ABSTRACT The capability to respond to wounding is a process shared by organisms of different kingdoms that can result in the regeneration of whole-body parts or lost structures or organs.

Filamentous fungi constitute a rich food source that ensures survival and reproduction of their predators and are therefore continuously exposed to mechanical damage. Nevertheless, our

understanding of how fungi respond to wounding and predators is scarce. Fungi like plants and animals respond to injury recognizing Damage- and Microbe-Associated Molecular Patterns

(DAMPs/MAMPs) that activate Ca2+ and Mitogen-Activated Protein Kinase dependent signaling for the activation of defense mechanisms. During herbivory, plants, in addition to activating

hyphal regeneration, and the fungal innate immune and chemical defense responses. We also provide mechanistic insight of this inhibition involving components of the _D. melanogaster_

salivary glands that repress the expression of a set of genes and block hyphal regeneration. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING

VIEWED BY OTHERS _AGASICLES HYGROPHILA_ ATTACK INCREASES NEROLIDOL SYNTHASE GENE EXPRESSION IN _ALTERNANTHERA PHILOXEROIDES_, FACILITATING HOST FINDING Article Open access 12 October 2020

MECHANOSENSORY TRICHOME CELLS EVOKE A MECHANICAL STIMULI–INDUCED IMMUNE RESPONSE IN _ARABIDOPSIS THALIANA_ Article Open access 08 March 2022 EARLY TRANSCRIPTOMIC RESPONSES OF RICE LEAVES TO

HERBIVORY BY _SPODOPTERA FRUGIPERDA_ Article Open access 03 February 2024 INTRODUCTION As cosmopolitan organisms, fungi share a niche with multiple organisms where they are exposed to

different competitors and predators [1]. Fungi are absorptive heterotrophs, playing a major role in recycling dead and decayed matter and represent a nutrient source rich in amino acids and

sugars for their predators [2]. Multicellular organisms have developed specific strategies to respond to mechanical injury and pathogen attack. A primary defense mechanism is the activation

of innate immunity, which involves proteins that recognize molecules produced by foreign organisms or by themselves [3, 4]. These molecules are classified in MAMPs, derived from microbes,

and DAMPs that are self-derived [5]. Pattern Recognition Receptors (PRRs) are in charge of recognizing MAMPs [6]. During the animal and plant innate immune responses, PRR-dependent signaling

pathways trigger cell death. In plants, this occurs via the hypersensitive response and in animals via inflammasome assembly [6,7,8]. The existence of fungal PRRs, with a similar structure

to plant and animal nucleotide-binding leucine-rich repeat (NLR) immune receptors, has been documented [8, 9]. Fungal NLRs have been associated with heterokaryon incompatibility during cell

fusion [10] and with a fungal innate immune system [11]. Fungi can recognize MAMPs [12, 13] and DAMPs [14, 15] that trigger defense responses. The main defense of fungi is chemical, i.e.,

the production of toxins impairing growth, development, or viability of their antagonists. In addition, fungi posses an innate immune system that allows them to distinguish self from

non-self and results in cell death or autophagy. The immune system restricts horizontal transmission of deleterious cytoplasmic elements, such as viruses, and prevents resource plundering by

parasitic genotypes and a pro-survival function in response to bacteria [12, 13, 16]. Recognition of these cues involves activation of ATP, calcium (Ca2+), and Reactive Oxygen Species (ROS)

dependent signaling pathways. As observed in plants and animals, activation of programmed cell death and reactivation of the cell cycle is part of the fungal damage response, which is

linked to the innate immune response [14, 15, 17]. _D. melanogaster_, the springtail _Folsomia candida_, and the nematode _Aphalenchus avenae_ have been used as fungivorous models in

interactions with _Aspergillus_ spp. and _Coprinopsis cinerea_, demonstrating the importance of the fungal chemical defense against predators [18,19,20,21,22,23,24]. In addition, _C.

cinerea_ initiates a transcriptional response to different fungivorous and non-fungivorous organisms such as bacteria, nematodes, and to wounding, including the activation of defense-related

genes [23]. The capacity of fungi to defend themselves against competitors and predators is also linked to the innate immune system. In _Fusarium graminearum_ bacterial MAMPs induce

processes associated with innate immunity in animals, such as mitochondrial activity, activation of ROS production-related genes, and the transcriptional upregulation of PRRs [12]. The

filamentous fungus _Trichoderma atroviride_ is a model to study regeneration due to the similarities of its response to mechanical injury to that of higher eukaryotes. _T. atroviride_

regenerates its hyphae upon mechanical injury, which triggers asexual reproduction in the damaged area [17]. Mitogen-Activated Protein Kinases (MAPKs) and Ca2+ mediated signaling, as well as

the transcriptional activation of NLR (HET) encoding genes are considered key for hyphal regeneration in _T. atroviride_ [14, 15]. Furthermore, the attack on _T. atroviride_ by _D.

