Fused expression of Sm1-Chit42 proteins for synergistic mycoparasitic response of Trichoderma afroharzianum on Botrytis cinerea

Sm1 and Chit42 of Trichoderma have been universally confirmed as crucial biocontrol factors against pathogen infection through induced resistance and mycoparasitism, respectively. However, not enough work has been conducted to understand the novel function of fused expression of these two proteins in Trichoderma. The results of this study demonstrated that Sm1-Chit42 protein (SCf) engineered T. afroharzianum strain OE:SCf exerted synergistic inhibition to Botrytis cinerea growth at multiple stages of mycoparasitic interaction of T. afroharzianum and B. cinerea including chemotropism sensing, hyphal coiling, hydrophobicity modulation, cell wall adhesion, virulence reduction and pathogen killing by ROS. These results highlight a novel mycoparasitic system in Trichoderma strains engineered with Sm1-Chit42 chimeric protein to combat B. cinerea growth and reproduction, which would lay a strong foundation for exploring a new engineered Trichoderma biofungicide created with chimeric proteins in the future. Supplementary Information The online version contains supplementary material available at 10.1186/s12934-023-02151-w.


Introduction
Microbial interactions are ubiquitous and versatile, ranging from mutualism to parasitism and competition.Fungi have numerous mechanisms to communicate with other organisms, including plants, animals, and other microorganisms [1].Interestingly, most fungal relations are competitive or combative [1,2].Trichoderma is one of the most widely used biocontrol fungi and is usually viewed as a green alternative to chemical pesticides in Trichoderma spp., Sm1 evolved the ability to modify the hydrophobicity of the fungal hypersphere and, thus, facilitate the nutritional versatility of Trichoderma [17,18].
In the case of mycoparasitic Trichoderma, the fungal pathogen cell wall is exposed to the action of Trichoderma hydrolases such as chitinases, which digest the fungal cell wall chitin as an antifungal mechanism and release chitin oligomers that can be recognized by the host plant [19][20][21].Fungal chitinases are therefore not only involved in exogenous chitin decomposition but also fungal cell wall degradation and remodeling [22,23].Chitinases have diverse sources, characteristics, and mechanisms, which present challenges to the development of optimization procedures and standardization techniques for enhancing practical applications.With the development of protein engineering techniques, these difficulties can be overcome by modifying or redesigning chitinases to improve specific features required for specific applications [24].Fungal chitinases consist of glycoside hydrolases 18 (GH 18), GH 19, and GH 20 modules; thus, they also include different carbohydrate-binding modules (CBMs) [25,26].Chitinase 42 (Mchit42) of Metarhizium anisopliae has been proven to be a major contributor to the antagonistic activity against B. cinerea [27] however, MaChit42 lacks CBM.The Sm1-like proteins have been proven to bind chitin [18,[28][29][30].It was speculated that the integration of Sm1 and Chit42 proteins may allow Sm1 as the CBM of Chit42 to complement the functions of Chit42, thereby potentially developing a synergistic mechanism against pathogenic infection.Therefore, constructing chimeric proteins from two different effectors through a linker has become a new strategy for Trichoderma to improve its biocontrol activity against fungal phytopathogen infection.This study was designed to elucidate the mechanism by which engineered Trichoderma with the chimeric proteins Sm1-Chit42 confers synergistic control of B. cinerea.

Interaction assays between engineered strains of T. afroharzianum and fungal phytopathogens
The Trichoderma and phytopathogens interaction were assessed by dual confrontation assays [17].Agar plugs of fresh engineered strains and the plant fungal pathogens were pregrown on PDA at 25 °C for 4 days, and were placed on opposite poles of a PDA plate.Images of each plate were recorded by an MF image system (Shineso, Hangzhou, China) after incubation at 25 °C in darkness for 4-7 days.Fungal biomass was collected from the interacting and non-interaction sides after connection for 24 h, which could be used to determine the expression levels of mycoparasitism and signal transition-related genes.A quick method for visualizing the interaction process of engineered strains and B. cinerea was accomplished by using FM4-64 (AAT Bioquest, Pleasanton, USA) and CDCFDA (AAT Bioquest, Pleasanton, California) probe and Leica TCS SPS-II (Wetzlar, Germany) confocal laser scanning microscopy.The engineered strains and B. cinerea were cultured on a PDA plate containing glass paper.Glass paper plugs of B. cinerea were incubated with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO4, pH = 7.2) diluted 2-20 µM FM4-64 probe, and which were kept on ice for 15 min.The glass plugs were put on the PDA plate after twice PBS washing.Glass paper plugs of engineered strains were incubated at 28 ℃ with PBS buffer diluted 0.5 ~ 25 µM CDCFDA probe for 15 min, and then incubate at 37 ℃ for 30 min after changing the 37 ℃ preheated CDCFDA probe.The glass plugs of engineered strains were put on the PDA plate close to B. cinerea after twice PBS washing.The interaction process of engineered strains and B. cinerea was observed on confocal laser scanning microscopy after 12 h.

