Agrobacterium Tumefaciens-mediated Transformation Method Research Articles

Visualization of the Infection and Colonization Process of Dendrobium officinale Using a Green Fluorescent Protein-Tagged Isolate of Fusarium oxysporum.

Dendrobium officinale soft rot is a widespread and destructive disease caused by Fusarium oxysporum that can seriously affect yield and quality. To better understand the fungal infection and colonization, we successfully created an F. oxysporum labeled with green fluorescent protein using the Agrobacterium tumefaciens-mediated transformation method. Transformants had varying fluorescence intensities, but their pathogenicity did not differ from that of the wild type. Fluorescence microscopy revealed that F. oxysporum primarily entered the aboveground portion of D. officinale through the leaf margin, stomata, or by direct penetration of the leaf surface. It then colonized the mesophyll and spread along its vascular bundles. D. officinale exhibited typical symptoms of decay and wilting at 14 days postinoculation, accompanied by a pronounced fluorescence signal in the affected area. The initial colonization of F. oxysporum in the subterranean region primarily involved attachment to the root hair and epidermis, which progressed to the medullary vascular bundle. At 14 days postinoculation, the root vascular bundles of D. officinale exhibited significant colonization by F. oxysporum. Macroconidia were also observed in black rot D. officinale tissue. In particular, the entire root was surrounded by a significant number of chlamydospore-producing F. oxysporum mycelia at 28 days postinoculation. This approach allowed for the visualization of the complete infection process of F. oxysporum and provided a theoretical foundation for the development of field control strategies.

Evaluation of Parameters Affecting Agrobacterium-Mediated Transient Gene Expression in Industrial Hemp (Cannabis sativa L.).

Industrial hemp Cannabis sativa L. is an economically important crop mostly grown for its fiber, oil, and seeds. Due to its increasing applications in the pharmaceutical industry and a lack of knowledge of gene functions in cannabinoid biosynthesis pathways, developing an efficient transformation platform for the genetic engineering of industrial hemp has become necessary to enable functional genomic and industrial application studies. A critical step in the development of Agrobacterium tumefaciens-mediated transformation in the hemp genus is the establishment of optimal conditions for T-DNA gene delivery into different explants from which whole plantlets can be regenerated. As a first step in the development of a successful Agrobacterium tumefaciens-mediated transformation method for hemp gene editing, the factors influencing the successful T-DNA integration and expression (as measured by transient β-glucuronidase (GUS) and Green Florescent Protein (GFP) expression) were investigated. In this study, the parameters for an agroinfiltration system in hemp, which applies to the stable transformation method, were optimized. In the present study, we tested different explants, such as 1- to 3-week-old leaves, cotyledons, hypocotyls, root segments, nodal parts, and 2- to 3-week-old leaf-derived calli. We observed that the 3-week-old leaves were the best explant for transient gene expression. Fully expanded 2- to 3-week-old leaf explants, in combination with 30 min of immersion time, 60 µM silver nitrate, 0.5 µM calcium chloride, 150 µM natural phenolic compound acetosyringone, and a bacterial density of OD600nm = 0.4 resulted in the highest GUS and GFP expression. The improved method of genetic transformation established in the present study will be useful for the introduction of foreign genes of interest, using the latest technologies such as genome editing, and studying gene functions that regulate secondary metabolites in hemp.

An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation

Cochliobolus heterostrophus is a plant pathogenic fungus of southern corn leaf blight, which has been regarded as a model necrotrophic plant pathogen. Many methods have been developed to knock out targeted genes in C. heterostrophus, of which the most widely-used one is protoplast-mediated transformation. However, there are several problems of this method associated with protoplast preparation, DNA product, time consumption, or high cost. In this study, a highly efficient target gene deletion approach in C. heterostrophus was established and optimized, based on Agrobacterium tumefaciens-mediated transformation (ATMT); the transformation efficiency of this approach was 85–88 transformants per 105 conidia, and the homologous recombination efficiency was approximately 68.3%. Furthermore, six gene knockout mutants of C. heterostrophus were obtained using this ATMT method. The phenotypes of this fungus altered in the mutant strains, and the virulence of the mutants significantly reduced compared to of the wild type strain. Taken together, this ATMT system established in this study can be used as a genetic manipulation tool for C. heterostrophus, to better understand the functions of genes and its relation to virulence.