melanogaster_ larvae represses the production of putative defense compounds and the expression of chemical-defense related genes [25]. Here we use _T. atroviride_ in a model system to study

antagonistic interactions of fungi with arthropods. We describe how _T. atroviride_ establishes a different transcriptional response to mechanical injury and to chewing arthropods. We show

that salivary gland components of _D. melanogaster_, during larval grazing on _Trichoderma_, inhibit hyphal regeneration and defense, by blocking the expression of genes critical to signal

perception and the fungal innate immune response. METHODS TRICHODERMA SPP. STRAIN _Trichoderma atroviride_ IMI 206040 used throughout was preserved as described [25]. ARTHROPOD MODELS To

compare the fungal response to arthropod grazing, we used _D. melanogaster_ SD-5 and the Collembolan _Orthonychiurus folsomi_ (obtained from the Faculty of Sciences, UNAM, Mexico)_. D.

melanogaste_r SD-5 reared as previously described [25], and the collembolans maintained in sterile soil at 23–25 °C, 70% humidity in darkness and fed mushrooms every seven days. RESPONSE TO

ARTHROPOD GRAZING To compare the morphological response of _T. atroviride_ to arthropods and mechanical injury, we inoculated 1 × 106 fresh conidia and incubated them in Petri dishes

containing PDA for 36 h in darkness at 27 °C. To examine the grazing response, 20 larvae or collembola were placed on the mycelia for 10 min in darkness and immediately withdrawn. Mechanical

injury was inflicted with a sterile star-shaped metal cookie cutter. After grazing or mechanical damage, plates were incubated 48 h in darkness at 27 °C and photographed. Five independent

biological replicates were performed, each with three technical replicates. RNA PREPARATION For differential gene expression analyses, RNA was extracted from _T. atroviride_ grown on PDA

plates covered with sterile cellophane sheets and incubated for 36 h in the dark at 27 °C. Interactions between _T. atroviride_ and the attackers were performed as mentioned above, but using

100 arthropods. For mechanical injury, the complete fungal colony was cut rapidly using a sterile scalpel. After arthropod attack or mechanical injury, the plates were incubated for an

additional 30 min, 90 min, four and eight hours, collected, and immediately frozen in liquid nitrogen. Control mycelia plates were opened for 2 min in the dark and incubated for an

additional 30 min and 8 h. Each treatment had three biological and three technical replicates. RNA was extracted using the TRIZOL method. SEQUENCING AND RNA-SEQ DATA ANALYSIS RNA sequencing

libraries were prepared following the Illumina TruSeq Stranded Total RNA Sample Preparation kit instructions. A total of 42 TruSeq libraries were sequenced using the NextSeq500 platform

(Illumina) in the 1 × 75 single-end mode, obtaining an average of 12 million raw reads per library (Supplementary Table 1). Raw RNA-seq data were processed with FastQC Version 0.11.6 [26],

obtaining about 10 million high-quality reads per library. Raw RNA-seq data are available at Gene Expression Omnibus (GEO) accession number: GSE152652. High-quality reads were aligned to the

_T. atroviride_ genome using HISAT2 version 2.1.0 [27] and counted using HTseq version 0.14.1 [28]. The genome sequence is available on NCBI genomes, accession number: JAEAGS000000000.

Differential expression analyses were carried out using _Edge R_ version 3.11 [29]. We detected no significant differences between control (T0 and T8) libraries, except for CT0-2, which was

discarded due to its poor quality. Contrasts between libraries were performed using an FDR < 0.005. Venn Diagrams were drawn using: http://bioinformatics.psb.ugent.be/webtools/Venn/. We

performed hierarchical clustering followed by the K-means analysis, supported by a Pearson correlation _P_ > 0.85 and 50 iterations per core. Codes used for RNA-seq data analysis are

available at https://github.com/Karina-atriztan/RNA-seq-data-analysis. MOTIF PREDICTION AND ORTHOLOGUES SEARCHING To determine if gene cores contained specific DNA binding motifs in their

promoters, we performed a motif prediction on 11 selected cores. We extracted 700 bp upstream of the ATG using the script: https://github.com/AgustinPardo/upDownStreamSeqsFromGbk. For

prediction of enriched ungapped motifs we used STREME of the MEME suite (http://meme-suite.org) with maximum and minimum lengths of 15 and eight nucleotides, respectively (_E_ value

threshold >0.05). As a control, we used the same number of random upstream sequences per core. De novo prediction was performed using MEME with a maximum of 20 motifs (_E_ value

