Cloning and Functional Analysis of tri14 in Trichoderma brevicompactum

Corresponding Author: Xiao-Ping Yu College of Life Sciences, China Jiliang University, Hangzhou, Zhejiang Province 310018, P.R. China Tel: +86-571-86836006 Fax: +86-571-86836006 E-mail: yxp@cjlu.edu.cn Abstract: Trichodermin, one of the trichothecenes, is a potential antifungal antibiotic and a protein synthesis inhibitor. Trichodermin is synthesized mainly by Trichoderma brevicompactum, but the biosynthetic pathway has yet been completely elucidated. Our earlier transcriptome assay on the trichodermin synthesis in T. brevicompactum 0248 identified a unique tri14 transcript, designated Tbtri14, that was co-regulated with other tri cluster genes. However, the function of this gene remains unknown. In this study, the full cDNA and DNA sequence of Tbtri14 was isolated from T. brevicompactum 0248. It was found the Tbtri14 gene encodes 364 amino acids with a 59 bp intron. For gene function study, the Tbtri14 was deleted in the 0248 strain. The silencing of Tbtri14 dramatically increased the transcription of Tbtri11, it had little influence on the expression of Tbtri4 and Tbtri5 as well as the production of trichodermin in △tri14 deletion mutants. This implied that Tbtri14 may not be directly involved in trichodermin biosynthesis. Instead, it may act as a gene regulator, such as a repressor for Tbtri11.


Introduction
Trichothecenes are a large group of sesquiterpenoidderived secondary metabolites and have mainly been synthesized by Fusarium and certain other fungal genera like Stachybotrys, Myrothecium, Trichoderma and Trichothecium (Shentu et al., 2014;Wilkins et al., 2003). The trichothecene biosynthetic pathway in Fusarium has been studied in detail and about 18 genes were found to be involved (Kimura et al., 2007). Most of the Fusarium genes directly involved in trichothecene biosynthesis are positioned at a locus designated the core trichothecene biosynthetic gene cluster (tri cluster) (Brown et al., 2004). Trichoderma species produce trichothecenes, most notably trichodermin and harzianum A (HA) that are synthesized mainly by T. brevicompactum and T. arundinaceum, respectively. Trichodermin and HA both have strong antifungal activities against phytopathogens such as Rhizoctonia solani and Botrytis cinerea (Moradi et al., 2012;Tijerino et al., 2011;Malmierca et al., 2012). Furthermore, HA shows extremely similar chemical structure to trichodermin. The biosynthetic pathway of trichothecenes by Trichoderma has yet been completely elucidated and the function of many genes in the Trichoderma is little known.
It was revealed that Trichoderma species also have a trichothecene biosynthetic gene cluster with orthologues of seven genes (tri3, tri4, tri6, tri10, tri11, tri12 and tri14) in the Fusarium tri cluster and tri4, tri11 are directly involved in trichothecenes biosynthesis . The tri5 gene (encoding the trichodiene synthase), located outside the cluster, is responsible for the first committed step in HA biosynthesis . It keeps unkown that other genes in tri cluster involve in trichothecenes biosynthesis or not.
The tri14 gene is located adjacent to the tri cluster in Trichoderma, it was considered as a part of the cluster because its transcription was co-regulated with the other cluster genes (Dyer et al., 2005;Brown et al., 2002). However, the function of tri14 remains unknown so far. In this study, the full sequence of Tbtri14 cDNA was cloned from T. brevicompactum 0248 and analyzed. For study of the gene function, Tbtri14 in strain 0248 was deleted. The influence on the trichodermin production as well as the expression levels of three genes directly involved in trichothecene biosynthesis Tbtri4, Tbtri5 and Tbtri11 were investigated.

Strains, Plasmids and Culture Conditions
Escherichia coli JM109 was used for plasmid construction and maintenance. The E. coli was cultivated at 37°C in Luria-Bertani (LB) medium containing 100 µg mL −1 ampicillin (Brayner et al., 2006).
T. brevicompactum 0248, isolated in our previous study and deposited in China General Microbiological Culture Collection Center (CGMCC No. 6985), was used as the original strain and transformation host. The cultivation of strain 0248 was carried out as described in the literature (Shentu et al., 2014).
The plasmid pSilent-1, kindly provided by Fungal Genetics Stock Center (FGSC) in USA, carries a hygromycin-resistant gene (hph) cassette. The plasmid pCAMBIA0380, obtained from Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, was used for ATMT.
All the primers used in this study were synthesized by Shanghai Sunny Biotechnology Co. Ltd and listed in Table 1.

Cloning and Sequencing of the tri14 Gene
Total RNA was extracted from fresh mycelia by using Spin Column Fungal Total RNA Purification Kit (Sangon, Shanghai, China). Genomic DNA of the strain 0248 was extracted by using Ezup Column Fungi Genomic DNA Purification Kit (Sangon, Shanghai, China). The Tri14 cDNA was amplified from the total cDNA by Polymerase Chain Reaction (PCR) with the primer 14-F/14-R and subcloned into the pMD18-T vector for sequence verification.

