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Keywords:

  • carbohydrate-binding protein;
  • CBM 50;
  • chitin;
  • germination;
  • LysM;
  • Trichoderma

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

LysM motifs are carbohydrate-binding modules found in prokaryotes and eukaryotes. They have general N-acetylglucosamine binding properties and therefore bind to chitin and related carbohydrates. In plants, plasma-membrane-bound proteins containing LysM motifs are involved in plant defence responses, but also in symbiotic interactions between plants and microorganisms. Filamentous fungi secrete LysM proteins that contain several LysM motifs but no enzymatic modules. In plant pathogenic fungi, for LysM proteins roles in dampening of plant defence responses and protection from plant chitinases were shown. In this study, the carbohydrate-binding specificities and biological function of the LysM protein TAL6 from the plant-beneficial fungus Trichoderma atroviride were investigated. TAL6 contains seven LysM motifs and the sequences of its LysM motifs are very different from other fungal LysM proteins investigated so far. The results showed that TAL6 bound to some forms of polymeric chitin, but not to chito-oligosaccharides. Further, no binding to fungal cell wall preparations was detected. Despite these rather weak carbohydrate-binding properties, a strong inhibitory effect of TAL6 on spore germination was found. TAL6 was shown to specifically inhibit germination of Trichoderma spp., but interestingly not of other fungi. Thus, this protein is involved in self-signalling processes during fungal growth rather than fungal–plant interactions. These data expand the functional repertoire of fungal LysM proteins beyond effectors in plant defence responses and show that fungal LysM proteins are also involved in the self-regulation of fungal growth and development.


Abbreviations
CBM

carbohydrate-binding module

GAP

glyceraldehyde-3-phosphate dehydrogenase

GlcNAc

N-acetylglucosamine

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

LysM motifs are carbohydrate-binding modules (CBMs) classified in the CAZy database as CBM family 50 and have general N-acetylglucosamine (GlcNAc) binding properties. They were first identified in bacteriophage and bacterial proteins as domains with binding function to the peptidoglycan of bacterial cell walls [1]. Later, LysM motifs were also discovered in eukaryotes and to date, based on the wealth of genome data from many different organisms, more than 4000 proteins that contain LysM motifs are known among prokaryotes and eukaryotes. In plants LysMs are able to recognize a variety of GlcNAc containing glycans and are associated with different functions. While some plant proteins with LysM motifs are involved in sensing of chito-oligosaccharides that activate plant immune responses, others bind to chito-oligosaccharide derivatives that trigger symbiotic interactions between plants and microbes [2].

The first identified fungal protein with LysM motifs was a killer-toxin chitinase from the yeast Kluyveromyces lactis. This chitinase is part of the heterotrimeric, plasmid-encoded killer toxin system, which inhibits the growth of a wide range of susceptible yeasts [3]. The protein architecture of killer-toxin chitinases is similar to subgroup C chitinases from filamentous fungi, which have LysM (CBM 50) and chitin-binding (CBM 18) domains located N-terminally of their catalytic glycoside hydrolase family 18 module [4, 5]. In addition to these LysM-containing fungal enzymes, there are fungal LysM proteins that have multiple LysM motifs but no catalytic modules [6]. The protein Ecp6 from the tomato pathogen Cladosporium fulvum is such a LysM effector protein and contains three LysM motifs. Ecp6 was shown to prevent chitin-triggered immunity in plants [7]. During infection, plant chitinases release chitin oligomers from the fungal cell wall. Ecp6 binds these GlcNAc-oligosaccharides and thereby prevents recognition of these molecules by the tomato plant, which otherwise would elicit its defence responses. A similar function was shown for the LysM protein Slp1 from the rice blast fungus Magnaporthe grisea [8]. In the wheat pathogen Mycosphaerella graminicola three homologues of Ecp6 are present, of which two, Mg1LysM and Mg3LysM, were found to be upregulated during symptomless leaf infection [9]. Only Mg3LysM blocked the elicitation of chitin-induced plant defences, whilst Mg1LysM and Mg3LysM, in contrast to Ecp6, also protected fungal hyphae against plant-derived hydrolytic enzymes. In C. fulvum the chitin-binding protein Avr4, which belongs to CBM family 14, has also been shown to protect fungi against plant chitinases [10].

LysM proteins can also be found in non-pathogenic fungi including many saprotrophic fungi and mycoparasites [4, 6]. Species from the fungal genus Trichoderma have a saprotrophic lifestyle and are opportunistic mycoparasites [11]. Genome analysis revealed that Trichoderma subgroup C chitinase genes are frequently clustered with genes encoding LysM proteins [4]. The mycoparasite Trichoderma atroviride has nine subgroup C chitinase genes. While the transcriptional regulation of most of these genes specifically responds to growth on fungal cell walls, one of them, tac6, is strongly expressed during hyphal network formation. These data indicate that the protein TAC6 is involved in self-cell-wall remodelling during fungal development and growth. The subgroup C chitinase gene tac6 is co-regulated with tal6, which encodes a secreted LysM protein with seven LysM motifs. The genes tal6 and tac6 are adjacent to each other in the genome and share their upstream regions. The amino acid sequences of the LysM motifs of TAL6 are markedly different from other so far investigated fungal LysM proteins [4].

