Differential Regulation of Orthologous Chitinase Genes in … · sion, starting from no expression (white boxes) to maximal expression levels detected in this study (black boxes)

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2011, p. 7217–7226 Vol. 77, No. 200099-2240/11/$12.00 doi:10.1128/AEM.06027-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Differential Regulation of Orthologous Chitinase Genes inMycoparasitic Trichoderma Species�†

Sabine Gruber, Christian P. Kubicek, and Verena Seidl-Seiboth*Research Area Gene Technology and Applied Biochemistry, Institute of Chemical Engineering,

Vienna University of Technology, Vienna, Austria

Received 29 June 2011/Accepted 12 August 2011

Mycoparasitic Trichoderma species have expanded numbers of fungal subgroup C chitinases that containmultiple carbohydrate binding modules and could thus be important for fungal cell wall degradation duringthe mycoparasitic attack. In this study, we analyzed the gene regulation of subgroup C chitinases in themycoparasite Trichoderma virens. In addition to regulation by nutritional stimuli, we found complex expressionpatterns in different parts of the fungal colony, and also, the mode of cultivation strongly influenced subgroupC chitinase transcript levels. Thus, the regulation of these genes is governed by a combination of colony-internal and -external signals. Our results showed completely different expression profiles of subgroup Cchitinase genes in T. virens than in a previous study with T. atroviride, although both fungi are potentmycoparasites. Only a few subgroup C chitinase orthologues were found in T. atroviride and T. virens, and eventhose showed substantially divergent gene expression patterns. Microscopic analysis revealed morphogeneticdifferences between T. atroviride and T. virens, which could be connected to differential subgroup C chitinasegene expression. The biological function of fungal subgroup C chitinases therefore might not be as clear-cut aspreviously anticipated. They could have pleiotropic roles and might be involved in both degradation ofexogenous chitinous carbon sources, including other fungal cell walls, and recycling of their own cell wallsduring hyphal development and colony formation.

Several species of the fungal genus Trichoderma (sexualform, Hypocrea) are mycoparasites and are thus able to antag-onize, parasitize, and kill other fungi. An integral part of themycoparasitic attack is degradation of the prey’s cell wall bychitinases, glucanases, and proteases (1). Genome analysis re-vealed that the numbers of chitinase-encoding genes arestrongly expanded in mycoparasitic Trichoderma spp. in com-parison to other filamentous fungi. Fungal chitinases belong toglycoside hydrolase family 18 (GH 18) and can be furthersubdivided into 3 different subgroups, A, B, and C (10, 11).Subgroup A chitinases in general contain no carbohydrate bind-ing modules (CBMs), but subgroup B chitinases frequentlyhave a CBM at the C-terminal end. All subgroup C (sgC)chitinases have multiple CBMs and have either two CBM 18(chitin binding) domains or one CBM 18 and two CBM 50(LysM) domains N terminal of their GH 18 module (5). CBMsenable efficient adherence of the enzyme to insoluble sub-strates and processive catalytic cleavage of the substrate (2).The additional chitinases in mycoparasitic Trichoderma spp.are members of subgroups B and C. While the saprotrophicspecies Trichoderma reesei has only 4 sgC chitinases, T. atro-viride has 9 and T. virens has 15, suggesting the potential in-volvement of these chitinases in mycoparasitism (7). Surpris-ingly, only five orthologue pairs can be found in the sgC

chitinases of T. atroviride and T. virens (TAC2/TVC2, TAC4/TVC4, TAC5/TVC5, TAC6/TVC6, and TAC7/TVC7) (5). Thissuggests the emergence of paralogues by gene duplications anda strong evolutionary pressure on this group of proteins.

Other families of glycoside hydrolases, e.g., cellulases in T.reesei, are largely coregulated on the transcriptional level (4),and a similar picture was found for sgC chitinase gene expres-sion in T. atroviride (5). All of these genes were induced duringmycoparasitism of the fungal prey Botrytis cinerea, but none byRhizoctonia solani, although both fungi can be well parasitizedby T. atroviride. Correspondingly, all of them were expressedduring growth on B. cinerea cell walls, but not on R. solani orT. atroviride cell walls. In contrast to cellulases, which are solelyinvolved in substrate degradation for nutritional purposes,chitinases have multiple functions in fungal biology. They areinvolved in exogenous chitin degradation or, in the case ofmycoparasites in a more aggressive form thereof, cell walldegradation during the mycoparasitic attack. However, chi-tinases are also important for the fungus during its life cycle bybeing involved in cell wall remodeling and recycling and hyphalbranching and fusion events. Thus, the functions of chitinasesrange from morphogenetic roles in fungal colony developmentto exogenous chitin degradation for nutritional purposes. It isnot yet understood how these functions are distributed amongor within the different chitinase subgroups.

