Introduction

Fungi that parasitize the mycelia of other fungi are termed mycoparasites. Mycoparasites of plant pathogenic fungi have received much attention due to their use as biocontrol reagents. Trichothercium roseum is a parasitic fungus that can parasitize Sclerotinia sclerotiorum and Pestalotiopsis funerea (Huang and Kokko 1993) and inhibit a variety of plant pathogenic fungi (Munshi et al. 2009; Jayaprakashvel et al. 2010; Shafat 2010).

Chitin is the second most abundant organic and renewable compound in nature after cellulose. Chitinases are chitin-degrading enzymes that have important biological functions and immense potential applications. In recent years, much research has focused on fungal chitinases, especially at the molecular level (Li 2006). Chitinases produced by mycoparasites can inhibit mycelial growth, spore germination, and germ tube elongation (Viterbo et al. 2001), degrading not only the tips of mature hyphae, but also the chitin-glucan complex of the cell wall and sclerotia (Lorito et al. 1993). Extracellular chitinase produced by Trichothecium roseum has been reported to be involved in the process of re-parasitic and antagonistic interactions (Li et al. 2004).

Chitinase genes isolated from different fungi have been used to enhance the resistance of crops and trees to fungal pathogens. The mechanism of chitinase action involves chitin degradation and cell wall disruption, leading to fungal cell lysis (Dahiya et al. 2006). Bolar et al. (2001) transformed ech42 encoding endochitinase and nag70 encoding exochitinase from Trichoderma atroviride into apple. The transgenic apple plants were highly resistant to Venturia inaequalis, the causal reagent of apple scab. Kern et al. (2010) demonstrated that expression of a chitinase gene from Metarhizium anisopliae in tobacco plants conferred resistance against Rhizoctonia solani. Hassan et al. (2009) observed that a family 19 chitinase (Chit30) from Streptomyces olivaceoviridis ATCC 11238 expressed in transgenic pea affected the development of Trichoderma harzianum in vitro. Jia et al. (2010) found that overexpression of the chitinase gene BbchiT1 from Beauveria bassiana resulted in enhanced resistance to Cytospora chrysosperma in Populus tomentosa Carr. Kumar et al. (2009) provided a powerful demonstration of the disease protection for Rhizoctonia solani using transgenic cotton plants that expressed an endochitinase gene from Trichoderma virens.

Trichothecium roseum is an important mycoparasitic fungus with significant antifungal activity but studies on chitinases of T. roseum have been limited. Purified chitinase from T. roseum strongly inhibits the growth and development of chitin-containing fungi in vitro (Li et al. 2004). Expression of chitinase genes in plants improves their defense responses against various fungal pathogens. In particular, chitinases from mycoparasitic Trichoderma spp. were overexpressed in several agriculturally important plants such as lemon, cotton, apple, and carrot (Dana et al. 2006; Kumar et al. 2009). For example, the chit42 gene of T. harzianum encoded a powerful endochitinase and was expressed constitutively in tobacco, and these transgenic plants showed broad resistance to fungal and bacterial pathogens, salinity, and heavy metals (Dana et al. 2006).

In this study, we cloned the chitinase gene Trchi1 from T. roseum and transferred it into tobacco plants by the Agrobacterium-mediated transformation. Our results indicate that Trchi1 may have efficacy for improving plant resistance to fungal pathogens.

Materials and methods

Bacterium and fungal strains, reagents, vectors, and plant materials

The M1 strain of Trichothecium roseum and plasmid pROKII (Sharma and Anjaiah 2000) were isolated and stored in our laboratory. E. coli DH5α strains, pMD 18-T Vector, RNA PCR Kit (AMV) Ver 3.0, TaKaRa EX Taq, EcoRI, and T4 DNA ligase were from TaKaRa Biotechnology Co., Ltd. (Dalian, China). XbaI and XmaI endonucleases were purchased from New England Biolabs (Ipswich, MA, USA). DIG High Prime DNA Labeling and Detection Starter Kit I was purchased from Roche Diagnostics GmbH (Mannheim, Germany). AMV Reverse Transcriptase was purchased from Promega (Madison, WI, USA). Agrobacterium tumefaciens strain LBA4404 and strains A. alternata and C. nicotianae were provided by Dr. Shao-Hui Chu, Dr. Yuancun Lian, and Dr. Guangmin Zhang, respectively, from the Institute of Plant Protection, Shandong Agricultural University, China. Tobacco (Nicotiana tabacum L.) cultivar NC89 plants were grown in the greenhouse at Shandong Agricultural University.