The expression of tal6 during fungal growth and development of T. atroviride raised our interest in the function and carbohydrate-binding properties of TAL6. Here we describe the properties of purified TAL6 with emphasis on its chitin-binding and germination-inhibitory properties. Our findings expand the functions of fungal LysM proteins from effectors in plant pathogenesis to self-regulatory roles in fungal growth.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Heterologous expression of TAL6-4 and TAL6-7 in Pichia pastoris

The T. atroviride protein TAL6 (Protein ID 297859 in the JGI T. atroviride genome database, http://genome.jgi-psf.org/Triat2/Triat2.home.html; GenBank/EMBL/DDBJ accession number EHK49205) has seven LysM motifs of which the four C-terminal motifs are highly similar to each other whereas the three N-terminal LysM motifs are more variable and also differ more strongly from the consensus patterns currently deposited in databases (e.g. SM00257, PF01476) (Fig. 1). We therefore studied the carbohydrate-binding properties of a full-length version of TAL6 and also a truncated version that consists only of the four strongly conserved LysM motifs. The proteins were heterologously produced in P. pastoris and were designated TAL6-7 (containing all seven LysM motifs) and TAL6-4 (containing only the four conserved LysM motifs).

image

Figure 1. LysM motifs of TAL6. (A) Model of the TAL6 protein architecture. The protein contains a signal peptide (circle) and seven LysM motifs (cylinders). The amino acid positions of the LysM motifs according to a prediction with interproscan (SM00257) are indicated. The overexpression constructs TAL6-7 and TAL6-4 generated in this study are also indicated. (B) Alignment of the LysM motifs of TAL6.

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The theoretical molecular masses are 38 kDa for TAL6-4 and 80 kDa for TAL6-7, but western blot analysis with His-tag antibodies of the supernatants from shake flask cultivations, as well as the purified proteins from fermentations, showed molecular masses of 70 kDa for TAL6-4 and two bands of 120 and 140 kDa for TAL6-7 (Fig. 2).

image

Figure 2. Analysis of heterologously expressed TAL6-7 and TAL6-4. SDS/PAGE: M, marker; lane 1, 5 μg purified TAL6-4; lane 2, 5 μg purified TAL6-7; lane 3, 1 μg TAL6-7 (for better visualization of the double bands).

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Glycoprotein staining of SDS gels showed that the proteins were glycosylated (Fig. S1a), and glycosylation was further confirmed by enzymatic N-deglycosylation with α-mannosidase and an endo-N-acetylglucosaminidase (Fig. S1b). Although the proteins on SDS gels were still larger than their theoretical molecular weight, the molecular weight of the proteins was reduced to a size below the theoretical molecular weight of TAL6 dimers. Thus, the proteins produced in P. pastoris were strongly glycosylated – and possibly also otherwise post-translationally modified – monomers. In addition, for TAL6-4 correct protein processing after the heterologous α-factor secretion signal was confirmed by N-terminal sequencing. 2D gel electrophoresis showed a train of spots for both proteins, which is also a typical feature of glycoproteins (Fig. S1c). The theoretical isoelectric points of TAL6-7 and TAL6-4 are 5.20 and 8.29, respectively. The observed pI ranges were 5.7–6.3 for the 140 kDa band and 7.2–7.7 for the 120 kDa band of TAL6-7 and 8.0–9.0 with the main protein spots at 8.5 for TAL6-4.

Chitin-binding properties of TAL6-4 and TAL6-7

In order to investigate the binding affinities of TAL6-7 and TAL6-4, pull-down assays were carried out with various insoluble carbon sources: chitin (fine and crude), colloidal chitin, chitosan, cellulose, Escherichia coli cell walls for peptidoglycan binding, and different fungal cell wall preparations (Table 1). As shown in Fig. 3, TAL6-7 bound to chitin beads, which are processed particles of reacetylated chitosan, but not to any of the other tested chitin forms. Moderate binding to chitosan (degree of acetylation ≤ 40 mol%) and no binding to cellulose were detected. Also, no binding of TAL6-7 to E. coli cell walls or fungal cell wall preparations from the fungi T. atroviride, Trichoderma reesei, Rhizoctonia solani and Botrytis cinerea was observed. Degradation of the TAL6-7 proteins was observed in binding assays with fungal cell walls, i.e. no bands in bound and unbound fractions (Fig. 3B), and therefore also autoclaved cell walls were included in the assays in order to inactivate cell-wall-bound proteases and thereby enhance the accessibility of these carbon sources for binding. However, binding was not observed in these cases either (Fig. 3C). Additional tests were performed with young and ageing cell walls from T. reesei and alkali-insoluble fractions of T. atroviride cell walls, which consist of the chitin- and β-glucan scaffold of the cell wall, but again no binding of TAL6-7 was observed in these experiments (Fig. 3D). Thus, in these assays TAL6-7 was only found to bind to chitin beads and weakly to chitosan, but not to any of the other tested carbon sources including fungal cell walls.

