Agrobacterium Tumefaciens-mediated Transformation Research Articles

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.

Highly efficient Agrobacterium rhizogenes-mediated hairy root transformation in citrus seeds and its application in gene functional analysis.

Highly efficient genetic transformation technology is beneficial for plant gene functional research and molecular improvement breeding. However, the most commonly used Agrobacterium tumefaciens-mediated genetic transformation technology is time-consuming and recalcitrant for some woody plants such as citrus, hampering the high-throughput functional analysis of citrus genes. Thus, we dedicated to develop a rapid, simple, and highly efficient hairy root transformation system induced by Agrobacterium rhizogenes to analyze citrus gene function. In this report, a rapid, universal, and highly efficient hairy root transformation system in citrus seeds was described. Only 15 days were required for the entire workflow and the system was applicable for various citrus genotypes, with a maximum transformation frequency of 96.1%. After optimization, the transformation frequency of Citrus sinensis, which shows the lowest transformation frequency of 52.3% among four citrus genotypes initially, was increased to 71.4% successfully. To test the applicability of the hairy roots transformation system for gene functional analysis of citrus genes, we evaluated the subcellular localization, gene overexpression and gene editing in transformed hairy roots. Compared with the traditional transient transformation system performed in tobacco leaves, the transgenic citrus hairy roots displayed a more clear and specific subcellular fluorescence localization. Transcript levels of genes were significantly increased in overexpressing transgenic citrus hairy roots as compared with wild-type (WT). Additionally, hairy root transformation system in citrus seeds was successful in obtaining transformants with knocked out targets, indicating that the Agrobacterium rhizogenes-mediated transformation enables the CRISPR/Cas9-mediated gene editing. In summary, we established a highly efficient genetic transformation technology with non-tissue-culture in citrus that can be used for functional analysis such as protein subcellular localization, gene overexpression and gene editing. Since the material used for genetic transformation are roots protruding out of citrus seeds, the process of planting seedlings prior to transformation of conventional tissue culture or non-tissue-culture was eliminated, and the experimental time was greatly reduced. We anticipate that this genetic transformation technology will be a valuable tool for routine research of citrus genes in the future.

Agrobacterium tumefaciens-mediated transformation of Nigrospora sp. isolated from switchgrass leaves and antagonistic toward plant pathogens

Nigrospora is a diverse genus of fungi colonizing plants through endophytic, pathogenic, or saprobic interactions. Endophytic isolates can improve growth and development of host plants, as well as their resistance to microbial pathogens, but exactly how they do so remains poorly understood. Developing a reliable transformation method is crucial to investigate these mechanisms, in particular to identify pivotal genes for specific functions that correlate with specific traits. In this study, we identified eight isolates of Nigrospora sp. internally colonizing the leaves of switchgrass plants cultivated in North Carolina. Using an Agrobacterium tumefaciens-mediated transformation approach with control and GFP-expressing vectors, we report the first successful transformation of two Nigrospora isolates. Finally, we demonstrate that wild-type and transgenic isolates both negatively impact the growth of two plant pathogens in co-culture conditions, Bipolaris maydis and Parastagonospora nodorum, responsible for the Southern Leaf Blight and Septoria Nodorum Blotch diseases, respectively. The GFP-transformed strains developed here can therefore serve as accurate reporters of spatial interactions in future studies of Nigrospora and pathogens in the plant. Finally, the transformation method we describe lays the foundation for further genetic research on the Nigrospora genus to expand our mechanistic understanding of plant-endophyte interactions.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris.

