Role of the methylcitrate cycle in growth,antagonism and induction of systemic defenceresponses in the fungal biocontrol agentTrichoderma atroviride

Mukesh K. Dubey,1 Anders Broberg,2 Dan Funck Jensen1

and Magnus Karlsson1

Correspondence

Mukesh K. Dubey

mukesh.dubey@slu.se

Received 19 June 2013

Accepted 2 October 2013

1Uppsala BioCenter, Department of Forest Mycology and Plant Pathology, Swedish University ofAgricultural Sciences, Box 7026, 75007 Uppsala, Sweden

2Uppsala BioCenter, Department of Chemistry, Swedish University of Agricultural Sciences,Box 7015, 75007 Uppsala, Sweden

Methylisocitrate lyase (MCL), a signature enzyme of the methylcitrate cycle, which cleaves

methylisocitrate to pyruvate and succinate, is required for propionate metabolism, for secondary

metabolite production and for virulence in bacteria and fungi. Here we investigate the role of the

methylcitrate cycle by generating an mcl deletion mutant in the fungal biocontrol agent

Trichoderma atroviride. Gene expression analysis shows that a basal expression of mcl is

observed in all growth conditions tested. Phenotypic analysis of an mcl deletion mutant suggests

the requirement of MCL in propionate resistance, growth, conidial pigmentation and germination,

and abiotic stress tolerance. A plate confrontation assay did not show a difference between the

WT and the Dmcl strain in antagonism towards Botrytis cinerea. However, the Dmcl strain

displays reduced antagonism towards B. cinerea based on a secretion assay. Furthermore, an in

vitro root colonization assay shows that the Dmcl strain had reduced ability to colonize

Arabidopsis thaliana roots, which results in reduced induction of systemic resistance towards B.

cinerea. These data show that MCL is important not only for growth and development in T.

atroviride but also in antagonism, root colonization and induction of defence responses in plants.

INTRODUCTION

The methylcitrate cycle is a carbon anaplerotic pathwaywhere propionyl-CoA is converted to pyruvate (Textoret al., 1997). Methylcitrate synthase (MCS), methylcitratedehydrogenase (MCD) and methylisocitrate lyase (MCL)are the signature enzymes of the pathway. MCS catalysescondensation of propionyl-CoA and oxaloacetate to methyl-citrate (Horswill & Escalante-Semerena, 1999), MCDcatalyses methylcitrate to methylisocitrate, and MCLcompletes the cycle by cleaving methylisocitrate to pyruvateand succinate (Brock et al., 2001). Succinate generated bythe pathway can go to the citric acid cycle, while pyruvatecan be used directly for energy and biomass production

(Brock et al., 2001). Propionyl-CoA used in the methyl-citrate cycle derives either from direct activation ofpropionate, an abundant carbon compound in soil, or fromthe degradation of odd-chain fatty acids and amino acidssuch as isoleucine, valine and methionine (Zhang et al.,2004). Besides being carbon anaplerotic, the methylcitratecycle also detoxifies propionate, which otherwise can exerttoxic effects by inhibiting several enzymes of primary metabolicpathways (Brock & Buckel, 2004; Limenitakis et al., 2013).

The role of the methylcitrate cycle in propionate metabol-ism, as well as in growth, conidiation and virulence, hasbeen demonstrated in bacterial and fungal systems (Brock,2005; Lee et al., 2009; Munoz-Elıas & McKinney, 2005;Upton & McKinney, 2007). In Aspergillus nidulans andAspergillus fumigatus, disruption of the methylcitrate cyclegene leads to accumulation of propionyl-CoA, and con-sequently growth retardation, impaired conidiation andreduced polyketide secondary metabolite synthesis (Brock,2005; Maerker et al., 2005; Zhang et al., 2004; Zhang &Keller, 2004). The importance of the methylcitrate cycle invirulence has been reported in As. fumigatus, wheredisruption leads to attenuated virulence in wax moth

Abbreviations: ATMT, Agrobacterium tumefaciens-mediated transformation;Ct, threshold cycle; CTAB, hexadecyltrimethylammonium bromide; ICL,isocitrate lyase; MCD, methylcitrate dehydrogenase; MCL, methylisocitratelyase; MCS, methylcitrate synthase; NAG, N-acetylglucosamine; qPCR,quantitative PCR; RT-PCR, reverse transcriptase-PCR; WGA, wheatgermagglutinin.

One supplementary table and three supplementary figures are availablewith the online version of this paper.

Microbiology (2013), 159, 2492–2500 DOI 10.1099/mic.0.070466-0

2492 070466 G 2013 SGM Printed in Great Britain

larvae (Maerker et al., 2005) and in immunosuppressedmice (Ibrahim-Granet et al., 2008). Furthermore, a Myco-bacterium tuberculosis mutant strain lacking MCS andMCD was unable to grow on propionate media or inmurine bone marrow-derived macrophages, but nochanges in virulence were observed in mice (Munoz-Elıaset al., 2006). The role of the methylcitrate cycle in growth,virulence and propionate detoxification has also beendemonstrated in the non-pathogenic bacterium Mycobac-terium smegmatis (Upton & McKinney, 2007).