Table 1. Binding of TAL6-7 and TAL6-4 to chitin-related polymers
Carbon sourceTAL6-7TAL6-4
Chitin flakes
Chitin powder
Colloidal chitin ++
Chitin beads++++++
Chitosan++++
Escherichia coli CW (autoclaved)+++
Trichoderma atroviride CW
T. atroviride CW (autoclaved)
T. atroviride CW (alkali-insoluble fraction)
Trichoderma reesei CW (young)
T. reesei CW (old)
R. solani CW
R. solani CW (autoclaved)+
B. cinerea CW
B. cinerea CW (autoclaved)
Cellulose
image

Figure 3. Carbohydrate-binding assays with TAL6-7: C, control (protein subjected to the steps of the binding assay without the addition of a carbon source); U, unbound protein; B, bound protein; (A) CH fine, fine chitin powder; CH flakes, flaked chitin; (B) CH B, chitin beads; Bc CW, B. cinerea cell walls; Rs CW, R. solani cell walls; Ta CW, Trichoderma atroviride cell walls; (C) coll CH, colloidal chitin; Bc aCW, B. cinerea autoclaved cell walls; Rs aCW, R. solani autoclaved cell walls; Ta aCW, T. atroviride autoclaved cell walls; Ec aCW, Escherichia coli autoclaved cell walls; (D) Tr CW 24 h, young Trichoderma reesei cell walls (from 24-h-old mycelium); Tr CW 48 h, old T. reesei cell walls (from 48-h-old mycelium); Ta CW alk. tr., alkaline insoluble fraction of T. atroviride cell walls.

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Carbohydrate-binding assays were also carried out with the truncated version TAL6-4 using the same carbon sources as for TAL6-7 (Fig. 4 and Table 1). Similar to TAL6-7, TAL6-4 also bound to chitin beads and moderately to chitosan, but in addition binding to colloidal chitin and weakly to autoclaved R. solani cell walls was found, thus showing less stringent binding specificities than TAL6-7. Further, TAL6-4, which contains the four more ‘traditional’ LysM motifs, interestingly also bound to E. coli cell walls, i.e. peptidoglycan.

image

Figure 4. Carbohydrate-binding assays with TAL6-4: C, control (protein subjected to the steps of the binding assay without the addition of a carbon source); U, unbound protein; B, bound protein; (A) CH B, chitin beads; CH fine, fine chitin powder; CH flakes, flaked chitin; CH coll, colloidal chitin; (B) Ta CW, Trichoderma atroviride cell walls; Ta aCW, T. atroviride autoclaved cell walls; Bc CW, B. cinerea cell walls; (C) Bc aCW, B. cinerea autoclaved cell walls; Rs CW, R. solani cell walls; Rs aCW, R. solani autoclaved cell walls; Ta CW alk. tr., alkaline insoluble fraction of T. atroviride cell walls; (D) Ec aCW, Escherichia coli autoclaved cell walls.

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Since the LysM effector protein Ecp6 from C. fulvum binds to short chito-oligosaccharides [7], this was also tested for TAL6-7 and TAL6-4 using isothermal titration calorimetry, but binding of (GlcNAc)3 and (GlcNAc)6 to TAL6-7 or TAL6-4 was not detected (Doc. S1). In order to evaluate a wider range of oligosaccharides, TAL6-7 and TAL6-4 were used for a glycan array screening with 611 different carbon sources including a GlcNAc oligomer, but also in this assay no binding to any of the targets was found (Doc. S2).

The results obtained therefore showed that the full-length version TAL6-7 binds only the highly processed form of polymeric chitin found in chitin beads and moderately to chitosan, but not to any other forms of chitin or (unmodified) chito-oligosaccharides, and that TAL6-4 has less stringent binding properties.

Effect of TAL6-4 and TAL6-7 on fungal spore germination

Although the TAL6-7 and TAL6-4 proteins did not bind to fungal cell wall preparations in the pull-down assays, we were interested to test whether they have an effect on fungal growth. The subgroup C chitinase gene tac6 was shown to be expressed during hyphal development in T. atroviride, which suggests a role of this protein in self-cell-wall remodelling. Since the tal6 gene is co-regulated with tac6, we were interested in testing whether TAL6 is involved in the self-regulation of fungal growth. This was tested in spore germination assays with T. atroviride and other fungi (T. reesei, Aspergillus niger and Neurospora crassa), alone or in combination with Trichoderma chitinases.