Cloning of the Trchi1 gene

According to the amino acid sequences of chitinase genes from 16 different fungi (Supplementary Fig. 1), three conserved domains having amino acid sequences LSIGGWT, DG-D-DWE, and MFWEASA, respectively were selected for primer design. The degenerate primers Ps446, Ps561, and Pa were designed to amplify the chitinase gene from T. roseum (Supplementary Table 1).

Trichothecium roseum was cultured in SMCS medium at 25 °C for 3–4 days (Li et al. 2004). Total RNA was isolated from cultured T. roseum using Trizol reagent. First-strand cDNA synthesis was conducted using RNA PCR Kit (AMV) Ver. 3.0. Using cDNA first strand as a template, we performed nested PCR amplification using Ps446 and Ps561 as upstream primers and Pa as a downstream primer. The PCR amplification was carried out for 34 cycles. The initial denaturation reaction was done at 94 °C for 4 min. 34 cycles were performed with denaturation (94 °C, 30 s), annealing (50 °C, 45 s), and extension (72 °C, 1 min), followed by 72 °C for 10 min.

The 3′-end flanking sequence of Trchi1 was isolated by 3′-RACE PCR using RNA PCR Kit (AMV) Ver. 3.0. We used specific primers M8501pa1 and M8501pa2 as forward primers, and M13 Primer M4 as reverse primer.

The 5′-flanking sequence of Trchi1 was cloned by TAIL-PCR (Liu et al. 1995). The first TAIL-PCR was performed by using 5MX8501Ps2, 5m7303ps2, and 5m7303ps1 as specific primers, and a random degenerate primer as forward primer. The annealing temperatures for specific amplifications were 60, 63, and 65 °C. The second TAIL-PCR amplification was performed using 5M8501RPs1, 5M8501RPs2, and 5M8501RPs3 as nested primers with annealing at 59, 63 and 60 °C over the three rounds of reactions.

Using T. roseum cDNA and genomic DNA as templates, psq8500 and paq8501 primers were used for PCR amplification. The PCR procedures were as follows: 94 °C for 4 min, 32 cycles of 94 °C for 1 min, 57 °C for 1 min, 72 °C for 2 min, and the final extension at 72 °C for 10 min.

For all above-mentioned PCR reactions, 2 μl cDNA, 2.5 μl of 10× Taq buffer, 2 μl of 10 mM dNTP, 0.5 μl (2.5 U) EX Taq, and 10 pmol of each pair of primers in 25 μl were used. The PCR products were separated by electrophoresis on 1 % agarose gels in TAE buffer and were observed by fluorescence with UV light after staining with ethidium bromide. PCR products were ligated to the cloning vector pMD18-T by T4 DNA ligase and transformed into E. coli DH5α. Positive colonies were confirmed by PCR and sequencing.

Construction of plant expression vector

Using T. roseum cDNA as the template, ycm8501bs and ycm8501ba primers were used for the amplification of the full-length Trchi1 gene, which was cloned into the pMD18-T vector using T4 DNA ligase, and the resulting vector was named pMD18-T/ycTrchi1 (Supplementary Fig. 2c). The vectors pROKII and pMD18-T/ycTrchi1 were digested with XbaI and XmaI, and ligated using T4 DNA ligase (Supplementary Fig. 2d). The final plant expression vector was named pROKII/ycTrchi1 (Fig. 1), and was confirmed by PCR and enzyme digestion.

Fig. 1
figure 1

T-DNA region of vector pROKII/ycTrchi1. LB left border, RB right border, tNos nos terminator, Trchi1 1,700-bp Trchi1 coding sequence, 35 s pro CaMV35 s promoter, NptII neomycin phosphotransferase II gene for kanamycin resistance, pNos nos promoter

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Genetic transformation of tobacco

The pROKII/ycTrchi1 was transferred into Agrobacterium tumefaciens LBA4404 using the freeze–thaw method (Chen et al. 1994). Leaves of in vitro cultured tobacco NC89 plants were cut into 0.5 cm × 0.5 cm leaf discs and immersed into the Agrobacterium culture for 10 min. The leaf discs were then cultured on MS medium (Murashige and Skoog 1962) in the dark for 2–3 days, then cultured on MS supplemented with 3 mg 6-BA/l, 0.2 mg NAA/l, 100 mg kanamycin/l, and 500 mg carbenicillin/l and solidified with Gelrite. When regenerated shoots were approx. 2 cm, the adventitious buds were moved into MS medium for root formation. When the seedlings were 5–7 cm high, they were transferred to soil and moved into a greenhouse for further growth. T1 transgenic seeds were harvested from different T0 transgenic lines, and T1 transgenic seedlings were used for molecular analysis and fungi bioassay.

