Trichoderma Histone Deacetylase HDA-2 Modulates Multiple ...Trichoderma Histone Deacetylase HDA-2 Modulates Multiple Responses in Arabidopsis1 Magnolia Estrada-Rivera,a,2 Oscar Guillermo

Trichoderma Histone Deacetylase HDA-2 ModulatesMultiple Responses in Arabidopsis1

Magnolia Estrada-Rivera,a,2 Oscar Guillermo Rebolledo-Prudencio ,a,2 Doris Arisbeth Pérez-Robles ,a

Ma. del Carmen Rocha-Medina,b María del Carmen González-López ,a and Sergio Casas-Floresa,3,4

aDivisión de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica (IPICYT), Caminoa la presa San José No. 2055, Colonia Lomas 4a sección. C.P. 78216, San Luis Potosí, MexicobLaboratorio Nacional de Biotecnología Agrícola, Médica y Ambiental, Instituto Potosino de InvestigaciónCientífica y Tecnológica (IPICYT), Camino a la presa San José No. 2055, Colonia Lomas 4a sección. C.P. 78216,San Luis Potosí, Mexico

ORCID IDs: 0000-0002-3579-2111 (M.E.-R.); 0000-0002-1042-5630 (O.G.R.-P.); 0000-0002-8314-9796 (M.d.C.R.-M.); 0000-0002-5407-4158(M.d.C.G.-L.); 0000-0002-9612-9268 (S.C.-F.).

Trichoderma spp. are a rich source of secondary metabolites and volatile organic compounds (VOCs), which may induce plantdefenses and modulate plant growth. In filamentous fungi, chromatin modifications regulate secondary metabolism. In thisstudy we investigated how the absence of histone deacetylase HDA-2 in the Trichoderma atroviride strain Dhda-2 impacts its effecton a host, Arabidopsis (Arabidopsis thaliana). The production of VOCs and their impact on plant growth and development wereassessed as well. The Dhda-2 strain was impaired in its ability to colonize Arabidopsis roots, thus affecting the promotion of plantgrowth and modulation of plant defenses against foliar pathogens Botrytis cinerea and Pseudomonas syringae, which normallyresult from interaction with T. atroviride. Furthermore, Dhda-2 VOCs were incapable of triggering plant defenses to counterattackfoliar pathogens. The Dhda-2 overproduced the VOC 6-pentyl-2H-pyran-2-one (6-PP), which resulted in enhanced root branchingand differentially regulated phytohormone-related genes. Analysis of ten VOCs (including 6-PP) revealed that three of thempositively regulated plant growth, whereas six had the opposite effect. Assessment of secondary metabolites, detoxification, andcommunication with plant-related genes showed a dual role for HDA-2 in T. atroviride gene expression regulation during itsinteraction with plants. Chromatin immunoprecipitation of acetylated histone H3 on the promoters of plant-responsive genes inDhda-2 showed, in the presence of Arabidopsis, low levels of epl-1 and abc-2 compared with that in the wild type; whereas ctf-1 presented high constitutive levels, supporting a dual role of HDA-2 in gene regulation. This work highlights the importance ofHDA-2 as a global regulator in Trichoderma to modulate multiple responses in Arabidopsis.

Phytohormones are small, signaling molecules,which occur at low concentrations and play pivotalroles in plants, including influencing plant growth anddevelopment. Classical phytohormones comprise auxins,abscisic acid, cytokinins, ethylene (ET), and gibberellicacid. However, brassinosteroids, jasmonates (JAs), andsalicylic acid (SA) are considered phytohormones as well.These signaling molecules not only integrate, but also

transmit environmental signals and modulate responsesto abiotic and biotic stresses (Pieterse et al., 2009; DeBruyne et al., 2014). In their natural environments, plantsinteract with a plethora ofmicroorganisms, establishingpathogenic or beneficial relationships. The plant re-sponse against these microbes lies primarily in an arrayof structural barriers or inducible defenses. The plantimmune system has the ability to perceive nonself byrecognizing pathogen- or microbe-associated molecu-lar patterns (PAMPs or MAMPs, respectively), andtranslate this perception into an appropriate adaptiveresponse (Pieterse et al., 2009). Plants have developedthe ability to enhance their basal resistance after PAMPsor MAMPs are detected, by triggering the systemicacquired resistance (SAR) or the induced systemic re-sistance (ISR), which are phenotypically similar butdifferent at the biochemical level. The SAR is associatedwith the accumulation of SA, and it is accompanied bythe direct activation of pathogenesis-related proteins,encoding genes (Dong, 2004); whereas in the ISR, JAand ET are accumulated, modulating the expression ofthe pathogen-inducible genes HEL (Potter et al., 1993),CHIB (Samac et al., 1990), and PDF1.2 (Penninckx et al.,1996). The SA-related defense response is triggeredagainst biotrophic and hemibiotrophic phytopathogens

1This work was supported by the National Council of Science andTechnology, Mexico grants to S.C.-F. (CB–2013–01–220791 and IFC2016–1538). Additionally, there were grants to M.E-R (193931),M.d.C.G.-L. (247751), and O.G.R.-P. (206894), who are indebted toCONACYT for doctoral fellowships.

2These authors contributed equally to this article.3Author for contact: [email protected] author.The author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Sergio Casas-Flores ([email protected]).

M.E.-R., O.G.R.-P, and S.C.-F. planned and designed the research.M.E.-R., O.G.R.-P., M.d.C.G.-L., and D.A.P.-R. performed the exper-iments. M.E.-R., O.G.R.-P. and M.d.C.R.-M. analyzed data. M.E.-R.and S.C.-F. wrote the manuscript with contributions from all authors.

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Plant Physiology�, April 2019, Vol. 179, pp. 1343–1361, www.plantphysiol.org � 2019 American Society of Plant Biologists. All Rights Reserved. 1343

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that feed on living host tissue, such as Pseudomonassyringae (Dong, 2004); whereas the JA/ET-related de-fense response is boosted by necrotrophic microor-ganisms, such as Botrytis cinerea (Pieterse et al., 1996),which kill host tissue at early stages of the invasion.Fungi belonging to the plant-beneficial Trichodermagenus are cosmopolitan inhabitants of soil. SomeTrichoderma species confer beneficial effects to plants bymeans of mycoparasitism, and the synthesis of antibi-otics against phytopathogens, inducing systemic dis-ease resistance, and promoting growth and fitness(Shoresh et al., 2010; Hermosa et al., 2012; Olmedo-Monfil and Casas-Flores, 2014). During root coloniza-tion, Trichoderma spp. produce and secrete a diversity ofMAMPs, such as xylanases (EIX), cellulases, and theproteinaceous Sm1/Epl1 (Martinez et al., 2001; Rotblatet al., 2002; Djonović et al., 2006; Salas-Marina et al.,2015) or secondary metabolites (SMs) such as alame-thicin and trichokonin (Engelberth et al., 2001; Luoet al., 2010). Trichoderma MAMPs are specifically rec-ognized by triggering simultaneously SAR and ISRresponses (Salas-Marina et al., 2011; Perazzolli et al.,2012). The plant growth promotion capability ofTrichoderma has been linked to its ability to colonize theplant roots (Hermosa et al., 2013). Some of the Tricho-derma mechanisms to promote plant growth includesynthesis of phytohormones, solubilization of soil nu-trients, increased uptake and translocation of nutrients,and enhanced root development (Baker, 1989; Harman,2000, 2006). VOCs released by Trichoderma spp. play apivotal role in plant growth promotion as well (Hunget al., 2013; Lee et al., 2015, 2016). These “semi-ochemicals” are low molecular mass and usually hy-drophobic compounds with high vapor pressure(Effmert et al., 2012). Approximately 479 TrichodermaVOCs have been reported (Siddiquee, 2014). One of themost common SMs is 6-PP, and the responsible VOC forthe coconut odor in some Trichoderma species. Plantgrowth is promoted by 6-PP, and it regulates rootarchitecture, inhibiting primary root growth and in-ducing lateral root formation (Vinale et al., 2008;Garnica-Vergara et al., 2016 Kottb et al., 2015). VOCsappear during both primary and secondarymetabolism(from intermediates of the primary metabolism; Korpiet al., 2009). The genes encoding for enzymes involvedin the synthesis of SMs are typically arrayed in geneclusters in filamentous fungi (Keller and Hohn, 1997;Walton, 2000; Keller et al., 2005), frequently regulatedby chromatin modifications, such as histone acetylationand deacetylation, performed by histone acetyltrans-ferases and histone deacetylases (HDACs) activity(Tribus et al., 2005; Shwab et al., 2007; Lee et al., 2009). Itis well known that addition of acetyl groups to histonesby histone acetyltransferases promote a relaxed chro-matin state, leading to gene expression, whereas re-moval of such groups by HDACs drives chromatincompaction and represses transcription. In this regard,it has been reported that HDACs are involved ingrowth and development, synthesis of SM, viru-lence, and invasive growth of plant pathogenic fungi