Agrobacterium tumefaciens-mediated transformation (AMT) serves as a widely employed tool for manipulating plant genomes. However, A. tumefaciens exhibit the capacity for gene transfer to a diverse array of species. Numerous microalgae species lack well-established methods for reliably integrating genes of interest into their nuclear genome. To harness the potential benefits of microalgal biotechnology, simple and efficient genome manipulation tools are crucial. Herein, an optimized AMT protocol is presented for the industrial microalgae species Chlorella vulgaris, utilizing the reporter green fluorescent protein (mGFP5) and the antibiotic resistance marker for Hygromycin B. Mutants are selected through plating on Tris-Acetate-Phosphate (TAP) media containing Hygromycin B and cefotaxime. Expression of mGFP5 is quantified via fluorescence after over ten generations of subculturing, indicating the stable transformation of the T-DNA cassette. This protocol allows for the reliable generation of multiple transgenic C. vulgaris colonies in under two weeks, employing the commercially available pCAMBIA1302 plant expression vector.

Agrobacterium tumefaciens-mediated transformation of the white-rot fungus Dichomitus squalens

Dichomitus squalens is an efficient white-rot fungus that generates a wide range of extracellular enzymes to degrade lignocellulose in nature. Although a protoplast-mediated transformation method for D. squalens has been developed, the transformation efficiency remains low. Here, we established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) procedure for D. squalens by transferring a binary vector harboring the neomycin phosphotransferase II (nptII) resistance gene fused with DsRed-Express2, under the control of the native glyceraldehyde-3-phosphate dehydrogenase (GPD) gene promoter. Key factors affecting the efficiency of transformation were tested. A. tumefaciens EHA105 strain with a cell density of 0.4 OD600nm and 96 h co-cultivation resulted in the highest transformation efficiency, with an average of 98 ± 11 transformants per co-cultivation plate. Besides, the strong expression of DsRed-Express2 indicates the effectiveness of the DsGPD promoter in driving gene expression in D. squalens. This ATMT system of D. squalens would be beneficial for its molecular genetic studies.

CRISPR/Cas9 gene editing in peanut by Agrobacterium tumefaciens-mediated pollen tube transformation

CRISPR/Cas9 gene editing in peanut by Agrobacterium tumefaciens-mediated pollen tube transformation

Co-expression of nitrogenase proteins in cotton (Gossypium hirsutum L.).

Chemical nitrogen fertilizer can maintain crop productivity, but overuse of chemical nitrogen fertilizers leads to economic costs and environmental pollution. One approach to reduce use of nitrogen fertilizers is to transfer nitrogenase biosynthetic pathway to non-legume plants. Fe protein encoded by nifH and MoFe protein encoded by nifD and nifK are two structural components of nitrogenase. NifB encoded by nifB is a critical maturase that catalyzes the first committed step in the biosynthesis of nitrogenase FeMo-cofactor that binds and reduces N2. Expression of the nifB, nifH, nifD and nifK is essential to generate plants that are able to fix atmospheric N2. In this study, the four genes (nifB, nifH, nifD and nifK) from Paenibacillu polymyxaWLY78 were assembled in plant expression vector pCAMBIA1301 via Cre/LoxP recombination system, yielding the recombinant expression vector pCAMBIA1301-nifBHDK. Then, the four nif genes carried in the expression vector were co-introduced into upland cotton R15 using Agrobacterium tumefaciens-mediated transformation. Homozygous transgenic cotton lines B2, B5 and B17 of T3 generation were selected by PCR and RT-PCR. qRT-PCR showed that nifB, nifH, nifD and nifK were co-expressed in the transgenic cottons at similar levels. Western blotting analysis demonstrated that NifB, NifH, NifD and NifK were co-produced in the transgenic cottons. Co-expression of the four critical Nif proteins (NifB, NifH, NifD and NifK) in cottons represents an important step in engineering nitrogenase biosynthetic pathway to non-legume plants.