CRISPR/Cas9-mediated Targeted Mutagenesis of Inulin Biosynthesis in Rubber Dandelion

Rubber dandelion (Taraxacum kok-saghyz) is a natural rubber-producing dandelion that has the potential to become an industrial crop. Inulin is a storage carbohydrate in rubber dandelion, and its synthesis competes with rubber production for assimilated carbon. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system to simultaneously target two sites in the gene that encodes 1-fructan:fructan-1-fructosyl transferase gene (1-FFT), a key enzyme in inulin biosynthesis. Agrobacterium rhizogenes and Agrobacterium tumefaciens-mediated plant transformation methods were used to generate transgenic plants with CRISPR/Cas9 elements. Transformation rates were 71% and 64% via A. rhizogenes and A. tumefaciens-mediated transformations, respectively. Mutagenesis was confirmed by the loss of restriction site method and Sanger sequencing. Of 13 transgenic plants obtained via A. rhizogenes, six showed editing of both target sites within the 1-FFT gene. Transgenic rubber dandelion plants were obtained within 10 weeks using A. rhizogenes-mediated transformation, which was much faster than the 6 months required for A. tumafaciens transformants. Of 11 transgenic plants obtained via A. tumefaciens, five showed mutations in both target sites. Reverse-transcription polymerase chain reaction confirmed Cas9 expression in all edited transformants. Both A. rhizogenes-mediated double-mutant transformants and A. tumefaciens-mediated double mutant transformants had lower levels of inulin than wild-type plants. Moreover, A. rhizogenes-mediated transformants had a higher rubber content than wild-type plants. Therefore, the present study validates the use of CRISPR/Cas9 gene editing as an efficient tool for the generation of useful mutations in rubber dandelion and could be implemented in future crop improvement approaches.

Breeding of virus-resistant transgenic sugarcane by the integration of the Pac1 gene

Various RNA viral diseases on sugarcane result in yield loss and decreased sugar content. Breeding new varieties with virus resistance is the main goal of the sugarcane breeding program. Both single-stranded and double-stranded RNA viruses generated a double-stranded RNA replicative form (RF) during the replication cycle progress. While double-stranded RNA-specific ribonuclease (PAC1) encoded by the Pac1 gene (from Schizosaccharomyces pombe) can recognize and degrade double-stranded RNA specifically without any sequence, the expression of PAC1 in transgenic sugarcane may successfully develop virus-resistant sugarcane. In this research, we first expressed the PAC1 in prokaryotic cells. Then, double-stranded RNA RF of sugarcane's streak mosaic virus (SCSMV) was artificially synthesized. The degradation activity of the PAC1 was successfully tested by mixing the PAC1 protein and the double-stranded RNA RF. After that, the Pac1 gene was ligated to a plant expression vector and was then introduced into a virus-sensitive sugarcane cultivar by using Agrobacterium tumefaciens-mediated transformation method. Transgenic plants were challenged by inoculating with SCSMV. Results showed that although all the transgenic lines were infected by SCSMV, the mosaic symptoms that appeared on the leaves were significantly milder than that of the wild type. All transgenic shoots showed significantly lower viral loads and attained greater heights than wild-type shoots. This research provided a new pathway for breeding new varieties of sugarcane with virus resistance.