>0.05), and maximum and minimum length of 15 and six nucleotides, respectively. For motif comparison, we used the Tomtom tool from MEME suite, using _S. cerevisiae_ and Yeastract database

with default options. An overlap <70% between motifs and a _ρ_ value >0.001 were used to select homologous motifs. To find TF orthologues, we extracted the sequences for _S.

cerevisiae_ and _A. nidulans_ from the Yeastract (http://www.yeastract.com/index.php) and AspGD (http://www.aspergillusgenome.org/) databases. Candidates were obtained by Bidirectional Blast

between the _T. atroviride_:_Saccharomyces cerevisiae_:_Aspergillus nidulans_ proteomes. Candidates were supported by Hidden Markov Model analysis using HMMER V.3.2.1 (hmmer.org).

Phylogenetic relationships between protein candidates were analyzed using Neighbor-Joining trees with 1000 Bootstrap resampling using MAFFT V.7 (https://mafft.cbrc.jp/alignment/server/).

Additional fungal sequences were obtained from UNIPROT (uniprot.org) and FungiDB (fungidb.org). GO ENRICHMENT ANALYSIS To determine which processes were enriched in selected gene sets, we

performed GO enrichment analysis using _topGO_ version 3.11 [30] (_P_ > 0.05) for enriched Biological process or Molecular Function. Redundancy was eliminated using REVIGO [31]. Heatmaps

were generated using the _Heatmap2_ function of the gplots package from Bioconductor R. REGENERATION INHIBITION ASSAYS To test larval salivary gland components we dissected 300 salivary

glands of 3rd instar _D. melanogaster_ larvae under a stereoscope Zeiss Stemi 2000. Ten glands were homogenized in 30 µl PBS and centrifuged at maximum velocity for 10 seconds at 4 °C. We

used approximately 0.024 µg/µl of total protein for regeneration inhibition assays [32]. For fungal microcultures, 50 conidia were inoculated on sterile slides containing 2 ml PDA and

incubated for 16 h at 27 °C in darkness. A sterile scalpel with 1 µl of homogenized tissue was used per cut on the mycelia (MI + SGE), after cutting microcultures were incubated at 27 °C in

darkness for one and two hours. We observed no significant differences when counting 1 or 2 h after the treatment. Hyphae were stained with lactophenol blue and observed under a Leica

DM6000-B microscope fitted with a 100x objective HCX PL Fluotar (0.75 N. A) and photographed with a Leica DFC 429 C camera. As a control, we used a clean scalpel to cut the mycelia (MI).

Again, all experiments used three independent biological and three technical replicates. We determined the proportion of damaged hyphae that regenerated out of two hundred (100%) using a

one-way ANOVA and Tukey test (_P_ < 0.05). RESULTS _T. ATROVIRIDE_ DISTINGUISHES BETWEEN MECHANICAL DAMAGE AND ARTHROPOD ATTACK We hypothesized that, like plants, _T. atroviride_

discriminates between chewing arthropods and mechanical injury. We, therefore, analyzed the response of _T. atroviride_ to two chewing arthropods (Fig. 1), namely _D. melanogaster_ (Dm)

larvae and the springtail _Orthonychiurus folsomi_ (Of), and compared it to that displayed upon mechanical injury (MI). As expected, attack by Drosophila larvae and mechanical injury

triggered conidiation in the damaged area (Fig. 1B, C) [25]. Interestingly, the collembolans caused no evident response (Fig. 1D); the fungal colony looked just like the undamaged control

(Fig. 1A). When we observed the arthropods’ behavior under a magnifying lens, it became evident that while Drosophila larvae chewed and pulled the fungal mycelium (Fig. 1F, Supplementary