Highly efficient gene knockout system in the maize pathogen Colletotrichum graminicola using Agrobacterium tumefaciens-mediated transformation (ATMT)

Colletotrichum graminicola, a hemibiotrophic pathogenic fungus, is the causal agent of anthracnose of maize, which causes significant yield losses worldwide, especially in warm and humid maize production regions. An efficient targeted genes knockout protocol is crucial to explore molecular mechanisms of fungal virulence to the host. In this study, we established a gene knockout transformation system by employing Agrobacterium tumefaciens-mediated transformation to knockout genes in M 1.001 strain of C. graminicola. The conidia germination status, induction medium type, and ratio of Agrobacterium cell and conidia suspension were optimized for the knockout of CgBRN1(OR352905), a gene relating to the fungal melanin biosynthesis pathway. Additionally, CgPKS18 (OR352906) and CgCDC25 (OR352903) were knocked out to test the applicability of the gene knockout transformation system. In this established system, transformation efficiency was 176 transformants per 1 × 105 conidia and the homologous recombination efficiency was 53.3 to 75%. Furthermore, disease index, lesion number and lesion size caused by the three above-mentioned mutant strains were found to be reduced significantly compared to the wild-type strain, which indicated reduction in fungal virulence due to the lack of those genes.

Agrobacterium tumefaciens-mediated transformation of embryogenic callus and CRISPR/Cas9-mediated genome editing in ‘Feizixiao’ litchi

Litchi (Litchi chinensis Sonn.) is a type of commercially prevalent subtropical and tropical fruit. Since litchi has a highly heterozygous genetic background and a long reproductive cycle, conventional breeding methods (such as hybridization) have limited ability to nurture new litchi cultivars. Here, an efficient and stable Agrobacterium tumefaciens-mediated genetic transformation of embryogenic callus was established in ‘Feizixiao’ litchi. Transgenic materials were verified using polymerase chain reaction (PCR) analysis, β-glucuronidase (GUS) assay, and green fluorescent protein (GFP) assay. To implement the technology of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) technology in ‘Feizixiao’ litchi and verify the validity of these transformation systems, the litchi polyphenol oxidase gene (LcPPO, JF926153) was knocked out. Various categories of mutations, covering base insertions, deletions, and substitutions, were found in transgenic materials via sequence analysis. The transformation system achieved high feasibility and efficiency, and the system of CRISPR/Cas9 was successfully employed to edit genes in ‘Feizixiao’ litchi. This work provides an essential foundation for investigating the functions of genes and accelerating litchi genetic improvement.

An Efficient Transformation System for Fast Production of VcCHS Transgenic Blueberry Callus and Its Expressional Analysis.

The Agrobacterium tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of cv. 'Legacy' and 'Northland' were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with A. tumefaciens was operated on WPM plus 100 μM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L-1 2,4-D, 0.4 mg·L-1 6-BA, 50 mg·L-1 hygromycin, and 200 mg·L-1 cefotaxime. The VcCHS transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of cv. 'Northland'. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.

FungalBraid 2.0: expanding the synthetic biology toolbox for the biotechnological exploitation of filamentous fungi.

Fungal synthetic biology is a rapidly expanding field that aims to optimize the biotechnological exploitation of fungi through the generation of standard, ready-to-use genetic elements, and universal syntax and rules for contributory use by the fungal research community. Recently, an increasing number of synthetic biology toolkits have been developed and applied to filamentous fungi, which highlights the relevance of these organisms in the biotechnology field. The FungalBraid (FB) modular cloning platform enables interchangeability of DNA parts with the GoldenBraid (GB) platform, which is designed for plants, and other systems that are compatible with the standard Golden Gate cloning and syntax, and uses binary pCAMBIA-derived vectors to allow Agrobacterium tumefaciens-mediated transformation of a wide range of fungal species. In this study, we have expanded the original FB catalog by adding 27 new DNA parts that were functionally validated in vivo. Among these are the resistance selection markers for the antibiotics phleomycin and terbinafine, as well as the uridine-auxotrophic marker pyr4. We also used a normalized luciferase reporter system to validate several promoters, such as PpkiA, P7760, Pef1α, and PafpB constitutive promoters, and PglaA, PamyB, and PxlnA inducible promoters. Additionally, the recently developed dCas9-regulated GB_SynP synthetic promoter collection for orthogonal CRISPR activation (CRISPRa) in plants has been adapted in fungi through the FB system. In general, the expansion of the FB catalog is of great interest to the scientific community since it increases the number of possible modular and interchangeable DNA assemblies, exponentially increasing the possibilities of studying, developing, and exploiting filamentous fungi.