Molecular identification of transgenic tobacco plants

Genomic DNA was isolated from T1 transgenic tobacco plants according to the methods of Dellaporta et al. (1983). Using tobacco genomic DNA as the template, PCR was performed to confirm the presence of transgenes using specific primers psq8500 and paq8501. Southern hybridization was used to identify the integration pattern of transgenes. Transgenic tobacco genomic DNA was digested with HindIII. We used a 432-bp fragment of Trchi1 amplified with specific primers ycm8501bs and 5M8501RPs4 as the template to synthesize probes labeled with digoxin. The procedures for Southern hybridization were referred to the DIG High Prime DNA Labeling and Detection Starter Kit I.

RT-PCR analysis of transgenic tobacco plants

Total RNAs from different transgenic tobacco plants were isolated using Trizol. For RT-PCR, 1 μg total RNA was reverse transcribed using AMV Reverse Transcriptase following the manufacturer’s instructions. We performed RT-PCR using the gene-specific primers psq8500 and paq8501 (Supplementary Table 1). PCR cycling comprised an initial step at 94 °C for 5 min, followed by 31 cycles at 94 °C for 1 min, 57 °C for 45 s, and 72 °C for 50 s.

Enzyme assay

Tansgenic tobacco leaves (1 g) were powdered in liquid N2, transferred to a microcentrifuge tube, mixed with 3 ml 0.05 M phosphate buffer (pH 6.5) and centrifuged for 20 min at 4 °C at ~10,000 g. The supernatant was used directly for chitinase activity measured as previously reported (McCreath and Gooday 1992) using 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotrioside as substrate.

Fungi resistance of transgenic tobacco plants

A. alternata and C. nicotianae were placed in the center of a potato dextrose agar (PDA) plate and cultured at 25 °C to cover the entire plate. Two mature leaves per plant from transgenic plants were taken and non-transgenic plants at similar growth stage. Three small wounds were made between secondary veins of the backside of half the leaf with a needle and the fungi were inoculated into the wound sites. Three wounds were also made in the other half of each leaf but were not inoculated to be used as controls. The leaves were sealed with plastic wrap and placed on 4 layers of wet gauze in a porcelain plate at 25 °C. After 9 days, we checked inoculated leaves for fungal growth.

Results and discussion

Cloning of T. roseum chitinase gene

A 656-bp DNA sequence of T. roseum chitinase gene was obtained using Ps561 and Pa primers. Starting from this sequence, by 3′-RACE PCR, a 481-bp sequence was obtained, with 108-bp overlapping with the original 656-bp sequence. After three rounds of TAIL-PCR using nested primers 5MX8501Ps2, 5m7303ps2, and 5m7303ps1, we achieved a 1,000-bp fragment with 286-bp overlapping with 656-bp sequence. For the second TAIL-PCR using specific primers 5M8501RPs1, 5M8501RPs2, 5M8501RPs3, we obtained a 650-bp fragment with 108-bp overlapping with the 1,000-bp sequence. These four fragments were assembled in silico. Finally, using cDNA and genomic DNA as templates, we amplified the full length T. roseum chitinase gene by PCR using specific primers psq8500 and paq8501 designed on the vitual sequence. A 1,700-bp and 1,818-bp fragments were obtained from cDNA and genomic DNA, respectively (Supplementary Fig. 2a, b), sequenced, and deposited in Genbank with accession numbers GU361768 and GU361767, respectively.

The sequence analysis of Trchi1 gene and putative protein

The Trchi1 gene contains a 1,278-bp ORF, which putatively encodes a 425 amino acid protein. The genomic sequence contains two introns of 62 and 56-bp.

The deduced amino acid sequences of Trchi1 gene were compared with those of other chitinases using the BLASTx program. Phylogenic relationships between the homologous genes were analyzed by the DNAMAN software (Supplementary Fig. 1). The results showed that Trchi1 had the highest similarity (76 %) with a chitinase from Bionectria ochroleuca (Genbank accession No. 3G6L_A). Surprisingly, Trchi1 had low similarity with chitinases from mycoparasites, indicating that Trchi1 may be a new chitinase gene. Sequence analysis using Scanprosite software (http://www.expasy.ch/tools/scanprosite/) showed that the deduced Trchi1 protein belongs to family 18, which was confirmed by the specific sequence FDGIDIDWE (position 165–173) for this family. Trchi1 also contains a highly conserved region DGXDXDXE, which is the substrate-binding domain. Sequence analysis of Trchi1 by SignalP-NN software (http://www.cbs.dtu.dk/services/SignalP) indicated that the putative amino acid sequence of Trchi1 contains a signal peptide formed by 20 amino acids, suggesting that Trchi1 encodes a secreted protein.

