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Keywords:

  • Trichoderma harzianum;
  • Endochitinase;
  • Filamentous fungus;
  • Mycoparasitism;
  • Biological control

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Trichoderma harzianum is a filamentous soil fungus known as an effective biocontrol agent of several plant pathogenic fungi. Its mycoparasitism involves a complementary action of antibiosis, nutrient competition and cell wall degrading enzymes such as β-1,3-glucanases, proteases and chitinases. Since chitin is the major component of most fungal cell walls, a primary role has been attributed to chitinases in the biocontrol activity of Trichoderma[1]. The chitinolytic system in T. harzianum consists of at least six distinct enzymes, two N-acetylglucosaminidases and four endochitinases [2].

The expression of chitinases is repressed by glucose and induced by chitin, fungal cell walls or starvation [3]. Because of the important applications of chitinases in the fields of pest control, pollution abatement, and basic and commercial biology [4], many have been characterized, mostly from plants and bacteria and in minor proportion from fungi. So far the chitinase genes which have been isolated from Trichoderma strains are the ones encoding the N-acetylglucosaminidases [5], and the endochitinases CHIT42 [6] and CHIT33 [7].

Studies on the molecular structure and characteristics of genes encoding enzymes of the chitinase complex in Trichoderma will contribute to the better understanding of the relationships of the different enzymes involved in the biocontrol mechanisms. In the present work we describe the isolation of a new endochitinase gene, named chit36, its overexpression in Trichoderma via biolistic transformation and its antagonistic effect on Botrytis cinerea, Fusarium and Sclerotium rolfsii.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Strains, media and culture conditions

T. harzianum Rifai strain TM was grown on potato dextrose agar (PDA). To recover chitinolytic activities T. harzianum was grown on SM medium [8] with 0.2% (w/v) colloidal chitin as the sole carbon source or with 5% glucose for detection of the constitutively overexpressed CHIT36 protein.

2.2Protein sequence and oligonucleotides

Gel slices with purified CHIT36 [9] were sent to the Bletterman Macromolecular Research Laboratory, Hebrew University, Faculty of Medicine, Jerusalem and analyzed with the Procise Protein Sequencer (Perkin Elmer ABD 492). Two degenerate primers were designed according to the sequences of the two peptides obtained: 1: GYWENWD, 5′-GG(N) TA(C/T) TGG GA(A/G) AA(C/T) TGG GA-3′ and 2: YDMQVPG, 5′-CC (N)GG (N)AC (T/C)TG CAT (G/A)TC (G/A)TA-3′.

2.3Plasmids

The vector pAN7 containing the Escherichia coli hph gene was purchased from Stratagene. The vector pRLMex-30 was kindly provided by Prof. R.L. Mach. pRL-36 designates a plasmid encoding the chit36 open reading frame (ORF) fused to the pki1 promoter in XbaI/NsiI sites of pRLMex.

2.4Transformation procedure and selection

Microprojectile bombardment of intact T. harzianum conidia was performed essentially as described in [10], with minor modifications. 0.4-μm SE tungsten particles were used and a total of 1 μg DNA was loaded for co-transformation with two plasmids: pAN7 and pRL-36. Co-transformants were screened for the presence of the selectable marker by plating on PDA supplemented with 300 μg ml−1 hygromycin B. Colonies were grown on SM minimal medium plus 5% glucose and after 2 days the growth media were tested for the presence of endochitinase activity. Single spores were isolated from selected colonies and spread on selective medium. This process was repeated twice. Transformants were screened also by polymerase chain reaction (PCR) for the presence of both plasmids into the genome. Primers pairs were: 1: 5′-aggtaccgatttaatagctccatgtcaac-3′ (5′ end of trpC in pAN7); 2: 5′-aggtaccgtctagaaagaaggattacctc-3′ (3′ end of trpC in pAN7); 3: 5′-cgtggcagctcgagataacg-3′ (region −744 to −725 of the pki1 promoter in pRLMex-36); 4: 5′-cggtgccatagtcaagccaaa-3′ (reverse of FGLTMAP in chit36).

2.5Nucleic acid isolation and manipulations

T. harzianum chromosomal DNA was isolated according to Raeder and Broda [11]. Total RNA was isolated with the EZ-RNA kit (Biological Industries, Israel) and mRNA was purified with the PolyATrac kit (Promega). 5′ and 3′ RACE PCR was performed according to the SMART RACE cDNA Amplification Kit (Clontech). All other nucleic acid manipulations were carried out as described in [12].

2.6Isolation of extracellular proteins and Western blots

Two-day-old cultures of T. harzianum were filtered and the supernatant was concentrated 20 times with Vivaspin concentrators, 10 kDa cut-off membrane (Vivascience). Western blots were performed according to standard procedures. The anti-chit36 [9] was used in a 1:1000 dilution. Protein concentration was determined with the Bradford reagent (Bio-Rad).

2.7Chitinase activity

Crude chitinolytic activity was determined according to Inbar et al. [13]. Identification of enzymatic activities on sodium dodecyl sulfate (SDS) gels was done according to Haran et al. [2].

