Marker selection and genetic transformation in the ATCC 42464 strain
To perform M. thermophila transformation mediated by A. tumefaciens, the sensitivity of this fungus to hygromycin B (Amresco, Solon, OH, USA) and phosphinothricin
(Sigma-Aldrich, St. Louis, MO, USA) was tested. Five concentrations of two antibiotics
were used. The mature conidia were harvested in 0.05 % Tween 80, and 103 conidia were spread on MM (Vogel’s minimal medium supplemented with 2 % sucrose)
agar plates supplemented with various concentrations of antibiotic (Hygromycin B:
25, 50, 75, 100, 150Â ?g/mL; phosphinothricin: 25, 50, 100, 150, 200Â ?g/mL). After
incubation at 45 °C for 5 days, Hygromycin B showed little inhibition for M. thermophila at more than 100 ug/mL, whereas phosphinothricin concentration completely inhibited
the growth of this fungus at 100 ug/mL. Thus, the bar gene conferring fungal resistance to phosphinothricin was employed as the selection
marker for M. thermophila transformation.
The Ti vector pPK2BarGFPD containing the trpC promoter from Aspergillus nidulans and the tef promoter from Aureobasidium pullulans (PtrpC and Ptef, respectively) developed in a previous study 23] was chosen as the test plasmid for genetic transformation in M. thermophila ATCC 42464. The vector contains a GFP reporter gene as well as a bar selection marker gene, which confers resistance to phosphinothricin. PCR analysis
of the transformants showed the T-DNA was integrated in the chromosomes. High levels
of green fluorescent protein (GFP) signal were clearly detected in the conidia and
mycelia of transformants (Fig. 1), whereas no autofluorescent signal was observed in the parental strain. This suggested
pPK2BarGFPD and its elements, the promoters and the GFP gene, were functioning and
could be used for gene over-expression and protein localization analysis in M. thermophila ATCC 42464.
Fig. 1. Confocal fluorescence imaging of mycelia (Top) and conidia (Bottom) of M. thermophila pPK2BarGFPD transformants
Optimization of M. thermophila transformation mediated by A. tumefaciens
In an attempt to develop a simple, high efficiency transformation system mediated
by A. tumefaciens in M. thermophila ATCC 42464, we performed transformation optimization as described previously 21]. Acetosyringone (AS) serves as an inducer of virulence (vir) genes, whose expression is a prerequisite for A. tumefaciens T-DNA transfer. AS concentration influences the frequency of A. tumefaciens-mediated transformation for Saccharomyces cerevisiae and several filamentous fungi 11], 20], 24], 25]. Therefore, the impact of AS on this approach for M. thermophila was first investigated. As shown in Fig. 2a, using the binary plasmid pPK2BarGFPD, the maximum transformation efficiency (145?±?10
transformants per plate) was obtained with AS at a concentration of 200Â ?M, and the
number of transformants was reduced when the concentration was increased or decreased.
The observation that the higher concentration of AS resulted in a reduction in transformation
efficiency is similar to that for Fusarium avenaceum. However, it is in contrast to that for Beauveria bassiana, where an increase in the concentration from 100Â ?M to 800Â ?M resulted in increasing
numbers of transformants 21], 26]. Based on these observation, the different fungus seems has different tolerance to
Acetosyringone (AS), the low frequency of transformation induced by the increased
AS might be attributed to the toxic effect of AS on M. thermophila, and the deficient level of vir gene expression at low AS concentrations might be responsible for poor efficiency
of A. tumefaciens T-DNA transfer.