The aim of this study was to analyze the potential coregu-lation of chitinases in T. virens. They form the largest glycosidehydrolase family in mycoparasitic Trichoderma spp., and T.virens has an especially rich arsenal of them. In this study, wefocused on the 15 sgC chitinases of T. virens. Our results showthat sgC chitinase transcription in T. virens not only is triggeredby nutritional stimuli, but also strongly varies within different

* Corresponding author. Mailing address: Research Area GeneTechnology and Applied Biochemistry, Institute of Chemical Engi-neering, Vienna University of Technology, Gumpendorfer Strasse 1a/166-5, 1060 Vienna, Austria. Phone: 43-1-58801-166554. Fax: 43-1-58801-17299. E-mail: [email protected].

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 19 August 2011.

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types of hyphae and modes of cultivation. This indicates thatcolony-internal signals due to hyphal differentiation andinteraction are significant parameters for the regulation sgCchitinase gene expression. Our results show that the two mainfunctional aspects of chitinases—morphological developmentand exogenous chitin degradation—cannot be viewed sepa-rately but are strongly linked on the regulatory level and thusprobably also on the functional level.

MATERIALS AND METHODS

Strains and cultivation conditions. T. virens (teleomorph, H. virens)strain Gv29-8 (http://genome.jgi-psf.org/TriviGv29_8_2/TriviGv29_8_2.home.html) (DDBJ/EMBL/GenBank accession number ABDF00000000) and T. atro-viride IMI206040 (http://genome.jgi-psf.org/Triat2/Triat2.home.html) (accessionno. ABDG00000000) were used in this study and were maintained on potatodextrose agar (PDA) plates (BD Difco, Franklin Lakes, NJ). Confrontationassays and liquid cultures were performed as described previously (5). Briefly,the plant pathogens R. solani and B. cinerea were used as fungal hosts forconfrontation assays on PDA. Growth in liquid cultures was performed in staticcultures in petri dishes in order to enable the fungus to efficiently adhere to theinsoluble substrates predominantly used, such as cell walls and chitin. T. virenswas inoculated in Vogel’s minimal medium (VMM) (15) and T. atroviride inminimal medium (MM) (12), both including 0.05% peptone for efficient germi-nation. For cell wall stress experiments, calcofluor white (F3543-1G; Sigma-Aldrich, St. Louis, MO) and Congo red (L456540; Merck, Darmstadt, Germany)were added after autoclaving of the medium to the final concentrations given inResults. All cultures were inoculated in darkness at 25°C, and mycelia wereharvested at different time points, as indicated in Results. At least two indepen-dent experiments were performed for each growth condition, with biologicalreplicates (i.e., agar plates or static cultures in petri dishes, respectively) for each

time point. Gene expression data from one representative experiment are shownfor the different time points and cultivation conditions.

RNA isolation and RT-PCR. Mycelia were ground to a fine powder underliquid nitrogen. Total RNA was isolated as previously described (5). Note thatfor cultivations on agar plates, no cellophane overlays were used, but total RNAwas extracted directly from the agar. All isolated RNAs were treated with DNaseI (Fermentas, St Leon-Rot, Germany) and purified using the RNeasy MinElutecleanup kit (Qiagen, Hilden, Germany). Reverse transcription (RT)-PCR wasperformed as described previously using 25 cycles per reaction (5) with thegene-specific primers listed in Table 1.

Bioinformatics analyses. For comparison of gene and protein sequences andsynteny analysis, the tools available at the JGI genome website (http://www.jgi.doe.gov/) and the NCBI BLAST tools (http://blast.ncbi.nlm.nih.gov/Blast.cgi)were used.

The results of the RT-PCRs were visualized by agarose gel electrophoresis andanalyzed semiquantitatively by densitometry using the software ImageJ (http:///rsb.info.nih.gov/ij). The expression levels of sgC chitinase genes were quanti-fied and normalized to the corresponding signals of tef1 (encoding translationelongation factor 1�; JGI database protein identifier [ID] 83874), which was usedas a control gene in our studies. The normalized values were subsequently usedfor multiple clustering.

