Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis of Fusarium Oxysporum

Background Fusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species. In this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and ecient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation eciency was as high as 250 mutants per 10 4 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences anking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined. The developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi. The F. oxysporum T-DNA insertion mutants were analyzed by PCR and touchdown-TAIL PCR [16] and the T-DNA insertion sites were determined. Moreover, the transformation eciency was substantially improved by using the plasmid pXEN. By analyzing the phenotypic changes in mutants, the associated genes were identied. The study data provide the basis for future investigations of the mechanisms underlying F. oxysporum pathogenesis and drug resistance and may be useful for detecting new drug targets. In this study, we constructed the recombinant plasmid the neomycin phosphatase gene (neo) and established a simple and ecient procedure for the transformation of resistant F. oxysporum mediated by A. tumefaciens. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. By analyzing of the anking sequence, the insertion sites and genes associated with the phenotypic changes could be determined. The data presented herein reveal that the ATMT system developed in this study can eciently insert T-DNA into the F. oxysporum genome. Because the insertion sites can be determined, our system may enable researchers to analyze gene functions and study biological characteristics. Furthermore, our method may be applicable to investigations of other important pathogenic fungi.


Abstract
Background Fusarium species are important pathogenic organisms, which can cause many diseases in plants and humans. Characterizing the mechanism underlying their pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated genetic transformation has been widely used for the molecular analysis of many species.

Results
In this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and e cient procedure for the transformation of resistant Fusarium oxysporum mediated by A. tumefaciens. The transformation e ciency was as high as 250 mutants per 10 4 conidia. A total of 1,450 stably transformed mutants were generated, resulting in a small-scale library of F. oxysporum mutants containing T-DNA tags. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. Additionally, the sequences anking the inserted T-DNA were obtained by touchdown-TAIL PCR, the insertion sites and genes associated with the phenotypic changes could be determined.

Conclusions
The developed method may enable to analyze gene functions and study biological characteristics, which will lay the foundation for future analyses of the mechanism underlying F. oxysporum pathogenicity and resistance. Furthermore, it may be applicable to investigations of other important pathogenic fungi.

Background
Fusarium species are widely distributed diverse fungal organisms. They have a broad host range and can infect humans, animals, and plants [1]. Fusarium spp. is the most common pathogens causing fungal keratitis, with a very high blindness rate [2,3]. Because antibiotic and hormone use as well as organ transplantations are relatively common, the number of patients with AIDS, malignant tumors, diabetes, or hematological diseases has recently been increasing. Consequently, there has been a yearly increase in the number of invasive and disseminated Fusarium infections, which are second only to Aspergillus infections as the most common infections caused by lamentous fungi [2,4]. Fusarium spp. is also naturally resistant to multiple drugs, including almost all antifungal drugs, such as azoles, echinococcins, and polyene drugs [5]. Thus, effectively treating Fusarium infections is extremely di cult, leading to a very high fatality rate [2,6].
In terms of their effects on agriculture, Fusarium spp. can infect important crops and are di cult to control, with the resulting extensive damages potentially leading to annual economic losses totaling billions of dollars [7]. They also produce many highly toxic mycotoxins, including deoxynivalenol, monoterpene toxin, and fumonisin [7,8]. These toxins can kill plants and accumulate in cereal grains. The consumption of food and feed contaminated with these mycotoxins can cause liver and kidney failure and increase the risk of cancer in humans and livestock [9].
Characterizing the mechanism underlying Fusarium spp. pathogenicity and drug resistance is critical. Agrobacterium tumefaciens-mediated transformation (ATMT), which is applicable to a wide range of species, is an inexpensive and highly e cient method for the genetically stable random insertion of a DNA segment into the genome, with a high single-copy rate, making it ideal for the functional analysis of genes [10]. T-DNA insertion mutant libraries have been constructed and applied for the functional characterization of genes from various fungi, including Aspergillus fumigatus [11], Sporothrix schenckii [12], Magnaporthe oryzae [13], Cryptococcus neoformans [14], and Aspergillus terreus [15]. Analyses of these mutant libraries have uncovered mutants related to pathogenicity, drug resistance, and morphology, and the related gene functions have been explored. Therefore, ATMT may be useful for the high-throughput screening of mutants with speci c phenotypes as well as for elucidating the functions of the associated genes.
During the development of an ATMT system, screening for resistance is an important process. In this study, hygromycin B, phleomycin, bleomycin, glyphosate, neomycin, and geneticin (G418) were evaluated, with G418 ultimately selected. A new plasmid, pXEN, was constructed and an ATMT system was established and optimized. A Fusarium oxysporum T-DNA insertion mutant library based on G418 resistance was preliminarily constructed. The F. oxysporum T-DNA insertion mutants were analyzed by PCR and touchdown-TAIL PCR [16] and the T-DNA insertion sites were determined. Moreover, the transformation e ciency was substantially improved by using the plasmid pXEN. By analyzing the phenotypic changes in mutants, the associated genes were identi ed. The study data provide the basis for future investigations of the mechanisms underlying F. oxysporum pathogenesis and drug resistance and may be useful for detecting new drug targets.