(Baidyaroy et al., 2001; Lee et al., 2009; Ding et al., 2010;Tribus et al., 2010). The histone deacetylase hda-2 en-coding gene from Trichoderma atroviride can be inducedby light and reactive oxygen species (ROS). Its productregulates growth, conidiation, blue-light perception,and oxidative stress responses (Osorio-Concepciónet al., 2017). In this work, we study the T. atroviridehda-2 deletion mutant (Dhda-2), whose gene codes in thewild type for the histone deacetylase HDA-2, during itsinteraction with Arabidopsis (Arabidopsis thaliana) plants.We show that Dhda-2 is impaired in its ability to colo-nize Arabidopsis roots, as well as in triggering SAR andISR responses by direct contact or through VOCs. Ananalysis of the root system architecture of Arabidopsisshowed that the presence of Dhda-2 or its VOCs pro-moted a strong lateral root branching. We character-ized the VOCs profile emitted by Dhda-2 using gaschromatography-mass spectrometry (GC-MS). Inter-estingly, the absence of hda-2 impaired the VOCs me-tabolism, resulting in an overproducing-6-PP strain. Invitro assays in a medium amended with 6-PP (or sup-plied as VOC) demonstrated that the plant growth-promoting effect is dependent on the Arabidopsisage and the application protocol. Split-plate assayswith nine different VOCs resulted in the discovery ofthree different VOCs with a plant growth-promotingeffect in Arabidopsis seedlings, whereas six of themprovoked the opposite effect. Transcription analysesand chromatin immunoprecipitations of acetylatedhistone H3 of plant-responsive genes in T. atrovirideshowed that HDA-2 could be regulating these genesdirectly or indirectly by its HDAC activity. Togetherthese results indicate that HDA-2 is a global regulatorin T. atroviride, which modulates multiple responsesin Arabidopsis.

RESULTS

Root Treatment of Arabidopsis with Dhda-2 Increases theLateral Root Number

To investigate whether the product of the hda-2 geneis involved in the interaction of the fungus with Ara-bidopsis, plants were root inoculated with T. atroviride.The mycelium was collected at the indicated times ofcoculture, total RNA was extracted, and the transcriptlevel of hda-2was determined. ThemRNA level of hda-2was induced early (24 h) by the presence of Arabidopsis(Supplemental Fig. S1).

To analyze in vitro the effect of Dhda-2 on the rootsystem architecture of Arabidopsis, plants were inoc-ulated with the wild type or Dhda-2. Plants treated witheither the wild type or Dhda-2 showed a statisticallysignificant increase in lateral root number; however, theDhda-2-inoculated seedlings showed more lateral rootsthan that of the wild-type–treated plants (Fig. 1, A andB). Primary root length of plants treated with the wildtype was barely but significantly larger than thosetreated with Dhda-2 (Fig. 1C); however, no significant

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differences were observed in fresh weight of plantstreated with these fungi (Fig. 1D). Moreover, dryweight of the wild-type–treated plants was higherthan that of Dhda-2-treated seedlings, and the lattershowed higher dry weight than that of control plants(Fig. 1E). These data show that the balance betweenthe extension of the primary root and production oflateral roots is responsible for biomass gaining inplants inoculated with Dhda-2.

Histone Deacetylase HDA-2 from T. atroviride IsNecessary to Effectively Colonize and Promote PlantGrowth in Arabidopsis

To study the role of the histone deacetylase HDA-2from T. atroviride on the promotion of plant growth in

pots, and to determine if it is compatible with thein vitro approach, Arabidopsis seedlings grown in potswere root inoculated with mycelia of the T. atroviridewild type or Dhda-2. Plants inoculated with Dhda-2showed diminished fresh and dry weights comparedwith those treated with the wild type; however, theplants treated with Dhda-2 showed significantly en-hanced growth comparedwith that of the control plants(Fig. 2, A to C). To investigate the capability ofDhda-2 tocolonize Arabidopsis roots, seedlings were root inocu-lated with the wild type or Dhda-2. Thereafter, the rootswere detached; total DNA was extracted, and abun-dance of the T. atroviride tef-1 gene versus theArabidopsisACT2 gene were quantified by real-time polymerasechain reaction (PCR; quantitative PCR, qPCR). Dhda-2capability to colonize the Arabidopsis roots was im-paired compared with that of the wild type (Fig. 2D).

Deletion of hda-2 in T. atroviride Compromises itsCapability to Induce the Arabidopsis Systemic DiseaseResistance Against Foliar Pathogens

To determine if HDA-2 is necessary to elicit the plantdefense responses by Trichoderma, the expression pro-files of the well-known Arabidopsis marker genes PR-1a (SAR) and PDF1.2 (ISR) were assessed by reversetranscription-qPCR (RT-qPCR). The expression of PR-1a and PDF1.2 was strongly induced by the wild type,whereas Dhda-2 barely triggered the expression ofPDF1.2 at 72 h and 96 h (Fig. 3, A and B). Indeed,mutant-induced expression of PR-1a was not detectedat any tested time (Fig. 3, A and B).Based on the expression analysis of PR-1a and PDF1.2

in response to Dhda-2, we asked whether the mutantprovides protection against the fungal pathogen B. cinereaand the bacterial pathogen Pst DC3000. Plants treatedwith Dhda-2 exhibited an enhanced disease susceptibilityto both pathogens compared with that in plants treatedwith the wild type (Fig. 3, C to E); however, in the case ofB. cinerea, the plants treatedwith themutant did not reachthe disease susceptibility of the Trichoderma untreatedplants (Fig. 3, C and D). On the other hand, the wild-type–treated seedlings postinoculated with Pst DC3000showed 3.895 3 106 61.054 3 106 colony forming units(CFU)/mL; whereas plants treated with Dhda-2 and in-oculated with the bacterial pathogen showed similarlevels of CFU/mL (7.985 3 106 6 1.753 106) as that forthe control seedlings (9.2323 106 6 2.4543 106; Fig. 3E).

The Dhda-2 VOCs Enhance the Growth ofArabidopsis Seedlings

To assess whether the VOCs of Dhda-2 promotegrowth in Arabidopsis, seedlings were grown on MSand cocultured with mycelia of the wild type or Dhda-2grown in small plates within a large MS plate (Fig. 4A).Plants exposed to VOCs of Dhda-2 exhibited a significantincrease in lateral root number (Fig. 4B) and in fresh and

Figure 1. Dhda-2 increases the lateral root number in Arabidopsis.Eleven-day-old Arabidopsis seedlings grown on MS medium werecoincubated for 10 d with the wild type (WT) or Dhda-2. A, Repre-sentative pictures of Arabidopsis grown under the indicated treatments.Bar = 1.5 cm (applies to all images). B, Lateral root number. C, Rootlength. D, Fresh weight. E, Dry weight. Data from B to E show the mean6SD of two technical replicates (10 plates with 12 plants each). Theexperiment was repeated twice with similar results. Results were vali-dated with an ANOVA statistical analysis using a Tukey multiple com-parison test (a = 0. 05). Lower case letters a, b, and c (B to E) representmeans statistically different at the 0.05 level.

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dry weights (Fig. 4, D and E) compared with that inplants exposed to the wild-type VOCs, whereas nodifferences in primary root length inhibition were ob-served with both strains (Fig. 4C).

The Dhda-2 VOCs Do Not Trigger the SAR and ISRResponses in Arabidopsis

We next tested the effect of Dhda-2 VOCs on thetriggering of ISR and SAR responses in Arabidopsisseedlings. The VOCs of the wild type increased theexpression levels of both PR-1a and PDF1.2 (Fig. 5, Aand B), although to a lesser extent compared with thatof the root-inoculated plants (Fig. 3, A and B). Similar tothe results of the Dhda-2 root-inoculated Arabidopsisseedlings (Fig. 3, A and B), the plants exposed to Dhda-2VOCs barely induced PR-1a or PDF1.2 at any of thetested times (Fig. 5).

The Expression of Auxin- and ET-Related Genes IsDifferentially Modulated in Arabidopsis by Dhda-2 DirectContact or by Exposure to its VOCs

We analyzed the expression of ET- and auxin-relatedgenes in Arabidopsis. Arabidopsis seedlings wereroot inoculated or exposed to the wild-type or Dhda-2VOCs for 24, 48, 72, and 96 h. Overall, the auxin-relatedgenes TIR1 (auxin receptor), AUX1 (auxin importer),and PIN3 and PIN7 (auxin efflux carriers) were upre-gulated to a different extent in the wild-type or Dhda-2

root-treated plants (with the exception of PIN7, whichwas downregulated by the presence of Dhda-2; Fig. 6, Ato D). Exposure of Arabidopsis to the wild-type VOCsupregulated TIR1 at 24, 48, and 72 h, but TIR1 expres-sion downregulated at 96 h, whereas Dhda-2 VOCsbarely induced that gene. Furthermore, AUX1 wasbarely induced by both thewild-type andDhda-2VOCs,whereas PIN3 and PIN7were upregulated by the wild-type and Dhda-2 VOCs to a different extent (Fig. 6, A toD). On the other hand, the ET pathway-related genesACO2 [1-aminocyclopropane-1-carboxylic acid (ACC)oxidase], ETR1 (ET receptor 1), ERS1 (ET responsesensor 1), and EIN2 (ET insensitive 2) were upregulatedby the presence of the wild type, whereas roots exposedto Dhda-2 induced EIN2 at 24 h, although ACO2, ETR1,and ERS1were barely induced or unaltered (Fig. 6, E toH). Exposure of Arabidopsis to the wild-type VOCs ledto the up-regulation of ETR1 and EIN2, whereas ERS1and ACO2 showed basal levels. Dhda-2 VOCs-treatedseedlings showed basal or barely up-regulation of allfour ET pathway-related genes (Fig. 6, E to H).