For semiquantitative illustration, the Hierarchical Clustering Explorer 3(HCE3) tool was used (13). This program supports five different linkage meth-ods: average, average group, complete, single, and one-by-one linkage. In orderto find meaningful groups of conditions under which sgC chitinases are upregu-lated, average group linkage was used to identify sgC genes similarly expressedunder various cultivation conditions. For all analyses, Euclidean Distance Mea-sure was used. Hierarchical-clustering results are displayed as grayscale mosaicsattached to dendrograms. Further, the k means clustering algorithm tool wasused to generate exactly k different clusters with the greatest possible distinction.Grouped cultivation conditions were visualized as a grayscale mosaic accordingto levels of expression. The grayscale color code corresponds to levels of expres-

TABLE 1. Overview of T. virens subgroup C chitinasesa

a Chitinases with orthologues in T. atroviride are highlighted in boldface. Protein IDs from the JGIdatabase are given. Modular structure: 2 CBM18 (white boxes) and 1 GH 18 (black box) or 2 CBM50(gray boxes), 1 CBM18 (white box), and 1 GH 18 (black box) (5).

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sion, starting from no expression (white boxes) to maximal expression levelsdetected in this study (black boxes).

Microscopic analyses. For morphological characterization, liquid cultures sup-plemented with carbon sources (chitin, cell walls, and glucose) were prepared.Two hundred fifty microliters of each culture was placed on microscope slides,incubated for 25 and 42 h, and imaged with an inverted T300 microscope (Nikon,Tokyo, Japan). An M420 Photomacroscope (Wild-Leica, Solms, Germany) wasused for imaging the mycelial mat in liquid static cultures. Images were capturedwith a Nikon DXM1200F digital camera and digitally processed using PhotoshopCS3 (Adobe, San Jose, CA).

RESULTS

Comparison of the orthologous pairs of sgC chitinases be-tween T. atroviride and T. virens. T. atroviride and T. virens have9 and 15, respectively, members of sgC chitinases but shareonly five orthologues (Tables 1 and 2) (5). We were thereforeinterested in finding parallels between the orthologous pairs inT. atroviride and T. virens in order to elucidate the inducingstimuli and regulation of sgC chitinases in more detail. SgCchitinases in Trichoderma have two types of modular architec-ture (Table 1) (5), and of the orthologue pairs in T. atrovirideand T. virens, only TAC7/TVC7 belong to the clade of sgCchitinases that have two LysM motifs and one CBM 18 Nterminal of the GH 18 module. TAC2/TVC2, TAC4/TVC4,TAC5/TVC5, and TAC6/TVC6 contain only two CBM 18modules N terminal of the GH 18 module. Interestingly, onlyfor TAC7/TVC7 has an orthologue also been detected in T.reesei, whereas T. reesei has no sgC chitinases of the type withno LysM module. T. reesei and T. virens, which are taxonomi-cally more closely related to each other than to T. atroviride,have two further orthologue pairs, TRCHI18-1/TVC1 andTRCHI18-9/TVC13, for which no corresponding orthologuewas detected in T. atroviride. Thus, there is no correlationbetween the modular architectures and orthologous pairs of T.atroviride and T. virens.

A closer comparison of the sequence similarities betweensgC chitinases of T. atroviride and T. virens showed that TAC2/TVC2 and TAC7/TVC7 are strongly conserved on the proteinlevel, while the other orthologous pairs, TAC4/TVC4, TAC5/TVC5, and TAC6/TVC6, show only a moderate level of con-servation (Table 3). It should be noted that in these cases, theCBMs and GH 18 module show a higher degree of conserva-

tion than the parts of the proteins outside these defined re-gions.

For the strongly conserved protein pairs TAC2/TVC2 andTAC7/TVC7, even at the DNA level, 93 to 96% identities wereobserved (Table 3). An alignment of 1,000 bp of the up- anddownstream regulatory regions of the respective genes showedthat for tac7-tvc7, 340 bp upstream of the ATG are stronglyconserved, and that after a gap of ca. 80 bp, the following �600bp are also almost identical between the two fungi. The con-served region reaches into the gene upstream of the tac7-tvc7locus, encoding a protein with a concanavalin A-like lectin/glucanase IPR008985 domain (T. virens protein ID 53596).Further, approximately 160 bp downstream of the tac7-tvc7locus are strongly conserved. For tac2-tvc2, the situation issimilar, with 160 bp with high similarity upstream of the ATG,followed by a nonhomologous gap of ca. 160 bp and then acontinuous conservation of �700 bp reaching into the neigh-boring gene, encoding a LysM protein (T. virens protein ID28703).