Fungal MCLs display sequence similarity to fungalisocitrate lyases (ICLs) (Brock, 2005; Muller et al., 2011).ICL, a signature enzyme of the glyoxylate cycle, is requiredfor metabolism of non-fermentable carbon compoundslike acetate or ethanol, and virulence in bacteria and fungi(Dunn et al., 2009). Phylogenetic analysis of MCLs andICLs suggests that fungal MCLs evolved from a geneduplication of an ICL-encoding gene (Muller et al., 2011).In spite of the similarity, ICL and MCL in filamentousfungi have unique substrate specificities and catalyticmotifs (Brock, 2005; Muller et al., 2011), and thus performnon-overlapping functions in their respective pathways(Brock et al., 2001; Brock, 2005). In contrast to that,studies in M. tuberculosis, M. smegmatis and Saccharomycescerevisiae suggest a dual role of ICL: as ICL in the glyoxylatecycle, as MCL in the methylcitrate cycle and jointlyrequired for acetate and propionate metabolism, growthand virulence (Gould et al., 2006; Munoz-Elıas &McKinney, 2005; Upton & McKinney, 2007). The situationis more complicated in the phytopathogenic fungusFusarium graminearum, where ICL and MCL performspecific roles in acetate and propionate metabolism,respectively; however, both enzymes have redundantfunctions for plant infection (Lee et al., 2009).

Trichoderma atroviride, a ubiquitous soil-borne fungus, iswidely used as a model species to study mechanisms ofmycoparasitism and biocontrol of plant diseases. It lives as asaprotroph, interacts with fungal pathogens through myco-parasitism, can colonize roots and can even live as anendophyte inducing plant defence responses (Druzhininaet al., 2011; Harman et al., 2004). Knowledge of whichmetabolic pathways are used for exploiting carbon sources byT. atroviride when adapting to different life styles is thereforeimportant to achieve optimal biological control performance.

By generating MCL deletion (Dmcl) and complementation(Dmcl+) strains in T. atroviride, we demonstrated the roleof the methylcitrate cycle in propionate resistance, vegetativegrowth, conidial pigmentation and germination, and inantagonism. In addition, the Dmcl strain has a reduced, ordelayed, ability for root colonization and for induction ofsystemic defence responses in Arabidopsis thaliana.

METHODS

Sequence analysis. The T. atroviride genome sequence v.2 (http://

genome.jgi-psf.org/Triat2/Triat2.home.html) was used for gene

sequence retrieval. Analyses for conserved domains were performed

using the SMART protein analysis tool (Letunic et al., 2009),

InterProScan (Quevillon et al., 2005) and Conserved Domain

Search (Marchler-Bauer et al., 2009).

Fungal strains and culture conditions. The WT T. atroviride strain

IMI206040 and mutants derived from it, Botrytis cinerea strain B05.10

and Rhizoctonia solani strain SA1 were maintained on potato dextrose

agar (PDA) (Oxoid) medium at 25 uC. SMS medium (Dubey et al.,

2012) supplemented with 1 % glucose was used for gene expression

and phenotypic screening unless otherwise specified. Culture media

for different carbon sources were prepared by substituting 1 %

glucose in SMS medium with sodium acetate (25 mM), sodium

citrate (25 mM), sodium butyrate (25 mM), sodium pyruvate

(25 mM), sodium propionate (10 or 25 mM), Tween 20 (0.1 %),

ethanol (1 %), colloidal chitin (1 %), R. solani cell walls (RsCW) (1 %),

glucose (5 %) or N-acetylglucosamine (NAG) (10 mM). Starvation for

carbon (C lim), nitrogen (N lim) and carbon+nitrogen (C+N lim)

was induced as described previously (Dubey et al., 2012). T. atroviride

mycelia for submerged liquid cultures were cultivated and harvested as

described previously (Dubey et al., 2012). Colloidal chitin was prepared

from crab-shell colloidal chitin (Sigma-Aldrich) as described by Seidl

et al. (2005). R. solani cell wall material was prepared as described by

Inglis & Kawchuk (2002) with minor modifications.

Gene expression analysis. For gene expression analysis in different

nutritional conditions (described above) and during dual culture

against B. cinerea (Ta-Bc) and R. solani (Ta-Rs), mycelia were cultivated

and harvested as described previously (Dubey et al., 2012). T. atroviride

confronted with itself (Ta-Ta) was used as a control treatment. Total

RNA was extracted as described previously (Dubey et al., 2012).