The Dhda-2 Strain Overproduces 6-PP

To analyze the VOCs produced by Dhda-2 and tocompare them with those of the wild type, fresh inoc-ulums of both strains were grown on MS plates for 5and 6 d. The analysis of VOCs was performed throughGC-MS. The VOCs identified were assigned to alcohols,ketones, unknown terpenes, and pyrones. Two unknownterpenes and 6-PP (Table 1) were present in both strains.

Figure 2. Histone deacetylase HDA-2 is necessary in T. atroviride for effective colonization and growth promotion in Arabi-dopsis. Ten-day old Arabidopsis seedlings grown in soil were inoculated with mycelium of the wild type (WT) or hda-2 andcocultured for three weeks for plant growth promotion assays or for twoweeks for root colonization. A, Representative pictures offour-week-old Arabidopsis plants inoculated with mycelium of the wild type or Dhda-2. Bar = 1.5 cm (applies to all images). B,Fresh weight. C, Dry weight. D, Arabidopsis root colonization by Trichoderma quantified by qPCR. The fungal DNA wasquantified by real-time PCR (qPCR) using the T. atroviride tef-1 gene versus the Arabidopsis ACT2 gene. Photographs showrepresentative individuals of 24 plants. Data from B and C show the mean6SD of one technical replicate (12 groups with 2 plantsin each one). The experiment was repeated three times with similar results. Data from D show the mean 6SD of two technicalreplicates (16 pooled plants for treatment). Results were validatedwith an ANOVA using a Tukey multiple comparison test (a = 0.05). Lower case letters a, b, and c (B to D) represent means statistically different at the 0.05 level.



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The 1-octen-3-ol and an unknown terpenewas only foundin the wild type, whereas the 2-undecanone, unknownketone, two unknown terpenes, and b-curcumene wereonly detected in Dhda-2 (Table 1). Furthermore, based onthe percentage of relative area, 6-PP together with an un-known terpene were the most abundant VOCs producedby Dhda-2 and the wild type grown on MS for 5 and 6 d(Table 1). This was confirmed by quantification of 6-PP,whereby Dhda-2 synthesized 970.4 and 847.7 mg mL21 atday 5 and 6, respectively, whereas thewild type produced129.1 and 89.9 mg mL21 by the same time (Table 2).

The Effect of 6-PP on Root System Architecture Dependson the Plant Age and the Application Method

To evaluate the effect of 6-PP on the plant’s growthpromotion and the root systemarchitecture ofArabidopsis,

6-PP was supplied into the medium or provided asvolatile in split-petri dishes (Fig. 7, A and B). Two-day-old Arabidopsis plants were treated with ethanol(control treatment) or with 25, 50, 75, and 100 mM 6-PP(4.15, 8.30, 12.46, and 16.62 mg mL21 respectively).After 13 d of treatments with 6-PP, lateral root numberwas increased in a dose-dependent manner (Fig. 7, Ato C). A strong primary root growth inhibition wasdetected when 50, 75, and 100 mM of 6-PP were sup-plied in the growing medium. A similar behavior wasobserved when 6-PP was applied as VOC, but, to aminor extent, at concentrations from 25 to 100 mMof 6-PP (Fig. 7, A, B, and D). Regarding the fresh weight,both treatments followed a similar behavior, exceptwhen 50 mM was applied as a volatile or in thegrowing medium, showing a better biomass gainwhen themediumwas amendedwith 6-PP (Fig. 7E). Incontrast, the lateral root number and dry weight of

Figure 3. Deletion of hda-2 in T. atroviride compromises the induction of Arabidopsis systemic disease resistance against foliarpathogens. A and B, Ten-day-old Arabidopsis seedlings grown on MS medium were root-inoculated with the wild type (WT) orDhda-2, and the expression levels of PR-1a (A) or PDF1.2 (B) were analyzed by RT-qPCR at 24, 48, 72, and 96 hpi. RT-qPCR resultsare reported as fold-change expression compared with that in Arabidopsis grown without the fungi. Arabidopsis ACT2 gene wasused as control to normalize the expression of PR-1a and PDF1.2 using the 2-DDCt method. C, Representative images of leaves from10-d-old Arabidopsis seedlings grown in soil that were inoculatedwith the wild type or Dhda-2; and twoweeks later, leaves wereinfected with B. cinerea or the inoculating buffer as a control. Bar = 1.5 cm (applies to all images). D, Lesion sizes of infectedplants with B. cinerea were determined using ImageJ at 6 dpi. E, CFU of Pst DC3000 at 0 and 3 dpi in leaves of treated anduntreated plants with the wild type or Dhda-2. Data from A and B show the mean 6SD of one technical replicate (5 plates with7 plants each). The experimentwas repeated twicewith similar results. Data fromD show themean6SD of one technical replicate(12 leaves each). The experiment was repeated three times with similar results. Data from E show the mean6SD of one technicalreplicate (12 leaves each). The experiment was repeated twice with similar results. Asterisks indicate significant difference(independent Student’s t test, *P , 0.05 and **P , 0.01).



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plants treated with 6-PP, supplied as volatile, did notdiffer significantly between 25, 50, 75 mM in that order,whereas 100 mM caused a negative effect on plantgrowth. However, plants treated with 75 mM showedsignificant differences in biomass gain compared withthat of 25 mM-treated seedlings (Fig. 7, B, C, E, and F).To determine whether the age of Arabidopsis plays akey role in the plant growth promotion effect by 6-PP,7-d-old Arabidopsis plants were exposed to 25, 50, 75,and 100 mM of 6-PP or ethanol as a control. After 13 d,the treated plants showed an increased lateral rootnumber (Fig. 7, G and H), whereas the primary rootgrowth was inhibited in a dose-dependent manner(Fig. 7I). However, we observed a dry weight gainonly at 50 and 75 mM of 6-PP (Fig. 7K). In summary,the reduction in primary root length and the aerial part

of the plant by 6-PP seems to be compensated by anincrease in lateral root number, leading to a biomassgain at the indicated concentrations.

2-heptanol, 2-heptanone, and 3-octanol VOCs PromotedPlant Growth in Arabidopsis Seedlings

Next, we analyzed the VOCs profile produced by theDhda-2 and wild-type strains grown on PDA. To thisend, both strains were grown for 5 to 7 d. A total of28 VOCswere detected. The VOCswere assigned to thecompounds classes of alcohols, ketones, mono- di- andsesquiterpenes, alkanes, and pyrones (Table 1). Themost abundant compounds for the wild type were theketones 2-heptanone and 3-octanone, whereas forDhda-2 it was 6-PP. Four exclusive VOCs were identified for thewild type, comprising 3-octanone, 2-heptanol, 3-octanol,and 1-octen-3-ol (Table 1), whereas 17 volatileswere found

Figure 4. The VOCs of Dhda-2 enhance plant growth in Arabidopsis.Eleven-day-old Arabidopsis seedlings grown on a MS medium wereexposed to the wild type (WT) or Dhda-2 VOCs for seven days. A,Representative pictures of Arabidopsis grown under the indicatedtreatments. Bar = 1.5 cm (applies to all images). B, Lateral root number.C, Root length. D, Fresh weight. E, Dry weight. Data from B to E showthe mean6SD of two technical replicates (6 plates with 12 plants each).Results were validatedwith an ANOVA statistical analysis using a Tukeymultiple comparison test (a = 0. 05). Lower case letters a, b, and c (B toE) represent means statistically different at the 0.05 level.

Figure 5. Dhda-2 VOCs fail to properly induce ISR and SAR in Arabi-dopsis. A and B, Ten-day-old Arabidopsis seedlings grown on petriplates containing MS were exposed to the wild-type (WT) or Dhda-2VOCs. A and B, The expression levels of defense-related genes PR-1a (A)and PDF1.2 (B) were analyzed by RT-qPCR. Results are reported as fold-change expression compared with that in Arabidopsis grown withoutthe fungi. The Arabidopsis ACT2 gene was used as a control to nor-malize the expression using the 2-DDCt method. Data from A and B showthe mean 6SD of one technical replicate (5 plates with 7 plants each).The experiment was repeated twice with similar results. Asterisks in-dicate significant difference (independent Student’s t test, *P, 0.05 and**P , 0.01).