Genome-scale metabolic model led engineering of Nothapodytes nimmoniana plant cells for high camptothecin production.

Camptothecin (CPT) is a vital monoterpene indole alkaloid used in anti-cancer therapeutics. It is primarily derived from Camptotheca acuminata and Nothapodytes nimmoniana plants that are indigenous to Southeast Asia. Plants have intricate metabolic networks and use them to produce secondary metabolites such as CPT, which is a prerequisite for rational metabolic engineering design to optimize their production. By reconstructing metabolic models, we can predict plant metabolic behavior, facilitating the selection of suitable approaches and saving time, cost, and energy, over traditional hit and trial experimental approaches. In this study, we reconstructed a genome-scale metabolic model for N. nimmoniana (NothaGEM iSM1809) and curated it using experimentally obtained biochemical data. We also used in silico tools to identify and rank suitable enzyme targets for overexpression and knockout to maximize camptothecin production. The predicted over-expression targets encompass enzymes involved in the camptothecin biosynthesis pathway, including strictosidine synthase and geraniol 10-hydroxylase, as well as targets related to plant metabolism, such as amino acid biosynthesis and the tricarboxylic acid cycle. The top-ranked knockout targets included reactions responsible for the formation of folates and serine, as well as the conversion of acetyl CoA and oxaloacetate to malate and citrate. One of the top-ranked overexpression targets, strictosidine synthase, was chosen to generate metabolically engineered cell lines of N. nimmoniana using Agrobacterium tumefaciens-mediated transformation. The transformed cell line showed a 5-fold increase in camptothecin production, with a yield of up to 5 µg g-1.

Heterologous gene expression system for the production of hydrolyzable tannin intermediates in herbaceous model plants.

Aluminum toxicity is the main factor limiting the elongation of plant roots in acidic soil. The tree species Eucalyptus camaldulensis is considerably more resistant to aluminum than herbaceous model plants and crops. Hydrolyzable tannins (HTs) accumulating in E. camaldulensis roots can bind and detoxify the aluminum taken up by the roots. However, in herbaceous model plants, HTs do not accumulate and the genes involved in the HT biosynthetic pathway are largely unknown. The aim of this study was to establish a method for reconstituting the HT biosynthetic pathway in the HT non-accumulating model plant Nicotiana benthamiana. Four E. camaldulensis enzymes were transiently expressed in N. benthamiana leaves via Agrobacterium tumefaciens-mediated transformation. These enzymes included dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDH2 and EcDQD/SDH3), which catalyze the synthesis of gallic acid, the first intermediate of the HT biosynthetic pathway that branches off from the shikimate pathway. The others were UDP-glycosyltransferases (UGT84A25 and UGT84A26), which catalyze the conversion of gallic acid to β-glucogallin, the second intermediate. The co-expression of the EcDQD/SDHs in transgenic N. benthamiana leaf regions promoted the synthesis of gallic acid. Moreover, the co-expression of the UGT84As in addition to the EcDQD/SDHs resulted in the biosynthesis of β-glucogallin, the universal metabolic precursor of HTs. Thus, we successfully reconstituted a portion of the HT biosynthetic pathway in HT non-accumulating N. benthamiana plants. This heterologous gene expression system will be useful for co-expressing candidate genes involved in downstream reactions in the HT biosynthetic pathway and for clarifying their in planta functions.

Optimization of Protocols for the Induction of Callus and Plant Regeneration in White Clover (Trifolium repens L.).