Total RNA (1 mg), post-DNase I treatment, was reverse transcribed in

a total volume of 20 ml using a Maxima first strand cDNA synthesis

kit (Fermentas). Transcript levels were quantified by quantitative PCR

(qPCR) using the SYBR Green PCR master mix (Fermentas) in an

iQ5 qPCR System (Bio-Rad) as described previously (Tzelepis et al.,

2012). Melt curve analysis was performed after the qPCRs to confirm

that the signal resulted from a single product amplification.

Expression of mcl was normalized by act1 expression for liquid

cultures and by sar1 expression for dual culture interactions, in

accordance with previous evaluations (Brunner et al., 2008). Relative

expression values were calculated from the threshold cycle (Ct) values

and the primer amplification efficiencies by using the formula

described by Pfaffl (2001). Gene expression analysis was carried out in

four biological replicates, each based on two technical replicates.

Primer sequences used for gene expression analysis are given in Table

S1 (available in Microbiology Online).

Construction of deletion and complementation vectors.Genomic DNA was isolated using a hexadecyltrimethylammonium

bromide (CTAB)-based method (Nygren et al., 2008). Phusion DNA

polymerase (Finnzymes) was used for PCR amplification of a 1091 bp

59 flanking region and a 1102 bp 39 flanking region of the mcl gene

(GenBank protein ID no. 154163) from genomic DNA of T. atroviride

using primer pairs MCLKO_1F/MCLKO_1R and MCLKO_2F/

MCLKO_2R (Table S1), respectively. The hygromycin resistance

gene (hygB) cassette of 1532 bp was amplified from the pCT74 vector

(Lorang et al., 2001) using the P3/P4 primer pair (Table S1). The

nourseothricin resistance gene (nat1) cassette was amplified from the

pD-NAT1 vector (Kuck & Hoff, 2006) using the NatF/NatR primer

pair (Table S1). Gateway (Invitrogen) entry clones of the purified 59

flanking region, 39 flanking region, and hygB and nat cassettes PCR

fragments, and thereafter the deletion vector (pPm43GW-MCL-ko)

using entry plasmids of the respective fragments and destination vector

pPm43GW (Karimi et al., 2005), were generated as described by the

manufacturer.

Role of methylcitrate cycle in T. atroviride

http://mic.sgmjournals.org 2493

An mcl complementation cassette was constructed by PCR amplifica-

tion of the full-length sequence of mcl (including 1151 bp upstream

and 749 bp downstream) from genomic DNA of T. atroviride WT

using MCL_Comp F/MCL_Comp R primers. The amplified DNA

fragments were purified and integrated into destination vector

pPm43GW as described above using Gateway cloning technology to

generate complementation vector pPm43GW-MCL-comp.

Agrobacterium tumefaciens-mediated transformation (ATMT).The deletion and complementation vectors were used to transform

Ag. tumefaciens strain AGL-1 following a freeze–thaw procedure, and

positive clones were selected on YEP (Dubey et al., 2013) plates

containing 35 mg rifampicin ml21 (Sigma-Aldrich) and 100 mg

spectinomycin ml21 (Sigma-Aldrich). ATMT was performed based

on a previous protocol for Trichoderma harzianum (Utermark &

Karlovsky, 2008). Transformed strains were selected on plates

containing hygromycin or in combination with nourseothricin in

the case of complementation. Putative transformants were tested for

mitotic stability as described previously (Dubey et al., 2012).

Mitotically stable colonies were purified by two rounds of single

spore isolation.

Validation of transformants. A PCR screening approach of putative

transformants was performed to validate the homologous integration

of the deletion cassette. The primers used were specific to the hygB

gene (P3/P4), sequences flanking the deletion construct (MCL_UPS/

MCL_DS) and in combination (HygF_qPCR/MCL_DS, HygR_qPCR/

MCL_UPS) (Table S1). Reverse transcriptase-PCR (RT-PCR) analysis

was conducted on WT, knockout and complemented strains using

RevertAid premium reverse transcriptase (Fermentas) and primer

pairs specific for hygB (HygF_qPCR/HygR_qPCR), nat1 (NatF_qPCR/

NatR_qPCR) and mcl (MCL_2F/MCL_2R) (Table S1).

Phenotypic analysis. A 3 mm agar plug from the growing mycelial

front was transferred to solid PDA or SMS media containing different

nutrient regimes as described above. Colony morphology and growth

diameter were recorded every day. For determination of conidial

germination, conidia were harvested in water from the surface of 7-

day-old PDA plates, inoculated on solid agar plates containing

different carbon compounds as the sole carbon source and

photographs were taken 16 h post-inoculation using a Zeiss

Axioplan microscope equipped with Leica application suite version

3.6.0. The frequency of conidial germination was determined at the

same time by counting the number of germinating and non-

germinating conidia using the microscope. Three hundred to five

hundred conidia were counted for each treatment. Each experiment

had three biological replicates and was repeated twice. Complemen-

tation strain Dmcl+ was included in all phenotype analyses to

exclude the possibility of phenotypes that derive from ectopic

insertions.