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Figure 6. The expression of auxin and ET synthesis and perception genes are altered in Arabidopsis by direct contact withDhda-2or exposure to its VOCs. A to H, Ten-day-old Arabidopsis seedlings grown on petri plates containing MSwere inoculated with thewild type (WT) or Dhda-2 (direct contact), or exposed to their VOCs. The expression levels of TIR1 (A), AUX1 (B), PIN3 (C), PIN7(D), ACO2 (E), ETR1 (F), ERS1 (G), and EIN2 (H) were analyzed by RT-qPCR. Results are reported as fold-change expressioncompared with that in Arabidopsis grown without the fungi. The Arabidopsis ACT2 gene was used as control to normalize theexpression using the 2-DDCt method. Data from A to H show the mean6SD of one technical replicate (5 plates with 7 plants each).



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in Dhda-2, including six unknown terpenes, 2-octanone,three unknown ketones, g-terpinene, a-zingiberene,b-sesquiphellandrene, two unknown alcohol, one un-known alkane, and one unknown phenol (Table 1).Compounds such as 2-heptanone, 2-pentylfuran, 2-nonanone, unknown terpene, 2-undecanone, 6-PP,and b-curcumene were common to both the wild typeand Dhda-2 (Table 1). Because Arabidopsis exposed toDhda-2 VOCs displayed a remarkable phenotype in plantgrowth, we decided to investigate the VOCs profile ofDhda-2 growing onPDA inmore detail.We found strikingdifferences in the production of 2-heptanol, 3-octanol, 1-octen-3-ol, 2-heptanone, 2-octanone, 3-octanone, 2-nonanone, 2-undecanone, and 2-pentylfuran betweenthewild type andDhda-2 (Tables 1 and 2). Therefore, thesecompounds were tested in plant-growth assays in vitro.Arabidopsis plants were exposed to individual VOCs atfour different concentrations: 10, 100, 1000, and 10,000 mgmL21. Addition of 2-heptanol (100 and 1000mgmL21), 3-octanol (100 mg mL21), and 2-heptanone (10, 100, and1000) as VOCs led to a significant effect on plant growth(Fig. 8 ). In contrast, 3-octanol, 1-octen-3-ol, 2-octanone, 3-octanone, 2-nonanone, 2-undecanone, and 2-pentylfuranwere highly phytotoxic at 10,000 mg mL21; the plantsshowed a yellow color and a bleaching phenotype(Fig. 8 ). Most of the VOCs did not result in a growtheffect at 10 mg mL21, whereas at 1000 mg mL21 theplants started to show a detrimental effect (Fig. 8).Interestingly, 2-heptanol and 2-heptanone were theonly VOCs that did not bleach the plants at 10,000 mgmL21 (Fig. 8, A and B).

Dhda-2 Has Misregulated Expression of SecondaryMetabolism-, Defense-, and PlantCommunication-Related Genes

To knowwhether Dhda-2 is affected in the expressionof genes described as important in Trichoderma to es-tablish a beneficial relationshipwith plants, aswell as inthe synthesis of SM, Dhda-2, and the wild type werecocultured with Arabidopsis seedlings for 72 and 96 h.Then, we analyzed the expression of genes involved indifferent traits of T. atroviride, such as communicationwith the plant (epl-1, epl-2, and pbs-1; Fig. 9, A to C),defense against toxic compounds (abc-2; Fig. 9D), andsynthesis of SM (ctf-1, tps-2, pbs-1, ggp-1, and fpp-1;Fig. 9, E to H). Indeed, epl-1, epl-2, pbs-1 (Fig. 9, A to C),and abc-2 (Fig. 9D) showed reduced expression levels inDhda-2 in the presence of Arabidopsis seedlings com-pared with that in the wild type, whereas ctf-1, tps-2,pbs-1, ggp-1, and fpp-1 showed enhanced levels oftranscription in Dhda-2 compared with that in the wildtype (Fig. 9, E to H).

HDA-2 Is Required in T. atroviride for Proper Acetylationof Histone H3 on the Promoter Region ofPlant-Responsive Genes

Chromatin immunoprecipitation (ChIP) assays wereperformed to determine the acetylation pattern of his-tone H3 at Lys-9/Lys-14/Lys-18/Lys-23/Lys-27 on thepromoter regions of epl-1, ctf-1, and abc-2(Supplemental Fig. S3) in the wild-type and Dhda-2backgrounds in the presence or absence of Arabi-dopsis. The coculture of the wild type with Arabidopsisincreased the acetylation of histone H3 to 19.88%,6.37%, and 21.38% on the promoters of epl-1 (Fig. 10A),ctf-1 (Fig. 10B), and abc-2 (Fig. 10C), respectively, com-paredwith that in the wild type grown in the absence ofthe plant (Fig. 10). However, histone H3 acetylation onepl-1 (Fig. 10A), ctf-1 (Fig. 10B), and abc-2 (Fig. 10C)promoters in Dhda-2 was barely higher when grownalone compared with that in the wild type (Fig. 10);however, during Dhda-2 interaction with plants, epl-1,ctf-1, and abc-2 histone H3 acetylation dropped to1.83%, 2.55%, and 2.81%, respectively, as comparedwith that in the wild type grown under the same con-ditions (Fig. 10).

DISCUSSION

Here, the hda-2 gene, which codes for an HDAC classI in T. atroviride, was induced by the presence of Ara-bidopsis. This result may indicate that the product ofthis gene could be involved in the Arabidopsis–T.atroviride interaction. Indeed, the absence of hda-2 inTrichoderma led to the loss of some of its beneficial ef-fects on Arabidopsis. For instance, Dhda-2 showed areduced effect in promoting plant growth and biomassgain, both when grown in vitro or in pots. Thisprompted us to hypothesize whether Dhda-2 was af-fected in its capability to colonize Arabidopsis roots.Root colonization assays revealed that Dhda-2 does notcolonize properly the plant roots. This evidence sug-gests that HDA-2 plays an important role in plant rootcolonization. In this regard, it has been reported forseveral phytopathogens that mutants in the hda-2 geneputative orthologwere strongly affected in virulence, asa result of reduced penetration efficiency (Proctor et al.,1995; Baidyaroy et al., 2001; Ding et al., 2010; Li et al.,2011). Furthermore, Dhda-2 is highly sensitive to oxi-dative stress (Osorio-Concepción et al., 2017), whichmay affect the colonization process.

At the beginning of plant root colonization by Trich-oderma, the fungus is recognized as foreign through itsMAMPs by the plant, triggering systemic disease re-sistance (Djonović et al., 2006; Viterbo et al., 2007;

Figure 6. (Continued.)The experiment was repeated twice with similar results. Asterisks indicate significant difference (independent Student’s t test, P,0.05); (*) significant difference between the control versus the wild type and versusDhda-2; (**) significant difference between thewild type versus Dhda-2.



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Brotman et al., 2008; Morán-Diez et al., 2009; Salas-Marina et al., 2015). Importantly, some of theseMAMPs are also proteins (swollenins, endopolyga-lacturonases, and hydrophobins) that work as coloni-zation factors (Brotman et al., 2008; Morán-Diez et al.,2009; Guzmán-Guzmán et al., 2017) or help the fungusto tolerate antimicrobial compounds accumulated bythe plant (Ruocco et al., 2009). Here, Dhda-2 was af-fected in its capability to induce the SAR and ISR inArabidopsis, probably as a consequence of its impair-ment to colonize Arabidopsis roots, and therefore wasunable to induce protection against the biotrophic andnecrotrophic pathogens Pst DC3000 and B. cinerea.Based on these results, it is tempting to hypothesizethat HDA-2 is a positive regulator of MAMPs and/orcolonization factors recognized as elicitors of plantdefenses and transporters to tolerate the plant antimi-crobial compounds as well. Indeed, Dhda-2 was se-verely affected in the expression of epl-1, -2, and pbs-1,which code for the cerato-platanin elicitor proteins -1and -2, and for the peptaibol synthetase PBS-1, respec-tively. These proteins are involved in the induction ofISR and SAR in plants (Djonović et al., 2006; Viterbo

et al., 2007; Salas-Marina et al., 2015). These results ex-plain in part the loss of induction of ISR and SAR inArabidopsis against the foliar pathogens B. cinerea andPst DC3000. Furthermore, the abc-2 gene that codes foran ABC transporter, involved in cell detoxification,showed dropped transcription levels, which could ex-plain the diminished capability of Dhda-2 to colonizeArabidopsis roots.Recently, it was reported that hda-2 codes for a class I

HDACs, such as HDA-2 and Hos2p of N. crassa and S.cerevisiae, respectively, which putatively remove acetylgroups from histone tails, leading to a compactedchromatin and therefore to gene repression (Osorio-Concepción et al., 2017). Interestingly, the presence ofArabidopsis increased the acetylation of H3 on thepromoters of epl-1, ctf-1, and abc-2; however, the acet-ylation of H3N-terminal tail on the promoters ofDhda-2was reduced, which suggests a positive role of HDA-2on the transcription of epl-1 and abc-2, as recentlyreported for blue light-responsive genes (Osorio-Concepción et al., 2017), and for HDA-2 putativeorthologs Rpd3 and Hos2p in S. cerevisiae (De Nadalet al., 2004; Sharma et al., 2007). In agreement with

Table 1. VOCs of the wild type and Dhda-2 detected by GC-MS in 1 3 MS and PDA

Mean values 6SE of the sum of three independent determinations are given. (–), No data; Rt, retention time.