In spore germination assays with T. atroviride a strong inhibitory effect of TAL6-7 on spore germination was found (Fig. 5 and Table 2). Germination of T. atroviride was completely inhibited at high protein concentrations (1.8 μm) of TAL6-7. In contrast, TAL6-4 had no effect on spore germination of T. atroviride.

Table 2. Effect of TAL6-7 and TAL6-4 on spore germination
 Control+TAL6-7+TAL6-4Germinated spores (%)
+CHI+TAL6-7 + CHI+TAL-4 + CHI
  1. a

    Significantly different from control (< 0.01).

Trichoderma atroviride88 ± 53 ± 3a79 ± 788 ± 56 ± 4a80 ± 5
Trichoderma reesei 85 ± 61 ± 1a85 ± 70a0a0a
Aspergillus niger 97 ± 197 ± 12 ± 1a0a0a0a
Neurospora crassa 94 ± 294 ± 23 ± 1a0a0a0a
image

Figure 5. Effect of TAL6-7 and TAL6-4 on spore germination of Trichoderma atroviride. Increasing protein concentrations of 15, 75 and 150 μg·mL−1 (left to right) were added. Scale bars 40 μm.

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TAL6-7 also showed a strong inhibitory effect on spore germination assays of T. reesei (Fig. 6A and Table 2). In T. reesei, complete inhibition of germination was found at even lower protein concentrations of TAL6-7 (0.18 μm) than in T. atroviride, and additionally aggregation of T. reesei spores was observed at higher protein concentrations (0.9–1.8 μm). Again, there was no effect of TAL6-4 on spore germination of T. reesei.

image

Figure 6. Effect of TAL6-7 and TAL6-4 on spore germination of Trichoderma reesei, A. nidulans and Neurospora crassa. Protein concentration 150 μg·mL−1. Scale bars 40 μm.

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With A. niger and N. crassa, spore germination was not influenced by TAL6-7 and TAL6-4 (Fig. 6B,C and Table 2). Thus, TAL6 has a pronounced effect on conidial germination of Trichoderma spp. but does not influence other fungi.

Chitinases from mycoparasitic Trichoderma spp. have been shown to have negative effects on the germination and growth of other fungi, but not on the mycoparasite itself [12, 13]. We showed recently that chitinolytic enzymes from T. atroviride did not influence the germination rate of T. atroviride but were able to inhibit germination of other fungi, including T. reesei [14]. In order to investigate any additive or subtractive functions of TAL6 in this process, i.e. whether TAL6 can possibly protect spores of other fungi from T. atroviride chitinases and whether chitinases dampen the inhibitory effect of TAL6 on T. atroviride spores, germination of T. atroviride, T. reesei, A. niger and N. crassa was tested upon addition of a chitinolytic enzyme mix to the growth medium and either TAL6-7 or TAL6-4 (Fig. S2). However, in T. reesei, A. niger and N. crassa, where germination was abolished due to the addition of chitinolytic enzymes, this stayed unchanged in the presence of TAL6-7 or TAL6-4, and in the case of T. atroviride also with chitinases germination rates were again decreased upon addition of TAL6-7 but not TAL6-4. Therefore, TAL6 is not able to counteract the activity of T. atroviride chitinases.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Recent genome-wide screening approaches showed that also filamentous fungi have large numbers of proteins containing LysM motifs [4, 6], but knowledge about the function of these proteins in fungi is so far limited to LysM proteins from plant pathogenic fungi. T. atroviride has a saprotrophic and mycoparasitic lifestyle and a well studied chitinase system, which is advantageous in view of the genomic association of genes encoding LysM proteins with genes encoding subgroup C chitinases [4]. In this study we focused on TAL6, a protein that contains seven LysM motifs. The amino acid sequences of the LysM motifs of TAL6 are markedly different from those of Ecp6 and Slp1, suggesting a different evolutionary origin and structure (Akcapinar GB, Sezerman OU and Seidl-Seiboth V, manuscript in preparation). TAL6 therefore represents a type of fungal LysM protein that has not been studied so far. The chitin-binding affinity of TAL6-7 and TAL6-4 was in general rather low and in the case of TAL6-7 was limited to chitin beads. Chitin beads are fine chitin particles (50–150 μm) and consist of pure chitin. Their manufacturing process contains a reacetylation step based on purified chitosan (information from manufacturer New England Biolabs). Both TAL6-7 and TAL6-4 showed only moderate binding to chitosan, which does not fully explain why out of the tested chitin forms preferentially chitin beads were bound. However, the weak binding to chitosan could indicate that the degree of (de)acetylation is indeed relevant for optimal binding. TAL6-4 bound well to E. coli cell walls, i.e. peptidoglycan, while TAL6-7 did not bind. The sequences of the LysM motifs of TAL6-4 are more closely related to the consensus pattern currently deposited in the PFAM database. This suggests that the three additional, more variable LysM motifs of TAL6-7 significantly alter the overall binding specificities of the protein. Further, TAL6-4 bound not only to chitin beads but also to colloidal chitin and autoclaved R. solani cell walls, indicating slightly less restrictive binding specificities. In contrast, the strong inhibitory effect on spore germination that was found for TAL6-7 was not observed for TAL6-4. This suggests that TAL6-7 – despite its weak chitin-binding properties – is a fully functional protein and suggests that the three more variable LysM motifs are possibly responsible for the specific binding properties of TAL6, thereby defining its biological function.