For abiotic stress analysis, PDA plates containing 1 % NaCl or

0.025 % SDS were inoculated with a 3 mm agar plug and incubated at

25 uC in darkness. Colony diameter was measured after 7 days of

growth. The cell walls of germinating conidia were stained using

fluorescence wheatgerm agglutinin (WGA) lectins bound to FITC

(Sigma-Aldrich). Conidia were germinated on thin slices of water agar

on glass microscope slides and incubated with FITC-conjugated WGA

(10 mg l21 in 10 mM phosphate buffer pH 7.5) for 30 min at room

temperature. After three washes with phosphate buffer, cell wall

fluorescence was analysed by fluorescence microscope at 640

magnification with a Leica DM5500B microscope, and images were

taken using a DFC295 digital camera. Each experiment had three

biological replicates and was repeated twice.

Antagonism test. Antagonistic behaviour against B. cinerea was

tested using an in vitro plate confrontation assay on acetate, Tween 20

or PDA medium. Secreted factors were assayed by growing T.

atroviride WT, mutants or complemented strains on PDA or acetate

medium, covered with cellophane at 25 uC in darkness. The

cellophane was removed when T. atroviride covered the plates,

followed by inoculation with B. cinerea agar plugs. Linear growth was

recorded daily in three replicates.

Analysis of secondary metabolites. Agar plugs with mycelia were

inoculated on acetate or PDA medium and allowed to grow for 10 days

at 25 uC. Agar plugs with mycelium from the centre of plates were

harvested in five replicates using 50 ml Falcon tubes and were pushed

to the bottom of the tube. The agar plugs were sonicated for 15 min in

20 ml methanol. Extract (1 ml) was transferred to a 1.5 ml centrifuge

tube for centrifugation at 10 000 g for 5 min. Supernatants were

collected in HPLC vials for liquid chromatography-mass spectrometry

(LC-MS) analysis. LC-MS was carried out on an HP1100 LC system

(Hewlett Packard) connected to an electrospray ionization–quadro-

pole/time of flight mass spectrometer (ESI-QTOF; Bruker maXis

Impact; Bruker Daltonik) operated in positive mode scanning m/z 50–

1000. For calibration of the mass spectrometer 20 ml sodium formate

solution [4 ml formic acid, 20 ml 1 M NaOH (aq.), 100 ml H2O and

100 ml 2-propanol] was injected with each sample, directly into the

ion-source at t50, using a software-controlled valve. The separation

was achieved on a Reprosil-Pur ODS-3 (C-18, 5 mm, 46125 mm; Dr

Maisch GmbH) column at a flow rate of 0.7 ml min21 and 20 ml of

each sample was injected. The mobile phases were water (A) and

acetonitrile (B), both with 0.2 % formic acid, and the gradient was: 8 %

B at 0 min; 70 % B at 50 min; 95 % B at 51 min; 95 % B at 55 min; 8 %

B at 56 min and 8 % B at 60 min. The MS data were calibrated and

analysed using the software Compass DataAnalysis 4.1. GC-MS was

carried out as described by Wickel et al. (2013).

Ar. thaliana root colonization assay. Ar. thaliana ecotype

Colombia-0 (Col-0) was used in this study. Surface-sterile seeds were

grown on 0.26 Murashige and Skoog (MS) agar plates following the

procedure described previously (Dubey et al., 2013). T. atroviride

conidia (56104) were inoculated under sterile conditions to the

middle of 10-day-old seedling roots grown on MS agar plates and

allowed to interact for 48 h. Water-inoculated roots were treated as a

control. For each set of experiments five biological replicates with 15

seedlings per replicate were used. Root colonization was determined by

quantifying the DNA level of T. atroviride strains in Ar. thaliana roots

using qPCR. DNA was isolated from the root samples as described

above using the CTAB method (Nygren et al., 2008), quantified

spectrophotometrically using NanoDrop (Thermo Scientific) and

100 ng was used for qPCR analysis. PR-2 was used as the target gene

for Ar. thaliana, and gpd1 was used as the target gene for T. atroviride.

Root colonization was expressed as the ratio between gpd1 and PR-2.

The specificity of primers for PR-2 and gpd1 was tested using the DNA

of Ar. thaliana and T. atroviride, respectively.

Bioassay with B. cinerea. B. cinerea conidia were collected from

15-day-old PDA plates with distilled water and a glass rod, filtered

and resuspended in 0.01 % (v/v) Tween 20 and 10 mM glucose

solution to induce germination. Ar. thaliana seeds were germinated,

grown and inoculated with T. atroviride strains as described

previously (Dubey et al., 2013). After 48 h of interaction, seedlings

were transferred to individual plastic pots (56565 cm) containing

sterilized peat moss soil (S-Jord; Hasselfors Garden) and the B. cinerea

bioassay was performed following the procedure described previously

(Dubey et al., 2013). To recover the T. atroviride strains, plants were

carefully removed from the pots, after washing in water the roots were

surface sterilized with 1 % NaOCl for 5 min and placed in Rose

Bengal medium (Vargas Gil et al., 2009). The bioassay experiment was

performed with five biological replicate and each replicate had three

plants for each treatment. The experiment was repeated twice.