Compound Name Rt (min)

Relative Area %

MS 1 3 PDA

Wild Type Dhda-2 Wild Type Dhda-2

5 dpi 6 dpi 5 dpi 6 dpi 5 dpi 7 dpi

2-heptanone 9.453 – – – – 46.08 6 0.02 0.20 6 0.00Unknown terpene 9.966 – – – – – 0.14 6 0.002-pentylfuran 12.976 – – – – 0.05 6 0.00 2.40 6 0.003-octanone 14.288 – – – – 20.12 6 0.03 –2-octanone 15.952 – – – – – 0.29 6 0.00Unknown ketone 19.303 – – – – – 0.15 6 0.002-heptanol 18.972 – – – – 3.66 6 0.00 –2-nonanone 21.926 – – – – 9.22 6 0.00 0.12 6 0.003-octanol 22.875 – – – – 0.80 6 0.00 –1-octen-3-ol 25.704 1.01 6 0.14 – – – 17.70 6 0.04 –Unknown terpene 30.644 5.06 6 1.62 7.24 6 1.94 0.46 6 0.00 1.79 6 0.19 0.12 6 0.00 2.63 6 0.0112-undecanone 31.644 – – 0.15 6 0.02 0.03 6 0.02 0.04 6 0.00 0.72 6 0.00Unknown ketone 32.247 – – – 0.25 6 0.05 – 0.36 6 0.00Unknown terpene 34.153 2.95 6 0.74 3.27 6 0.97 – 0.22 6 0.06 – –Unknown terpene 34.443 – 3.01 6 1.16 – – – 2.05 6 0.00g-terpinene 35.091 – – – – – 0.06 6 0.00a-zingiberene 36.371 – – – – – 0.30 6 0.00Unknown terpene 36.573 – – – – – 0.27 6 0.00b-Sesquiphellandrene 38.182 – – – – – 1.29 6 0.00Unknown ketone 42.392 – – – – – 0.10 6 0.00Unknown alcohol 42.569 – – – – – 0.30 6 0.00Unknown alkane 42.896 – – – – – 0.09 6 0.00Unknown phenol 44.06 – – – – – 0.39 6 0.00Unknown terpene 49.15 – – – – – 0.11 6 0.00Unknown terpene 51.374 – – – 2.22 6 0.46 – 0.33 6 0.00Unknown terpene 52.304 – – – 1.08 6 0.24 – –6-pentyl-2H-pyran-2-one 53.22 90.90 6 2.51 39.14 6 2.07 90.58 6 0.17 76.78 6 2.42 0.44 6 0.00 72.32 6 0.02b-curcumene 54.089 – – – – 1.06 6 0.00 1.43 6 0.00Unknown terpene 55.507 – 47.31 6 2.05 8.79 6 0.19 17.30 6 2.12 – 13.22 6 0.02Unknown alcohol 59.214 – – – – – 0.39 6 0.00



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such results, the lack of hda-2 led to the down-regulation of epl-1 and abc-2 transcripts. On the otherhand, acetylation of H3 N-terminal tail on the promoterof ctf-1 reached high basal levels in Dhda-2 (comparedwith epl-1 and abc-2 promoters) in either the presence orabsence of the plant, which correlates with high levelsof transcription, suggesting a direct regulation of ctf-1 by HDA-2.

Several reports on plant growth-promoting rhizo-bacteria and plant growth-promoting fungi, includingTrichoderma, have shown the role of their VOCs intriggering SA- and JA-signaling pathways (Ryu et al.,2004; Park et al., 2013; Naznin et al., 2014; Kottb et al.,2015). In agreement with these reports, we demon-strated the induction of SAR and ISR by the wild-typeVOCs. Interestingly, decreased levels of PR-1a andPDF1.2 were observed at 72 h of Arabidopsis exposureto the wild-type VOCs, compared with that at 48 and96 h, a phenomenon that did not happen by directcontact with the fungus. In this regard, it is known thatthe circadian clock controls secondary metabolism infungi, and therefore accumulation of VOCs might notbe continuous through all developmental stages(Bayram and Braus, 2012). Circadian rhythms have notbeen described in Trichoderma spp.; however, their ge-nomes contain the central components of the circadianclock (Casas-Flores and Herrera-Estrella, 2013, 2016).

Conversely, Dhda-2 VOCs failed to properly induceISR and SAR. Probably some VOCs in Dhda-2 wereabsent or in diminished amounts owing to the absenceof HDA-2, which points to a positive role of HDA-2 onthe synthesis of VOCs that could be functioning aselicitor molecules to induce plant responses. Interest-ingly, we analyzed several SM metabolism-relatedgenes, such as ggp-1 (hexaprenyl pyrophosphate syn-thase); fpp-1 (farnesyl pyrophosphate synthase), in-volved in the biosynthesis of isoprenoids; ctf-1 (cutinasetranscription factor 1 beta protein), a putative positiveregulator of the biosynthesis of 6-PP (Rubio et al., 2009);and tps-2 (terpene synthase-2), whose product is

putatively involved in the synthesis of terpenes. All ofthese were upregulated both in the presence and ab-sence of the plant, which supports our hypothesisstated above. A striking observationwas themagnitudeof responses between plants exposed to TrichodermaVOCs and Arabidopsis roots treated with Trichoderma,whereby the latter provoked stronger responses. Thesedata indicate that Trichoderma triggers the plant de-fense responses mainly by root colonization (probablydelivering elicitors of ISR and SAR), and secondly bythe secretion of VOCs.

Previous studies have shown that 6-PP triggers thesystemic resistance (Vinale et al., 2008; Kottb et al.,2015). Interestingly, Dhda-2 produced 7.5 to 9.5-foldmore 6-PP than that in the wild type growing on MS,which cannot explain the impairment of Dhda-2 to in-duce SAR and ISR, thus suggesting that 6-PP does notplay or could be playing aminor role on triggering suchpathways. In addition, we propose that HDA-2 couldbe exerting its role as a negative regulator of the pro-moter activity of 6-PP biosynthetic genes or through apositive regulator, whose transcription depends onHDA-2. Here, we demonstrated that ctf-1 transcript,which codes for a positive regulator of the synthesis of6-PP (Rubio et al., 2009), was upregulated in a Dhda-2background at all tested conditions. These resultsstrongly support our hypothesis about the regulation ofa positive regulator of 6-PP biosynthesis by HDA-2.

Dhda-2 presented a diminished ability to promoteplant growth and biomass gain in Arabidopsis. Thisreduction might be the result of Dhda-2 inability tocolonize the plant root; however, Arabidopsis seedlingstreated with Dhda-2 VOCs presented a greater biomassgain compared with that of Arabidopsis treated withthe wild-type VOCs. Stimulation of lateral root numberin plants treated with Dhda-2 or exposed to its VOCswas consistent in both treatments, together with ashortening of the primary root length. Together, theseresults indicate that in our working conditions, rootcolonization plays a minor role in plant growth

Table 2. Quantification of the wild type and Dhda-2 VOCs in MS and PDA by GC-MS

Mean values 6SE of the sum of three independent determinations are given. (–), No data.

Compound NameCalibration Curve

(r2)

Concentration (mg mL21)

MS 1 3 PDA

Wild Type Dhda-2Wild Type

Dhda-2Wild Type

Dhda-2

(5 dpi) (5 dpi) (6 dpi) (6dpi) (5 dpi) (7 dpi)

2-heptanone 0.99 – – – – 3189.6 6 189.9 184.8 6 17.62-pentylfuran 1.00 – – – – 5.0 6 1.9 43.6 6 18.73-octanone 0.99 – – – – 1388.0 6 3.2 –2-octanone 0.99 – – – – – 221.4 6 24.32-heptanol 0.99 – – – – 771.7 6 74.8 51.5 6 2.72-nonanone 0.99 – – – – 768.4 6 22.2 38.3 6 4.32-octanol 0.99 – – – – 339.3 6 80.2 –1-octen-3-ol 0.98 269.4 6 0.5 – – – 3354.0 6 238.6 –2-undecanone 0.98 – – 262.9 6 0.8 262.4 6 0.9 387.0 6 0.6 474.1 6 12.26-pentyl-2H-pyran-2-

one0.99 129.1 6 30.2 970.4 6 42.6 89.9 6 6.9 847.7 6 148.3 53.2 6 3.9 9579.1 6 1945.8



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Figure 7. The 6-PP effect on root system architecture is dependent on plant age and the application method. Two- or seven-d-oldArabidopsis seedlings were grown on 13 MS supplemented with increasing concentrations of 6-PP or onto sterile cottons ofcompartmented petri dishes for 13 d. A, Representative pictures of 2-d-old Arabidopsis grown on supplemented medium with 6-PPat the indicated concentrations. Bar = 1.5 cm (applies to all images). B, Representative pictures of 2-d-old Arabidopsis exposedto the 6-PP VOC at the indicated concentrations. Bar = 1.5 cm (applies to all images). C, Lateral root number. D, Root length. E,Fresh weight. F, Dry weight. G, Representative pictures of 7-d-old Arabidopsis exposed to 6-PP at the indicated concentrations.Bar, 1.5 cm (applies to all images). H, Lateral root number. I, Root length. J, Fresh weight. K, Dry weight. Data for C to FandH to Kshow the mean6SD of three technical replicates (15 plates with 5 plants each). Results were validated with an ANOVA statisticalanalysis using a Tukey multiple comparison test (a = 0. 05). Lower case letters a, b, and c (C to F and H to K) represent meansstatistically different at the 0.05 level.