White clover is a widely grown temperate legume forage with high nutritional value. Research on the functional genomics of white clover requires a stable and efficient transformation system. In this study, we successfully induced calluses from the cotyledons and leaves of 10 different white clover varieties. The results showed that the callus formation rate in the cotyledons did not vary significantly among the varieties, but the highest callus formation rate was observed in 'Koala' leaves. Subsequently, different concentrations of antioxidants and hormones were tested on the browning rate and differentiation ability of the calluses, respectively. The results showed that the browning rate was the lowest on MS supplemented with 20 mg L-1 AgNO3 and 25 mg L-1 VC, respectively, and the differentiation rate was highest on MS supplemented with 1 mg L-1 6-BA, 1 mg L-1 KT and 0.5 mg L-1 NAA. In addition, the transformation system for Agrobacterium tumefaciens-mediated transformation of 4-day-old leaves was optimized to some extent and obtained a positive callus rate of 8.9% using green fluorescent protein (GFP) as a marker gene. According to our data, by following this optimized protocol, the transformation efficiency could reach 2.38%. The results of this study will provide the foundation for regenerating multiple transgenic white clover from a single genetic background.

Agrobacterium tumefaciens-Mediated Plant Transformation: A Review.

Agrobacterium tumefaciens-mediated plant transformation is the most dominant technique for the transformation of plants. It is used to transform monocotyledonous and dicotyledonous plants. A. tumefaciens apply for stable and transient transformation, random and targeted integration of foreign genes, as well as genome editing of plants. The Advantages of this method include cheapness, uncomplicated operation, high reproducibility, a low copy number of integrated transgenes, and the possibility of transferring larger DNA fragments. Engineered endonucleases such as CRISPR/Cas9 systems, TALENs, and ZFNs can be delivered with this method. Nowadays, Agrobacterium-mediated transformation is used for the Knock in, Knock down, and Knock out of genes. The transformation effectiveness of this method is not always desirable. Researchers applied various strategies to improve the effectiveness of this method. Here, a general overview of the characteristics and mechanism of gene transfer with Agrobacterium is presented. Advantages, updated data on the factors involved in optimizing this method, and other useful materials that lead to maximum exploitation as well as overcoming obstacles of this method are discussed. Moreover, the application of this method in the generation of genetically edited plants is stated. This review can help researchers to establish a rapid and highly effective Agrobacterium-mediated transformation protocol for any plant species.

Quantitative and qualitative plant-pathogen interactions call upon similar pathogenicity genes with a spectrum of effects.

Septoria leaf blotch is a foliar wheat disease controlled by a combination of plant genetic resistances and fungicides use. R-gene-based qualitative resistance durability is limited due to gene-for-gene interactions with fungal avirulence (Avr) genes. Quantitative resistance is considered more durable but the mechanisms involved are not well documented. We hypothesize that genes involved in quantitative and qualitative plant-pathogen interactions are similar. A bi-parental population of Zymoseptoria tritici was inoculated on wheat cultivar 'Renan' and a linkage analysis performed to map QTL. Three pathogenicity QTL, Qzt-I05-1, Qzt-I05-6 and Qzt-I07-13, were mapped on chromosomes 1, 6 and 13 in Z. tritici, and a candidate pathogenicity gene on chromosome 6 was selected based on its effector-like characteristics. The candidate gene was cloned by Agrobacterium tumefaciens-mediated transformation, and a pathology test assessed the effect of the mutant strains on 'Renan'. This gene was demonstrated to be involved in quantitative pathogenicity. By cloning a newly annotated quantitative-effect gene in Z. tritici that is effector-like, we demonstrated that genes underlying pathogenicity QTL can be similar to Avr genes. This opens up the previously probed possibility that 'gene-for-gene' underlies not only qualitative but also quantitative plant-pathogen interactions in this pathosystem.

