22 transformants, 9 albA deletion mutants were constructed in the A. niger N1 background using the protoplast-mediated transformation method and the marker-free CRISPR/Cas9 system developed here. The genome editing methods improved here will accelerate the elucidation of the mechanism of glucoamylase hyperproduction in these industrial fungi and will contribute to the use of efficient targeted mutation in other industrial strains of A. niger.