Molecular identification of T1 transgenic tobacco plants

We obtained transgenic tobacco plants by Agrobacterium-mediated transformation with the binary vector pROK and most of them grew normally and set seeds. PCR amplification of genomic DNA from putative transgenic plants showed that the expected 1.5-kb Trchi1 amplicon could be obtained from 86 % of the plants (data not shown). Southern hybridization showed that in all PCR-positive transgenic tobacco plants, the Trchi1 gene was integrated into the tobacco genome. In addition, most of the transgenic plants had one copy of the transgene (Fig. 2).

Fig. 2
figure 2

Southern blot analysis. A 432-bp fragment of the Trchi1 gene was used as the template to synthesize probes labeled with digoxin. 3, 5, 7, 15, and 17: T 1 transgenic lines, WT non-transgenic plants, P positive control, vector pROKII/ycTrchi1 (1 ng)

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Trchi1 expression analysis in T1 transgenic tobacco plants

We isolated RNA from 10 different positive transgenic tobacco plants, as confirmed by Southern blot analysis. RT-PCR analysis showed that the expected 1,500 bp band was obtained from all of them, whereas it was not amplified from the non-transgenic plants, demonstrating that the Trchi1 gene was transcribed in the transgenic lines (Fig. 3). All 10 transgenic plants displayed much higher chitinase activity than the control plants, with a 2.2- to 5.8-fold increase (Fig. 4), indicating that the active Trchi1 protein was produced.

Fig. 3
figure 3

RT-PCR identification of transgenic tobacco plants M: DL2000 marker; 3, 5, 7, 15-17, 23-25, 33: T 1 transgenic lines, WT non-transgenic tobacco, CK positive control, vector pROKII/yctrchi1 (1 ng)

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Fig. 4
figure 4

Chitinase activity of wild-type and transgenic tobacco plants. The data were analyzed by Data Processing System v7.05, and comparison between different transgenic lines and the wild type (Relative activity = 100 %) were performed with SSR Duncan multiple range test. 3, 5, 7, 15–17, 23–25, 33: T 1 transgenic lines, WT wild type

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Resistance of transgenic T1 tobacco plants to A. alternata and C. nicotianae

A. alternata and C. nicotianae were applied to the in vitro leaves of transgenic T1 tobacco plants. Nine days after inoculation owith A. alternata, only 2.8 % (1 out of 36) of transgenic plants showed significant lesions, whereas all the negative control plants produced significant lesions (Fig. 5). Similarly, 9 days after C. nicotianae inoculation, only 7.1 % (3 out of 42) of transgenic plants showed significant lesions, whereas all the non-transgenic plants had significant lesions. These experiments indicate that overexpression of the Trchi1 gene greatly enhanced tobacco resistance against A. alternata and C. nicotianae Table 1.

Fig. 5
figure 5

Resistance to two pathogenic fungi of tobacco plants overexpressing the Trthi1 gene A3, A7, and A17 are T1 transgenic lines #3, #7, and #17, respectively, which were inoculated with Alternaria alternata. NA non-transgenic plants were inoculated with A. alternata. C3, C7, and C17 represented T1 transgenic lines #3, #7, and #17, respectively, which were inoculated with Colletotrichum nicotianae. NC non-transgenic plants inoculated with C. nicotianae

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Table 1 Analysis of fungal resistance of transgenic plants
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One approach to enhancing plant resistance to fungal diseases is to provide the host plant with an exogenous transgene, just as we have done in this study with the overexpression of T. roseum Trchi 1 in tobacco. This strategy has been applied successfully to crops such as tobacco, potato, and apple (Li 2006); however, the chitinase genes are cloned mainly from plants, and only a few are originated from mycoparasites. In this study, overexpression of the Trchi1 gene cloned from T. roseum significantly improved tobacco resistance on two severe fungal pathogens, A. alternata and C. nicotianae, as estimated by artificial inoculum in lab conditions. If confirmed in field conditions, this new source of resistance could broaden the resources for long-term and durable resistance to fungi in crops.