M. K. Dubey and others

2494 Microbiology 159

Statistical analysis. Analysis of variance (ANOVA: one way or twoway) was performed on gene expression and phenotype data using a

general linear model approach implemented in Statistica version 10(StatSoft). Pairwise comparisons were made using the Tukey–Kramer

method at the 95 % significance level.

RESULTS

Identification of a MCL-encoding gene inT. atroviride

The MCL sequences of As. fumigatus (CAW40752), As.nidulans (CAI65406), F. graminearum (XP_380352), Neuro-spora crassa (EAA35839), S. cerevisiae (NP_015331), Schizo-phyllum commune (XP_003038428), Ustilago maydis (XP_758039) and Yarrowia lipolytica (XP_506117) were used astemplates to screen the T. atroviride genome sequence v.2.0(http://genome.jgi-psf.org/Triat2/Triat2.home.html). BLASTP

searches using the listed MCLs resulted in high similarity(E values50) to protein ID 154163. The 561 amino acidsequence of protein ID 154163 was encoded by a 1683 bpORF, interrupted by a single intron. Conserved domainanalysis of protein ID 154163 identified a single ICL superfamily module in between amino acid positions 36 and 560.NCBI BLAST searches showed highest sequence similarity withMCLs from Trichoderma reesei (100 %) and Verticilliumdahliae (99 %). Furthermore, active sites alignment ofprotein ID 154163 with ICL and MCL enzymes where activesites are known (Muller et al., 2011) revealed the presence ofthe MCL characteristic amino acid residues leucine andserine, similar to known MCLs, in protein ID 154163 at theplace where phenylalanine and threonine residues are thecharacteristics of ICL. Based on this information, protein ID154163 was putatively identified as an MCL and thecorresponding gene named mcl.

Gene expression of mcl is not influenced bycarbon sources or antagonistic interactions

qPCR was used to analyse the gene expression of mcl in T.atroviride grown on medium containing different ferment-able or non-fermentable compounds, under nutritionalstress and during fungal–fungal interactions. Carbon sourcesrelated to fungal cell wall material were also included [NAG,chitin, R. solani cell wall material (Rs-CW)]. Expression ofmcl was induced (P¡0.007) when NAG was used as the solecarbon source (Fig. 1a). In addition, the transcript level ofmcl was not significantly induced 24 h post-contact witheither B. cinerea or R. solani in dual cultures (Fig. 1b).

Generation of deletion and complementationstrains

Successful mcl gene replacement in mitotically stablemutants was confirmed by PCR using primers locatedwithin the hygB cassette together with primers locatedupstream and downstream of the construct (Fig. S1a). Theexpected size of PCR fragments were amplified in all

selected Dmcl strains, while no amplification was observedin WT (Fig. S1b, c). The complete deletion of mcl wasfurther confirmed by PCR amplification of fragments ofthe expected size using primer pairs located outside theconstruct borders, from mutant and WT strains (Fig. S1d).Furthermore, RT-PCR experiments using primers specificto mcl sequences demonstrated the complete loss of mcltranscripts in the Dmcl strain (Fig. S1e). In addition, RT-PCR product from the hygB gene was obtained in the Dmclstrain, whereas no transcripts were found in the WT strain(Fig S1e).

The deletion mutant was complemented with the WT mclgene through ATMT. Successful integration of the comple-mentation vector in mitotically stable mutants was con-firmed by PCR amplification of the nat1 cassette (data notshown). RT-PCR of randomly selected nat1-positive mcl-complemented (Dmcl+) strains using mcl-specific primersdemonstrated restored mcl transcription, while no transcriptswere detected in the parental deletion strains (Fig. S1f).

Phenotypic characterization of Dmcl strains

Growth and conidial germination of the Dmcl strain wascompletely abolished on media with 10 mM propionate asthe sole carbon source, whereas WT and Dmcl+ strainswere able to grow (Fig. 2a, b). In addition, we evaluated thegrowth and development of the mutant and complementedstrains in medium containing either non-fermentablecompounds, such as acetate or ethanol (C2), pyruvate(C3), butyrate (C4), citrate (C6) or fatty acids like Tween20 (C58), or fermentable carbon compounds like glucose(C6) or NAG or chitin as the sole carbon source, and PDA.A significant reduction (P,0.001) in the growth rate of theDmcl strain was found in all tested media. Besides a 100 %reduction in growth rate on propionate medium, the Dmclstrain displayed maximum reduction of growth rate onbutyrate (80 %), followed by ethanol (69 %), citrate (65 %)and Tween 20 (57 %) (Fig. 2c).