We also analyzed whether the orthologue pairs are locatedin syntenic regions in T. atroviride and T. virens. Interestingly,this was not the case. All five of them are located in small,nonsyntenic clusters of 2 to 5 genes within large syntenic re-gions (Fig. 1). It was also conspicuous that all five chitinaseswere close to the ends of scaffolds in T. virens. TVC5 andTVC2 were located near the beginning and end, respectively,of scaffold 6 in the genome database, and TVC4, TVC6, andTVC7 were found on relatively short scaffolds.

Gene expression of sgC chitinases in T. virens. We focusedon three different topics in the investigation of sgC chitinasegene expression in T. virens: (i) mycoparasitism-related growthconditions (confrontation assays and growth on cell walls), (ii)consumption of chitinous carbon sources under nonmycopara-sitic conditions (growth on chitin and starvation), and (iii)hyphal development and colony formation. T. atroviride sgCchitinase genes were found to be inducible only by a small setof the screened growth conditions in our previous study (Fig.2a). We expected a similar on/off situation for these genes in T.

TABLE 2. Summary of the properties ofT. atroviride sgC chitinasesa

a For the modular structure, see Table 1, note a.

TABLE 3. Comparison of the orthologous sgC chitinase pairs inT. virens, T. atroviride, and T. reesei

Compared proteins orgenes

% Alignmentcoverage

% Identity% Positive

(aa)Aminoacid DNA

Proteinsa

TVC2 vs. TAC2 97 93 95TVC4 vs. TAC4 97 39 53TVC5 vs. TAC5 95 67 79TVC6 vs. TAC6 98 69 79TVC7 vs. TAC7 100 93 96TVC7 vs. CHI18-8 99 88 94

Genesb

tvc2 vs. tac2 95 96tac6 vs. tvc6 50 75tvc7 vs. tac7 99 93

a Protein IDs: TVC7, 53606; CHI18-8, 108346; TAC7, 247300; TVC2, 191688(updated); TAC2, 348134; TAC4, 348132; TVC4, 81573; TVC5, 112098; TAC5,348128; TAC6, 53627; TVC6, 348129.

b The DNA genomic sequences of the complete genes, including 200 bp of theup- and downstream regions, were used.

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FIG. 1. Synteny of the genomic regions containing sgC chitinase orthologues in T. atroviride and T. virens, generated with the synteny tool inthe JGI database. Three hundred fifty kilobases up- and downstream of the respective chitinase genes in T. atroviride located on the indicated contigwere compared to the T. virens genome. The scaffolds covering the respective regions are shown in color-coded bars. The regions containing thechitinases indicated above the respective diagrams are circled.

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FIG. 2. Gene expression profiles of sgC chitinases in T. virens and T. atroviride. The grayscale ranges from white (no expression) to black(maximum expression). CA, confrontation assays; CW, cell walls; BC, before contact; C, contact; AC, after contact. (a) Gene expression profilesof sgC chitinases in T. atroviride. In order to facilitate the comparison between T. atroviride and T. virens, data from reference 5 that are directlycomparable with data presented in this study were converted to a grayscale mosaic. Note that none of the T. atroviride sgC chitinase genes was inducibleby R. solani or T. atroviride. (b to d) Gene expression profiles of sgC chitinases in T. virens. (b) k means clustering (k � 3) of growth conditions. min,minimum; max, maximum. (c) Clustering of genes using average group linkage. (d) Phylogenetic tree of sgC chitinases of T. reesei, T. atroviride, and T.virens (data from reference 5) in which the overall expression rates of the respective T. virens and T. atroviride genes are indicated as follows: ��, strongexpression under many growth conditions; ��, strong to moderate expression under many growth conditions; �, moderate to weak expression undera few growth conditions; �, no expression in our data set. Note that the genes of orthologue pairs are not necessarily expressed under the same growthconditions. The two subclades containing a strongly active and an inactive T. virens sgC chitinase are encircled.