Deciphering the genome of Simplicillium aogashimaense to understand its mechanisms against the wheat powdery mildew fungus Blumeria graminis f. sp. tritici

AbstractSimplicillium spp. are mycoparasites that exert growth-inhibitory effects on phytopathogenic fungi. However, limited studies have examined the effects of Simplicillium spp. on powdery mildews. In this study, morphological and molecular analyses revealed that S. aogashimaense is a mycoparasite of the wheat powdery mildew fungus, Blumeria graminis f. sp. tritici (Bgt), under field conditions. The inoculation of Bgt colonies with S. aogashimaense significantly impaired Bgt colony formation and conidial distribution and markedly decreased the biomass of Bgt. To examine the interaction between Simplicillium and Bgt, an S. aogashimaense strain that constitutively expresses green fluorescent protein (GFP) was constructed using the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The hyphae of GFP-expressing S. aogashimaense directly penetrated the B. graminis structures. These findings indicate that ATMT can be employed to reveal the biocontrol activities of physiologically and phylogenetically diverse Simplicillium spp. In vitro, S. aogashimaense exudates compromised Bgt conidial germination and appressorial formation. Thus, S. aogashimaense functions as a potential biological control agent by impeding the development of Bgt and can be a viable alternative for controlling the wheat powdery mildew. To gain further insights into the mechanism underlying this mycoparasitism, the genome of S. aogashimaense was sequenced and assembled. S. aogashimaense harbored seven chromosomes comprising 8963 protein-coding genes. Additionally, two putative effector-coding genes (Sao008714 and Sao006491) were identified. The expression levels of Sao008714 and Sao006491 in S. aogashimaense were dramatically upregulated during the mycoparasitic interaction. In addition, 41 gene clusters putatively involved in the production of secondary metabolites, which exhibit insecticidal, antifungal and antibacterial activities, were identified using genome-wide identification, annotation and analysis of secondary metabolite biosynthesis gene clusters. These results suggest that S. aogashimaense parasitizes Bgt and hence, can be considered for phytopathogen management.

Screening and Functional Verification of Selectable Marker Genes for Cordyceps militaris

The selectable marker genes are necessary resistance genes for gene knockout, gene complementation, and gene overexpression in filamentous fungi. Moreover, the more sensitive the filamentous fungi are to antibiotics, the more helpful it is to screen the target transformants. In order to obtain the antibiotic (or herbicide) which can effectively inhibit the growth of Cordyceps militaris and verify the function of the corresponding resistance gene in C. militaris, the sensitivity of C. militaris to hygromycin and glufosinate ammonium was compared to determine the resistance gene that was more suitable for the screening of C. militaris transformants. The binary vector of the selectable marker gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the selectable marker gene in C. militaris was verified by the Agrobacterium tumefaciens-mediated transformation method. The results showed that C. militaris was more sensitive to glufosinate ammonium than hygromycin. The growth of C. militaris could be completely inhibited by 250 μg/mL glufosinate ammonium. The expression cassette of the glufosinate ammonium resistance gene (bar gene) was successfully constructed by DJ-PCR. The binary vector pCAMBIA0390-Bar was successfully constructed by homologous recombination. The bar gene of the vector pCAMBIA0390-Bar was successfully integrated into the C. militaris genome and could be highly expressed in the transformants of C. militaris. This study will promote the identification of C. militaris gene function and reveal the biosynthetic pathways of bioactive components in C. militaris.

Improved production of β-glucan by a T-DNA-based mutant of Aureobasidium pullulans.

To improve β-1,3-1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • β-Glucan produced by the mutant was significantly improved.

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

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

Calcium-dependent protein kinase 32 gene maintains photosynthesis and tolerance of potato in response to salt stress