Video 1) _O. folsomi_ walked on _T. atroviride_ without causing any apparent damage (Fig. 1E, Supplementary Video 2), and made no attempt to ingest mycelium. Furthermore, the fungus emerged

from dead Drosophila bodies but we found no evidence of its presence in collembolans’ bodies, which would indicate ingestion of Trichoderma mycelium (Supplementary Fig. 1). We analyzed the

fungus’ transcriptomic response when exposed to the two arthropods and upon mechanical injury in a time course. The collembolans induced a weak transcriptional response at 30 min (nine

upregulated and 127 downregulated genes), and none later (90 min to 8 h; Table 1; Supplementary Data 1). In contrast, mechanical injury and grazing by _D. melanogaster_ provoked significant

changes in the expression level of thousands of genes at 30 and 90 min, and hundreds after 4–8 hours, sharing a substantial number of genes and displaying specific modifications of the

transcriptional landscape (Table 1; Supplementary Fig. 2A). We, therefore, analyzed in detail only the fungal response to Drosophila attack and to mechanical injury (Supplementary Table 2).

MECHANICAL INJURY AND ATTACK BY DROSOPHILA DIFFERENTIALLY REGULATE THE EXPRESSION OF SPECIFIC GENE SETS We visualized all (5561) Differentially Expressed Genes (DEG) in the comparison of the

responses to Drosophila attack and mechanical injury in a heatmap (Fig. 2A). Hierarchical clustering followed by K-means analysis resulted in 65 gene cores, of which 11 were unique for a

time or condition (Fig. 2A; Supplementary Data 2). We decided to focus on four of the 11 clusters, which contain the earliest responsive genes and are more likely part of the primary

response to the challenge. Clusters (C10 and C49) contain the most extensive sets of genes (675 and 446, respectively) that modify their expression only at 30 min after mechanical injury but

are not altered in any other condition analyzed (Fig. 2B & D). Similarly, the expression of clusters C16 (102 genes) and C56 (175 genes) was modified only 30 min after attack by the

arthropod (Fig. 2C & E). We observed other, smaller, and/or less specific gene clusters (Supplementary Data 2). Transcription Factor (TF) binding motif predictions for the promoters of

the genes belonging to the 11 clusters (Supplementary Data 3) revealed the enriched motifs for the contrasting cores C10, C16, C56, and C49 (Fig. 2F–I). All of them contain a recognition

sequence for an _Aspergillus nidulans_ TF for which we found an orthologue in _T. atroviride_. Genes in C10 contain four enriched motifs recognized by NDT80-pho (Tatro_010668-T1,

Tatro_003887-T1 and Tatro_001315), Sfr1 (Tatro_003671-T1), Aft1 (Tatro_009572-T1), and Met32 (Tatro_001433-T1) (Fig. 2F; Supplementary Figs. 3A–C). C16 genes contain motifs recognized by

Fkh1 (Tatro_011463-T1) (Fig. 2G; Supplementary Fig. 4A). C49 contains two regulatory elements recognized by Mat alpha2 (Tatro_004149-T1) and AreB (Tatro_003849-T1) (Fig. 2H; Supplementary

Fig. 4B, C). The core of downregulated genes at 30 min in response to Drosophila attack contains a motif recognized by the orthologue of StuA (Tatro_009561-T1) (Fig. 2I; Supplementary Fig. 

4D). Using MEME for a de novo approach we found as significant (_E_ value <0.05) the novel “GAAGAAGAARA” motif, present in 2264 sites in the 645 sequences for C10 (Fig. 2F) that could be

specifically related to the injury response. We performed Gene ontology and enrichment analyses for the 11 selected clusters (Supplementary Data 2 and Supplementary Fig. 5). For the four

clusters shown in Fig. 2, mechanical injury-induced, within minutes, the expression of genes related to DNA metabolic process, DNA repair, and response to stress, among other processes

(C10). In contrast, the expression of genes related to cell redox homeostasis was downregulated (C49), as previously reported [15, 16], (Supplementary Fig. 5A). C16 contains genes

upregulated only at 30 min after exposure to Drosophila, related to DNA packaging, protein DNA assembly, oxidation-reduction, DNA conformation change, and response to oxidative stress

(Supplementary Fig. 5B). In contrast, C56 contains 179 genes with decreased expression only at 30 min of exposure to Drosophila, which is related to transcription regulation, cellular

metabolic process, and macromolecule metabolic process, among others (Supplementary Fig. 5B). In summary, the first minutes after mechanical injury appear critical to transcriptionally

activate genes, like those involved with cell homeostasis and DNA damage, to protect against damaging effects of mechanical injury. In contrast, attack by Drosophila induced genes related to