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virens, but surprisingly, we obtained complex expression pat-terns under most growth conditions. The expression profiles ofthe 15 T. virens sgC chitinase genes from 35 different growthconditions and time points were analyzed by RT-PCR, mea-sured by densitometry, and converted into a grayscale mosaic(see Fig. S1 in the supplemental material). Cluster analysis ofthe results (see Materials and Methods for details) is shown inFig. 2b and c. When we used k means clustering to partition thegrowth conditions into three groups (Fig. 2b; k � 3), all con-frontation assays, with a defined set of four expressed chitinasegenes, grouped together in cluster II, but clusters I and IIIcontained strongly mixed growth conditions with fragmentedexpression patterns. This showed that in T. virens, nutritionalstimuli did not appear to be the main regulators of sgC chi-tinase gene expression under most tested growth conditions.Dendrograms obtained by cluster analysis of the growth con-ditions were too divided for interpretation (data not shown).Clustering of genes (Fig. 2c), using the average group linkagealgorithm, resulted in clades of chitinase genes with similarexpression patterns. The results obtained from the expressionprofiles are evaluated in detail below, but overall, it can beclearly stated that T. virens sgC chitinase genes have com-pletely different expression patterns than T. atroviride sgCgenes. Not even for the orthologue pairs were correlationsobserved, although, as stated above, two of the genes, namely,tvc2 and tvc7, are 96 and 93% identical to those of T. atrovirideat the DNA level. Under most of the tested conditions, expres-sion of tvc4 was detected, and the genes tvc1, tvc8, tvc13, andtvc15 were not found to be expressed at all. Figure 2d shows aschematic representation of the expression patterns of T. virenssgC chitinases in their phylogenetic context. TVC1 and TVC13have apparent orthologues in T. reesei, but their genes are notexpressed; however, in both cases, a T. virens chitinase in thesame subclade is strongly expressed. TVC8 is closely related toTVC14, but again, only one of them is expressed. For TVC15,no protein model could be developed, and it is therefore notincluded in the tree. This indicated that tvc1, tvc8, and tvc13 areprobably remnants from gene duplications and are thus inac-tive pseudogenes.

Mycoparasitism. SgC chitinase gene expression during my-coparasitism was tested in confrontation assays with the asco-mycete B. cinerea and the basidiomycete R. solani. As controls,T. virens was (i) confronted with itself and (ii) grown alone ona plate without a host. For comparison with other growthconditions described below, it is important to note that byanalogy to previous experiments with T. atroviride (5), only ca.5 mm of the hyphal area that was growing toward the preyfungus was harvested (see Fig. S2a in the supplemental mate-rial). Expression patterns in T. virens were substantially differ-ent from those previously reported for T. atroviride (5). In T.atroviride, all sgC chitinase genes were induced during confron-tation with B. cinerea but none with R. solani or in the control.In T. virens, a defined subset of sgC chitinase genes (tvc2, tvc3,tvc4, and tvc10) was induced in all confrontation assays, includ-ing in the control confrontations of T. virens against itself, but,interestingly, not when T. virens was grown alone on an agarplate under the same conditions, even including the asymmet-rical placement of the colony (Fig. 2).

Growth on cell walls of B. cinerea, R. solani, and T. virens ledto expression patterns that were completely different from

those in the confrontation assays (Fig. 2). Eleven out of the 15T. virens sgC chitinase genes (tvc2, tvc3, tvc4, tvc5, tvc6, tvc7,tvc9, tvc10, tvc11, tvc12, and tvc14) were found to be induced byB. cinerea cell walls. Since the other four genes (tvc1, tvc8,tvc13, and tvc15) were not expressed under any of the testedgrowth conditions, it can be said that “all” sgC chitinase geneswere induced by B. cinerea cell walls, similar to T. atroviride.However, in T. virens, different subsets of sgC genes were alsoinduced by R. solani cell walls (tvc4, tvc6, tvc7, and tvc10) andT. virens cell walls (tvc2, tvc3, tvc4, tvc7, and tvc9). The genetvc10 was found to be induced under mycoparasitism-relatedconditions, but not when grown on glucose or cell walls of T.virens itself as a control.

Chitin and starvation. T. virens sgC gene expression wasanalyzed with colloidal chitin and the nonpretreated and thusless accessible practical-grade chitin (which also may containprotein impurities) as carbon sources (Fig. 2). In contrast togrowth on cell walls, where strong mycelial development wasobserved, growth on chitin was poor (see Fig. S3 in the sup-plemental material). Clear differences in expression sets be-tween colloidal chitin and crude chitin were observed (tvc2,tvc3, tvc4, tvc5, tvc6, tvc7, and tvc10 for colloidal chitin versustvc4, tvc7, and tvc10 for crude chitin). SgC gene expressionanalysis of T. virens cultures under carbon starvation showedthat several sgC genes were induced by starvation, but again, adifferent subset (tvc3, tvc4, tvc5, tvc6, tvc7, tvc9, tvc10, andtvc12) than under all previously tested growth conditions.

Hyphal development and colony formation. We also testedthe expression of sgC chitinase genes during growth and hyphaldevelopment in two types of cultivation (Fig. 2): (i) Startingfrom a spore suspension and harvesting the mycelial mat atdifferent time points and (ii) starting from an agar plug withmycelium and harvesting the peripheral and central hyphalzones of the colony (see Fig. S2b in the supplemental mate-rial). By analogy to growth on cell walls and chitin, both cul-tivations were carried out in liquid static cultures, but withglucose as the carbon source. Additionally, gene expression inconidia was tested.