Calcium-dependent protein kinase 32 (StCDPK32) of potato is identified as a mono-molecular Ca2+-sensor/kinase-effector protein encoding by StCDPK32. This study aims to understand the mechanism of StCDPK32 involved in resisting to salinity stress. StCDPKs were examined in Solanum tuberosum L. cv. Zihuabai treated with 200 mM NaCl for 0–48 h. To investigate the ability of potato to resist salt stress, Zihuabai was cultured in NaCl-conditioned medium. To explore the effects of StCDPK32 on physiochemical activities and gas exchange, StCDPK32-upregulated and StCDPK32-downregulated potato plants were developed with Agrobacterium tumefaciens-mediated transformation method. Subcellular location of StCDPK32 protein was detected using confocal scanning laser microscopy. It was found that NaCl treatment resulted in transcription change of StCDPKs. Salinity treatment promoted StCDPK32 transcription in potato plants. StCDPK32-transgenic plants are tolerant to NaCl while StCDPK32-repressed plants are sensitive to NaCl in comparison with the untransgenic plants. StCDPK32 overexpression elevates genes expression and enzyme activities associated with salt stress. Additionally, photosynthesis is promoted by StCDPK32 overexpression under salt stress conditions. StCDPK32 protein localizes in nucleus, cytoplasm and cellular membrane. StCDPK32 positively modulates physiological properties and photosynthesis in response to salinity stress. StCDPK32 is a potential candidate gene for growth improvement under salt conditions.

Cloning of the ben gene and its functional identification in Cordyceps militaris

Selectable marker genes are very important for the screening of target mutants. At least two selectable marker genes are usually required to identify the function of a gene by gene knockout, gene complementation, and gene overexpression. However, there are few selectable marker genes that can be applied to Cordyceps militaris. In order to obtain the selectable marker gene which can be used to C. militaris, the sensitivity of C. militaris to benomyl as a fungicide was studied by the concentration gradient method. Then the benomyl resistance gene (the ben gene) was cloned from Neurospora crassa. The binary plasmid of the ben gene was constructed by combining the double-joint PCR (DJ-PCR) method and the homologous recombination method, and the function of the ben gene in C. militaris was identified by the Agrobacterium tumefaciens-mediated transformation (ATMT) method. The results showed that 3 μg/mL benomyl could completely inhibit the growth of C. militaris. The ben gene with a length of 1344 bp was successfully cloned from N. crassa. The binary plasmid pCAMBIA0390-Ben containing the ben gene expression cassette was efficiently constructed by the DJ-PCR method and the homologous recombination method. The ben gene of the plasmid pCAMBIA0390-Ben was successfully integrated into the genome of C. militaris by the ATMT method, and the C. militaris transformants with resistance to benomyl were obtained. Moreover, the ben gene could be highly expressed in C. militaris. It was confirmed that the ben gene derived from N. crassa could be used as a selectable marker gene in C. militaris. These results provide technical support for the identification of C. militaris gene function and the elucidation of the biosynthetic pathways of bioactive components in C. militaris.

An efficient Agrobacterium tumefaciens-mediated transformation method for Simplicillium subtropicum (Hypocreales: Cordycipitaceae).

Filamentous fungi are the organisms of choice for most industrial biotechnology. Some species can produce a variety of secondary metabolites and enzymes of commercial interest, and the production of valuable molecules has been enhanced through different molecular tools. Methods for genetic manipulation and transformation have been essential for the optimization of these organisms. The genus Simplicillium has attracted increased attention given several potential biotechnological applications. The Simplicillium genus harbors several entomopathogenic species and some isolates have been explored for bioremediation of heavy metal contaminants. Furthermore, the myriad of secondary metabolites isolated from Simplicillium spp. render these organisms as ideal targets for deep exploration and further biotechnological mining possibilities. However, the lack of molecular tools hampered the exploration of this genus. Thus, an Agrobacterium tumefaciens-mediated transformation method was established for Simplicillium subtropicum, employing the far-red fluorescent protein TURBOFP635/Katushka, as a visual marker, and the selection marker SUR gene, that confers resistance to chlorimuron ethyl. Notably, one round of transformation using the established method yielded almost 400 chlorimuron resistant isolates. Furthermore, these transformants displayed mitotic stability for, at least, five generations. We anticipate that this method can be useful for deep molecular exploration and improvement of strains in the Simplicillium genus.