Fig. 2. Optimization of A. tumefaciens-mediated M. thermophila transformation. a The impact of acetosyringone (AS) on A. tumefaciens-mediated transformation efficiency. Co-cultivation was conducted in induction medium
containing AS on indicated concentrations for 2Â days. b The effect of co-culture time on transformation efficiency. Co-cultivation was conducted
in induction medium containing 200Â ?M AS for various times indicated. Error bars indicate
the standard deviation of three independent experiments
The transformation efficiency is also dependent on co-culture time of M. thermophila and A. tumefaciens. The largest number of transformants was observed with a co-culture time of 2Â days,
and alterative co-culture times impaired the transformation efficiency (Fig. 2b). Similar results were obtained in Metarhizium anisopliae, Aspergillus terreus and Beauveria bassiana transformation mediated by A. tumefaciens2], 18], 27], whereas the co-culture time had no influence on a Ganoderma lucidum transformation system 28]. In conclusion, with the optimized approach, a high transformation efficiency of
145 transformants per 105 conidia was obtained. This was higher compared with the amount of transformants obtained
in fungi using other approaches, typically less than 100 transformants per 105 conidia 18], 29]–31].
Mitotic stability analysis of transformants
The mitotic stability of the T-DNA in M. thermophila transformants was examined next. Twenty randomly selected transformants containing
pPK2BarGFPD were cultured on media without phosphinothricin for five generations.
After five generations, 19 transformants (95Â %) of 20 tested strains retained the
resistance to phosphinothricin. Satisfyingly, PCR analysis showed the presence of
bar in the 19 transformants, and GFP signal was observed under the fluorescence microscope.
ku70 disruption of M. thermophila ATCC 42464
The high transformation frequency, together with the precision and simplicity of T-DNA,
make A. tumefaciens-mediated transformation a suitable genome mutagenesis approach in filamentous fungi,
such as Trichoderma, Aspergillus, Beauveria and Metarhizium22], 27], 32], 33]. Ku70 and Ku80 make up the Ku heterodimer, which binds to the ends of double-stranded
DNA breaks and is required for the non-homologous end joining (NHEJ) pathway of DNA
repair 34]. In some fungi, including the M. thermophila C1 strain, Trichoderma reesei and Neurospora crassa, mutation of ku70 results in a dramatically increased homologous integration frequency with short homologous
flanks 9], 35], 36]. Therefore, disruption of ku70 of the ATCC 42464 strain was performed to test the efficiency of this approach for
knocking out of a specific gene in wild-type M. thermophila.
The binary vector pPK2BarGFPD was employed for DNA manipulation and the ku70 knockout cassette (Fig. 3) was constructed as described in the Methods section. The gene encoding green fluorescence
protein on the binary vector served as the marker to differentiate between ectopic
insertion and correct gene replacement. Fluorescence should not be detected in the
transformants where the target gene (ku70) was properly replaced by the bar cassette (Fig. 4a). Conversely, if the fluorescent signal is detected in a transformant, this suggests
the bar-gfp segment of the T-DNA was ectopically integrated elsewhere in the genome (random insertion)
rather than at the correct gene locus. Using this strategy, we performed the ku70 disruption experiment. One hundred sixteen primary transformants were obtained. Forty-six
(39.7Â %) transformants were filtered out by an obvious fluorescent signal, indicating
they were ectopic insertion mutants. The remaining 70 transformants (60.3Â %) with
no detectable fluorescent signal under the microscope were likely free of ectopic
insertions and passed the first round screening. Subsequent PCR analysis showed the
ku70 gene was properly replaced by the bar cassette in 68 of these 70 transformants. To further confirm this observation, we
randomly selected eleven PCR-confirmed transformants to perform Southern blotting
analysis, and the result showed all 11 tested mutants were correct (Fig. 4b and c). Taken together, these results indicate as many as 58 % (68 of 116) of the
transformants could be correct ku70 disruption mutants, indicating a very high rate of homologous recombination. Previous
studies reported that the efficiencies of genetic targeted disruption via transformation
mediated by A. tumefaciens varied greatly in fungal species, such as 0.04Â % for Blastomyces dermatitidis, 29Â % for Aspergillus awamori, 74Â % for Fusarium avenaceum and 85Â % for Fusarium graminearum37], 38]. Compared with transformation by direct electroporation transformation of protoplasts
or conidia, the transformation mediated by A. tumefaciens seems more likely to achieve a higher successful gene disruption rate, at least for
some of the species. For example, in Neurospora crassa the homologous recombination rate was usually low (10Â %) without NHEJ disruption
using electroporation 35]. Although future studies with more precise statistical analyses are needed to support
the improved homologous recombination rate, if validated, it will be a boost for genetic
manipulation in this strain, and will speed up strain engineering for industrial applications.
