Isolation of sequences flanking the Trichoderma asperellum task1 gene using a single specific primer PCR and their use for gene knockout

Abstract

Gene recombination has been widely used in bacteria, yeast and other lower organisms for gene function research, but its application in filamentous fungi is uncommon because long homologous sequences are required. In this study, first, the task1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was cloned, and then we devised a novel and efficient PCR-based technique to amplify unknown regions adjacent to known genes using a single, specific primer. The feasibility of this technique was demonstrated by isolating sequences flanking the task1 gene of Trichoderma asperellum. The flanking regions obtained were used to construct a binary vector to knock out T. asperellum task1 gene by a Agrobacterium tumefaciens-mediated transformation method.

Introduction

Trichoderma spp. have been extensively studied because they have the capacity for efficient biological control of soil-borne plant pathogens, and can degrade the chemical pesticide dichlorvos, which has caused environmental pollution in recent years (Sun et al. 2010). This fungus protects crop plants against a variety of phytopathogenic fungi, including Rhizoctonia solani (Lewis and Lumsden 2001), Sclerotinia sclerotiorum (Rabeendran et al. 2006), Botrytis cinerea (Olson and Benson 2007) and Phytophthora parasitica (Hanada et al. 2009), by releasing various cell wall-degrading enzymes and secondary metabolites. Although utilising this fungus is not as harmful to the environment as chemical pesticides, biocontrol is less efficacious than using chemical agents. In addition, application of Trichoderma spp. must be restricted because they are susceptible to external environmental conditions, and biological activity decreases rapidly (Guigon-Lopez et al. 2010). It is important to master Trichoderma spp. molecular mechanisms to effectively improve its biological control capacity and environmental adaptability. Numerous Trichoderma spp. genes related to biological control have been cloned (Vizcaíno et al. 2005; Cardoza et al. 2006), but their functions and relationships are unclear. The most direct and effective way to study gene function is to generate gene knockouts by homologous recombination and observe changes of phenotype, gene expression, and metabolite. Gene recombination has been extensively used in bacteria (Hishida et al. 1996), yeast (Khrebtukova et al. 1998) and other lower organisms, but its application in filamentous fungi is limited because it requires long homologous sequences.

Since the invention of PCR, various adapted techniques have been developed to clone unknown fragments. These methods can be divided into three main types of technology: reverse PCR (Prod’hom et al. 1998), foreign joint-mediated PCR (Nthangeni et al. 2005) and semi-random primer PCR (Limansky and Viale 2002).

Reverse PCR depends on the efficiency of intramolecular cyclisation of a restriction fragment near a known locus. This method uses random connections between segments, which can lead to simultaneous amplification of multiple products. Moreover, it can be difficult to form circular products near known sequences if suitable restriction sites are lacking (Wang et al. 2006). Numerous factors are responsible for the high failure rates of foreign joint-mediated PCR include. These include a dependence on the availability of favourable restriction sites around the known locus, a low efficiency of ligation between the cassette and the restricted target DNA fragment, a high rate of mispriming, high cost, and technical difficulties (An et al. 2010).

Both PCR strategies mentioned above require a large number of complicated operations, which are time consuming and labour intensive. In contrast, semi-random primer PCR for cloning flanking sequences is a newer technique that does not require the same troublesome steps. However, it is rarely used because the use of random primers can create many false-positive PCR products.

In this study, we first used genomic DNA as a template and performed PCR using a specific primer at a high annealing temperature to allow specific combination of the primer and template. A large number of complementary strands to the target template were created through multiple amplification cycles. A random combination of single-stranded DNA were converted into double-stranded DNA and, subsequently, the second PCR reaction was performed using the same primer but with the products of the first PCR as the templates and performing the cycles at a low annealing temperature. This ensured the combining of primers and complementary strands from the first round of PCR. Finally, multiple loops were performed at a high annealing temperature to remove the non-specific fragments and specifically amplify the flanking segment.

This technology overcomes all the previously discussed drawbacks because it has high specificity, eliminates the need for restriction enzymes and ligases, is an easier technical procedure, is faster and is generally reliable. Using this method, we isolated the regions flanking the task1 gene of T. asperellum. The flanking sequences identified were used to construct a binary vector for knocking out T. asperellum task1 gene.

Materials and methods

Strains and plasmids

Trichoderma asperellum was isolated from a soil sample from the Peide country, Mishan city, China. It was identified by the Institute of Microbiology (Chinese Academy of Sciences) and was cultivated at 28°C on potato dextrose agar (PDA) until sporulation. Agrobacterium tumefaciens strain AGL-1 was used for fungal transformation and Escherichia coli TOP10 cells were used to propagate fungal transformation vectors.