2.8Biological assays

2.8.1Conidia germination

The bioassay was performed essentially as described in [14]. The assay mixture (60 μl) containing 2000–3000 conidia of B. cinerea in PDB was incubated in a flat-bottomed ELIZA plate at 22°C. The culture filtrates from transformant C, wild-type (WT) grown on glucose-rich medium or 0.2% colloidal chitin were added to the reaction mixture at a final concentration of ×1 after dialysis against buffer potassium acetate 0.1 M pH 5.5. The plates were observed after 20 h using an inverted microscope and the percentage of germinating spores was determined for each field. The experiment was repeated twice in three replicates.

2.8.2Growth inhibition

Petri dishes with agar SM medium supplemented with 5% glucose were covered with cellophane disks (Scotch, Cergy-Pontoise, France) and inoculated in the center with a 5 mm diameter mycelial disk of T. harzianum WT, transformant C or D. After 2 days incubation at 28°C the cellophane was removed and the plates were inoculated with a 5 mm diameter mycelial disk of different plant pathogenic fungi. Chitinase secretion on replicate plates was checked with the fluorescent substrate 4-MU-(GlcNAc)2[2] and visualized under UV light. Non-pre-inoculated plates were used as control for normal growth.

2.8.3Interactions in dual cultures and greenhouse experiments

The antagonistic ability of the transformants was tested against the pathogens S. rolfsii, Rhizoctonia solani, and B. cinerea on solidified SM medium, as in [15]. Cotton (Gossypium barbadenso L.) seedlings were used in greenhouse experiments which were performed in six replicates as described in Carsolio et al. [16].

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

3.1Gene isolation and characterization

Fractions containing CHIT36 activity which were eluted from the Q-Sepharose column [9] were pooled and separated on SDS–PAGE for sequencing. Since the N-terminus was found to be blocked the protein was digested with trypsin and internal amino acid sequences of two peptides were determined: (1) VLMGYWENWDGASNGVHPGF and (2) IPYDMQVPGLPAQNG.

Using the primer pairs described in Section 2 a 700-bp DNA fragment was isolated from both genomic DNA and cDNA of T. harzianum. The complete ORF was obtained by 3′ and 5′ RACE PCR. The chit36 encoding cDNA (accession number AY028421) is 1235 bp in length including a 3′ untranslated region of 200 bp. The ORF encodes an 344-amino acid protein with a 16-residue putative signal peptide, with an expected molecular mass of 36 kDa after processing. From previous data CHIT36 from T. harzianum TM has been shown to be an endochitinase with apparent molecular mass of 33 kDa, heat-resistant, with a pI value of 4.8. CHIT36 has been shown also to be induced by chitin or antagonistic interaction with R. solani and repressed by glucose [2,9]. Another endochitinase protein of molecular mass about 37 kDa and similar pI value was purified from T. harzianum Rifai CECT 2413 [17] and more recently also from T. harzianum isolate 1051 [18]. The chemical and physical similarities of these partially characterized proteins suggest that they might be identical or related to Chit36. Sequence alignment (Blast search) shows a high similarity (87%) of CHIT36 to a putative chitinase (CAB69724) from Streptomyces coelicolor, to CHIA from Bacillus cereus (50%), to Bacillus circulans CHITD (48%) and other prokaryotic chitinases. No significant similarity can be found with other eukaryotic chitinases and with another endochitinase (chit33), of close apparent molecular mass, isolated from Trichoderma Rifai [7] (Fig. 1). According to a Blast CD-Browser search, CHIT36 belongs to chitinase family 18, subfamily II, with a typical (β-α)8 fold barrel. Surprisingly, 76% identity is found at the nucleic acid level with the putative chitinase from S. coelicolor. From genomic DNA sequencing, no introns were identified in the coding region and Southern blot analysis of genomic DNA cut with EcoRI, which does not cut in the gene sequence, showed that chit36 is a single-copy gene (Fig. 2C).

image

Figure 1. Amino acid sequence comparison of CHIT36 from T. harzianum Rifai TM with CHIT33 from T. harzianum Rifai CECT2413 [7] and the putative chitinase CAB69724 from S. coelicolor.

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image

Figure 2. A: Northern blot analysis of RNA isolated from mycelia of T. harzianum WT and transformants. Hybridization of total RNA (15 μg) with the full-length ORF of chit36 as probe. B: Methylene blue staining of the filter shown as loading control. C: Southern blot analysis showing integration of the pki1-chit36 transgene into the genome of T. harzianum transformants. 10 μg of DNA was digested with EcoRI which cuts only once in plasmid pRL36 and probed with the chit36 ORF.