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Figure 8. In Arabidopsis seedlings, 2-heptanol, 3-octanol, and 2-octanone VOCs promote plant growth. Seven-day-old Arabi-dopsis seedlings, grown on split-plates containing MS on one side, were exposed for 7 d to the indicated VOC by our placing animpregnated cotton swab at the opposite side of the split-dish. A, Representative pictures of Arabidopsis exposed to increasingconcentrations of the indicated alcohols and furan. Bar = 1.5 cm (applies to all images). B, Representative pictures of Arabidopsisexposed to increasing concentrations of the indicated ketones. Bar = 1.5 cm (applies to all images). C, Dry weight of seedlingsexposed to the indicated alcohols and furan. D, Dry weight of seedlings exposed to the indicated ketones. Data from C and Dshow the mean6SD of three technical replicates (15 plates with 5 plants each). Results were validated with an ANOVA statisticalanalysis using a Tukeymultiple comparison test (a = 0. 05). Different letters representmeans statistically different at the 0.05 level.Asterisks indicate significant difference (independent Student9s t-test, *P , 0.05), between control H2O versus VOC treatment.



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promotion; but this process could play an importantrole in ISR and SAR induction, pointing to a more rel-evant role of T. atroviride VOCs in the promotion ofplant growth and biomass gain.In this respect, it has been reported that Arabidopsis

root colonization by T. atroviride promotes plant growthassociated with short root length and lateral rootgrowth (Salas-Marina et al., 2011; Hermosa et al., 2013).Contreras-Cornejo et al. (2009) proposed that auxin-likemolecules from Trichoderma promote plant growth andlateral root branching. They detected transcriptionalactivity of the DR5::GUS auxin reporter in Arabidopsisafter 5 d of fungus-plant interaction in both primaryand lateral roots. However, contradictory reports showthat Trichoderma represses the auxinic primary root tipresponses at 5 d of cocultivation (Nieto-Jacobo et al.,

2017; Pelagio-Flores et al., 2017). Nieto-Jacobo et al.(2017) attributed such phenotype to impaired auxinsignaling, whereas Pelagio-Flores et al. (2017) proposedthat acidification of the medium by Trichoderma leads tothe loss of root meristem functionality (after 72–96 h ofinteraction). Interestingly, they also showed that thebeneficial effects provided by T. atroviride took placeduring the first 60 h. Contrastingly, we inoculatedArabidopsis seedlings at the root tipswith thewild typeand Dhda-2, and observed the inhibition of primaryroots and the emergence of lateral roots and branching,but not the negative effects reported by Pelagio-Floreset al. (2017). Additionally, the medium amended with50% and 25% of free-mycelium culture filtrates ofTrichoderma showed an enhanced inhibition of plantgrowth, whereas 12.5% and 6.25% showed enhanced

Figure 9. As shown, Dhda-2 displays mis-regulated expression of genes related to secondarymetabolism, defense, and communication withthe plant. A to H, Ten-day-old Arabidopsis Col-0seedlings were root-inoculated with the wild type(WT) andDhda-2, and themyceliawere collectedat 72 and96hpi. Thewild type andDhda-2 grownin the MS medium alone were included as con-trols. The expression levels of epl-1 (A), epl-2 (B),pbs-1 (C), abc-2 (D), ctf-1 (E), tps-2 (F), ggp-1 (G),and fpp-1 (H) were analyzed by RT-qPCR. The RT-qPCR results are reported as fold-change expres-sion compared with that in the fungi grownwithout Arabidopsis. Data from A to H show themean 6SD of one technical replicate (fungi my-celia pooled and collected from 5 plates with 7plants each). The experiment was repeated twicewith similar results. Asterisks indicate significantdifference (independent Student’s t test, *P, 0.05and **P, 0.01).



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biomass gain in pepper plants (Olmedo-Monfil andCasas-Flores, 2014). These results support, at least inpart, the proposal of Pelagio-Flores et al. (2017), becauseaddition of water to an acidic solution renders it lessacidic and raises the pH; however, plant growth andbiomass gain have been observed using low concen-trations of mycelium-free culture filtrates (Olmedo-Monfil and Casas-Flores, 2014), indicating that solublecompounds could be acting also as plant growth

regulators or as molecules that modify hormonehomoeostasis provoking such phenotypes.

Based on our results usingDhda-2VOCs, we attributethe overstimulation of lateral root emergence mainly tothe overproduction of 6-PP; however, we do not dis-card that other VOCs could be involved. In this respect,the wild-type and Dhda-2 VOCs were more effective instimulating the emergence of lateral roots, comparedwith that of the different concentrations of 6-PP appliedas VOC or in the growing medium. The wild typeproduced from 5.3- to 7.7-fold compared with that ofthe highest concentration of 6-PP used (100 mMor 16.62mg mL21), whereas Dhda-2 synthesized from 50.9- to58.3-fold. Recent studies show that 6-PP regulates theroot architecture, formation of lateral roots, plantgrowth, and inhibition of the primary root (Kottb et al.,2015; Garnica-Vergara et al., 2016. Our results supportsuch works and add further knowledge, because weshowed that the effect of 6-PP is dependent on both theage of the plant and how it is applied. We proposethat 6-PP applied as VOC could be acting as an ef-fector molecule, which modulates plant phytohor-mones, such as auxins and ET, to promote abeneficial relationship with Trichoderma; whereas 6-PP applied in the culture medium exerts a role as astressor. In this regard, stresses such as phosphoruslimitation, exposure to heavy metals, irradiationwith UV light, mechanical stress (Potters et al., 2007),and medium salt stress (Zolla et al., 2010) reduceprimary root length and induce lateral rootsformation.

Auxins and ET coordinately regulate several devel-opmental programs in plants. These phytohormonesregulate apical hook formation (Lehman et al., 1996;Raz and Ecker, 1999), root hair differentiation (Masucciand Schiefelbein, 1994), root hair elongation (Pitts et al.,1998), root growth (Rahman et al., 2001), and hypocotylphototropism (Harper et al., 2000). On the other hand,changes in the root architecture have been attributed todiffusible Trichoderma compounds such as ET, indole-3-acetic acid , and indole-3-acetaldehyde (Gravel et al.,2007; Contreras-Cornejo et al., 2009; Hoyos-Carvajalet al., 2009; Salas-Marina et al., 2011; Nieto-Jacoboet al., 2017). In this work, most of the auxin pathway-related genes (TIR1, AUX1, PIN3, and PIN7), whoseproducts participate in the perception, efflux, influx,and homeostasis of auxins in Arabidopsis, were up-regulated in the presence of the wild-type or Dhda-2strains, or following exposure to their VOCs (albeit toa different extent), pointing to a positive regulation ofthis pathway in Arabidopsis at the beginning of theinteraction with the fungus. Although loss of HDA-2induced the auxin-related genes only to a minor extent,this result indicates that such increased transcription isenough to promote root branching. Another possibleexplanation is that 6-PP produced by Dhda-2 and/or itsVOCs are promoting this phenotype in the plant bymodulating a different pathway.

In this study, the ET response and biosynthesis-related genes (ACO2, ETR1, ERS1, and EIN2) were

Figure 10. A lack of HDA-2 leads to a misregulation of histone H3acetylation of plant-responsive genes in T. atroviride. A to C, The cross-linked chromatin of the wild type (WT) orDhda-2 after 96 h of coculturewith 10-d-old Arabidopsis seedlings was immunoprecipitated withantibodies against anti-H3, and against anti-H3ac (acetylated-histoneH3Lys9Lys14Lys18Lys23Lys27). Fungi grown in absence of the plantwere used as controls. Specific primers were designed on conservedregions of epl-1 (A), ctf-1 (B), and abc-2 (C) promoters to quantify inputDNA and immunoprecipitated chromatin by qPCR. Enrichment wascalculated as a percentage of DNA control input. The experiment wasrepeated twice with similar results.