The PMD18-T plasmid (Takara) was used to clone the task1 gene and its flanking sequences. The pcambia1301 vector, which contains a hygromycin B-resistance gene (hph) driven by the cauliflower mosaic virus CAMV35S promoter and terminator, was used to clone hph cassette and was kept in our laboratory. The binary vector ppzptk8.10, which contains the herpes simplex virus thymidine kinase (HSVtk) gene, was used for A. tumefaciens-mediated transformation of fungi and was kindly provided by Professor Seogchan Kang, Department of Plant Pathology, Pennsylvania State University, USA.

Cloning and sequencing of the T. asperellum task1 gene

Total genomic DNA was extracted following a protocol adapted from the CTAB method described previously (Sambrook 1989). To research the molecular mechanisms of T. asperellum biological control, we constructed a cDNA library and isolated an EST of the task1 gene. The primers, F1 (5′-CCAGCCCGACCACCATGTCT-3′) and R1 (5′-CACCCGAAGCCAGTGTATTGA-3′), were designed based on the sequence of the homologous gene (tmk1) from Trichoderma atroviride. Afterwards, using T. asperellum genomic DNA as template, we PCR amplified in a final volume of 50 μL with the following components: 0.4 μM of each primer, 0.2 mM dNTPs, 1.5 U TaKaRa ExTaq DNA polymerase, 5 μL specific buffer (containing 2 mM MgCl₂), and 2.5 ng DNA. The PCR product was purified using the QIAquick PCR purification kit (Qiagen) and was ligated into the PMD18-T vector. The ligation product was transformed into E. coli TOP10. After cultures were grown, the constructs were purified and sequenced.

Cloning and sequencing of sequences flanking the T. asperellum task1 gene

The unknown fragments next to known sequences can be cloned directly using single specific primer PCR as illustrated in the Fig. 1. A specific primer (T1: 5′-AGAAGAAGGTGCCATTCCG-3′) was designed according to the lower half of the task1 gene and two rounds of PCR were performed to clone the region downstream of the T. asperellum task1 gene. The first round PCR was performed at high annealing temperature to enrich the targeted template DNA strand. The total PCR reaction volume was 25 μL and consisted of 0.4 μM primer T1, 0.1 mM dNTPs, 0.75 U of TaKaRa ExTaq DNA polymerase, 2.5 μL specific buffer (containing 2 mM MgCl₂), and 1.25 ng DNA. The mixture was initially denatured at 94°C for 5 min, and then 30 cycles were run as follows: denaturising at 94°C for 1 min, annealing at 70°C for 1 min, extension at 72°C for 3 min. This was followed by a final elongation step for 7 min at 72°C. For the second-round PCR, 1 μL of the first-round PCR product was used as template, but other components were the same as those in the first reaction. The PCR was initially denatured at 94°C for 5 min, followed 10 cycles of 94°C denaturing for 1 min, 55°C annealing for 1 min, 72°C extending for 3 min. Then, we performed 30 cycles of denaturising at 94°C for 1 min, annealing at 70°C for 1 min, extension at 72°C for 3 min. A final elongation step was run for 7 min at 72°C. The PCR product was purified using the QIAquick PCR purification kit (Qiagen) and was ligated to the PMD18-T vector. The ligation product was transformed into E. coli TOP10. After cultures were grown, the constructs were purified and sequenced. The same method was used to isolate sequences upstream of the task1, except we used a different gene using specific primer (T2: 5′-TTCATCTCTCGCAGGGTTCT-3′) that was designed according to the upper half of task1 gene.

Fig. 1

Schematic representation for the isolation of unknown sequences next to a known gene In the picture , represents a specific primer, represents a sequence complementary to the specific primer, and represents a known sequence

Construction of the recombinant plasmid ppzptk8.10Δtask1

For the construction of ppzptk8.10Δtask1, a 1.3-kb XbaI/HindIII fragment, containing sequences flanking the 5′ of the T. asperellum task1gene, was obtained by PCR amplification using T. asperellum genomic DNA as template and primers F2/XbaI (5′-ATGGCAGATGGCTATTGTTC-3′) and R2/HindIII (5′-CCCAAGCTTTGGCGACTTGGGCCTGAAT-3′). After digestion with the XbaI and HindIII restriction enzymes, the fragment was inserted into XbaI/HindIII cut ppzptk8.10. Subsequently, a 1.8-kb hygromycin B-resistance gene (hph) cassette obtained by PCR amplification using pcambia1301 vector as template and primers F3/KpnI (5′-CGGGGTACCTAATTCGGGGGATCTGGATT-3′), R3/ HindIII (5′-CCCAAGCTTATGGTGGAGCACGACACTCT-3′) was cut with HindIII/KpnI and inserted into the plasmid obtained above after digestion with HindIII/KpnI. The resulting plasmid was digested with KpnI and a 1.2-kb fragment containing the 3′ flanking sequences of the task1 gene was obtained by PCR amplification with primers F4/KpnI (5′-CGGGGTACCTAGGGAACTGATGAAGCA-3′) and R4/KpnI (5′-CGGGGTACCAAGGGACTCAGTGTGCGGAT-3′) and inserted.