The Dmcl strain showed significantly reduced (P¡0.023)numbers of germinating conidia in all the media testedexcept NAG and PDA (Fig. 2d). Germ-tube growth andmorphology of mutant strains were similar to the results ofconidial germination (Fig. S2). Delayed conidial pigmenta-tion of the Dmcl strain was observed in comparison to theWT when grown on media that contained non-fermentablecarbon compounds as the sole carbon source. Nevertheless,normal conidial pigmentation was observed when mutantstrains were grown on glucose, NAG, chitin or PDA plates(Fig. 3). In addition, visual and microscopy observation ofgrowth plates indicated no differences in conidiation in theDmcl strain (Fig. 3).

Deletion of mcl results in decreased tolerance toosmotic stress

A significant decrease (P,0.001) in growth rate wasrecorded for the Dmcl strain [0.8±0.03 mm per day

Role of methylcitrate cycle in T. atroviride

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(±SD)] in PDA medium that contained NaCl as an osmoticstress agent, compared with the WT (3.1±0.03 mm perday). The Dmcl+ strain grew like WT (3.0±0.04 mm per

day). The 76 % reduction in the growth rate of the Dmclstrain compared with the WT on PDA+NaCl mediumwas more severe (P,0.001) than the 10 % growth rate

(a) (b)

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Fig. 1. Expression analyses of mcl in T.atroviride mycelium grown with different car-bon sources (a) and during mycoparasiticconditions (b). Total RNA was extracted frommycelia after 24 h incubation in submergedshake-flask cultures representing differentnutritional/stress and mycoparasitic condi-tions. Relative expression levels for mcl inrelation to act1 (a) or sar1 (b) were calculatedfrom the Ct values and the primer amplificationefficiencies by using the formula described byPfaffl (2001). Error bars represent SDs basedon four biological replicates. Different letters(A, B) indicate statistically significant differ-ences (P¡0.05) within experiments based onthe Tukey–Kramer test.

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Fig. 2. Growth (a, c), and conidial germination (b, d) of T. atroviride strains grown on media containing different compounds asthe sole carbon source. (a) Photos of growth on solid medium, (b) photos of the conidia and conidial germ-tubes, (c) growthrate, (d) conidial germination frequency. For growth rate, strains were inoculated on solid agar medium, incubated at 25 6C indarkness and the growth diameter was recorded at 42 h post-inoculation. For propionate medium, photos were taken at 72 hpost-inoculation at the same distance. Frequency of germination was determined for each treatment by counting 300 to 500conidial germ-tubes or conidia under a microscope. Error bars represent SD based on three biological replicates. Differentletters (A, B, C) indicate statistically significant differences (P¡0.05) within media based on the Tukey–Kramer test. Eachexperiment was repeated twice.

M. K. Dubey and others

2496 Microbiology 159

reduction recorded on PDA (Fig. 2). In contrast, nodifferences were found between the Dmcl strain and theWT strain when grown on SDS medium (data not shown).Cell wall staining with FITC-conjugated WGA did notshow visible differences in cell wall structure between theWT and the mutant strain (data not shown).

Deletion of mcl reduces in vitro antagonismagainst B. cinerea

No differences in the ability to overgrow B. cinerea betweenT. atroviride WT and the Dmcl strain were observed in dualculture tests (Fig. 4a). A secretion assay was further used totest the antagonistic ability. Growth of B. cinerea wascompletely inhibited on acetate or PDA plates where the T.atroviride WT or complemented strain Dmcl+ had beenpre-grown (Fig. 4b). In contrast, B. cinerea was able togrow where the Dmcl strain had been pre-grown (Fig. 4b).The growth rate of B. cinerea on pre-grown Dmcl strainplates was significantly (P,0.001) lower than on controlplates (Fig. 4b).

These data suggested that the reduced antagonistic abilityof the mutant strain may be partly due to reducedproduction of secreted secondary metabolites. An analysisof extracts from acetate media colonized by the Dmcl strainrevealed the absence of a compound with the molecularformula C10H14O2, although the same compound wasdetected in WT (Fig. S3). The antifungal compound 6-pentyl-2H-pyran-2-one (6-PP) has the molecular formulaC10H14O2, and since this compound is known to beproduced by T. atroviride (Mukherjee et al., 2012), thecompound C10H14O2 was tentatively identified as 6-PP.However, extracts from PDA medium colonized by eitherWT or Dmcl contained 6-PP at similar concentrations (Fig.S3a). The identity of 6-PP was further verified by GC-MSanalysis as in Wickel et al. (2013), but with direct injectionof a methanol extract of a culture. The resulting MS datawere in agreement with the data presented by Wickel et al.(2013) (Fig S3b).

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Fig. 3. Colony morphology of T. atroviride strains grown on media containing different non-fermentable and fermentablecompounds as the sole carbon source. The names of the different carbon sources used are given above the figure. Theexperiments were carried out in three biological replicates and photographs of representative plates were taken at 10 dayspost-inoculation.

