Agrobacterium Tumefaciens-mediated Transformation System Research Articles

Tissue culture and Agrobacterium-mediated genetic transformation of the oil crop sunflower

Sunflower is one of the four major oil crops in the world. ‘Zaoaidatou’ (ZADT), the main variety of oil sunflower in the northwest of China, has a short growth cycle, high yield, and high resistance to abiotic stress. However, the ability to tolerate adervesity is limited. Therefore, in this study, we used the retention line of backbone parent ZADT as material to establish its tissue culture and genetic transformation system for new variety cultivating to enhance resistance and yields by molecular breeding. The combination of 0.05 mg/L IAA and 2 mg/L KT in MS was more suitable for direct induction of adventitious buds with cotyledon nodes and the addition of 0.9 mg/L IBA to MS was for adventitious rooting. On this basis, an efficient Agrobacterium tumefaciens-mediated genetic transformation system for ZADT was developed by the screening of kanamycin and optimization of transformation conditions. The rate of positive seedlings reached 8.0%, as determined by polymerase chain reaction (PCR), under the condition of 45 mg/L kanamycin, bacterial density of OD600 0.8, infection time of 30 min, and co-cultivation of three days. These efficient regeneration and genetic transformation platforms are very useful for accelerating the molecular breeding process on sunflower.

Establishment of an Efficient Genetic Transformation System in Sanghuangporus baumii.

(1) Background: Sanghuangporus baumii, a valuable medicinal fungus, has limited studies on its gene function due to the lack of a genetic transformation system. (2) Methods: This study aimed to establish an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for S. baumii. This study involved cloning the promoter (glyceraldehyde-3-phosphate dehydrogenase, gpd) of S. baumii, reconstructing the transformation vector, optimizing the treatment of receptor tissues, and inventing a new method for screening positive transformants. (3) Results: The established ATMT system involved replacing the CaMV35S promoter of pCAMBIA-1301 with the gpd promoter of S. baumii to construct the pCAMBIA-SH-gpd transformation vector. The vectors were then transferred to A. tumefaciens (EHA105) for infection. This study found that the transformation efficiency was higher in the infection using pCAMBIA-SH-gpd vectors than using pCAMBIA-1301 vectors. The mycelia of S. baumii were homogenized for 20 s and collected as the genetic transformation receptor. After 20 min of co-culture and 48 h of incubation in 15 mL PDL medium at 25 °C, new colonies grew. (4) Conclusions: These colonies were transferred to PDA medium (hygromycin 4 μg/mL, cefotaxime 300 μg/mL), and the transformation efficiency was determined to be 33.7% using PCR.

Agrobacterium tumefaciens-Mediated Transformation of the Aquatic Fungus Phialemonium inflatum FBCC-F1546.

Phialemonium inflatum is a useful fungus known for its ability to mineralise lignin during primary metabolism and decompose polycyclic aromatic hydrocarbons (PAHs). However, no functional genetic analysis techniques have been developed yet for this fungus, specifically in terms of transformation. In this study, we applied an Agrobacterium tumefaciens-mediated transformation (ATMT) system to P. inflatum for a functional gene analysis. We generated 3689 transformants using the binary vector pSK1044, which carried either the hygromycin B phosphotransferase (hph) gene or the enhanced green fluorescent protein (eGFP) gene to label the transformants. A Southern blot analysis showed that the probability of a single copy of T-DNA insertion was approximately 50% when the co-cultivation of fungal spores and Agrobacterium tumefaciens cells was performed at 24-36 h, whereas at 48 h, it was approximately 35.5%. Therefore, when performing gene knockout using the ATMT system, the co-cultivation time was reduced to ≤36 h. The resulting transformants were mitotically stable, and a PCR analysis confirmed the genes' integration into the transformant genome. Additionally, hph and eGFP gene expressions were confirmed via PCR amplification and fluorescence microscopy. This optimised transformation system will enable functional gene analyses to study genes of interest in P. inflatum.

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.

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.

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.

An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens

This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

A newly constructed Agrobacterium-mediated transformation system based on the hisB auxotrophic marker for genetic manipulation in Aspergillus niger.

The filamentous fungus Aspergillus niger is widely exploited as an industrial workhorse for producing enzymes and organic acids. So far, different genetic tools, including CRISPR/Cas9 genome editing strategies, have been developed for the engineering of A. niger. However, these tools usually require a suitable method for gene transfer into the fungal genome, like protoplast-mediated transformation (PMT) or Agrobacterium tumefaciens-mediated transformation (ATMT). Compared to PMT, ATMT is considered more advantageous because fungal spores can be used directly for genetic transformation instead of protoplasts. Although ATMT has been applied in many filamentous fungi, it remains less effective in A. niger. In the present study, we deleted the hisB gene and established an ATMT system for A. niger based on the histidine auxotrophic mechanism. Our results revealed that the ATMT system could achieve 300 transformants per 107 fungal spores under optimal transformation conditions. The ATMT efficiency in this work is 5 - 60 times higher than those of the previous ATMT studies in A. niger. The ATMT system was successfully applied to express the DsRed fluorescent protein-encoding gene from the Discosoma coral in A. niger. Furthermore, we showed that the ATMT system was efficient for gene targeting in A. niger. The deletion efficiency of the laeA regulatory gene using hisB as a selectable marker could reach 68 - 85% in A. niger strains. The ATMT system constructed in our work represents a promising genetic tool for heterologous expression and gene targeting in the industrially important fungus A. niger.