It should be noted that the spacing between the LysM motifs is rather small (10–30 amino acids), but there is a gap of 170 amino acids between the second and third LysM motifs. blast analysis using this sequence yielded only hits to fungal LysM proteins, but did not indicate any other domains. Manual inspection of this amino acid stretch showed weak similarity to LysM motifs, albeit with larger spacing between the conserved residues. It is therefore possible that this region contains another LysM motif or another CBM that is even more distantly related to the current LysM consensus pattern.

In fungi high spore densities can lead to the autoinhibition of germination through self-produced chemical signals [15]. These self-inhibitors have been reported for more than 60 fungal species and comprise different classes, but all of them are small organic molecules. Therefore, this type of autoinhibition is quite different from our observations with the protein TAL6. Spore germination assays are a sensitive method to analyse the involvement of a protein or substance in fungal growth and development. However, since tal6 is mainly expressed at later growth stages, i.e. in mature hyphae, it is possible that in vivo TAL6 has a self-regulatory role in growth and development of mature hyphae, which would be difficult to detect in an assay with exogenously added protein. Therefore, germination assays are a good model to study such functions. There are two main possibilities about how TAL6 may exert its inhibitory effect: either TAL6 binds to the polymer, i.e. the fungal cell wall, or it binds to (modified) soluble chito-oligosaccharides. We did not detect binding to fungal cell wall preparations or the chitin/glucan fraction of cell walls in our assays, but it is possible that the harsh treatment of the fungal cell wall that is necessary for its extraction destroyed a structural feature that is essential for binding. With respect to oligosaccharide binding using two different approaches, isothermal titration calorimetry and glycan arrays, we were not able to show binding to unmodified chito-oligosaccharides. Possibly a substitution on these oligosaccharides is important for specific binding of TAL6. Such modifications have already been reported for mediating the specificity in binding and thus interaction between plants and their symbiotic microorganisms, which is communicated via LysM motifs [2]. Since TAL6-7 was biologically active, but we were not able to detect strong binding to fungal cell walls or chito-oligosaccharides, we hypothesize that this protein binds to a type of oligosaccharides that are fungal specific chitin derivatives and act as signalling molecules during germination. Binding of TAL6 to such a molecule would interfere with signalling processes during germination. While the polymeric chitin-glucan network in the fungal cell wall is likely to be similar between different fungi, specific oligosaccharide-signalling molecules might be – in analogy with other self-regulators that have been described [15] – more variable among different fungal genera. This would explain why the inhibitory function of TAL6-7 was observed for T. atroviride and T. reesei but not A. niger and N. crassa.

The gene encoding TAL6 is located next to the subgroup C chitinase gene tac6 (protein ID 53627 in the JGI T. atroviride genome database) and a corresponding gene cluster can also be found in Trichoderma virens [16]. It should be noted that, although tac6 belongs to ‘subgroup C chitinases’, a detailed analysis of its catalytic residues showed that it has a deletion in its substrate binding site, which can also be found in its T. virens homologue. Thus TAC6 is probably a chitin-binding protein rather than an active chitinase. Nonetheless, knockout strains of tac6 exhibit faster growth than the wild-type (Gruber and Seidl-Seiboth, manuscript in preparation). This suggests a function of TAC6 in fungal development that decelerates hyphal growth. Such a function would be in agreement with our observations of TAL6-7, which also slowed down germination and at large concentrations inhibited it. Binding assays of TAL6-7 with fungal cell walls showed that even in extracted cell walls cell-wall-bound proteases can degrade TAL6-7, because no TAL6-7 bands were detected anymore in the bound and unbound fractions in cell wall assays unless the cell walls were autoclaved (Fig. 3). Therefore, rather high protein concentrations were used in the spore germination assays to overcome this effect. We think it likely that under native conditions the inhibitory effect of TAL6 is less pronounced, possibly spatially restricted, and that TAL6, together with TAC6, is involved in the fine-tuned regulation of fungal growth and hyphal development in T. atroviride. The results obtained in this study reveal a so far not described function for fungal LysM proteins and shed a completely new light on the functional repertoire of the many genes encoding LysM proteins that can be found in fungal genomes.