In conidia, only tvc4 was expressed, which was also foundunder almost all other tested conditions. In young mycelium(15 h), starting from a spore suspension, we found that all butthe four seemingly inactive sgC chitinase genes were expressed.Expression patterns decreased to only tvc4, tvc6, tvc7, and tvc9at 25 h, and their transcript abundances decreased strongly atlater time points. The observed expression pattern at 65 h,where the onset of autolysis can be assumed, differs stronglyfrom that during starvation (0.05% peptone but no carbonsource) but bears some similarity to growth on T. virens cellwalls, as tvc4, tvc7, and tvc9 were also expressed under thesegrowth conditions.

When the inner and peripheral hyphal zones were inves-tigated, again, all 11 apparently active chitinases were ex-pressed in the inner zone of the fungal colony but only a fewof them in the peripheral hyphal zone, and those were atlower levels. In the context of these results, it should be takeninto account that in the confrontation assay controls, only thefirst few millimeters of the hyphae were harvested from agarplates (see Fig. S2a and b in the supplemental material), andno sgC chitinase genes were expressed. The results from geneexpression analysis during hyphal development and colony for-


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mation, therefore, clearly showed that in T. virens, sgC chi-tinase genes are abundantly expressed during “normal”growth, i.e., in the absence of a potentially inducing carbonsource, but their expression patterns vary strongly with themode of cultivation and the hyphal zone that is harvested.Since the same cultivation conditions were used for T. atro-viride, the possibility that the observed differences are due tothe experimental setup can be excluded.

Differences in biomass formation, hyphal growth, and col-ony morphology between T. virens and T. atroviride. The mostconspicuous detail of the gene expression analysis in T. virenswas the abundant expression of several chitinases duringgrowth without the presence of potentially inducing carbonsources. In order to elucidate why the respective sgC geneexpression patterns of T. virens are so substantially differentfrom those of T. atroviride, we investigated a number of pa-rameters, such as the influence of the medium composition,morphology, and biomass formation, in more detail. Differentstandard media are usually used for cultivation of T. atrovirideand T. virens (3, 12), and attempts to grow T. virens in T.atroviride MM led to low biomass formation (see Fig. S4 in thesupplemental material). Upon cultivation of T. virens in MMwith glucose, we found a slightly altered expression profile: tvc4was not expressed, but tvc6, tvc7, and tvc9 were still expressed,and in addition, tvc2, tvc3, tvc12, and tvc14 were very weaklyinduced (data not shown). This indicates that the slow growthon this medium slightly altered the expression patterns but re-sulted in expression of even more sgC chitinase genes. In contrast,when T. atroviride was grown in VMM, the expression patterns ofsgC genes were identical to those during growth in MM (data notshown). Therefore, although change of the medium revealedsome differences in sgC expression profiles, it did not explain thecomplex and abundant expression patterns observed in T. virensin comparison to the simple patterns in T. atroviride. Thus, weconcluded that the medium composition itself cannot explain theobserved differences between T. atroviride and T. virens. Further,it should be noted that for confrontation assays, the same medium(PDA) was used for both fungi.

Next, we investigated the morphological development of thetwo fungi at different time points and with various media andcarbon sources. Microscopic analysis revealed clear differencesin hyphal development between T. atroviride and T. virensgrown on glucose independent of the medium used. Young T.virens hyphae were strongly branched and showed irregularconstrictions, whereas T. atroviride hyphae grew as straight,long, thin tubes, with very few branches (Fig. 3a). Strong mac-roscopic differences in the formation of the mycelial mat instatic cultures were also observed. While T. atroviride formed auniform mycelial mat when starting from a spore suspension,T. virens initially formed small clumps and clusters (Fig. 3b),indicating different hyphal network formation due to morpho-genetic parameters.

Cell wall stress is not a significant regulatory center for sgCchitinases. In order to test whether stimuli involved in cell wallformation and remodeling could influence sgC chitinase ex-pression, we tested the influence of cell wall stress on sgC geneexpression. The dyes Congo red and calcofluor white interca-late with glucan and chitin polymers and therefore interferewith fungal cell wall formation. Addition of these agents to themedium causes cell wall stress and was shown to lead to in-