Agrobacterium tumefaciens-mediated transformation of T. asperellum

Agrobacterium tumefaciens-mediated transformation (ATMT) of strain T. asperellum was based on previous protocols (Sun et al. 2009), with modifications. A fresh colony of A. tumefaciens strain AGL-1 containing ppzptk8.10Δtask1 was cultured at 28°C for 2 days with shaking at 240 rpm in LB broth supplemented with 50 μg/mL kanamycin and 50 μg/mL streptomycin. The A. tumefaciens cells were collected by centrifugation and diluted to an optical density at 660 nm (OD₆₆₀) of 0.15 in liquid induction medium (IM) (g/L: K₂HPO₄, 1.45, KH₂PO₄, 2.5, NaCl, 0.15, MgSO₄, 0.5, CaCl₂, 0.066, FeSO₄, 0.0008, NH₄SO₄, 0.5, glucose, 1.8, pH 5.3, 0.5% glycerol (w/v), and 200 μM acetosyringone), and induced for 6–12 h at 28°C with shaking.

After growing on PDA for 5 days at 28°C, conidia of strain T. asperellum were collected and diluted to 10⁷ spores/mL using a sterilised physiological saline solution. For co-incubation, 200 mL of the T. asperellum conidia suspension was mixed with an equal volume of A. tumefaciens cells, spread on the surface of round filter papers, and placed horizontally on IM plates containing the ingredients of liquid IM medium except for 0.9 g/L instead of 1.8 g/L glucose and 15 g/L agar. The cells were co-cultivated at 24°C in the dark. After 2 days, the filter papers were transferred to solid M-100 medium supplemented with 300 μg/mL cefotaxime, to inhibit growth of A. tumefaciens cells, and 300 μg/mL hygromycin B, as the selection agent for fungal transformants. Putative transformants formed within 3∼7 days. The colonies were then transferred to M-100 plates that contained 300 μg/mL hygromycin B and were incubated at 24°C. A. tumefaciens AGL-1 without the binary vector was used as a negative control.

The secondary screening of transformants

The transformants obtained above were subcultured to selective medium PDA containing 300 μg/mL hygromycin, 300 μg/mL cefotaxime and 20 μM thymidine analogues (F2dU), and continued to cultivate 2∼3 days. The HSVtk gene from insertion mutants of T. asperellum produces thymidine kinase, which converts nucleoside analogs F2dU to toxic compounds. Thus, the insertion mutants cannot grow and any colonies grown on these plates should be recombinant task1 gene mutants.

The PCR analyses for the recombinant mutants of task1 gene

The T. asperellum and its recombinant mutants were inoculated into 50 mL PD medium and were incubated in a rotary shaker at 180 rpm for 3 days at 28°C. The mycelia were collected by filtration through lens paper and rinsed twice with sterile water. Fungal genomic DNA was extracted from mycelia using the CTAB method (Sambrook et al. 1989). Two PCR methods were used to screen for homologous recombination transformants. In the first, a pair of primers [F5 (5′-GCACAGAGTTATCCGTACCCA-3′) and R5 (5′-CAGATAAGGGTGTCGGTGAAT-3′)] were designed to amplify a 400-bp fragment of the task1 gene. The DNA fragment would not be amplified if the task1 gene was knocked out. In the second PCR analysis, one primer was designed on the chromosome sequence outside the task1 gene knockout cassette (F6, 5′-ATGGCAGATGGCTATTGTTC-3′), while another is on the selection marker hph gene (R6, 5′-AGAGTGTCGTGCTCCACCAT-3′). If the task1 gene was knocked out, the pair of primers should amplify a product of 1,350 bp.

Results

Cloning of T. asperellum task1 gene

The task1 gene is amplified with the F1 and R1 primers. The length of the task1 gene is 1,757 bp, as shown in Fig. 2. The deduced protein sequence comprises 357 amino acids and is 90% identical to Trichoderma virens TmkA and 92% identical to T. atroviride Tmk1. The sequence was submitted to GenBank under accession number JN035617.