Material and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

Strains and cultivation conditions

The filamentous fungi T. atroviride (teleomorph Hypocrea atroviridis) strains P1 (ATCC 74058) and IMI 206040, T. reesei (teleomorph Hypocrea jecorina) QM9414, N. crassa ATCC N402 and A. niger N402 were used and maintained on potato dextrose agar. P. pastoris strain X33 (purchased from Invitrogen, Paisley, UK) was used for protein overexpression. E. coli JM 109 cells were used for plasmid propagation.

PCR-aided methods

For PCR approaches GoTaq polymerase (Promega, Madison, WI, USA) and Phusion polymerase (Finnzymes; Thermo Fisher Scientific, Waltham, MA, USA) were used, following the recommended protocols. cDNA was produced with the Revert Aid H minus Kit (Fermentas; Thermo Fisher Scientific). Isolation of plasmids was performed according to standard protocols [17]. Preparation of DNA and RNA from T. atroviride was performed as described previously [5] and for preparation of DNA and RNA from P. pastoris the guidelines in the EasySelect™ Pichia Expression Kit manual (Invitrogen) were followed.

Plasmid construction and overexpression of TAL6 protein constructs in Pichia pastoris

Overexpression of the TAL6 protein (protein ID 297859 in the JGI T. atroviride genome database, http://genome.jgi-psf.org/Triat2/Triat2.home.html; GenBank/EMBL/DDBJ accession number EHK49205) was based on the pPICZα B plasmid from the EasySelect™ Pichia Expression Kit (Invitrogen) and the manufacturer's instructions regarding Zeocin selection. For generation of the TAL6-7 overexpression plasmid, the respective coding region was amplified from T. atroviride cDNA with the primer pair for tal6-7, 6LysM-Fw 5′-GTACCTGCAGCATCAAAGCTAGGCGTACCC-3′ and 6LysM-Rv 5′-GTACGCGGCCGCTCCTGTAACACGAGCAGCG-3′, which contain PstI and NotI restriction sites, respectively. This PCR fragment was cloned into the corresponding sites of pPICZBα transformed into E. coli JM109 cells, which were selected positively for Zeocin resistance. The tal6-7 expression cassette contains the coding region for a 6x-His-tag at the C-terminus of the protein. Since no efficient protein production could be achieved with this plasmid, the AOX1 (alcohol oxidase) promoter was exchanged with the constitutive GAP (glyceraldehyde 3-phosphate dehydrogenase) promoter. For this the primers GAPBglII_Fw 5′-GATAGATCTGATCTTTTTTGTAGAAATGTCTTGGTG-3′ and AlphaPstI_Rv 5′-GATCCTGCAGCTTCGGCCTCTCTCTTCTCGA-3′ were used to amplify the GAP promoter from pGemTGAP (kindly provided by C. Ruth and A. Glieder, Graz University of Technology, Austria). The PCR fragment was digested with BglII and PstI and cloned into the corresponding sites of the pPICZαB already containing tal6-7. The resulting plasmid was named pTAL6-7. In analogy to that, plasmid pTAL6-4 was constructed for overexpression of the four conserved LysM motifs of TAL6 in P. pastoris. The corresponding DNA fragment was amplified with the primers 4LysM-Fw 5′-GTACCTGCAGACAGCATCCCATCTATTTCGC-3′ and 4LysM-R5′-GTACTCTAGAGCTCCTGTAACACGAGCAGCGG-3′ and cloned in pPICZαB via the PstI and XbaI restriction sites. The AOX1 promoter was exchanged with the GAP promoter as described for pTAL6-7 above, resulting in plasmid pTAL6-4. The plasmids were linearized with BstXI. Preparation of competent P. pastoris cells and transformation were performed as described by Lin-Cereghino et al. [18]. Transformants were screened with colony PCR and strains containing the overexpression constructs were cultivated in YPD medium and BM medium (EasySelect™ Pichia Expression Kit manual) in baffled shake flasks at 28 °C and 250 r.p.m. for screening of protein production. In order to enhance the induction of the GAP promoter as recommended by Hohenblum et al. [19], every 24 h 10 g·L−1 glucose was added. Samples were taken every 24 h to test for protein production.