creased chitin polymerization and thicker cell walls (9). Theaim was to analyze whether chitinase expression patterns arealtered during cell wall stress. T. virens was found to be verysensitive to Congo red and calcofluor white, and while forother fungi concentrations in the range of 50 to 200 �g/ml ofthese substances are used (9, 14, 17), for T. virens, addition ofmore than 5 �g/ml Congo red to the medium with the sporesuspension in liquid, static cultures led to such a drastic de-crease in growth that comparison with normal growth was notpossible. Unfortunately, growth on calcofluor white was com-pletely abolished even at 1 �g/ml under these growth condi-tions. The results with Congo red (Fig. 4a) showed that chi-tinase patterns were not different but that the expression levelswere higher under cell wall stress. In this experiment, the sgCchitinase genes tvc4, tvc6, and tvc7, which were found to beinduced in glucose cultivations (see above), showed enhancedexpression. Starting from an agar plug in a static liquid culture,somewhat higher concentrations of the cell wall stress reagentswere tolerated, but growth was strongly delayed and colonieswith an irregular shape were formed; therefore, only the cen-tral hyphal zones were harvested (Fig. 4b). Similar to the con-trol, a large set of genes was expressed, and the response ofmost sgC chitinase transcript levels to calcofluor white wasdifferent from that to Congo red. Only tvc4 was induced byboth cell wall stress reagents, and only tvc5 and tvc11 wereclearly downregulated by both. These results show that cellwall stress has an influence on sgC chitinase expression in T.virens, supporting our hypothesis that sgC genes are regulatedby colony-internal morphological parameters. However, cellwall stress does not appear to be the main factor for upregu-lation of these genes. Further, these data showed that Congored and calcofluor white have different modes of action duringthis process.

DISCUSSION

In this study, we investigated the gene expression of sgCchitinases in T. virens. Mycoparasitic Trichoderma spp. havesignificantly higher numbers of sgC chitinases than, e.g., thesaprotrophic species T. reesei, and it was thus previously hy-pothesized that they are involved in the mycoparasitic attack(6). The results presented in this study shed a completely newlight on sgC chitinase regulation and showed only very fewparallels between T. atroviride and T. virens. Even orthologousgenes showed completely different expression patterns. All fiveorthologous sgC chitinase pairs are located in small, nonsyn-tenic islands within regions of high synteny, and in T. virens,they are located rather close to the ends of scaffolds. This couldindicate their proximity to repetitive regions that favorgenomic rearrangements. For tvc1, tvc8, and tvc13, no expres-sion was detected, but phylogenetically closely related sgCchitinases were (strongly) expressed. These data point to geneduplications with subsequent inactivation of one of the copies,and tvc1, tvc8, and tvc13, and possibly also tvc15, could there-fore be inactive pseudogenes.

With respect to mycoparasitism, confrontation assays ofT. virens with itself led to the induction of strictly the samechitinases that were also induced by mycoparasitism of R. so-lani and B. cinerea, whereas when T. virens was alone on anagar plate, no expression of sgC chitinases was observed in the


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hyphal front. This suggests that in T. virens, nonmycoparasiticinducers derived from living fungal cell walls triggered theexpression of these genes. This is in strong contrast to T.atroviride, where during confrontation with itself and with R.solani, none of the sgC chitinase genes was induced. Thus, theexpression pattern observed during the confrontation assays inT. virens is, interestingly, not specific to a mycoparasitic inter-action but, rather, for sensing the presence of another fungus

in a more nonspecific way. In this context, it should be men-tioned that, while T. atroviride can always (partially) overgrowitself, T. virens colonies do not come into physical contact whengrown in constant light or in a 12-h light/12-h darkness cyclebut are able to (partially) overgrow each other when cultivatedin constant darkness (see Fig. S5 in the supplemental mate-rial). This further underlines the different lifestyles and sensingmechanisms of the two mycoparasites.

FIG. 3. Growth morphology of T. atroviride and T. virens in static cultivations after 25 h. (a) Microscopic images. (b) Photographic images.


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In T. virens, growth on fungal cell walls as carbon sources ledto sgC expression profiles substantially different from thoseobserved in confrontation assays. This suggests a different uti-lization potential of the cell walls that leads to different induc-ing stimuli, which could be due to, e.g., limited accessibility ofthe chitin, melanization of the cell wall, or general differencesin cell wall composition.