Fig. 2

The PCR products for task1 gene using the primers of F1 and R1

The determination of sequence flanking task1 gene from T. asperellum

The flanking segments of the task1 gene were isolated using single specific primer PCR. The downstream segment of the gene was 2,336 bp as shown Fig. 3(a) and the upstream sequence of the gene was 2,014 bp as shown in the Fig. 3(b).

Fig. 3

The PCR products for regions flanking the task1 gene. a The downstream sequence was amplified using the primer T1. b The upstream sequence was amplified using the primer T2

The acquisition of T. asperellum transformants

Agrobacterium tumefaciens, a plant-pathogenic bacterium, has the ability to transfer a fragment of DNA (T-DNA) from its tumor-inducing plasmid to a recipient’s genome. According to this, we constructed task1 gene knockout cassette by using hygromycin resistance (hph gene) as the selectable marker. Our results indicate that A. tumefaciens harbouring the ppzptkΔtask1 binary vector efficiently transferred T-DNA to cells as shown (Fig. 4a) by active fungal cell growth on PDA medium containing 300 μg/mL hygromycin and 300 μg/mL cefotaxime. The conversion rate was about 50–60 transformants per 10⁷ spores.

Fig. 4

a The transformants of T. asperellum. b The growth of T. asperellum transformants on the plates containing of F2du

Isolation of recombinant transformants of the task1 gene

The recombinant transformants were identified by growth on PDA medium containing 300 μg/mL hygromycin, 300 μg/mL cefotaxime and 20 μM thymidine analogues (F2dU). The HSVtk gene from insertion mutants of T. asperellum produces thymidine kinase, which converts nucleoside analogues F2dU to toxic compounds. Insertion mutants could not grow on this media; therefore, the colonies grown on the plate shown in Fig. 4b should be the recombinant mutants of the task1 gene. The recombination rate was about one recombinant transformants per eight colonies.

PCR confirmation of the recombinant transformants

The confirmation of transformants was done with two PCR reactions using DNA extracted from cultures of the transformants and the wild-type strain as negative control. Firstly, a fragment of 400 bp from the task1 gene was amplified with primers F5 and R5, shown in Fig. 5a. The wild-type strain yielded the predicted 400-bp amplicon, but the transformants did not. This is a preliminary indication that gene task1 was knocked out. Secondly, to further identify that transformants were task1 gene recombinant mutants, PCR was performed using F6 and R6 oligonucleotide primers. One primer, F6, was located on the chromosome outside the gene knockout cassette, while the other primer, R6, was on the selection marker hph gene. It was possible to amplify a PCR product of the expected size (1,350 bp) from the recombinant transformants tested. The 1,350-bp fragment from ten transformants was generated as shown in Fig. 5b. In contrast, the band was not amplified from the wild-type control strain. The result further illustrates that the T. asperellum task1 gene was knocked out.

Fig. 5

PCR analysis of recombinant transformants

Discussion

Agrobacterium tumefaciens-mediated transformation of filamentous fungi has been used in numerous studies in recent years. The experimental conditions including induction by acetosyringone (Figueiredo et al. 2010), A. tumefaciens concentration, induction time, co-culture time and temperature (Rafat et al. 2010), concentration of fungal spores, and spore growth period (Zhang et al. 2008) have been optimised. Therefore, this experiment did not research the optimal conditions.

The key to success for this method was binding affinity between the primer and the specific single-stranded DNA from the first round PCR in the second round PCR reaction. Therefore, primer and annealing temperature are two important parameters, which were altered at times to promote synthesis of the fragments we wanted to obtain. In this study, only one specific primer was applied to acquire the sequences downstream of the task1 gene. However, four primers were separately tested to amplify upstream sequences, but only one of them was effective in obtaining the upstream sequence. Thus, to get the flanking sequences, several experiments may be required to determine the optimum primer and annealing temperature. However, the program is much simpler than previous strategies. In this study, the flanking sequences were used for gene knockout. This technique could also be used for other studies, such as the separation of gene regulatory region, looking for efficient promoters, or analysis of transgenic insertion site.

Conclusion

In this study, first, task1 gene encoding a mitogen-activated protein kinase (MAPK) during fungal growth, mycoparasitic interaction, and biocontrol was cloned, and then we proposed and used a novel and efficient PCR-based technique to amplify unknown regions adjacent to known genes using a single, specific primer. The feasibility of this technique was demonstrated by isolating sequences flanking the task1 gene of Trichoderma asperellum. Finally, we used the flanking sequences obtained to design a binary vector for knocking out task1 gene in T. asperellum by an Agrobacterium tumefaciens-mediated transformation method.