DNA protection. At (4–8 h), processes related to protein degradation and secondary metabolism were activated (Supplementary Data 2). ATTACK BY _D. MELANOGASTER_ AFFECTS THE TRANSCRIPTIONAL

RESPONSE OF GENES RELATED TO HYPHAL REGENERATION AND IMMUNE RESPONSE Mechanical injury induces hyphal regeneration during the first two hours after damage, triggering the expression of genes

related to regeneration and the putative innate immune system (Fig. 3A; [15, 16]). Therefore, we analyzed the impact of attack by Drosophila on the expression of genes activated early after

the challenges. Figure 3B shows the sets of up- and downregulated genes 30 min after mechanical injury and insect attack. The expression of 940 genes was upregulated exclusively 30 min

after mechanical injury, while Drosophila attack induced the expression of a different set of genes (381), and 755 genes were upregulated by both stimuli (Fig. 3B). A similar pattern existed

for the downregulated genes at 30 min. Mechanical injury resulted in the specific downregulation of 742 genes, attack by the arthropod resulted in repression of 707, and 961 genes were

downregulated in response to both treatments (Fig. 3B). At 90 min, mechanical injury resulted in the upregulation of 737 genes: 165 for Drosophila attack and 485 genes upregulated in common

(Fig. 3C). Similarly, we found 577 specifically downregulated at 90 min for mechanical injury and 422 genes for Drosophila attack, with 899 genes in common (Fig. 3C). GO term enrichment

analysis of the DEG at 30 min showed upregulation of genes involved in RNA processing and modification, ncRNA metabolic processes, macromolecule metabolism, and methylation in response to

the two challenges (Fig. 3D, Group 4). At 30 min, mechanical injury resulted in upregulation of processes related to gene expression, DNA metabolism, cell cycle, mRNA metabolism, primary

metabolism, and response to ion transport (Fig. 3D, Group 5). Interestingly, within DNA metabolism, genes related to DNA damage checkpoint, histone H3-K79 methylation, DNA replication

initiation, RNA splicing, snoRNA processing, response to light, and protein biosynthesis were enriched (Fig. 3D, Group 6). Processes such as protein metabolism, reproduction, actin

filament-based process, and cellular component organization were downregulated in response to both challenges (Fig. 3D, Group 1). Cytokinesis, glycoprotein metabolism, aromatic compounds

biosynthesis, reproduction, and response to oxidative stress and steroid metabolism were downregulated only at 30 min after mechanical injury (Fig. 3D, Group 2). While only after arthropod

grazing, the Biological Processes related to cell wall organization, cell wall protein metabolism, drug catabolism, ergosterol metabolism, intracellular signal transduction, and

morphogenesis were downregulated (Fig. 3D, Group 3). GO enriched terms for groups shown in Fig. 3D are presented in Supplementary Data 4. Ninety minutes after the challenges, the number of

Biological Processes common to the two conditions, represented by cellular component organization, regulation of translation, macromolecule metabolism, and biosynthesis, diminished (Fig. 3E,

Group 1). Mechanical injury provoked the upregulation of genes related to nitrogen metabolism, cell wall organization, RNA processing and modification, rRNA metabolism, ncRNA metabolism,

calcium transport, cell cycle checkpoint, response to light, nucleic acid metabolism, aromatic metabolic process, which were not induced upon attack by Drosophila (Fig. 3E, Group 2). After

arthropod attack, genes involved in DNA damage checkpoint, histone H3-K79 methylation, and DNA replication were still upregulated. Processes such as cAMP-mediated signaling, regulation of

programmed cell death, antibiotic metabolism, and phosphorelay signal transduction system were upregulated only 90 min after attack (Fig. 3E, Group 3), among other less represented

processes. Ninety minutes after the challenges, almost 900 genes were downregulated by both treatments, genes involved in establishing and maintaining localization, cell division,

reproduction, cytokinetic process, catabolism lipid homeostasis, and cell polarity (Fig. 3E, Group 4). At this stage of the response, mechanical injury resulted in downregulation of genes

related to growth, development, response to chemicals, signaling, cellular localization, and actin polymerization (Fig. 3E, Group 6). Whereas larval grazing negatively impacted the

expression of genes involved in actin filament-based process, glucan metabolism, lipid metabolism, autophagy control, membrane budding, and vesicle-mediated transport (Fig. 3E, Group 5). GO

enriched terms for groups from Fig. 3E are presented in Supplementary Data 4. Overall, early (30-90 min) after the challenges, we observed that critical processes for hyphal regeneration and

chemical response, such as heterokaryon incompatibility, calcium signaling, cell death, DNA metabolism, secondary metabolism, and cell cycle do not respond to Drosophila’s attack