Engineering 4-hydroxyisoleucine biosynthesis by ectopic expression of bacterial IDO gene in fenugreek and tobacco

There is enough evidence that 4-hydroxyisoleucine (4-HIL), the main ingredient of fenugreek plant for the treatment of type 2 diabetes, has significant influences on the secretion of and resistance to insulin in human cells. However, its content in the leaves and seeds of fenugreek seems insufficient for industrial and pharmaceutical exploitation. In order to boost 4-HIL content in fenugreek and promote its heterologous expression in the model tobacco plant, the encoding gene of isoleucine dioxygenase (IDO), a key enzyme in the 4-HIL biosynthesis pathway, was cloned from Bacillus thuringiensis and ectopically expressed under the control of CaMV-35S promoter in fenugreek (Trigonella foenum-graecum L.) using a viral vector (pER2L5R5) and in tobacco (Nicotiana tabacum L.) via Agrobacterium tumefaciens-mediated transformation method. Transgene expression was confirmed at the mRNA level through RT-PCR and qRT-PCR, and 4-HIL content was measured by High Performance Liquid Chromatography (HPLC). The obtained results showed that the amount of 4-hydroxyisoleucine in transfected fenugreek plants was higher than that in the control plants. Also, heterologous production of 4-HIL in transgenic tobacco plants was achieved and foliar application of isoleucine on these plants could increase the amount of this valuable amino acid derivative.

Overexpression of nicotinamide mononucleotide adenylyltransferase (nmnat) increases the growth rate, Ca2+ concentration and cellulase production in Ganoderma lucidum.

Identifying new and economical means to utilize diverse lignocellulosic biomass is an urgent task. Ganoderma lucidum is a well-known edible and medicinal basidiomycete with an excellent ability to degrade a wide range of cellulosic biomass, and its nutrient use efficiency is closely related to the activity of extracellular cellulase. Intracellular nicotinamide adenine dinucleotide (NAD+) biosynthesis is controlled in response to nutritional status, and NAD+ is an essential metabolite involved in diverse cellular processes. Nicotinamide mononucleotide adenylyltransferase (NMNAT) is a common enzyme in three NAD+ synthesis pathways. In this study, a homologous gene of nmnat was cloned from G. lucidum and two G. lucidum overexpression strains, OE::nmnat4 and OE::nmnat19, were constructed using an Agrobacterium tumefaciens-mediated transformation method. The G. lucidum nmnat overexpression strains showed obviously increased colony growth on different carbon sources, and intracellular Ca2+ concentrations in the G. lucidum OE::nmnat4 and OE::nmnat19 strains were increased by 2.04- and 2.30-fold, respectively, compared with those in the wild-type (WT) strains. In the G. lucidum OE::nmnat4 and OE::nmnat19 strains, endo-β-glucanase (CMCase) activity increased by approximately 2.8- and 3-fold, while β-glucosidase (pNPGase) activity increased by approximately 1.9- and 2.1-fold, respectively, compared with the activity in the WT strains. Furthermore, overexpression of NAD+ synthesis pathways was found to elicit cellulase production by increasing the intracellular Ca2+ concentration. In summary, this study is the first to demonstrate that increased intracellular NAD+ contents through overexpression of the nmnat gene of NAD+ synthesis pathways may increase cellulase production by increasing intracellular Ca2+ concentrations in G. lucidum. KEY POINTS: • The concentration of NAD+influences cellulase production in G. lucidum. • The concentration of NAD+influences the intracellular Ca2+concentration in G. lucidum. • The concentration of NAD+influences cellulase production by eliciting a change in intracellular Ca2+in G. lucidum.