Construction of Recombinant Vector
A 1-kb upstream DNA fragment before the tri14 gene start codon was amplified using total DNA as template and following primers pf1 and pr2, the forward primer pf1 containing BstXI site and the reverse primer pr2 containing a 22 bp reverse complementary sequence of hph cassette. To obtain the hph expression cassette, hygromycin resistance gene was amplified by PCR using plasmid pSilent-1 as the template with the primers pf3 and pr4. Similarly, another DNA fragment downstream of the tri14 gene stop codon was amplified using total DNA as template and following primers pf5 and pr6, the forward primer pf5 containing a 22 bp overlapping sequence of hph gene and the reverse primer pr6 containing a BglII site (Fig. 1). Then, the three linearised fragments were equimolarly mixed and cyclised in a fusion PCR to generate a gene knockout fragment (Cao et al., 2014). The gene knockout fragment was cloned into the BstXI/XmaI sites of pCAMBIA0380 to generate a vector designated pCAMBIA0380-kt.

Transformation of T. brevicompactum 0248
The expression vector pCAMBIA0380-kt was introduced into A. tumefaciens AGL-1 by heat shocking at 37° for 5 min after pretreatment with liquid nitrogen for 3 min. The primer H-F/H-R was used to screen the positive cloning that was named as A. tumefaciens AP03HS. The AP03HS-mediated transformation of T. brevicompactum 0248 was then performed as described earlier (Almeida et al., 2007;dos Reis et al., 2004).

Genetic Stability Analysis of Transformants
The transformants were purified by single-spore isolation and analyzed by PCR and qRT-PCR to confirm the deletion in the Tbtri14 gene. The selected transformants were cultured in PDA plates without hygromycin B for 72 h. The fresh mycelia was picked up and inoculated into new PDA plates without hygromycin B and incubated for another 60 h. After this procedure was repeated for five times, mycelia from each transformants were inoculated in PDA plates with 100 µg mL −1 hygromycin B and their growth was observed.

Analysis of Gene Expression by using qRT-PCR
Total cDNA was extracted as described above and diluted 10 times before used. qRT-PCRs were performed in a Applied Biosystems StepOnePlus Real-Time PCR System using a SYBR ® Premix Ex Taq TM (Tli RHaseH Plus) reagent (TaKaRa, Dalian, China). The primers used in qRT-PCRs were given in Table 1. All qRT-PCRs for each gene were carried out in triplicate within a plate and the β-tubulin gene of T. brevicompactum 0248 was used as the reference gene . The quantification of the Tbtri4, Tbtri5, Tbtri11 and Tbtri14 gene expression was analyzed by the 2 -∆∆Ct method.

Analysis of Trichodermin by Using GC
The fermentation broth was separated from the mycelia by filtration using a Buchner funnel and extracted exhaustively with ethyl acetate (v/v, 1:2). The organic fractions were combined and evaporated to dryness in a vacuum at 50°C. The recovered residues were re-suspended in methanol and subjected to Gas Chromatographic (GC) analysis for the quantification of trichodermin .

Results
Sequence Analysis of Tbtri14 from T. brevicompactum 0248 The full cDNA sequence (1,092 bp) and DNA sequence (1,151 bp) of Tbtri14 from T. brevicompactum 0248 were obtained in this study. The nucleotide sequence of tri14 cDNA (Supplementary 1) and its predicated protein has been submitted to GenBank under accession no. KJ957748. It was predicted that the molecular mass of TbTRI14 is 40.11 kDa and the isoelectric point is 4.62 by using ProtParam of ExPASy. The analysis of the TbTRI14 sequence by using InterPro of EMBL-EBI indicated that there was a noncytoplasmic domain, a signal peptide and a polypeptide belonging to the calcium-dependent phosphotriesterase superfamily. A tertiary structure analysis of TbTRI14 by using CPHmodels of CBS predicted that it may be a protein with a barrel structure. The deduced protein sequence of TbTRI14 was compared with those of the Tri14 from T. arundinaceum, S. chlorohalonata and F. sporotrichioides, the identities were 99, 67 and 64%, respectively. However, the function of Tri14 proteins in these three strains was also unknown Brown et al., 2002).

Genetic Stability Analysis of Transformants
With ATMT, a total of 58 hygromycin-resistant transformants were obtained. Screened by PCR, three were identified which had the hygromycin cassette replacing the ORF of Tbtri14 (data not show), designated kt26, kt31, kt52. The genetic stability analysis of the three transformants showed that the lossof-function mutations could be passed down steadily to the next generation. The mutants were identical to each other with respect to the phenotypes observed.