Quantification of T. afroharzianum chemotropism
This experiment is modified according to the method of Turrà et al. [35]. 2 mL of sterile water was added to the mature culture plates of T. afroharzianum wild-type and engineered strains (OE:Sm1, OE:Chit42, OE:SCf), the conidial suspensions were filtered through a sterile glass funnel and gauze into a sterile 15 mL falcon tube.Freshly obtained conidial spores of T. afroharzianum wild-type and engineered strains (OE:Sm1, OE:Chit42, OE:SCf) were embedded in 5 ml water agar (WA; 1%, w/v) (Oxoid, Thermo Fisher, MA, USA) at a final concentration of 2 × 10 5 /ml and poured into a standard Petri dish (Fig. 2A).A central scoring line was drawn on the bottom of the plate, and two parallel wells were cut into the WA layer on both sides at 5 mm distance from the scoring line.Then, 50 µl of the test compound solution or the solvent control was added to the wells at both sides of the scoring line.In gradient competition experiments, solutions of the two different test compounds were applied at both sides of the scoring line.Tested compounds and standard concentrations were: chitohexaose and chitotriose at 50 mM; or cellulose, B. cinerea cell wall, and colloidal chitin, all at 1% (w/v); or unconcentrated B. cinerea culture broth.Cellulose was used as solvent control.To measure the chemotropism of T. afroharzianum towards test compounds, plates were maintained in a plastic box at 28 °C in the dark for the indicated periods (13 h unless otherwise stated).Chemotropism of conidial germ tubes was quantified with a Leica binocular microscope (200 × magnification), by counting the number of hyphal tips pointing toward the test compound and those pointing toward the solvent control.The chemotropic index was calculated as Eq. ( 1): where H test is the number of hyphae growing towards the test compound, H solvent is the number of hyphae growing towards the solvent control, and H total is the total num- ber of hyphae counted.For each test compound, a total of 100 hyphal tips were scored.All experiments were performed at least twice.Statistical analysis was conducted using a t-test.

Chitinase activity assay
Chitinase activity was measured using the Micro Chitinase Assay Kit (SolarBio, China) following the manufacturer's instructions.The T. afrohazianum overexpression strains were cultivated in Czapek-Dox medium.Secretion analysis was performed as described.Briefly, 7-dayold engineered strains culture supernatants (1 mL culture supernatant per sample) were collected.The chitinase activities can be measured fluorometrically at a wavelength of 585 nm.Measurements were done by incubating 100 ng of recombinant protein for 60 min at 37 °C.
Fluorescence was detected with a Microplate Reader (SpectraMaxi3x, Molecular device) at room temperature.Each measurement was performed in triplicate and experiments were repeated at least four times with independent batches of recombinant proteins.Each treatment was replicated three times.

Gene expression analyses by qPCR
The transcript levels of the TaSm1 gene were determined by qPCR in T. afroharzianum engineered strains.And the transcript levels of mycoparasitism and signal transitionrelated genes were determined in the interaction process of engineered strains (Wild-type, OE:Sm1, OE:Chit42, OE:SCf ) and B. cinerea.Transcript levels of B. cinerea genes affected by T. afroharzianum OE:SCf engineered strain were determined by qPCR.For quantification of gene expression, total RNAs were extracted from different tissues using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, USA). 1 µg RNA was reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (Vazyme).The cDNAs were used as templates for PCR with gene-specific primers (Table S1).Quantitative real-time RT-PCR analysis (qRT-PCR) was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) on Roche lightcycler96 (Basel, Switzerland).All experiments were repeated three times independently.

Quantification and statistical analysis
All statistical analyses described in this study were performed using Origin 2022b.The number of replicates for each experiment is reported in the figure legends.For the chitinase activity assays, WCA, and qPCR, statistical comparison among groups was performed by one-way ANOVA with the Fisher's Least Significant Difference (LSD) post hoc test.