Fig. 3. Vector map of the pPK2-ku70 vector constructed based on binary vector pPK2BarGFPD.
Kan, kanamycin resistance gene; Ptef, promoter of translation elongation factor gene from Aureobasidium pullulans; egfp, enhanced green fluorescence protein; PtrpC, promoter of tryptophan synthetase gene from Aspergillus nidulans; bar, phosphinothricin resistance gene; 3?-flank and 5?-flank, 3? and 5? flanking fragments
of ku70, respectively; RB and LB, right and left border of T-DNA, respectively
Fig. 4. ku70 disruption of wild-type M. thermophila ATCC 42464. a Pattern of the ku70 knockout cassette integrating into the chromosomes of M. thermophila ATCC 42464 via ectopic insertion or homologous recombination. b PCR analysis of ku70 deletion transformants with one primer (ku70KO-F) located in the bar gene cassette and the other (ku70KO-R) located in the downstream of 3? flank in the
genomic DNA (expected product of 1872 bp) and c Southern blotting analysis of ku70 mutants with XhoI digested genomic DNA and the probe amplified from the 3? flank. Lines 1–11 in (b) and (c), genomic DNA from ku70 mutants (expected product of 5056 bp). WT, genomic DNA from wild-type as negative
control (expected product of 1703Â bp). Primers are represented by solid black arrows.
Abbreviations: gDNA, genomic DNA; RB and LB, right and left border of T-DNA, respectively
There was no noticeable difference between ku70 disruption mutants and the wild-type strain during our analysis (Fig. 5). After five generations of sub-culturing on MM agar medium, 10 randomly selected
ku70 mutants were shown to be mitotically stable. Taken together, these results indicate
the approach we developed is an efficient system for gene disruption in M. thermophila ATCC 42464.
Fig. 5. Phenotypic characterization of ku70 mutant. a Protein concentration in supernatants from wild-type (WT) and ku70 mutant cultures
grown on 1 % Avicel for 3 d at 45 °C. b SDS-PAGE gel of wild-type and ?ku70 strains grown at 45 °C for 3 d on 1 % Avicel. c Conidiation of wild-type and ?ku70 strains growth at 45 °C for 9 d on agar plate without selective agent. d The hyphae of wild-type and ?ku70 strains growth at 45 °C for 24 h on liquid MM without selective agent
In order to check whether the gene homologous recombination rate was improved in ku70 mutant compared to that in wild type strain, we chose the potential marker gene pyrG (MYCTH_2311494) as a test to disrupt in both wild-type and ku70 deletion strain. As expected, 97Â % (29/30) of transformants were correct pyrG deletion mutants under ku70 background, whereas only 30Â % (12/40) of transformants were correct in wild type
background (Fig. 6). This result clearly shown the deletion of KU70 gene can improve the gene homologous
recombination in this strain, similar as reported before 9].
Fig. 6. pyrG gene deletion in wild type and ?ku70 mutant of M. thermophila ATCC 42464. The experiment design (a), PCR analysis of transformants under wild-type background (b) and ?ku70 mutant (c) with one primer (pyrGout-F) located in the neo gene cassette and the other (pyrGout-R)
located in the downstream of 3? flank in the genomic DNA. The genomic DNA from the
wild-type (WT) and ?ku70 strains was used as the negative control
Taken together, these results indicate the approach we developed is an efficient system
for gene disruption in M. thermophila ATCC 42464 and will speed up its strain engineering for industrial applications.