(Supplementary Data 5). Thus, we wondered if attack by Drosophila could affect hyphal regeneration. LARVAE SALIVARY GLAND EXTRACTS BLOCK HYPHAL REGENERATION IN _T. ATROVIRIDE_ When looking

at the upregulated genes considered necessary for hyphal regeneration [14] that did not respond to Drosophila’s attack, we found genes related to heterokaryon incompatibility, calcium

signaling, cell death, and cell cycle (Supplementary Data 5). We selected two genes belonging to each of these processes for RT-qPCR analysis. As expected, all selected genes were induced by

mechanical injury, five of them showing maximum expression level 15 min after mechanical injury and the remaining three at 90 min after the treatment (Fig. 4A). In contrast, only one

encoding a cyclin-dependent kinase was slightly induced in response to Drosophila, confirming the RNAseq results. The fact that a set of genes considered necessary for hyphal regeneration

did not respond to attack by Drosophila suggested that products of their salivary glands could block the activation of gene expression. Therefore, we decided to determine the effect of

salivary glands extracts on hyphal regeneration and gene expression. Using a scalpel soaked in Salivary Gland Extract (SGE), we injured hyphae, quantified hyphal regeneration, and compared

these numbers with those obtained upon damage with a clean scalpel. As previously reported [17], upon hyphal injury, cytoplasm leakage stops immediately, regardless of the presence of

salivary gland extract (Fig. 4B). However, hyphal regeneration dropped from 70% to 30% when the scalpel was soaked in the extract (Fig. 4B & C). To test if SGE blocked the activation of

gene expression, we selected four genes induced by mechanical injury and repressed by Drosophila attack (Het-1, Het-2, cyclin, and calmodulin) for RT-qPCR analysis (Supplementary Table 3).

As hypothesized, the presence of the salivary gland extract blocked the transcriptional activation of regeneration-related genes (Fig. 4D). Thus, a component of the larval salivary glands

blocks the transcriptional response and consequently hyphal regeneration. To determine the nature of the product of the salivary glands that could exert the blocking effect, we quantified

regeneration frequency upon treating the extract with heat, proteases, and protease inhibitors. Heat treatment of the extract allowed almost twice as many hyphae to regenerate compared to

the untreated SGE (Supplementary Fig. 6), indicating that the inhibitory molecules are heat sensitive. Protease treatment of the extract and the addition of protease inhibitors produced

similar results (Supplementary Fig. 6), suggesting that the inhibitory component present in the extracts is proteinaceous, very likely proteases. DISCUSSION Here we describe how _T.

atroviride_ responds specifically to different types of damage. Attack by _D. melanogaster_ larvae resulted in a strong transcriptional response. In contrast, exposure to the springtail _O.

folsomi_ resulted in a feeble transcriptional response, which could be explained by nearly complete inhibition of any response from the fungus. Nevertheless, no fungi emerged from

collembolan bodies. As fungi emerged from dead _D. melanogaster_, bodies, it suggests that the collembolans did not ingest _T. atroviride_ mycelia. In this regard, collembolans are

considered generalist feeders but in most laboratory studies they are selective when given fungi as a food source, feeding on specific fungal taxa or structure [33]. Thus, the mere presence

of a potential predator is insufficient to trigger a response. Possibly the transient, weak transcriptional response to the collembolan being triggered by the mechanical perturbation from

walking on the mycelial mat. Drosophila attack inhibited the transcriptional activation of genes involved in ROS production, Ca2+ and MAPK mediated signaling pathways, programmed cell death,

and the innate immune system, previously reported as key processes for the injury response ([14, 15, 17]; Fig. 5). We showed that mechanical injury and Drosophila attack provoke specific

transcriptional responses, allowing us to propose regulatory elements present in genes co-expressed in response to damage, which resembles the differential response of plants to mechanical

wounding and herbivory [34,35,36,37]. As in plant herbivory, larval oral secretions could be an important factor for the adverse effects observed on fungal fitness when challenged with

Drosophila larvae [25, 38]. Larval saliva at the instar used here (late third instar) is composed mainly of proteins, sugars, and RNA, including glycoproteins, proteases, and protease

inhibitors, among other components [39, 40]. Fungi recognize MAMPs and DAMPs to activate their innate immune system in response to injury or predation [1, 8, 12]. We proposed that salivary

gland contents could inhibit the fungal response. Consequently, DAMPs released after the insect attack would not activate the signaling pathways necessary to trigger the transcriptional

response required to initiate hyphal regeneration. In this regard, our transcriptional analysis showed that genes of the putative immune response acting as NLRs, like those encoding HET,