Construction of engineered T. afroharzianum strains with Sm1-Chit42
An expression vector harboring a copy of the Sm1-Chit42 gene with its trpC terminator under the control of the trpC promoter from Aspergillus nidulans was used to overexpress Sm1-Chit42 in T. afroharzianum.MaChit42, TaSm1, SCf, CSf, SCr, CSr, SCnl, and CSnl (Fig. 1A, Figure S1A-D) overexpression vectors were constructed by using the same method.At least 15 putative transformants of each of T. afroharzianum engineered strains were found to have the expected overexpression cassette.OE:MaChit42-6, OE:TaSm1-1, OE:SCf-7, OE:CSf-11, OE:SCr-5, OE:CSr-3, OE:SCnl-2 and OE:CSnl-7 transformants of T. afroharzianum were identified as single copy strains by using Southern blotting (Figure S1E).Therefore, the engineered strains were designated MaChit42, TaSm1, SCf, CSf, SCr, CSr, SCnl, and CSnl by removing the number mark in further studies.The expression of the Sm1 gene in the above mutants was detected by qPCR.
The results showed that the expression level of Sm1 in OE:SCf strain remained high in PDA and PD, and the Sm1 expression of other engineered strains on the PDA plate was lower than OE:SCf (Figure S1F).As MaChit42 and TaSm1 proteins are similarly secreted from M. anisopliae and T. afroharzianum respectively, six chimeric protein engineered strains were selected for further verification by Western Blot analysis (Fig. 1E), and the results indicated that chimeric proteins are targeted to the extracellular space.

Chitinase activities of Sm1-Chit42 engineered T. afroharzainum strains
The chitinase activity (U/ml) of wild-type (T30) and engineered strains with colloidal chitin as substrate were T30 (  1B).The chitinase activity of OE:SCf was higher than that of other engineered strains, which was a significant (P < 0.05) increase of 58.36% compared to the wildtype (T30).

Chimeric protein offers synergistic inhibitory effects in trichoderma against phytopathogenic fungi
A dual confrontation test of the engineered T. afroharzianum strains against multiple plant fungal pathogens (including B. cinerea, M. oryzae, F. graminearum, F. oxysporum, F. verticillioides, R. solani, B. maydis, M. anisopliae, and C. lunata) was performed as shown in Fig. 3.No significant morphological difference was observed due to the overexpression of chimeric proteins, except that the OE:SCf engineered strains of T. afroharzianum showed a higher growth rate than the other engineered strains (Fig. 1C-D).Overexpression of chimeric protein in the Trichoderma strain showed different antagonism against different pathogens (Table 1), and the inhibitory effects of OE:SCr and OE:CSr were 74.38 ± 4.88 and 70.00 ± 26.46 respectively.However, the inhibitory effect of these two strains was lower than that of OE:MaChit42

SCf chimeric protein triggered early sensing in the interaction between T. afroharzianum engineered strain and B. cinerea
Mutually sensitive sensing between biocontrol fungi and phytopathogenic fungi is usually an early response necessary for initiating a follow-up recognition and mycoparasitism [36].The mycelium of T. afroharzianum wild-type, OE:TaSm1, OE:MaChit42, and OE:SCf strains (Fig. 4A) showed parallel hyphal growth around B.  Letters represent conditions with significant differences according to the post hoc ANOVA Fisher's test (p < 0.05) cinerea and no cell wall degradation debris was detected in the interaction zone.In the T. afroharzianum wildtype and OE:MaChit42 strain interaction zone, a sparse mycelial mass attached to the mycelium of B. cinerea was observed with no hyphal degradation (Fig. 4A).In the T. afroharzianum OE:TaSm1 and OE:SCf strain interaction zone, more mycelia adhered around the mycelium of B. cinerea with the preliminary hyphal degradation (Fig. 4A).The interaction between engineered T. afroharzianum and B. cinerea was also observed using confocal laser microscopy (Fig. 4B).The typical Trichoderma mycoparasitic action with hyphal coiling around the pathogen was observed only in the T. afroharzianum OE:SCf strain treatment but not in wild-type T. afroharzianum wild-type, OE:TaSm1, or OE:MaChit42.RT-qPCR results showed that the expression of Sm1 gene in the engineered strain was more significantly increased in the presence of B. cinerea, M. oryzae, F. graminearum, F. oxysporum, F. verticillioides, R. solani, B. maydis, M. anisopliae, and C. lunata, revealing that specific inductive response occurred between the engineered strains and B. cinerea (Figure S2).That implies that SCf chimeric protein triggered early sensing at the early mutual interaction stage.