Fermentation setup for recombinant Pichia pastoris strains

Fermentations were performed in a stirred Labfors glas bioreactor (Infors HT, Bottmingen, Switzerland) with a working volume of 2 L in a total volume of 3 L. The reactor was controlled at 28 °C and 1200 r.p.m. Initial volume was 1 L of 4-fold concentrated YPD medium. The airflow was kept constant at 2 L·min−1 to maintain the dissolved oxygen (dO2) (Hamilton, Switzerland) at a level higher than 40% during the whole fermentation and thus to avoid oxygen limitation. The pH was maintained at 5.0 by the addition of 2.0 m NH4OH. The preculture (100 mL of YPD medium) was inoculated with a single colony of P. pastoris TAL6-7 and TAL6-4 transformations, respectively, incubated for 20 h at 28 °C, 250 r.p.m., and transferred aseptically to the bioreactor. After a sharp increase of dO2 was observed, pulses of 500 mL 4-fold concentrated YPD medium containing 20 g·L−1 glucose were carried out to further increase the biomass concentration and concomitantly the product content. After 72 h of cultivation, the fermentation broth was centrifuged for 20 min at 4 °C and 4500 g to obtain the cell-free fermentation supernatant.

Protein extraction and purification

Despite optimized cultivation conditions and testing of different media the protein yield was in general rather low (~ 20 mg·L−1) and we therefore tested whether the proteins are not fully secreted or stay non-covalently attached to the cell wall of P. pastoris. In order to verify that TAL6-7 and TAL6-4 are secreted proteins, total intracellular and cell-wall-bound proteins were extracted from P. pastoris biomass; 2 g of the harvested biomass was resuspended in 20 mL of 20 mm Tris/HCl buffer pH 7.5. The cells were lysed using an EmulsiFlex-C3 Homogenizer (Avestin, Mannheim, Germany). Cell debris was removed by centrifugation for 20 min and 5000 g at 4 °C. Afterwards the cell debris was consecutively extracted with different detergents – 1% (w/v) Triton, 2% (w/v) CHAPS, 1% (w/v) Tween – and the protein extracts were analysed by SDS/PAGE and western blot analysis (see below), but yielded no signal. This showed that the TAL6-7 and TAL6-4 proteins were fully secreted but expressed at rather low levels in P. pastoris. We also attempted to overexpress TAL6-7 in the filamentous fungus T. reesei, but also in T. reesei the yields were low and the unpurified TAL6-7 protein showed a molecular mass above 170 kDa on SDS gels (data not shown). Because of these findings and the easier upscaling of P. pastoris fermentations, TAL6-7 and TAL6-4 were produced in larger quantities in P. pastoris bioreactor cultivations for further biochemical characterizations.

The culture supernatants from P. pastoris fermentations of the respective strains (see above) were harvested and filtered through Steritop Filter Units 0.22 μm (Millipore, Billerica, MA, USA); 250 mL of the filtered supernatants were concentrated in Amicon Ultra centrifugal filter units Ultra-15, MWCO 50 kDa (Millipore) at 4 °C and the buffer was changed to 20 mm Tris/HCl buffer pH 7.5. The concentrated protein solutions were purified with the ÄKTATM purifier (GE Healthcare, Little Chalfont, UK) and HisTrapTM HP columns 1 mL (GE Healthcare), making use of the His-tags of the heterologously produced proteins. The columns were used following the manufacturer's instructions using an imidazole gradient for protein elution. Protein purification was monitored by measuring the absorbance at 280 nm and TAL6-7 and TAL6-4 eluted as single peaks at 200 mm imidazole.

Carbohydrate-binding assays

Protein pull-down assays with insoluble carbon sources were performed using 10 mg of the following carbon sources: fungal and E. coli cell walls and colloidal chitin (prepared as described in [5]), chitin from crab shells fine and crude (Sigma, St Louis, MA, USA), chitosan (molecular weight 140 000–220 000, degree of acetylation ≤ 40 mol%, Sigma), Arbocel cellulose fibres (JRS, Rosenberg, Germany) and chitin beads (New England Biolabs, Ipswich, MA, USA). Thus 200 μg protein were mixed with 10 mg of the different carbon sources and 20 mm Tris/HCl, pH 7.5, buffer in a total volume of 1 mL. The samples were incubated for 4 h at room temperature with gentle agitation and then centrifuged for 5 min at 10 000 g. The supernatant was transferred into a new tube and the pellets were washed three times with buffer. Then the pellet was suspended in 1 mL 20 mm Tris/HCl buffer pH 7.5 containing 1% (w/v) SDS and incubated for 10 min at 99 °C. After centrifugation for 5 min at 10 000 g the supernatant was transferred into a new tube; 250 μL of the supernatants were precipitated using chloroform and methanol precipitation [20]. Unbound and bound protein fractions were analysed with SDS/PAGE and western blotting. As controls proteins were submitted to the treatment described above without the addition of a carbon source.