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Fig. 4. (a) Plate confrontation assay against B. cinerea. The namesof the different carbon sources used are given on the left side ofthe figure. Agar plugs of T. atroviride strains (right side of the plate)and B. cinerea (left side of the plate) were inoculated on oppositesides of 9 cm agar plates and incubated at 25 6C. The experimentwas performed in three replicates and photographs of represen-tative plates were taken at 15 days post-inoculation. (b) Secretionassay of T. atroviride strains. Agar plugs were inoculated onacetate or PDA plates covered with cellophane and incubated at25 6C in darkness. After reaching the same diameter the colonywas removed together with the cellophane disc. Plates werereinoculated with a B. cinerea agar plug and incubated at 25 6C.The growth diameter was recorded at 5 days post-inoculation.Error bars represent SD based on three biological replicates.Different letters (A, B) indicate statistically significant differences(P¡0.05) within media based on the Tukey–Kramer test.

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Deletion of mcl delays root colonization

Ar. thaliana roots were inoculated with T. atroviride conidiaand allowed to interact for 48 h using an in vitro system onPDA plates. Roots were detached from the shoot, washed inwater or sodium hypoclorite and placed on PDA to recoverthe fungus. Growing mycelia were recovered from rootswashed with water and from surface-sterilized roots for WTand mutant strains (data not shown). Furthermore, theroot colonization ability was quantified by measuringT. atroviride and Ar. thaliana DNA with qPCR. Our datashowed a significant (P,0.001) ~90-fold reduction in the T.atroviride Dmcl/Ar. thaliana DNA ratio compared to T.atroviride WT/Ar. thaliana or T. atroviride Dmcl+/Ar.thaliana DNA ratio, suggesting reduced colonization of Ar.thaliana roots by the T. atroviride Dmcl strain (Fig. 5).

Deletion of mcl affects induction of systemicresistance in Ar. thaliana

A plant protection assay in pots was employed to assesswhether the delay in root colonization in the Dmcl strainaffects the ability to induce systemic defence in Ar. thalianaagainst the necrotrophic fungus B. cinerea. Leaves of Ar.thaliana plants, with roots pre-inoculated with either WTor mutant strains 2 weeks before, were inoculated with B.cinerea and the necrotic lesions area was measured 4 dayspost-inoculation. A significant (P,0.001) increment innecrotic leaf lesion area was recorded when plant rootswere colonized by the Dmcl strain in comparison to WT-colonized plant roots, although the lesion area of themutant-strain-inoculated plants was in turn smaller(P,0.001) than the uninoculated control plants (Fig. 6).Recovery of WT, Dmcl and Dmcl+ strains was confirmedfrom surface-sterilized root fragments in selection plates.

DISCUSSION

ICL and MCL are placed in the same ICL superfamily (Liuet al., 2005), and cleave the C-C bond in tricarboxylic acids

at the C2 position; ICL hydrolyses isocitrate to succinateand glyoxylate (Dunn et al., 2009), while MCL cleavesmethylisocitrate to pyruvate and succinate (Brock et al.,2001). The main difference between the two enzymes is thereplacement of conserved amino acid residues phenylala-nine and threonine in the ICL active site by leucine andserine in MCL (Brock, 2005; Muller et al., 2011). This twoamino acid replacement allows acceptance of an additionalmethyl group in MCL (Liu et al., 2005; Gould et al., 2006).In our study, the active site of T. atroviride MCL containsleucine and serine, which indicates MCL activity.

As expected for a key enzyme in the methylcitrate cycle,deletion of mcl in T. atroviride completely inhibits growthon propionate. This shows that the T. atroviride MCLfunctions in the methylcitrate pathway and that ICL is notable to compensate for the function of MCL. Hence, notonly the toxic metabolite propionate-CoA will accumulatein the cells of the mcl deletion strain, but also methylcitratepathway intermediates, such as methylcitrate. Methylcitrateis proposed to inhibit growth and conidiation throughseveral mechanisms: inhibition of the pyruvate dehydro-genase complex; inhibition of aconitase, which may slowdown glycolysis; and by the trapping of oxaloacetate, whichrequires energy-consuming de novo synthesis (Brock &Buckel, 2004; Brock, 2005).

The role of the methylcitrate cycle in bacteria and fungi isthe b-oxidation of propionate to pyruvate. It is similar tothe step in the glyoxylate cycle where acetate is oxidized toglyoxylate. There are several reports that show the uniquefunction of ICL and MCL in their respective pathways(Brock et al., 2001; Brock, 2005; Liu et al., 2005). However,a dual role of ICL is shown in the bacteria M. tuberculosisand M. smegmatis, and the yeast S. cerevisiae (Gould et al.,2006; Munoz-Elıas & McKinney, 2005; Upton &McKinney, 2007). Furthermore, MCS activity from As.