Establishment of the 'imbibed seed piercing' method for Agrobacterium-mediated transformation of jute and flax bast fibre crops via phloem-specific expression of the β-glucuronidase Gene

Jute (Corchorus capsularis) and flax (Linum usitatissimum) have been established as important sources of bast fibres (phloem fibres) worldwide. These long and lustrous natural fibres are used for various commercial and industrial purposes and hold immense future prospects. To meet the worldwide demand for bast fibres, improvement in the production and quality of jute and flax is essential. Molecular breeding is an expeditious approach for varietal improvement and requires an efficient gene transformation system. The available methods for jute and flax transformation are highly specific to species and variety, requiring a long time and rigorous preparation of media, explants, and screening procedures. In this study, we used a fast and stable method for Agrobacterium tumefaciens-mediated transformation of jute (variety JRC321) and flax (variety FT-897), known as the ‘imbibed seed piercing’ method (ISPM), where seeds were used as explants. A phloem-specific Arabidopsis sucrose-proton H+ symporter 2 (AtSUC2) promoter-driven gus gene (β-glucuronidase) was used for the assessment of genetic transformation of jute and flax plants. Transformed seedlings were selected following three consecutive selection pressure tests conducted at two-week intervals using kanamycin-supplemented culture media. Transgene integration was verified using Southern blotting and PCR-based molecular characterization of jute and flax plants. The gus gene expression levels in the transgenic jute and flax tissue samples were analysed by semi-quantitative and quantitative RT-PCR. The GUS protein expression in transgenic plant tissues was confirmed by the development of blue colour in the histochemical GUS assay. Blue colouration was observed only in phloem cells of the tested plant tissues. Transgenic progeny plants of subsequent generations followed the 3:1 transgene segregation pattern and exhibited phloem-specific GUS expression. This fast, easy, and stable ISPM could also be applied to other fibre producing industrial crops and could form a part of a modern CRISPR/Cas9-based genome editing tool by efficiently delivering CRISPR vectors in jute and flax plants.

Supplementation of Aspergillus glaucus with gfdB gene encoding a glycerol 3-phosphate dehydrogenase in Aspergillus nidulans.

In Aspergillus nidulans, there are two putative glycerol 3-phosphate dehydrogenases encoded by the genes gfdA and gfdB, while the genome of the osmophilic Aspergillus glaucus harbors only the ortholog of the A. nidulans gfdA gene. Our aim was to insert the gfdB gene into the genome of A. glaucus, and we reached this goal with the adaptation of the Agrobacterium tumefaciens-mediated transformation method. We tested the growth of the gfdB-complemented A. glaucus strains on a medium containing 2 mol l-1 sorbitol in the presence of oxidative stress generating agents such as tert-butyl hydroperoxide, H2 O2 , menadione sodium bisulfite, as well as the cell wall integrity stress-inducing agent Congo Red and the heavy metal stress eliciting CdCl2 . The growthof the complemented strains wassignificantly higher than that of the wild-type strain on media supplemented with these stress generating agents. The A. nidulans ΔgfdB mutant was also examined under the same conditions and resulted in a considerably lower growth than that of the control strain in all stress exposure experiments. Our results shed light on the fact that the gfdB gene from A. nidulans was also involved in the stress responses of the complemented A. glaucus strains supporting our hypothesis on the antioxidant function of GfdB in the Aspergilli. Nevertheless, the osmotolerant nature of A. glaucus could not be explained by the lack of the gfdB gene in A. glaucus, as we hypothesized earlier.

The Role of Glyceraldehyde-3-Phosphate Dehydrogenases in NADPH Supply in the Oleaginous Filamentous Fungus Mortierella alpina.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly conserved enzyme within the glycolytic pathway. GAPDH catalyzes the transformation of glyceraldehyde 3-phosphate to glycerate-1, 3-biphosphate, a process accompanied by the production of NADH. Its role in the NADPH production system of the oleaginous filamentous fungus Mortierella alpina was explored. Two copies of genes encoding GAPDH were characterized, then endogenously overexpressed and silenced through Agrobacterium tumefaciens-mediated transformation methods. The results showed that the lipid content of the overexpression strain, MA-GAPDH1, increased by around 13%. RNA interference of GAPDH1 and GAPDH2 (MA-RGAPDH1 and MA-RGAPDH2) greatly reduced the biomass of the fungus. The lipid content of MA-RGAPDH2 was found to be about 23% higher than that of the control. Both of the lipid-increasing transformants showed a higher NADPH/NADP ratio. Analysis of metabolite and enzyme expression levels revealed that the increased lipid content of MA-GAPDH1 was due to enhanced flux of glyceraldehyde-3-phosphate to glycerate-1, 3-biphosphate. MA-RGAPDH2 was found to strengthen the metabolic flux of dihydroxyacetone phosphate to glycerol-3-phosphate. Thus, GAPDH1 contributes to NADPH supply and lipid accumulation in M. alpina, and has a distinct role from GAPDH2.