The T-DNA integration characteristics of monokaryotic mutant library in Lentinula edodes

At present, proteomic and transcriptomic analysis are generally used in the study of gene function of edible fungi, and there are few reports of gene function using mutant library. In this study, the Agrobacterium tumefaciens-mediated transformation (ATMT) system of monokaryotic Lentinula edodes was used for constructing a T-DNA insertional mutant library, and over 800 transgenic strains were collected. Forty-eight strains were randomly selected for T-DNA integration characteristic analysis by whole genome resequencing, and the insertion and integration model of T-DNA in L. edodes were presented. These results lay a foundation for gene function research of L. edodes, and can make it better to understand the insertion and integration mode of exogenous T-DNA in basidiomycetes. By screening the mutant library, 38 mutants with altered thermotolerance were screened out from 221 mutants. According the results of whole genome resequencing, the T-DNA flanking sequences in eight thermotolerancechanged mutants were analyzed and annotated. The further studies for these genes disrupted T-DNA insertion would reveal the molecular mechanisms of high temperature response of L. edodes.

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.

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.

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.

A strategy for interfering with the formation of thick cell walls in Haematococcus pluvialis by down-regulating the mannan synthesis pathway

The challenges associated with effective cell wall disruption remain an important bottleneck that has restricted efforts to extract astaxanthin from Haematococcus pluvialis. Here, available transcriptomic data and an Agrobacterium tumefaciens-mediated transformation system were used to establish an H. pluvialis strain in which the key cell wall formation-related enzyme α-1,6-mannosyltransferase (HpOCH1) was downregulated in an effort to thin cell walls and thereby simplify the astaxanthin extraction process. The cell wall remodeling activity observed in these HpOch1 knockdown H. pluvialis cells resulted in dramatic reductions in the mannan organization and protective ability of the established cell walls. The cell fragmentation rate increased by 58% in HpOch1− group relative to the control group. Critically, astaxanthin synthesis was not altered in the HpOch1 knockdown cells. Overall, this study highlights a novel technical approach to artificial cell wall thinning, offering a foundation for further efforts to more effectively leverage the astaxanthin resources of H. pluvialis.

An efficient overexpression method for studying genes in Ricinus that transport vectorized agrochemicals

BackgroundPlant plasma membrane transporters play essential roles during the translocation of vectorized agrochemicals. Therefore, transporters associated with phloem loading of vectorized agrochemicals have drawn increasing attention. As a model system, castor bean (Ricinus communis L.) has been widely used to detect the phloem mobility of agrochemicals. However, there is still a lack of an efficient protocol for the Ricinus seedling model system that can be directly used to investigate the recognition and phloem loading functions of plasmalemma transporters toward vectorized agrochemicals.ResultsHere, using vacuum infiltration strategy, we overexpressed the coding gene for enhanced green fluorescent protein (eGFP) in R. communis seedlings by Agrobacterium tumefaciens-mediated transformation system. Strong fluorescence signals were observed in leaves, demonstrating that exogenous genes can be successfully overexpressed in seedlings. Subsequently, gene expression time and vacuum infiltration parameters were optimized. Observation of fluorescence and qRT-PCR analysis showed that eGFP strength and expression level reached a peak at 72 h after overexpression in seedlings. Parameter optimization showed Agrobacterium concentration at OD600 = 1.2, and infiltration for 20 min (0.09 MPa), return to atmospheric pressure, and then infiltration for another 20 min, were the suitable transformation conditions. To test the application of vacuum agroinfiltration in directly examining the loading functions of plasma membrane transporters to vectorized agrochemicals in seedlings, two LHT (lysine/histidine transporter) genes, RcLHT1 and RcLHT7, were overexpressed. Subcellular localization showed the strong fluorescent signals of the fusion proteins RcLHT1-eGFP and RcLHT7-eGFP were observed on the cell membrane of mesophyll cells, and their relative expression levels determined by qRT-PCR were up-regulated 47- and 52-fold, respectively. Furthermore, the concentrations of l-Val-PCA (l-valine-phenazine-1-carboxylic acid conjugate) in phloem sap collected from seedling sieve tubes were significantly increased 1.9- and 2.3-fold after overexpression of RcLHT1 and RcLHT7, respectively, implying their roles in recognition and phloem loading of l-Val-PCA.ConclusionsWe successfully constructed a transient expression system in Ricinus seedlings and laid the foundation for researchers to directly investigate the loading functions of plasma membrane transporters to vectorized agrochemicals in the Ricinus system.

Protoplast Isolation and Transfection in Rice.