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upregulated by the wild-type and Dhda-2 VOCs andduring direct contact; however, Dhda-2 caused this re-sponse to a minor extent. The ET receptor-encodinggenes ERS1 and ETR1 were upregulated by the wildtype and Dhda-2 through their VOCs and by direct-contact, together with the up-regulation of ACO2,whose products are involved in the synthesis of ET.This likely promotes the dephosphorylation andcleavage of the EIN2 C terminus and translocation tothe nucleus to exert positive regulation on EIN3, lead-ing to the ET responses, including a reduction in pri-mary root growth and emergence of lateral roots. Thissituation points to a key role of ET in primary root in-hibition in Arabidopsis seedlings and to a differentmodulation of this pathway by Dhda-2. In agreementwith our results, whereby ISR and ET-related genes werecoexpressed with EBP (ET-responsive element bindingprotein; Supplemental Fig. S4) in plants inoculatedwithT.atroviride, or in the presence of the wild-type VOCs, it hasbeen reported that ACC,MeJA, or infectionwithB. cinereainduced the expression of EBP,which correlates with theinduction of PDF1.2 (Li et al., 2008).Taken together, these results suggest that T. atroviride

modulates the auxin–ET pathways in Arabidopsis topromote root branching. In this regard, it has beendemonstrated that ET–auxin interactions regulate lat-eral root initiation, emergence, and elongation in Ara-bidopsis (Ivanchenko et al., 2008). Moreover, it wasdescribed that, at low doses, ET promotes auxin bio-synthesis leading to lateral root initiation (Ivanchenkoet al., 2008). Furthermore, indole-3-acetic acid and ETsynthesized by Tuber spp. act additively on plant roots,provoking root shortening, increased branching, androot hair elongation (Splivallo et al., 2009).Trichoderma spp. are major producers of numerous

bioactive SMs, many of which are part of large, bio-synthetic gene clusters tightly regulated by chromatinmodifications (Schmoll et al., 2016). In this study, wereport that the absence of the histone deacetylase HDA-2 in a beneficial microorganism resulted in an alteredproduction of VOCs. Our results suggest both a nega-tive and a positive role for HDA-2 on VOCsmetabolismin T. atroviride. In this regard, deletion of genes encod-ing for HDACs class II in Aspergilli resulted in over-production of several SMs, which correlated withincreased gene expression of secondary metabolism-related genes (Shwab et al., 2007). Here, we demon-strated that the production and amount of the VOCsemitted by the wild type and Dhda-2 are dependent onthe fungal age and the composition of the culture me-dium. Sixteen compounds were produced only byDhda-2 in PDA, whereas 6-PP was overproduced. Fur-thermore, the expression of genes involved in the syn-thesis of SM was upregulated. These results suggestthat HDA-2 is a key global positive and negative reg-ulator of the synthesis of VOCs.In our results, we report 2-heptanol, 3-octanol, and

2-heptanone as plant growth promoters; whereas 3-octanol, 1-octen-3-ol, 2-pentylfuran, 3-octanone, 2-nonane, and 2-undecanone showed a phytotoxic effect

at higher concentrations. In 2016, the VOCs emitted by20 Trichoderma strains were identified and the strainsclassified based on their impact on plant growth aspromoters, neutrals, or detrimental (Lee et al., 2016).Identified in promoting and neutral strains were 3-octanol, 3-octanone, and 2-pentylfuran, whereas 2-heptanone, 2-octanone, 2-nonanone, and 2-undecanonewere identified in promoting, neutral, and negativestrains; however, none of the VOCs tested in this workwere detrimental to Arabidopsis growth at concentra-tions from 10 to 100 mg mL21, suggesting that the doseapplied in the plant environment will determine theoutcome of the plant. Furthermore, all the C8 compoundsand furan tested in thisworkwere phytotoxic at 10,000mgmL21. The phytotoxic effects of 1-octen-3-ol, 3-octanol,and 3-octanone were demonstrated by Splivallo et al.(2007), who also reported that the phytotoxic effect of1-octen-3-ol was due to an increased ROS-scavengingenzyme activity and/or increased H2O2 concentrations.

CONCLUSION

Our results revealed that histone deacetylase HDA-2from T. atroviride is necessary to effectively colonize andpromote plant growth in Arabidopsis, as well as to in-duce the systemic disease resistance against foliarpathogens. Moreover, Dhda-2 VOCs enhanced thegrowth of Arabidopsis seedlings and did not trigger theSAR and ISR responses in the plant. The absence of hda-2 impaired VOCs metabolism, resulting in an over-production of 6-PP. In vitro assays using mediumamended with 6-PP or 6-PP supplied as VOC demon-strated that the plant growth-promoting effect is de-pendent on the Arabidopsis age and the applicationprotocol. Split-plate assays with nine different VOCsrevealed that 2-heptanol, 2-heptanone, and 3-octanolpromoted plant growth in Arabidopsis seedlings. Ourexpression analysis led to the conclusion that the ex-pression of auxin- and ET-related genes is differentiallymodulated in Arabidopsis by Dhda-2 direct contact orby exposure to its VOCs, which may explain the dif-ferent phenotype of the wild -type–treated and Dhda-2-treated plants. Our work highlights the importance ofHDA-2 as global regulator of multiple processes in T.atroviride and highlights the relevance of maintaining ahistone acetylation balance to properly respond to thepresence of Arabidopsis seedlings.

MATERIALS AND METHODS

Organisms and Growth Conditions

A Trichoderma–plant interaction was fostered on a 13MS medium (Murashigeand Skoog, 1962; PhytoTechnology Laboratories). Arabidopsis (Arabidopsis thaliana)ecotype Col-0 was used in this study. Arabidopsis seeds were sterilized bysoaking in 75%ethanol for 3min, treatedwith 10%bleach (HOCl) inwater for 7min,and rinsed three times with sterile, distilled water. Seeds were stratified for 2 d at4°C, germinated on MS agar plates, and grown under a 16-h/8-h light/dark pho-toperiod at 22°C 61°C, 65% relative humidity, and 150 mM m22 s21 light.



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Trichoderma–plant interaction in pots: Arabidopsis seeds were sown in potscontaining peat moss (Lambert peat moss) as a substrate and stratified for 2 d at4°C. One-day-old seedlings were transplanted into pots containing sterile peatmoss and grown as just described.

The trichoderma atroviride IMI 206040 wild type, its isogenic Dhda-2 mutant(Osorio-Concepción et al., 2017), and Botrytis cinerea B05.10 (Amselem et al.,2011) were used throughout this study. All fungal strains were routinely grownat 25°C on potato dextrose agar (PDA; DIFCO), under a 12-h/12-h light/darkregime, unless otherwise specified. The bacterium Pseudomonas syringae pv.Tomato, strain DC3000 (Cuppels, 1986) was grown at 28°C in Kings B medium,supplemented with rifampicin 50 mg/mL (King et al., 1954).

Effect of the T. atroviride Wild Type and Dhda-2 on PlantGrowth Promotion and Root Colonization in Soil

Fifteen 10-d-old Arabidopsis plants were root inoculated with mycelium ofthe wild type or Dhda-2, as described in the "Organisms and Growth Con-ditions"section. At three weeks postinoculation, fresh and dry weights weredetermined. For root colonization, the roots were detached from at least 10plants and rinsed with sterile distilled water. Total DNA was extractedaccording to (Dellaporta et al., 1983) and subjected to relative quantification(qPCR) of the Trichoderma tef-1 gene (Supplemental Table S1), which codes forthe translation elongation factor, and the Arabidopsis ACT2 gene, which codesfor the actin protein 2 (Supplemental Table S1).

Effect of the Wild Type and Dhda-2 on Plant Biomass andRoot System Architecture In Vitro

Arabidopsis seedswere germinatedandplacedonpetri dishes containing 13MS. Eleven days thereafter, the seedlings were inoculated with mycelial plugsof the wild type or Dhda-2, sealed with plastic film, and cocultured for 10 dunder a 16-h/8-h light/dark photoperiod at 22 °C 61°C. Control plates wereinoculated with a PDA plug without the fungi. The length of primary roots andthe number of lateral roots were determined. Fresh and dry weights were alsodetermined on an analytical scale.

Botrytis cinerea Infection Assay

Taking into account that Dhda-2 does not undergo conidiation, flasks con-taining 100 mL of potato dextrose broth (PDB, DIFCO) were inoculated withthree mycelial plugs of the wild type or Dhda-2 and grown at 25°C, 200 rpm, inthe dark for 72 h. Mycelia of the wild type or Dhda-2 were vacuum-harvestedonto 0.2mmmembrane filters (Whatman). The collectedmyceliumwas cut witha 0.6 mm diameter cork borer and used to inoculate the roots of 10-d-oldArabidopsis seedlings, and allowed to interact for two weeks. Three Arabi-dopsis leaves per plant were inoculated with 10 mL of 53 105 conidia/mL of B.cinerea diluted in inoculation buffer (per 40 mL of stock solution: 1.37 g Suc,400 mL of 1 M KH2PO4, 80 mL of 12.5% Tween 20). Lesioned areas of infectedleaves were quantitatively measured at 3 and 6 d postinoculation (dpi) usingImageJ software (http://rsb.info.nih.gov/ij/index.html).

Pseudomonas syringae Infection Assay

Two-week-old Arabidopsis seedlings were grown and inoculated withmycelium of the wild type or Dhda-2 as described in the "Organisms andGrowth Conditions" section. Thereafter, three leaves per plant were infiltratedwith Pst DC3000 in 10 mM MgCl2 (OD600 = 0.0004) using a needleless syringe.Twelve leaves of control and treated plants were collected at 0 and 3 dpi, andground in 10 mMMgCl2. Samples were serial-diluted and plated onto a King’sB medium containing the appropriate antibiotics to determine the CFU.