Glycan arrays were carried out by Core H of the Consortium of Functional Glycomics (http://www.functionalglycomics.org) according to their standard protocols. His-tag monoclonal IgG2b antibody was used for protein detection (Invitrogen). Isothermal titration calorimetry was carried out with a VP-ITC (Microcal, GE Healthcare) according to the manufacturer's instructions. In analogy to previously reported data (e.g. [7]), protein ranges from 20 to 100 μm and chito-oligomer ranges – (GlcNAc)3 and (GlcNAc)6 – from 0.8 to 3 mm were tested.

Protein analysis

Proteins were analysed with SDS/PAGE and colloidal Coomassie staining and western blot analysis using standard protocols [17]. For protein detection an His-tag(C-term) antibody (Invitrogen) was used as primary antibody and an anti-mouse IgG alkaline phosphatase conjugate (Promega) was used as secondary antibody. Glycosylated proteins were stained with the periodic acid–Schiff method using the GelCode Glycoprotein Staining Kit (Thermo Fisher Scientific). For enzymatic deglycosylation α-mannosidase from jack bean (Sigma) and Endo H (New England Biolabs) were used following the instructions from the Endo H manual. N-terminal sequencing was performed at the Protein Sequencing Facility at the Biocenter of the Medical University Innsbruck, Austria. For 2D gel electrophoresis the protein samples were dissolved in 2D sample buffer [9 m urea, 2% CHAPS, 1% dithiothreitol, 0.5% carrier ampholytes (Biorad, Hercules, CA, USA)]. IPG strips (Biorad) with a linear pH gradient from pH 3 to pH 10 were rehydrated overnight with 300 μL of 2D sample buffer containing 50 μg TAL6-4 and TAL6-7, respectively. Isoelectric focusing was performed in an IEF cell (Bio-Rad). The focusing programme included a linear ramp to 250 V over 1 h, a linear ramp to 4000 V over 2 h and 10 000 V h at 4000 Vmax with a limit of 50 μA per IPG strip.

The IPG strips were equilibrated for 15 min in equilibration buffer (6 m urea, 2% SDS, 0.05 m Tris/HCl, pH 8.8, 20% glycerol) containing 2% (w/v) dithiothreitol, and for 15 min in equilibration buffer containing 2.5% (w/v) iodoacetamide. The strips were then mounted on 12% SDS/polyacrylamide gels for SDS/PAGE and proteins were visualized with colloidal Coomassie staining.

Spore germination assays

Spore suspensions of T. atroviride, T. reesei, A. niger and N. crassa were prepared at a final spore concentration of 1 × 106 spores·mL−1 in potato dextrose broth (Difco; Thermo Fisher Scientific). Chambered coverglass devices (Lab-Tek II, Nunc; Thermo Fisher Scientific) were inoculated with 170 μL of the spore suspensions and different concentrations (3 μg, 15 μg and 30 μg) of TAL6-7 or TAL6-4, in 20 mm Tris/HCl buffer, were added. All samples were filled up to 200 μL with buffer. As control only 30 μL buffer was added to the 170 μL PDB-spore suspension. T. atroviride, T. reesei and A. niger were incubated for 12 h at 28 °C and N. crassa was incubated for 6 h.

In another set of experiments a chitinolytic enzyme mix of T. atroviride was additionally added to the spore suspensions. In this case 33% of the medium were replaced with a chitinolytic enzyme mix from T. atroviride [culture supernatant from ageing T. atroviride cultivation, buffer changed to 20 mm Tris/HCl buffer pH 7.5 with Amicon Ultra centrifugal filter units Ultra-15, MWCO 10 kDa (Millipore), and filtered with a 0.22 μm Steritop filter unit (Millipore)]. Germination behaviour of the different spores was investigated on an inverted TE300 microscope (Nikon, Tokyo, Japan) by using differential interference contrast optics and imaged with a DXM1200F digital camera (Nikon).

For numerical and statistical evaluation of the results the percentage of germinated and ungerminated spores was determined by counting. At least two biological replicates and several images/replicate, corresponding to ~ 200 spores per replicate, were evaluated. Statistical analysis was performed using graphpad instat software (San Diego, CA, USA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information

We would like to thank Anton Glieder (ACIB, Austria) for his helpful suggestions for protein overexpression in P. pastoris. The glycan arrays were carried out by Core H of the Consortium of Functional Glycomics. This work was funded by the Austrian Science Fund (FWF), grants P20559 and T390 to V.S.-S.

References

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
febs12113-sup-0001-FigureS1-S2-FileS1-S2.zipapplication/ZIP5807K

Fig. S1. Glycoprotein analysis and 2D gel electrophoresis of TAL6-7 and TAL6-4.

Fig. S2. Effect of chitinolytic enzymes of Trichoderma atroviride and TAL6-7 or TAL6-4 on spore germination.

Doc. S1. Isothermal titration calorimetry data.

Doc. S2. Glycan array data.

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