CRISPR/Cas9-mediated mutagenesis of Clpsk1 in watermelon to confer resistance to Fusarium oxysporum f.sp. niveum.

CRISPR/Cas9-mediated editing of Clpsk1 enhanced watermelon resistance to Fusarium oxysporum. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be an effective genome-editing tool for crop improvement. Previous studies described that Phytosulfokine (PSK) signalling attenuates plant immune response. In this work, we employed the CRISPR/Cas9 system to knockout Clpsk1 gene, encoding the PSK precursor, to confer enhanced watermelon resistance to Fusarium oxysporum f.sp. niveum (FON). Interactions between PSK and FON were analysed and it was found that transcript of Clpsk1 was significantly induced upon FON infection. Meanwhile, application of exogenous PSK increased the pathogen growth. Then, one sgRNA, which targeted the first exon of Clpsk1, was selected for construction of pRGEB32-CAS9-gRNA-Clpsk1 expression cassette. The construct was then transformed to watermelon through Agrobacterium tumefaciens-mediated transformation method. Six mutant plants were obtained and three types of mutations at the expected position were identified based on Sanger sequencing. Resistance evaluation indicated that Clpsk1 loss-of-function rendered watermelon seedlings more resistant to infection by FON. These results indicate that CRISPR/Cas9-mediated gene modification is an effective approach for watermelon improvement.

Cmfhp Gene Mediates Fruiting Body Development and Carotenoid Production in Cordyceps militaris.

Cordyceps militaris fruiting bodies contain a variety of bioactive components that are beneficial to the human body. However, the low yield of fruiting bodies and the low carotenoid content in C. militaris have seriously hindered the development of the C. militaris industry. To elucidate the developmental mechanism of the fruiting bodies of C. militaris and the biosynthesis mechanism of carotenoids, the function of the flavohemoprotein-like Cmfhp gene of C. militaris was identified for the first time. The Cmfhp gene was knocked out by the split-marker method, and the targeted gene deletion mutant ΔCmfhp was obtained. An increased nitric oxide (NO) content, no fruiting body production, decreased carotenoid content, and reduced conidial production were found in the mutant ΔCmfhp. These characteristics were restored when the Cmfhp gene expression cassette was complemented into the ΔCmfhp strain by the Agrobacterium tumefaciens-mediated transformation method. Nonetheless, the Cmfhp gene had no significant effect on the mycelial growth rate of C. militaris. These results indicated that the Cmfhp gene regulated the biosynthesis of NO and carotenoids, the development of fruiting bodies, and the formation of conidia. These findings potentially pave the way to reveal the developmental mechanism of fruiting bodies and the biosynthesis mechanism of carotenoids in C. militaris.

Agrobacterium-mediated Transformation of Sweet Basil (Ocimum basilicum).

Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we present a detailed protocol for a highly efficient Agrobacterium tumefaciens-mediated transformation method for sweet basil, which was established based on a previously reported method by other researchers, with modifications on several aspects, including growth of sweet basil, age of plants used for explants, preparation and concentration of Agrobacteria. This protocol allows researchers in academia and agroindustry to generate transgenic sweet basil plants in an easy, quick and highly reproducible manner. In addition, this protocol may be applicable to transform other species within the genus Ocimum.