ROS release to kill pathogens in the interaction of the T. afroharzianum OE:SCf engineered strain and B. cinerea
ROS have been demonstrated to have dual functions in biocontrol (killing pathogens) and ISR to host plants.The CDCFDA fluorescence probe is also used to detect the ROS burst in the microorganism.Interestingly, the ROS burst could be observed in the mycelium of the T. afroharzianum OE:SCf strain in the mycoparasitic interaction between the T. afroharzianum OE:SCf strain and B. cinerea (Fig. 4B), implying that the higher ROS release level induced by the chimeric protein makes the engineered strain more active in killing pathogens or triggering plant immunity.Similar behavior also occurred in the interaction between T. afroharzianum strains and R. solani (data not shown).

The SCf chimeric protein triggered mycoparasitism and signal transduction genes to modulate the interaction between T. afroharzianum engineered strain and B. cinerea
Confrontation assays were performed between T. afroharzianum wild-type, OE:TaSm1, OE:MaChit42, and OE:SCf strains and B. cinerea.Mycelial RNAs from the confrontation area were extracted from hyphal contact and mycoparasitism.The relative expression levels of Sm1, Chit42, and eleven mycoparasitism gene markers were analyzed to determine the effect of the SCf gene on the modulation of mycoparasitism gene expression (Fig. 4C).In the process of hyphal contact between T. afroharzianum OE:TaSm1 and B. cinerea, nine genes were upregulated, of which Sm1, Lys, Kss1, and Slt2 were prominent.Seven genes were upregulated, of which BC1G_12298, exg1, Chit42, Tga1, and Slt2 were more prominent in the process of hyphal contact between T. afroharzianum OE:MaChit42 and B. cinerea.Thirteen analyzed genes showed a marked difference in their expression levels, indicating SCf fusion gene altered the T. afroharzianum perception of B. cinerea.The expression levels of signal transduction genes were increased by hyphal contact of T. afroharzianum engineered strains and B. cinerea.The signal transduction genes of the T. afroharzianum OE:TaSm1 and OE:SCf strains were better activated in the hyphal contact process, of which the Kss1 and Slt2 genes were more important in this process.Despite the relatively stable gene expressions with slight variations, significant differences were noted in the expression modulation of the transduction genes in the process of hyphal contact and mycoparasitism.The signal transduction genes play an important role in hyphae contact before the mycelium of T. afroharzianum engineered strains covered B. cinerea.In the confrontation between T. afroharzianum engineered strains and B. cinerea, the gene expression level of mycoparasitism genes was divided into 3 clusters according to their expression pattern: Group 1 (PRA1, mutAW, PM28, and exg1), Group 2 (Chit42, Sm1, sprT, nag1, and papA) and Group 3 (Lys and Lyase7).The expression levels of genes in Group 2 were higher when T. afroharzianum OE:SCf interacted with B. cinerea.The results described above demonstrate that the SCf genes can modulate the expression of mycoparasitism and signal transduction genes to improve the antagonistic effects on B. cinerea.

SCf chimeric protein improved the chemotropism of the engineered T. afroharzianum strain for scavenging chitin fragments
The chemotropism between Trichoderma and pathogenic fungi is also crucial for the successful contact of engineered strains over pathogens.In this study, we sought to understand whether chimeric protein in engineered stains cause faster growth toward chitin or derivatives from pathogen cell walls.First, we investigated what chemoattractants from B. cinerea sense chimeric proteins of engineered T. afroharzianum strains (Fig. 5B-C).The results showed that the B. cinerea cell wall is the chemoattractant for the pathogen to sense T. afroharzianum OE:TaSm1 and OE:Chit42.The order of the CI value of T. afroharzianum OE:SCf was colloidal chitin, B. cinerea cell wall, chitohexaose, chitotriose.The CI value of B. cinerea cell wall of T. afroharzianum OE:SCf was higher than OE:TaSm1 and OE:Chit42.The chitin derivates are the specific chemoattractants for T. afroharzianum OE:SCf.It was indicated that chitin and its derivatives are the chemoattractants for the pathogen to sense T. afroharzianum OE:SCf.The comparison of chemoattractants confirmed that the B. cinerea cell wall and colloidal chitin were the strongest chemoattractants.To evaluate the morphological responses of engineered T. afroharzianum strains to pathogen chemotropism, assays were conducted based on the changes in plate colonies.Distinct morphological responses to positive or negative chemotropic compounds were detected at this stage (Fig. 5D).The B. cinerea cell wall and colloidal chitin notably promoted colony extension of engineered strains, and the colony edge of T. afroharzianum OE:Sm1 and OE:SCf grew more quickly toward the B. cinerea cell wall and colloidal chitin than the wild-type strain.Thus, the attraction of engineered strains to chitin and its derivatives in the pathogen make it more closely attached to the surface of the pathogen, thereby allowing T. afroharzianum OE:SCf to establish a stronger mycoparasitic relationship with the pathogen.