Influence of the Wild Type and Dhda-2 VOCs on PlantBiomass and Root System Architecture In Vitro

Exposure of Arabidopsis plants to the wild-type and Dhda-2 VOCs wasachieved using a double plate-within-a-plate system (Olmedo-Monfil andCasas-Flores, 2014). Small petri dishes containing 13 MS (35 3 10 mm) wereembedded into large petri dishes containing 13MS aswell (1003 15mm). Ten-day-old Arabidopsis seedlings were grown onto large petri dishes. Ten daysthereafter, the seedlings were inoculated with a mycelial plug of the wild type

or Dhda-2 onto the small petri dishes, sealed with plastic film, and grown asdescribed in the "Organisms and Growth Conditions" section. Control plantswere inoculated with an MS plug. Root length, number of lateral roots, freshand dry weights were determined as described in the "Effect of the Wild Typeand Dhda-2 on Plant Biomass and Root System Architecture In Vitro" section.

Analysis of Gene Expression by Quantitative ReverseTranscription PCR (RT-qPCR)

Total RNA extraction was performed by the Trizol method, as described bythe vendor (Invitrogen). Total RNAwas DNase I (RNase-free; Ambion) treated,followed by cDNA synthesis using SuperScript II Reverse Transcriptase (Invi-trogen), following the manufacturer’s recommendations. cDNAs were used astemplate for RT-qPCR reactions with gene-specific primers (SupplementalTable S1) and the Fast SYBRGreenMasterMix (Applied Biosystems), accordingto manufacturer’s recommendations.

The reaction mixtures were as follows: 10 mL SYBR Green Master Mix(Applied Biosystems), 200 ng cDNA template, and 0.3 mL of gene-specificprimers (150 nM). The qPCR program consisted of one cycle at 95°C for5min, 40 cycles at 95°C for 30 s, 65°C for 30 s, and 72°C for 40 s. Relative expressionwas normalized against the level of tef-1 for Trichoderma samples or ACT2 for Ara-bidopsis samples using the 2-DDCt method (Livak and Schmittgen, 2001).

Expression Analysis of Auxin, ET, and Defense-RelatedGenes in Arabidopsis Inoculated with the Wild Type orDhda-2 or Exposed to their VOCs

Plantswere germinated and grown for 9 d onpetri dishes containing 13MS.For direct contact, the root tips were inoculated with disks of fresh mycelium ofthe wild type or Dhda-2; whereas for exposure to VOCs, the mycelial disk was in-oculated into the opposite compartment of Arabidopsis seedlings grown in split-petri dishes. Plants were harvested at 0, 24, 48, 72, and 96 hpi (hours postinocula-tion), frozen in liquid nitrogen, and stored at 280°C until total RNA extraction.Plants growing without the fungi were used as controls. Total RNA extraction,cDNA synthesis, and RT-qPCR were performed as described in the "Analysis ofGene Expression by Quantitative Reverse Transcription PCR (RT-qPCR)" section.

Expression Analysis of Trichoderma Genes in Coculturewith Arabidopsis Seedlings

Arabidopsis Col-0 seedlings were germinated and grown for 10 d on petriplates containing a MS medium. At day 10, the seedlings were root-inoculatedwith the wild type or Dhda-2, and the fungi-samples were collected at 72 and 96hpi. The wild type and Dhda-2 growing in MS medium alone were included ascontrols. Total RNA extraction, cDNA synthesis, and RT-qPCRwere performedas described in the "Analysis of Gene Expression by Quantitative ReverseTranscription PCR (RT-qPCR)" section.

Effect of 6-PP on Plant Growth of 2- and 7-D-Old Plants

Two-day-old seedlings were placed on petri dishes containingMS amendedwith 25, 50, 75, and 100 mM 6-PP (Sigma Aldrich; Garnica-Vergara et al., 2016);whereas 2- or 7-d old seedlings’ exposure to 6-PP, asVOC,wasperformedaccordingto Splivallo et al. (2007), with some modifications. Briefly, seedlings were placed insplit-petri dishes containing MS in one side to sow the seedlings, and 300 mL 6-PPwas added to a piece of sterile cotton at the opposite side at the same concentrationsas above. Ethanol (Sigma Aldrich) was used as solvent at a final concentration of1.3% in water (higher concentrations promote plant growth) and used as control aswell. Petri plates containing five plants were closedwith sealing film and incubatedfor 13 d under a 16-h/8-h light/dark photoperiod at 22 °C 61°C.

Identification of VOCs through GC-MS

For VOCs analysis, the T. atroviride wild type and Dhda-2 were grown onPDA plates at 25°C for 5 and 7 d, respectively. Noninoculated PDA plates wereincluded as controls. Compounds were collected as described (SupplementalFig. S2) for 1 h with a blue SPME fiber (PDMS/DVB; Supelco Inc.), and des-orbed at 180°C for 30 s in the injector port of a gas chromatograph (Agilent7890B; Agilent), equipped with a mass spectrometry detector (5977A; Agilent)and Mass Hunter Workstation Software (Agilent Technologies) for data



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acquisition and processing. In the operating conditions, heliumwas used as thecarrier gas (1 mL min21) and the detector temperature was 250°C. The columnwas held for 1 min at 60°C, and then programmed to rise at a rate of 3°C min21

to a final temperature of 180°C.

Quantification of Fungal VOCs

The concentration of fungal VOCs (Table 2) contained in the cultures wascalculated with their corresponding standards (all from Sigma-Aldrich), basedon the calibration curves determined independently for each compound. TheSPME vials were filled with 1 mL of the corresponding standard diluted inmethanol. The compounds were adjusted to 10, 100, 1000, 5000, and 10,000 mgmL21and analyzed under the same conditions as used for the quantification ofthe fungal VOCs mixtures.

Exposure of Arabidopsis to Individual VOCs

The effect of Trichoderma individual VOCs on plant growth promotion wasassessed using a split-plate system. Arabidopsis plants were grown, sterilized,and stratified as described in the "Organisms and Growth Conditions" section.The different VOCswere supplied in sterile cottons as described in the "Effect of6-PP on Plant Growth of 2- and 7-D-Old Plants" section; 200 mL of 10, 100, 1000,and 10,000 mg mL21 of each selected VOC was applied to the cottons. Waterwas included as control because it was used as solvent for most VOCs (except 6-PP). Plates were closed with plastic film and incubated at 22°C under 12-h-light/12-h-dark cycles for 7 d. Fresh and dry weights were determined for eachgroup on an analytical scale.

Chromatin Immunoprecipitation Assays

The Trichoderma–Arabidopsis interaction experiment was carried out asdescribed in the "Expression Analysis of Trichoderma Genes in Coculture withArabidopsis Seedlings" section. Mycelia from interaction with Arabidopsis andcontrol in absence of the plant were fixed in 10 mL of 13 crosslinking buffer (103 crosslinking buffer: 0.5mL of 5MNaCl, 25mL of 0.5MEGTA [pH 8], 50mL of0.5 M EDTA [pH 8], 1.25 mL of 1 M HEPES [pH 8], 7.45 mL of 37% formalde-hyde, 15.75 mL water. Mycelia were incubated for 10 min at room temperatureon a rocking platform and neutralized by adding 1.25MGly for fivemin at 4°C.Cross-linked chromatin was immunoprecipitated using 2 mL of anti-H3 anti-body (Abcam; Ab1791), and 5 mL of antihistone-H3 acetyl K9K14K18K23K27(Abcam; ab47915); 10% of the chromatin was used as input. Immunoprecipi-tated chromatin was analyzed by qPCR using specific primers (SupplementalTable S1) on the promoter region of epl-1, ctf-1, and abc-2.

Data Analysis and Statistics

For all Arabidopsis experiments treated with the wild type and Dhda-2, theoverall data were statistically analyzed using SPSS 10 software (IBM Corp).Tukey’s post hoc test was used to assess the significance of differences in plantgrowth promotion and root system architecture between treatments. Differentletters are used to indicate means that differ significantly (P , 0.05). Student’st test was used to evaluate differences in the relative level expression of genes inpathogenesis assays, as well as in the plant growth promotion by 10different VOCs.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL datalibraries under accession numbers (Supplemental Table S1).

Supplemental Material

The following supplemental materials are available.

Supplemental Figure S1. The mRNA levels of hda-2 were slightly in-creased in Trichoderma in the presence of Arabidopsis Col-0 seedlings.

Supplemental Figure S2. Trichoderma VOCs exposure system.

Supplemental Figure S3. Chromatin immunoprecipitation (ChIP) assay onthe promoter regions of T. atroviride plant-responsive genes.

Supplemental Figure S4. The expression of EBP was induced in Arabidop-sis by direct contact with Dhda-2 or exposure to its VOCs.

Supplemental Table S1. List of primers used in this study

ACKNOWLEDGMENTS

The authors wish to thank Nicolás Gómez-Hernández, Norma AngelicaRamírez Pérez, Mitzuko Dautt Castro, Edith Elena Uresti-Rivera, and MaríaGuadalupe Ortega Salazar for their technical support. We also thank the Na-tional Biotechnology, Agricultural andMedical Laboratory for providing accessto LANBAMA’s GC-MS.

Received September 10, 2018; revised December 10, 2018; accepted January 9,2019; published January 22, 2019.

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