Effect of Tbtri14 Deleting on the Expression Levels of Tbtri4, Tbtri5, Tbtri11
The relative expression levels of Tbtri14 and other three key genes involved in trichothecenes biosynthesis Tbtri4, Tbtri5 and Tbtri11 in △tri14 knockout mutant (kt26) and wild strain 0248 were detected. As shown in Fig. 2A, the expression of the Tbtri14 gene in the wild strain changed with time (40, 52, 64, 76 and 88 h), with the highest expression level observed at 40 h. At each time point, the Tbtri14 expression level was not detected in △tri14 knockout mutants. This indicated that the Tbtri14 gene was completely silenced in △tri14 deletion mutants.
Surprisingly, there was a significant increase in the Tbtri11 expression in △tri14 deletion mutants than that in wild strain at 40, 52, 64, 76 and 88 h (Fig. 2B). In contrast to Tbtri11, the expressions of Tbtri4 (Fig. 2C) and Tbtri5 (Fig. 2D) were hardly affected by the deleting of the Tbtri14 gene; no significant difference in the expression levels of these three genes between the transformant and wild strain was detected.

Effect of tri14 Deleting on the Production of Trichodermin
Fermentation broths from transformant kt26 and wild strain 0248 were assayed for trichodermin production. As shown in Fig. 3, the trichodermin yields produced by kt26 transformant was similar to those produced by wild strain at different time. For both transformant and wild strain, the trichodermin was rapidly synthesized from 40 to 64 h. After 64 h, no increase in the trichodermin concentrations was found.

Discussion
Transcriptome sequencing and gene expression analysis of the 0248 strain identified a unique Tbtri14 transcript that is highly similar to the Fusarium tri14 gene. Furthermore, Tbtri14 was shown to have a significant differential expression under the trichodermin-producing condition. The cDNA and DNA sequence of Tbtri14 was cloned and analyzed in this study. Subsequently, the function of the Tbtri14 gene was also researched in the 0248 strain.
It was found that while the expression levels of Tbtri14 in the three △tri14 deletion mutant was completely silenced, the yields of trichodermin were comparable between the mutant and the wild strain (Fig.  3). This indicated that Tbtri14 may be indirectly involved in the trichodermin synthesis in T. brevicompactum. Similar to this finding, deletion of the Fstri14 gene in F. sporotrichioides was found not to change the synthesis of T-2 toxin that also belongs to the trichothecenes family, which implied that Fstri14 may not be required for the T-2 toxin synthesis in F. sporotrichioides (Brown et al., 2002). The study of the tri14 function in F. graminearum showed that Fgtri14 was required for the wild-type levels of virulence on wheat, but not for the synthesis of deoxynivalenol (another trichothecene mycotoxin) in cracked corn culture.
With the deleting of Tbtri14, the expression levels of Tbtri11 were significantly increased in the transformant. This implied that the expression of Tbtri14 may repress the transcription of Tbtri11 in T. brevicompactum 0248. This result was consistent with the earlier assumption that the Fgtri14 gene in F. graminearum may act as a regulator of the deoxynivalenol synthesis by affecting the transcription of other relevant genes (Dyer et al., 2005).
The tri11 gene in T. arundinaceum (Tatri11) has been shown to encode a requisite enzyme, TaTRI11, in the HA biosynthetic pathway; it catalyzes the C-4 hydroxylation of trichothecene . As we know that both trichodermin and HA can be synthesized by the Trichoderma species and their chemical structure are extremely similar (Degenkolb et al., 2008). The only difference is the side group at the C-4 position; trichodermin has an acetyl group while HA has an octa-2,4,6-trienedioic acid (Malmierca et al., 2012). Thus, it is very likely that the trichodermin in T. brevicompactum shares the similar biosynthetic pathway with the HA in T. arundinaceum. In other words, the Tbtri11-encoded TbTRI11 may also catalyze the C-4 hydroxylation of trichothecene in the biosynthetic pathway of trichodermin, as does the TaTRI11 in HA biosynthesis. However, with the significant enhancement of the Tbtri11 expression in the T. brevicompactum transformant, the yield of trichodermin was not improved. This implied that the expression of Tbtri11 is probably not a rate-limiting step in trichodermin biosynthesis. Other tri cluster genes (e.g., Tbtri3, Tbtri4 or Tbtri5), of which the expression was not affected by the deleting of Tbtri14, may play critical roles in trichodermin biosynthesis. However, it was recently reported that overexpression of Tatri4 in T. arundinaceum did not enhance the HA biosynthesis either (Malmierca et al., 2012). In contrast, overexpression of Tbtri5 in T. brevicompactum IBT 40841 resulted in an increased production of trichodermin. These results indicated that Tbtri5 is most likely a critical gene that limits the trichodermin synthesis in Trichoderma (Tijerino et al., 2011).

Conclusion
Tbtri14 may not be required for trichodermin biosynthesis in T. brevicompactum 0248. Instead, it may act as a regulator of other relevant genes, such as a repressor for Tbtri11. The results obtained from this research provide new information for future study on the biosynthesis pathway of trichodermin or other trichothecenes.