SCf protein is secreted into the extracellular space to modulate surface hydrophobicity
The ability to modify the surface hydrophobicity of Sm1, Chit42, and SCf as surface-active proteins was illustrated by the results of the water contact angle (WCA) measurement with the proteins extracted from T. afroharzianum engineered strains.The data shown in Fig. 6A demonstrate the potential of recombinant Sm1, Chit42, and SCf protein to change the surface hydrophobicity of materials.Specifically, a coating of 50 µg/ml recombinant Sm1, Chit42, and SCf proteins on PET (hydrophobic) surface significantly reduced the surface hydrophobicity.However, the WCAs of the recombinant Sm1, and SCf proteins were 83.94° and 87° of the control, respectively, compared to the Chit42 protein, which was 67.64° of the control (Fig. 6B).The effect on glass (hydrophilic) was not significant (P > 0.05) (Fig. 6C).The hydrophobicity modulation ability of recombinant Sm1, Chit42, and SCf was investigated in more detail by AFM imaging.High-resolution imaging of the protein layers in PET with AFM revealed that SCf and Sm1 protein modulated rather irregular particle structures at concentrations of 50 µg/ml compared to sterile water and Chit42 protein (Fig. 6D).It was possible to measure the roughness of recombinant Sm1, Chit42, and SCf protein-treated PET surfaces by Gwyddion.Sm1 and SCf treated PET had a similar surface roughness (Fig. 6E), and the surface irregular particle height changed more than that of Chit42 and sterile water-treated PET.In addition, there was no significant difference in the effect of overexpressed TaSm1, MaChit42, and SCf on the hydrophilicity of the mycelia surface of engineered strains (Figure S3A-B).The hydrophobicity of the spore suspensions of T. afroharzianum OE:TaSm1, OE:MaChit42, and OE:SCf was not remarkably changed (Figure S3C-D).These results suggested that the SCf protein is secreted into the extracellular space to modulate the surface hydrophobicity of the host/pathogen.

SCf chimeric protein can better adhere to the cell wall of B. cinerea
The Sm1, Chit42, and SCf proteins were labeled by FITC staining, and the mycelium of B. cinerea was stained by FM4-64 to observe the B. cinerea hyphal attachment ability of the Sm1, Chit42, and SCf proteins (Fig. 7).Confocal laser microscopy results showed that more Sm1 FITC and SCf FITC were attached to B. cinerea hyphal than Chit42 FITC protein.The Sm1 FITC and SCf FITC proteins showed more uniform adhesion to the surface of B. cinerea hyphae.This indicated that the chimeric protein improved the potential of Trichoderma to bind chitin and its derivatives from the pathogen compared to Chit42 protein.

SCf fusion affected the expression of cell wall and virulence genes of B. cinerea
The expression levels of chitin synthesis (Bcchs1, Bcchs3a, and Bcchs5) virulence-related genes (BcOAH, BcC-FEM, BcBMP1, BcPLC1, and BcPLS1) in B. cinerea during the interaction with T. afroharzianum OE:Sm1, OE:Chit42, and OE:SCf strains are shown in Fig. 8.The T. afroharzianum OE:SCf strain more effectively inhibited the synthesis of chitin, however, there was no significant difference between the T. afroharzianum OE:Chit42 and OE:SCf strains.The expression levels of virulence-related genes (BcOAH, BcCFEM, BcBMP1, BcPLC1, and BcPLS1)