Our data showed that in T. virens, sgC chitinase genes aredifferentially expressed in various zones of the fungal colony(i.e., the central versus the peripheral hyphal zone in liquid,static cultures) without a potentially inducing carbon source,thus indicating that colony-internal signals derived from hyphaldifferentiation influence the expression of these genes. It canbe expected that these internal stimuli overlap with externalnutritional stimuli and together regulate sgC chitinase geneexpression. We were therefore not able to find typical “induc-ing” growth conditions as is usually the case for glycosidehydrolases. Why was this strong influence of morphogeneticparameters apparently not observed in T. atroviride? There aretwo possible explanations, which are not mutually exclusive:either the regulation of these genes is substantially different inthe two species or the observed differences in growth morphol-ogy are pivotal for the regulation of sgC chitinase genes. Someof the results are probably due to differences in the regulatoryregions of the respective genes. It should be considered, how-

ever, that tvc2 and tvc7 are, even in their regulatory regions,almost identical at the DNA sequence level to their T. atro-viride orthologues, with the exception of a small gap in thepromoter regions. Nonetheless, we found strongly divergenttranscript patterns. In view of the fact that the complete genesand large parts of the regulatory regions show �90% identity,we consider it unlikely that the strong differences in the ob-tained expression profiles could be explained solely by theshort nonhomologous gaps in the promoter regions. Species-specific transcription factors could therefore be responsible forthe differential regulation. Genome analysis showed large setsof transcription factors that were unique to either of the twospecies. Differences in signal transduction and transcriptionalactivation could therefore be an important factor in the ob-served differences in sgC chitinase gene regulation between T.atroviride and T. virens. In order to test whether the regulationof sgC chitinases is species specific, heterologous expression ofthese genes in the other species could be carried out. Thisapproach could also show whether the differential transcrip-tion of genes of orthologue pairs has an effect on the growthmorphology of each species. In addition to the regulatory as-pects, we hypothesize, based on our observations, that theobserved morphogenetic differences between T. atroviride andT. virens are connected to sgC chitinase regulation via colony-internal inducing stimuli. Further supporting this hypothesis,

FIG. 4. Gene expression analysis of sgC chitinases during cell wall stress. (a) Static cultivations starting from a spore suspension in VMM withglucose (control) and supplemented with Congo red (5 �g/ml). Cultures were harvested after 48 h. Expression levels were measured by ImageJsoftware (optical density [OD]) and normalized to tef1. (b) Static cultivations starting from a conidiated agar plug in VMM with glucose (control)and supplemented with Congo red (5 �g/ml) and calcofluor white (10 �g/ml). The central hyphal zones were harvested, and gene expression levelswere analyzed as described for panel a.


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we found that cell wall stress influences the expression of thesegenes. Cell wall stress has an effect on the cell wall composi-tion, i.e., increased chitin content, and is also accompanied byaltered hyphal morphology and colony formation (as also ob-served in this study). Calcofluor white and Congo red arenonspecific fluorochromes that bind to cellulose and chitinpolymers in the cell walls of fungi (9, 16). The strong toxicity ofcalcofluor white and Congo red for T. virens was somewhatsurprising in view of the fact that the organism is a myco-parasite and thus potentially has well-protected cell walls.However, for the biopolymer chitosan, which has antibacte-rial and antifungal properties due to its free amino groups,an increased sensitivity was also already reported for myco-parasitic Trichoderma spp. in comparison to, e.g., ento-mopathogenic or plant-pathogenic fungi (8). Calcofluor whiteand Congo red cause the formation of thicker cell walls andlead to the upregulation of chitin synthases (9). The fact thatwe found altered, but not strictly increased, sgC expressionpatterns in the presence of these cell wall stress agents suggeststhat sgC chitinase expression is not coupled to chitin synthesisrates.

The growth of T. virens on glucose led to abundant sgCchitinase expression, which indicates that sgC chitinases areactually multipurpose chitinases and are involved in the deg-radation and recycling of (exogenous) chitin from dead andliving hyphal material. This is in agreement with the fact thatfungi with a less aggressive life style, e.g., saprotrophic fungi,also have several sgC chitinases. Although increased numbersof sgC chitinases were found in the genomes of mycoparasiticTrichoderma spp., they are not exclusively induced during my-coparasitism, but rather, their expression data indicate a gen-eral involvement in various degradation processes of chitinouscarbon sources, including their own and other fungal cell walls.Our data link for the first time the two main functional aspectsof chitinases—morphogenetic development and exogenouschitin degradation—and also underline a potential interplay ofthese two aspects in the mycoparasitic attack.

ACKNOWLEDGMENTS

This work was supported by the FWF Austrian Science Fund (T390and P20559 to V.S.-S.). The work conducted by the U.S. Department

of Energy Joint Genome Institute is supported by the Office of Scienceof the U.S. Department of Energy (contract no. DE-AC02-05CH11231).

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Differential Regulation of Orthologous Chitinase Genes in … · sion, starting from no expression (white boxes) to maximal expression levels detected in this study (black boxes)