Results
Minimum inhibitory concentration of G418 against F. oxysporum F.oxysporum growth was completely inhibited on PDA medium containing G418 (50 µg/ml). Therefore, this concentration was used as the minimum inhibitory concentration for screening the mutants.

Optimization of the transformation conditions
Increases in the co-culture time resulted in gradual increases in the transformation e ciency (Fig. 1a). However, overlapping colonies were observed at 60 h, making it di cult to isolate single mutants. Additionally, at each analyzed co-culture time-point, the transformation e ciency was highest at 25 °C (Fig. 1a).
Therefore, a 48-h culture at 25 °C was considered optimal for the transformation of F. oxysporum.
The transformation e ciency was highest when the OD 600 of the A. tumefaciens culture was 0.8 (Fig. 1b). Increases in the F. oxysporum conidia concentration gradually increased the transformation e ciency (Fig. 1b). However, excessively high transformation e ciencies were not conducive to the subsequent selection of individual mutants. Therefore, an A. tumefaciens culture with an OD 600 of 0.8 and an F. oxysporum conidia concentration of 1 × 10 4 CFU/ml were used for co-culturing. The resulting transformation e ciency was 250 mutants per 10 4 conidia.

Screening mutants with phenotypic changes
The mutants were cultured and compared with the wild-type strain to detect changes to colony morphology (Fig. 2). The Mut1, Mut2, Mut3 and Mut4 mutants grew more slowly than the wild-type, whereas Mut5 exhibited increased hyphal growth. Additionally, Mut4 produced a dark-blue pigment, Mut3 and Mut5 produced less pigment. These changes may be related to the pathogenicity of the mutants. Accordingly, the mutants obtained in this study may be valuable materials for future research on the pathogenicity of Fusarium spp.

Analysis of T-DNA insertion sites
To con rm the T-DNA fragment was inserted in the genomes of the putative mutants, we randomly selected 15 mutants for a PCR ampli cation of the neomycin phosphatase II gene (neo) fragment. The ampli ed 700-bp fragment was 99% similar to the neo fragment. After ve rounds of inoculations of PDA lacking G418, the mutants were still able to grow on PDA medium containing G418 (50 µg/ml) and tested positive for the neo fragment in one PCR ampli cation (Fig. 3a). These observations implied the T-DNA was stably inserted into the F. oxysporum genome.
The T-DNA anking sequences were ampli ed by touchdown-TAIL PCR (Fig. 3b). The T-DNA insertion site was located via an alignment with the pXEN sequence and the F. oxysporum genome sequence ( Table 1). The mutated genes and the resulting phenotypic changes may be useful for the functional characterization of F. oxysporum genes.

Discussion
The e cient and simple detection of mutants is a critical part of fungal transformation systems. The wild-type F. oxysporum analyzed in the current study is resistant to multiple drugs and chemicals used for screening mutants. Regarding the commonly used G418 [17], we determined that G418 (50 µg/ml) can completely inhibit F. oxysporum growth. However, there is currently no plasmid carrying a G418-resistance gene that can be used to transform F. oxysporum. In this study, we constructed the pXEN recombinant plasmid containing the G418-resistance neo gene. This plasmid can be used for the ATMT of F. oxysporum. Because it has two multiple cloning sites between the LB and RB and resistance genes, it is also useful for gene knockout experiments. Furthermore, it may be applicable for functional analyses of genes in other strains sensitive to geneticin.
The transformation e ciency of ATMT is restricted by many factors. The optimal ATMT conditions for F. oxysporum were determined based on analyses of the co-culture time and temperature as well as the ratio of A. tumefaciens to F. oxysporum. We determined that an A. tumefaciens culture with an OD 600 of 0.8, an F. oxysporum conidia concentration of 1 × 10 4 CFU/mL, and a 48-h co-culture at 25 °C are the optimal conditions for transforming F. oxysporum. The resulting transformation e ciency was 250 mutants per 10 4 conidia, which was approximately 30-times higher than that reported for other transformation systems [18]. The higher transformation e ciency of the current study may be due to the use of G418 resistance to detect mutants. To the best of our knowledge, this is the rst study to identify the mutants based on G418 resistance. Thus, our method may be a better option for the ATMT of F. oxysporum than previously described procedures. Additionally, changing the method used to screen mutants may enhance the transformation of some strains with low transformation e ciencies or special resistance, which may enable the study of gene functions in these strains.