Discussion
The biocontrol fungus T. afroharzianum is an avirulent symbiont with the ability to control plant disease through the production of antibiotic compounds, induction of plant resistance to pathogens, and mycoparasitism of other fungi [37][38][39][40], Mycoparasitism is a well-established mode of biocontrol microbes against fungal pathogens, for instance, Trichoderma uses the mechanism to offer antagonistic action against a group of fungal pathogens [41,42].The mycoparasitism process conducted by Trichoderma involves chemotropism-based sensing, contact, and recognition, coiling and lysing of the target pathogen hyphae [43].To enhance the biocontrol efficiency of Trichoderma, the construction of engineered Trichoderma strains with overexpressed functional genes is an alternative approach to improve Trichoderma agent performance in biological control [44].Owing to their complementary advantages, Sm1 and Chit42 were linked and cotransformed into T. afroharzianum to develop a more powerful Trichoderma agent available to use for highly effectively parasitizing pathogens and inducing plant resistance to pathogen infection.Chitin, one of the most widespread polysaccharides in nature, is a linear polymer of β-1-4-linked N-acetyl glucosamine (NAG) units [45].This biopolymer is part of insect exoskeleton, crustacean shells, and fungal cell walls [46,47].Trichoderma chitinases degrade pathogen cell wall chitin components into low molecular weight chitooligomers, which have been demonstrated to induce local and systemic plant resistance [20,48].The chitinase activity of OE:SCf was higher than that of other engineered strains, showing a significant (P < 0.05) increase of 58.36% compared to that of the wild-type.The T. afroharzianum OE:SCf was proven to effectively control B. cinerea and R. solani.The chitinase from Trichoderma sp.drastically affected the cell walls of phytopathogens [48], indicating that the increase of chitinase activities can stimulate the antagonistic ability of the engineered T. afroharzianum strain.
The first evidence regarding the role of SCf genes in the T. afroharzianum OE:SCf strain indicates the generation of a sensing mechanism by developing more aerial hyphae to overgrow the B. cinerea aerial hyphae even though the sensing factors remain unclearly.Although positive chemotropism in response to prey-derived stimuli has been recognized as a key initiating feature of mycoparasitism early on [49], the cellular and molecular details of prey sensing are still poorly understood.Interspecies recognition in fungi has been linked to remote sensing of cell wall components [50][51][52].It is generally accepted that fungi permanently secrete low amounts of extracellular chitinases that cause target fungi in the vicinity to release cell wall oligochitosan.Oligochitosan, in turn, is recognized as a ligand by the fungal mycoparasite and further induces the massive release of cell wall degrading enzymes from the mycoparasites [42,53] promote degradation of the chitin in the target fungal pathogen cell wall.This cascade of events is considered the starting point for the activation of a mycoparasitic attack program in various Trichoderma species [50,54].To initiate the mycoparasitic process, early sensing or chemotropism plays a crucial role in the interaction of Trichoderma and fungal pathogens.In our study, B. cinerea cell wall and colloidal chitin were confirmed as the strongest chemoattractants for T. afroharzianum OE:SCf.Chemotropism between Trichoderma and fungal pathogens has been viewed as an important factor for effective mycoparasitism against fungal pathogens [55].Interestingly, SCf from the engineered Trichoderma strain generated more activity to promote the growth of the engineered Trichoderma strain toward chitin and its derivatives from B. cinerea.In addition, the SCf protein is a complex hydrophobic protein that can modulate surface-surface hydrophobic contact between Trichoderma and pathogens, and between Trichoderma and plants, which would benefit the colonization of Trichoderma over pathogen mycelium and host plant.We assumed that the enhanced function of the SCf complex could be attributed to the changes in MaChit42 hydrophobicity resulting from partner Sm1 protein because Sm1 belongs to cerato-platanin (CP), a protein closely related to hydrophobin [18] Sm1 is characterized as an amphoteric protein membrane formed at the hydrophilic-hydrophobic interface, which can improve Trichoderma attachment on the surface of pathogenic fungus and plant leaf [18].Therefore, we assumed that OE:SCf enhanced the destruction of the pathogen cell wall and induced plant resistance by secreting SCf protein into the extracellular space to modulate the surface hydrophobicity of the host/pathogen.After contact of Trichoderma mycelia on the surface of pathogen mycelia, the former typically coil around the latter and form of helix-shaped hyphae [42,56], and this phenomenon is dependent on the recognition of lectins (CBM) from the fungal prey [57].In our study SCf protein enhanced Trichoderma mycelial coiling around B. cinerea mycelium relative to the Trichoderma engineered strains with a single engineered protein.
SCf was demonstrated to promote reactive oxygen species (ROS) release from engineered Trichoderma, which was supposed to enhance the killing effects of pathogen or chitin-triggered immunity.Recently studies showed that T. guizhouense NJAU 4742 can use protease [58] and reactive oxygen species to kill pathogens such as F. odoratissimum (formerly known as FOC4) [2].The key reactions of chitin biosynthesis are catalyzed by chitin synthase, a membrane-integrated glycosyltransferase that transfers GlcNAc from UDP-GlcNAc to a growing chitin chain [59][60][61].Chitin synthase from the devastating soybean root rot pathogenic oomycete Phytophthora sojae (PsChs1) reveals the directional multistep mechanism of chitin biosynthesis and provides a structural basis for the inhibition of chitin synthesis.The homologous gene of PsChs1 in B. cinerea was identified, however, the expression of BcChs1 in interaction with T. afroharzianum OE:SCf was inhibited compared to that in interaction with T. afroharzianum OE:Chit42, implying T. afroharzianum OE:SCf strains could affect chitin synthesis.
Virulence proteins secreted by B. cinerea such as BcC-FEM, BcOAH, BcBMP1, BcPlc1, and BcPls1 were affected by T. afroharzianum OE:SCf.These genes were proven to modulate the virulence of B. cinerea [62][63][64].Other research also showed that in the interaction zone of the aerial hyphae of F. odoratissimum and T. guizhouense contain proteolytic enzymes, hydrogen peroxide, and other metabolites [65,66], those substances may also directly or indirectly participate the antagonistic process to B. cinerea by chimeric protein from engineered strains.It is well known that the great advantage of Trichoderma, based on accumulating knowledge of fungi-triggered plant immunity, is the induction of broad-spectrum pathogenic fungal resistance, which promises to be an effective and eco-friendly avenue to combat fungal diseases [5].
Overall, mycoparasitic interaction between the Trichoderma Sm1-chit42 engineered strain and B. cinerea involved the chemotaxis sensing, hyphal coiling, hydrophobicity modulation, cell wall adhesion, virulence reduction, and pathogen killing by ROS (Fig. 8).In conclusion, the chimeric protein SCf composed of Sm1 and Chit42, can enable synergistic action against B. cinerea as the following model.