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3.2Isolation and characterization of transformants

Transformation frequencies achieved by co-bombardment of pAN7 and pRL-36 were relatively low (1–10 transformants μg−1 total DNA). However, the efficiency was very high, seven of nine transformants selected on hygB medium were also positive in the PCR screening for the pki1-chit36 transgene construct (data not shown). Transformants which showed chitinolytic activity in growth medium with 5% glucose were chosen for further characterization. Southern analysis of genomic DNA digested with EcoRI, which cuts only once in the pRL-36 plasmid, revealed that the stable transformants showed up to two integrations of pki1-chit36 in the genomic DNA (Fig. 2C). These were in the same locations, indicating a preferred recombination site of the transgene. The signal in the D1 and D2 transformants is more intense indicating a probable multicopy insertion of the construct. This correlates with the higher chitinolytic activity of these transformants (Table 1). Moreover, the two transformants C1 and D1 have higher levels of mRNA expression in glucose-rich growth medium (Fig. 2A) whereas the WT remains silent. The secreted protein can be detected in activity SDS gels and Western blots as the only chitinolytic enzyme (Fig. 3) and at the expected molecular mass as in the WT upon induction. The transformants have greater (up to 40 times) specific chitinase activities than the WT on glucose-rich medium (Table 1). With chitin as carbon source there was no significant difference from the WT (data not shown) as was previously reported for other chitinase-overexpressing mutants [15].

Table 1.  Specific chitinase activity in cultures of the transformants and Trichoderma WT
 Specific activity (U g−1 dry weight)
  1. Strains were cultured in SM medium plus 5% glucose for 48 h and the mycelium was collected and the dry weight recorded for the calculation of the specific activity. Filtered cultures (440 μl) were assayed with p-nitrophenyl-β-d-N,N-diacetylchitobiose according to [13]. One unit of chitinase activity was defined as the amount of enzyme required to increase absorbance at 400 nm by 1 OD unit ml−1 h−1.

WT10±1.7
A112±4
B378±22
C1257±35
D1360±45
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Figure 3. SDS–PAGE (12%) of extracellular proteins from growth media of transformants C, B and D and WT (chitin 0.2%, lane 1; 5% glucose, lane 2). 40 μl of ×20 concentrated medium was loaded on the gel. A: Activity staining. B: Western blot.

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3.3Biological activity of CHIT36

3.3.1Conidia germination

The transformant culture filtrate was used for testing the biological effect of CHIT36 compared to the WT culture filtrate, in which no chitinolytic activity could be detected in a state of carbon repression. After 20 h an almost complete inhibition in the germination of B. cinerea conidia could be seen, whereas germination in WT culture filtrate was normal (Fig. 4). Similar experiments with purified CHIT36 will be necessary to verify if this effect comes from the sole chitinolytic activity of the endochitinase or, more probably, from its synergistic activity with other hydrolytic enzymes which are secreted in glucose-rich media [14,19]. WT filtrate from chitin-inducing medium also had a complete inhibitory effect (Fig. 4).

image

Figure 4. Spore germination of B. cinerea in the presence of cultures filtrates of Trichoderma transformant C and WT. WT-C: WT grown with chitin 0.8 OD unit ml−1 h−1; WT-G: WT grown with 5% glucose 0.003 OD unit ml−1 h−1; C: transformant C grown with 5% glucose 0.34 OD unit ml−1 h−1. Pictures were taken at a ×150 magnification.

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3.3.2Growth inhibition

Trichoderma WT inhibits pathogen growth, perhaps due to the release of toxic secondary metabolites in glucose medium. Nonetheless, inhibition of the growth of both Fusarium and S. rolfsii was much stronger by transformants than by WT (Table 2). This difference is most likely due to the constitutive secretion of Chit36 on glucose plates where the transformants were previously grown. Chitinase secretion could be detected by the fluorescent substrate 4-MU-(GlcNAc)2 in pre-inoculated transformants plates and not in WT plates (data not shown).

Table 2.  Growth inhibition of Fusarium oxysporum and S. rolfsii on SM (5% glucose) agar plates previously inoculated with transformants C, D, or T. harzianum WT Thumbnail image of
3.3.3In vitro dual cultures and greenhouse test

We further attempted to observe in vitro and in vivo activities of Chit36 transformants versus different plant pathogens. No differences could be observed in plate confrontation assays between transformants and Trichoderma WT. Tests of R. solani control under greenhouse conditions suggested a higher efficacy of the transformants, but this did not reach statistical significance (data not shown). As already reported by Carsolio et al. [16] the levels of chitinases naturally secreted by Trichoderma might be high enough for efficient biocontrol of the phytopathogenic fungi tested. Apparently, only when Chit36 is constitutively overexpressed under repressive conditions for the whole chitinolytic machinery can its antifungal activity be discerned.

In conclusion, the biolistic co-transformation of Trichoderma conidia is a very quick and simple method for obtaining transformants which selectively express proteins under a constitutive promoter in glucose-repressive conditions. In this work we showed that transformants overexpressing CHIT36 have an increased antifungal effect compared to the WT. This new enzyme therefore might have an important role in the mycoparasitic activity of Trichoderma and could be used to genetically manipulate other biocontrol agents or valuable agricultural crops.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by grants from the BMBF (Israel-Germany collaboration) and by the Horowitz foundation.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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