Development of a Transformation System for the Medicinal Fungus Sanghuangporus baumii and Acquisition of High-Value Strain

To further explore the molecular mechanism of triterpenoid biosynthesis and acquire high-value strain of Sanghuangporus baumii, the Agrobacterium tumefaciens-mediated transformation (ATMT) system was studied. The key triterpenoid biosynthesis-associated gene isopentenyl diphosphate isomerase (IDI) was transformed into S. baumii by ATMT system. Then, the qRT-PCR technique was used to analyze gene transcript level, and the widely targeted metabolomics was used to investigate individual triterpenoid content. Total triterpenoid content and anti-oxidant activity were determined by spectrophotometer. In this study, we for the first time established an efficient ATMT system and transferred the IDI gene into S. baumii. Relative to the wild-type (WT) strain, the IDI-transformant (IT) strain showed significantly higher transcript levels of IDI and total triterpenoid content. We then investigated individual triterpenoids in S. baumii, which led to the identification of 10 distinct triterpenoids. The contents of individual triterpenoids produced by the IT2 strain were 1.76–10.03 times higher than those produced by the WT strain. The triterpenoid production showed a significant positive correlation with the IDI gene expression. Besides, IT2 strain showed better anti-oxidant activity. The findings provide valuable information about the biosynthetic pathway of triterpenoids and provide a strategy for cultivating high-value S. baumii strains.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Developing a UV-visible reporter-assisted CRISPR/Cas9 gene editing system to alter flowering time in Chrysanthemum indicum.

Heterokaryosis, the main obstacle in the generation of PPO1-edited Agaricus bisporus by CRISPR/Cas9 system

PPO1 gene editing in Agaricus bisporus was carried out by CRISPR/Cas9 through Agrobacterium tumefaciens-mediated transformation (ATMT). The ATMT to the gill tissue yielded 52 transformants, however, sequencing of which failed to detect any distinct PPO1 modification. Instead, two transformants, N9 and N14, were identified to have mixed sequences at the vicinity of the protospacer adjacent motif (PAM), which was attributed to the presence of multiple nuclei in the mycelial cell. Heterokaryosis of N9 was disrupted to generate 24 homokaryotic and 29 heterokaryotic isolates. PPO1 sequencing of the 53 isolates identified three edited homokaryotic isolates with two single base deletions (L18 and L22) and a four-base deletion (L41) at the 4th base from PAM, and six isolates with mixed sequences. The mixed sequences were resolved to consist of the edited and unedited PPO1s, indicating the presence of both edited and unedited nuclei, thus emphasizing that heterokaryosis is a major obstacle in generating edited nuclei in A. bisporus. Notably, the unedited PPO1s in the homokaryotic L32 and heterokaryotic L49 were discovered to be edited by single base deletion after prolonged incubation, suggesting the transfer of the Cas9-gRNA complex to neighboring nuclei in the common cytoplasm. Lastly, the PPO1-edited heterokaryon Het1, created by mating the edited homokaryotic L18 with the wild-type NH1, exhibited significantly less browning than the wild-type strain, indicating a functional role for PPO1 in the button mushroom browning.

Salicylic acid promotes terpenoid synthesis in the fungi Sanghuangporus baumii

Sanghuangporus baumii is a medicinal fungi with anti-inflammatory, liver protection and antitumour effects. Terpenoids are one of the main medicinal ingredients of S. baumii. However, terpenoid production by wild-type S. baumii cannot meet the market demand, which affects its application in medical care. Therefore, exploring how to increase terpenoid content in S. baumii is a promising path in this research field. Salicylic acid (SA) is a secondary metabolite. In this study, a concentration of 350 μmol/L SA was added into fungal cultivations for 2 and 4 days, and then the transcriptome and metabolome of untreated mycelia and treated with SA were analysed. The expression of some genes in the terpenoids biosynthesis pathway increased in SA-induced cultivations, and the content of isopentenyl pyrophosphate (IPP) and geranylgeranyl-PP (GGPP) increased significantly as well as the contents of triterpenoids, diterpenoids, sesquiterpenoids and carotenoids. The gene FPS was considered to be a key gene regulating terpenoid biosynthesis. Therefore, FPS was overexpressed in S. baumii by Agrobacterium tumefaciens-mediated genetic transformation. The gene FPS and its downstream gene (LS) expression levels were confirmed to be increased in the FPS overexpressing transformant, and terpenoid content was 36.98% higher than that of the wild-type strain in the evaluated cultivation conditions.