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trovi

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thal

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Fig. 5. Determination of T. atroviride root colonization in Ar.thaliana roots by quantifying DNA level using qPCR. T. atroviride

colonization is expressed as the ratio between T. atroviride DNAand Ar. thaliana DNA. For DNA quantification gpd1 and PR-2 wereused as the target genes for T. atroviride and Ar. thaliana,respectively.

3A

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Fig. 6. Effect of T. atroviride root colonization on systemic defencein Ar. thaliana. Measurement of B. cinerea necrotic lesions onleaves of Ar. thaliana plants with roots colonized by T. atroviride

strains. Water-inoculated Ar. thaliana roots served as thetreatment control. A total of 5�103 B. cinerea conidia wereinoculated on the leaf surface and the area of the necrotic lesionswas determined at 5 days post-inoculation under the microscopeusing a DeltaPix camera and software. The lesion area is presentedin relation to WT-inoculated plants.

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2498 Microbiology 159

nidulans has been shown to be active not only withpropionyl-CoA but also with acetyl-CoA (Brock & Buckel,2004). These previous reports prompted us to analyse thegrowth and development of the Dmcl strain in mediacontaining different non-fermentable or fermentable car-bon compounds as the sole carbon source. In agreementwith the gene expression study where basal expression ofmcl was observed under all growth conditions tested, themcl mutant displayed severely reduced growth rates onmost tested carbon sources. However, this is in contrastwith results in F. graminearum where deletion of theGzMCL1 gene reduced growth rates on glucose but not onacetate or fatty acids (Lee et al., 2009). The induction ofmcl during growth on NAG-containing media may suggesta connection between the methylcitrate cycle and chitincatabolism. Steady-state levels of cellular propionyl-CoAand MCL activities are reported from several filamentousfungi when grown on propionate-free media (Miyakoshi,et al., 1987; Zhang et al., 2004). The traditional use ofpropionate in semi-selective media for isolation of Tricho-derma spp. (Papavizas & Lumsden, 1982) may suggest thatthe methylcitrate cycle is highly efficient in these fungi andtherefore the T. atroviride Dmcl strain is especially sensitiveto internal propionyl-CoA levels.

Trichoderma spp. produce several hydrolytic enzymes andantifungal compounds, such as peptaibols, isoprenoids andpyrones, during the mycoparasitic attack (Druzhinina et al.,2011), and alteration in production in any of thesemetabolites/enzymes may affect the normal activity. Ourresults from the secretion assay show that the Dmcl strainsecretes fewer enzymes or secondary metabolites than theWT or Dmcl+ strains, which allows growth of B. cinerea.The interpretation of these data must take into account thelower growth rate of the Dmcl strain compared with theWT or Dmcl+ strains. At least for the secretion assay onPDA, the difference in growth was very small and B. cinereawas inoculated at the centre of the agar plate that wasexposed to T. atroviride during the whole experimentalperiod, which suggests that the effect is not due to thedifference in growth rate.

We found that production of a secondary metabolite,putatively identified as 6-PP, was altered in the Dmcl strainduring growth on acetate medium, but not on PDAmedium. It is therefore unlikely that 6-PP is the onlycompound responsible for the reduced antagonism in thesecretion assay, as reduced antagonism was observed onboth growth media. Disruption of the methylcitratepathway in As. nidulans results in increased propionyl-CoA levels that inhibit polyketide synthesis (Brock &Buckel, 2004; Zhang et al., 2004; Zhang &Keller, 2004).Our data show that impairment of the methylcitrate cycleresults in delayed conidial pigmentation in the Dmcl strainon media that contain non-fermentable carbon com-pounds as the sole carbon source. This phenotype issimilar to those observed for MCL deletions in As. nidulans(Brock, 2005; Brock & Buckel, 2004; Zhang et al., 2004;Zhang & Keller, 2004), and double deletion of GzICL1 and

GzMCL1 in F. graminearum (Lee et al., 2009), whereproduction of secondary metabolites related with conidialpigmentation is inhibited. Again, this indicates that thesecondary metabolite production in T. atroviride isinfluenced by the methylcitrate cycle. Secondary metaboliteproduction in fungi is in turn coupled with morphologicaldevelopment including sporulation and pigmentation(Calvo et al., 2002).

In agreement with published reports (Contreras-Cornejoet al., 2011; Salas-Marina et al., 2011), our results showsystemic protection against B. cinerea in WT-colonized Ar.thaliana plants compared with control plants. However,this effect is compromised, but not abolished, in plantscolonized by the Dmcl strain. It is possible that the reducedability of the Dmcl strain to induce systemic defence isrelated to the delay in root colonization.

ACKNOWLEDGEMENTS

This work was financially supported by the Department of Forest

Mycology and Plant Pathology, and the Swedish Research Council for

Environment, Agricultural Sciences and Spatial Planning (FORMAS;

grant number 229-2009-1530).

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Edited by: A. Herrera-Estrella

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2500 Microbiology 159