WD40, and NACHT domain proteins, were not induced upon insect attack [8, 15]. In addition, specific _het_ genes were induced within minutes upon mechanical injury, and only those activated

late (8 h) responded to both mechanical injury and arthropod attack. In this sense, _het_ genes, induced late could prepare the cell for future damage or participate in developmental

processes, and those induced early after mechanical injury could be related to the innate immune system [15]. Consistently, salivary gland extracts repressed the expression of _het_ genes

and inhibited hyphal regeneration. Transcriptomic response of the basidiomycete fungus _C. cinerea_ to biotic and abiotic stress revealed the induction of lectin encoding genes during its

interaction with the fungivorous nematode _A. avenae_ [23]. This toxicity extended to the nematode _Caenorhabditis elegans_ [41], the mosquito _Aedes aegypti_, and the amoeba _Acanthamoeba

castellani_ [24]. Thus, fungal lectins are part of the first barrier of fungal defense against predators. We found the early upregulation of six genes encoding Concanavalin A-like

lectin/glucanases (galectins) after challenge with Drosophila (30 and 90 min). It is exciting that a gene encoding a Ricin-B lectin, a member of the lectin family related to the fungal

defense against fungivorous nematodes [23, 24], was induced early by arthropod attack. Galectins, like Ricin-B lectins, have been linked to immune responses in fungi, plants, and animals

recognizing carbohydrates present in pathogens or antagonists [23, 42,43,44]. Thus, Trichoderma lectins could serve as a natural defense against the larvae, as entomotoxic proteins as

previously proposed [23, 24], causing larval death and developmental delays [25]. Calcium signaling is critical for initiating the regeneration processes [15, 45]. The lack of activation or

repression of Ca2+ signaling related genes upon attack by Drosophila and injury by larval salivary gland extract is consistent with the severe reduction in hyphal regeneration capacity in

_T. atroviride_ (Fig. 5). ROS participate as signal molecules at low levels to induce an immune response or to eliminate invading pathogens [46, 47] or to promote regenerative events in

wounded Zebrafish and Xenopus [48, 49]. In _Trichoderma_, NADPH oxidase-dependent ROS production is essential to trigger conidiation in the damaged area after mechanical injury [17].

However, at high concentration ROS induce severe DNA damage, leading to cell death, affecting tissue repair [50]. Here we show that mechanical injury induced the expression of 11 genes

related to the DNA repair pathway, encoding four proteins involved in DNA mismatch repair (MutS-like), five genes encoding the DNA repair proteins (Rad18, Rad50, Rad52, Rad21/Rec8-like

protein and RecA), and two DNA replication/checkpoint proteins MRC1 (homologs of the human Mediator protein MDC1). In contrast, Drosophila attack resulted in the downregulation of five DNA

damage response genes, two encoding Rad, and three MutS-like proteins. The genes related to the DNA damage response pathways could respond to the previously described production of ROS after

mechanical injury [17] and the subsequent activation of processes related to hyphal regeneration (Fig. 5, [51]). In contrast, damage caused by the larvae could activate the production of

high concentrations of ROS, increasing DNA damage and leading to the activation of a protective mechanism mediated by the superoxide dismutase and chloroperoxidase induced by Drosophila

attack (Fig. 5). In conclusion, it appears that Drosophila attack does not activate the necessary mechanisms to repair possible DNA damage caused by an increase of ROS production and

consequently, affects the hyphal regeneration process. The first barrier of fungal defense against predators is the production of secondary metabolites [18, 19, 52]. Previously we reported

that attack by Drosophila affected the production of secondary metabolites presumably related to _T. atroviride_ chemical defense [25]. We found induced 12 secondary metabolism-related genes

after mechanical injury. Among these genes, putative regulators of secondary metabolism, such as the methyltransferase _Talae_1 and the bZIP transcription factors _Tanap_A and _Tamet_R were

induced by mechanical injury and repressed by arthropod attack [25]. Attack by Drosophila resulted in the upregulation of 15 secondary metabolism genes, mainly related to the production of

non-ribosomal peptides and the orthologue of a gene coding for the _Clostridium difficile_ insecticidal toxin TcdB. These genes may constitute a defense mechanism against an attacker,

possibly even killing the predator. At late stages of the responses, mechanical injury and Drosophila attack activated many non-ribosomal peptide synthase and polyketide synthase encoding

genes, that could play a role in conidiophore development and conidia pigmentation [53,54,55]. In regenerative organisms, the expression of genes related to cell cycle control and activation

is vital to injury response and regeneration [56, 57]. Here, the transcript of the _T. atroviride_ gene (Tatro_009561-T1) orthologue of StuA decreased within minutes during the response to