Rice (Oryza sativa) is an important cereal crop and a model monocot plant for biology research. The reliable system of foreign DNA transformation and expression is a valuable strategy for basic research and molecular breeding application in rice. The Agrobacterium tumefaciens-mediated foreign DNA transformation system was a powerful tool for genetic research. However, it needs a long period to obtain the stable transformants for further analysis and the transformation rate limits in some organism. Protoplasts are plant cells without a cell wall, and it is much easier for foreign DNA transformation and expression. It has been widely applied in transient expression. Here, we describe a simple method for efficient protoplast isolation and transfection in rice.

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.

Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae.

Genetic engineering of the filamentous fungus Aspergillus oryzae still requires more suitable selection markers for fungal transformation. Our previous work has shown that Agrobacterium tumefaciens-mediated transformation (ATMT) based on the uridine/uracil auxotrophic mechanism with pyrG as the selection marker is very efficient for gene transfer in A. oryzae. In the present study, we delete the hisB gene, which is essential for histidine biosynthesis, in A. oryzae via homologous recombination and demonstrate that hisB is a reliable selection marker for genetic transformation of this fungus. Under optimal conditions, the ATMT efficiency of the histidine auxotrophic A. oryzae reached 515 transformants per 106 spores. Especially, we have succeeded in constructing a new ATMT system based on dual auxotrophic A. oryzae mutants with two different selection markers including hisB and pyrG. This dual auxotrophic ATMT system displayed a transformation efficiency of 232 transformants per 106 spores for the hisB marker and 318 transformants per 106 spores for the pyrG marker. By using these selectable markers, the co-expression of the DsRed and GFP fluorescent reporter genes was implemented in a single fungal strain. Furthermore, we could perform both the deletion and complementation of the laeA regulatory gene in the same strain of A. oryzae to examine its function. Conclusively, the ATMT system constructed in our work represents a promising genetic tool for studies on recombinant expression and gene function in the industrially important fungus A. oryzae.

Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum

Purpureocillium lilacinum (formerly Paecilomyces lilacinus) is widely commercialized for controlling plant-parasitic nematodes and represents a potential cell factory for enzyme production. This nematicidal fungus is intrinsically resistant to common antifungal agents used for genetic transformation. Therefore, molecular investigations in P. lilacinum are still limited so far. In the present study, we have established a new Agrobacterium tumefaciens-mediated transformation (ATMT) system in P. lilacinum based on the uridine/uracil auxotrophic mechanism. Here, uridine/uracil auxotrophic mutants were simply generated via UV irradiation instead of a complicated genetic approach for the pyrG gene deletion. A stable uridine/uracil auxotrophic mutant was then selected as a recipient for fungal transformation. We further indicated that the pyrG gene from Aspergillus niger can be used as a selectable marker for genetic transformation of P. lilacinum. Under optimized conditions for ATMT, the transformation efficiency reached 2873 ± 224 transformants per 106 spores. Using the constructed ATMT system, we succeeded in expressing the DsRed reporter gene in P. lilacinum. Additionally, we have identified a very promising mutant for chitinase production from a collection of T-DNA insertion transformants. This mutant possesses a special phenotype of hyper-branching mycelium and produces more conidia in comparison to the wild strain. Conclusively, our ATMT system can be exploited for overexpression of target genes or for T-DNA insertion mutagenesis in the agriculturally important fungus P. lilacinum. The genetic approach in the present work may also be applied for developing similar ATMT systems in other fungi, especially for fungi that their genome databases are currently not available.

Establishment of Agrobacterium tumefaciens-Mediated Transformation of Cladonia macilenta, a Model Lichen-Forming Fungus.

Despite the fascinating biology of lichens, such as the symbiotic association of lichen-forming fungi (mycobiont) with their photosynthetic partners and their ability to grow in harsh habitats, lack of genetic tools manipulating mycobiont has hindered studies on genetic mechanisms underpinning lichen biology. Thus, we established an Agrobacterium tumefaciens-mediated transformation (ATMT) system for genetic transformation of a mycobiont isolated from Cladonia macilenta. A set of combinations of ATMT conditions, such as input biomass of mycobiont, co-cultivation period with Agrobacterium cells, and incubation temperature, were tested to identify an optimized ATMT condition for the C. macilenta mycobiont. As a result, more than 10 days of co-cultivation period and at least 2 mg of input biomass of the mycobiont were recommended for an efficient ATMT, owing to extremely slow growth rate of mycobionts in general. Moreover, we examined T-DNA copy number variation in a total of 180 transformants and found that 88% of the transformants had a single copy T-DNA insertion. To identify precise T-DNA insertion sites that interrupt gene function in C. macilenta, we performed TAIL-PCR analyses for selected transformants. A hypothetical gene encoding ankyrin repeats at its C-terminus was interrupted by T-DNA insertion in a transformant producing dark-brown colored pigment. Although the identification of the pigment awaits further investigation, this proof-of-concept study demonstrated the feasibility of use of ATMT in construction of a random T-DNA insertion mutant library in mycobionts for studying genetic mechanisms behind the lichen symbiosis, stress tolerance, and secondary metabolite biosynthesis.

Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana.

The online version contains supplementary material available at 10.1007/s13205-021-02750-7.