Conclusions
In this study, we constructed the pXEN recombinant plasmid carrying the neomycin phosphatase II gene (neo) and established a simple and e cient procedure for the transformation of resistant F. oxysporum mediated by A. tumefaciens. Some of the mutants exhibited phenotypic changes in growth, metabolism, and development. By analyzing of the anking sequence, the insertion sites and genes associated with the phenotypic changes could be determined. The data presented herein reveal that the ATMT system developed in this study can e ciently insert T-DNA into the F. oxysporum genome. Because the insertion sites can be determined, our system may enable researchers to analyze gene functions and study biological characteristics. Furthermore, our method may be applicable to investigations of other important pathogenic fungi.

Strains and plasmids
Wild-type F. oxysporum JLCC31768, which was isolated from patients with clinical infections in Jilin province, China, was used as the parent strain for transformations. Agrobacterium tumefaciens AGL-1 carrying the pBHT1 plasmid, A. tumefaciens Agr0, as well as the pEGFP-N3 and pXEH plasmids was used in this study. All strains are preserved in the Fungi Research Center of Jilin University.

Construction of the G418 resistance plasmid
The trpC promoter region in pBHT1 was ampli ed with the FtrpC-f and FtrpC-r primers. The neo fragment in pEGFP-N3 was ampli ed with the FNeor-f and FNeor-r primers. The FtrpC-f and FNeor-r primers were then used to generate the complete trpC promoter-neo expression cassette in a staggered extension process. The trpC promoter-neo sequence was ampli ed with the tRPCNf and tRPCNr primers, after which the amplicon was digested and linked to pXEH. The resulting pXEN recombinant plasmid was sequenced ( Table 2) and then inserted into A. tumefaciens Agr0 cells to produce the AgrN strain.
F.oxysporum was transformed according to an optimized version of a previously described method [10,12]. A. tumefaciens AgrN cells carrying the pXEN plasmid were activated, collected, and resuspended in liquid induction medium containing 200 μM acetosyringone [10].

Screening of phenotypic mutants
The center of Petri plates containing PDA medium was inoculated with the mutants. After incubating at 25 ℃ for 7 days, the colony morphology was analyzed.

Analysis of the genetic stability of the T-DNA insertion
The stability of the T-DNA insertion was assessed based on the G418 resistance of the mutants. Speci cally, 15 randomly selected mutants were examined for the presence of the neo fragment via the ampli cation with the neoF and neoR primers ( Table 2). The mutants were then cultured on PDA lacking G418 for 3 days. An inoculation needle was used to transfer mycelia from the colony edge to fresh PDA lacking G418 for another 3-day culture. After ve rounds of inoculation, the mycelia were transferred to screening medium and the presence of the neo fragment was con rmed by PCR.

Analysis of the T-DNA anking sequences
The putative mutants were added to PDB medium containing G418 (50 μg/ml) and cefotaxime sodium (200 μM) and then incubated at 25 °C with shaking (120 rpm) for 72 h. Genomic DNA was extracted from the cultured mutants [19], and used as the template for the ampli cation of the neo fragment to determine whether the T-DNA was inserted into the genome of these putative mutants.
The sequences anking the inserted T-DNA in the mutant genomes were ampli ed by touchdown-TAIL PCR [16] ( Table 2). The left and right arms of the T-DNA were ampli ed by two rounds of semi-nested PCR, sequenced, and aligned with the pXEN plasmid. The anking sequence was compared with the F. oxysporum f. sp. lycopersici 4287 genome (GCF_000149955.1) to determine the insertion site.