Drosophila. Consistently, genes required for cell cycle activation were downregulated, and, consequently, hyphal regeneration inhibited. Accordingly, the StuA DNA binding motif was enriched

in the promoters of genes downregulated early after insect attack. Other TFs important for fungal development are the members of the NDT80 family. In _A. nidulans_ NdtA, a member of this

family, is required for sexual reproduction [58, 59]. In _N. crassa_ Vib-1, one of the three members of the Ndt80 family, is required for expression of genes involved in

heterokaryon-incompatibility and programmed cell death [59,60,61]. Here we reported three NDT80 homologues in _T. atroviride_, Tatro_010668-T1 being the more closely related to Vib-1 of _N.

crassa__,_ and Tatro_003887-T1 to NdtA of _A. nidulans_. Although these TFs are not differentially expressed, we found an enrichment in the motif recognized by these TFs in the promoter of a

large set of genes induced early after mechanical injury, containing genes related to heterokaryon-incompatibility. The _T. atroviride_ Srf1 and Fkh1 orthologues are induced by mechanical

injury. In _A. nidulans_ Srf1 is involved in sexual/asexual development [62], while the _S. pombe_ orthologue of Fkh1 participates in cell cycle and sexual differentiation [63]. Thus, these

TFs are likely involved in the regulation of hyphal regeneration in _T. atroviride_. Furthermore the _T. atroviride_ orthologue of Atf1 is repressed by _Drosophila_ attack and a set of genes

enriched in its recognition motif are induced by mechanical injury. In _A. nidulans_ Atf1 and SakA (the orthologue of the _T. atroviride_ Tmk3) regulate oxidative and osmotic stress

responses [64]. Earlier we described the participation of Tmk3 in the chemical and developmental responses to injury [14, 15, 25]. In conclusion, _T. atroviride_ responds differently to

damage caused by an attacker or mechanically and activates a developmental program leading to conidiation to permit its survival. Thus, fungi display an inducible defense that allows

“economically friendly” allocation of resources, likely to influence multitrophic interactions. We also provide transcriptomic evidence of the specific response of _T. atroviride_ to injury

and attack by Drosophila and how the latter affects processes related to chemical defense, the putative innate immune system, DNA damage repair, and hyphal regeneration (Fig. 5). In general

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ACKNOWLEDGEMENTS We thank Pedro Martínez-Hernández and Nestor Nazario-Yepiz for technical assistance. We also wish to thank Dr. Therese Markow for critical reading of the manuscript and

providing _D. melanogaster_ and, José Palacios-Vargas and Blanca Estela Mejia-Recamier for providing the collembollan used in this study. This work was support in full by Cinvestav

institutional funds. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Advanced Genomics Unit-Langebio, Guanajuato, Mexico Karina Atriztán-Hernández & Alfredo Herrera-Estrella Authors *

Karina Atriztán-Hernández View author publications You can also search for this author inPubMed Google Scholar * Alfredo Herrera-Estrella View author publications You can also search for

this author inPubMed Google Scholar CONTRIBUTIONS KA-H carried out the experiments, analyzed data, and contributed to the experimental design. AH-E supervised K A-H, designed experiments,

and obtained financial support. AH-E and KA-H wrote the manuscript. CORRESPONDING AUTHOR Correspondence to Alfredo Herrera-Estrella. ETHICS DECLARATIONS COMPETING INTERESTS The authors

declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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ARTICLE CITE THIS ARTICLE Atriztán-Hernández, K., Herrera-Estrella, A. Drosophila attack inhibits hyphal regeneration and defense mechanisms activation for the fungus _Trichoderma

atroviride_. _ISME J_ 16, 149–158 (2022). https://doi.org/10.1038/s41396-021-01068-9 Download citation * Received: 14 January 2021 * Revised: 30 June 2021 * Accepted: 09 July 2021 *

Published: 19 July 2021 * Issue Date: January 2022 * DOI: https://doi.org/10.1038/s41396-021-01068-9 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this

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