Fig. 1
Fig. 1 Construction and enzyme activity of T. afroharzianum engineered strains.(A) Illustration of open reading frame of chimeric protein; (B) Chitinase activities of T. afroharzianum wild-type (T30) and engineered strains.The chitinase activity of OE:SCf was higher than that of other engineered strains and wild-type (T30); Diameter of mycelium growth of T. afroharzianum engineered strains cultured in PDA medium with different inoculation methods of the colony plugs (C) and spore suspension (D); (E) Western blot analysis of chimeric protein in the culture broth of T. afroharzianum.Letters represent conditions with significant differences according to the post hoc ANOVA Fisher's test (p < 0.05)

Fig. 2
Fig. 2 Chemotropism of T. afroharzianum engineered strains.(A) Schematic representation of the plate chemotropism assay.Test compound and solvent control are applied to opposite sides of a Petri dish containing a layer of water agar with 2 × 10 5 /ml conidia spore of T. afroharzianum wild-type (T30), OE:Sm1, OE:Chit42, and OE:SCf at a distance of 0.5 cm from the central scoring line; (B) Directed growth of germ tubes after exposure to the indicated compounds.Letters represent conditions with significant differences according to the post hoc ANOVA Fisher's test (p < 0.05); (C) Germination of conidia spore of T. afroharzianum over time.Germ tube emergence sites are visualized by inserted the GFP protein into T. afroharzianum wild-type (T30), OE:Sm1, OE:Chit42, and OE:SCf.DIC, differential interference contrast.Scale bar, 50 µm; (D) Mycelium chemotropism of T. afroharzianum towards cellulose (control solvent), B. cinerea cell wall, and colloidal chitin.The left filter slice is B. cinerea cell wall or colloidal chitin; The right filter slice is cellulose (91.33 ± 15.01).The overexpression of SCf chimeric protein enhanced antagonism against B. cinerea (80.00 ± 4.10), F. oxysporum (44.73 ± 11.26), F. verticillioides (48.65 ± 3.33), and R. solani (86.98 ± 11.30) compared to that of the wild-type (T30), OE:TaSm1 and OE:MaChit42 strains.However, the antagonistic effect of OE:SCf on F. oxysporum and F. verticillioides was not significantly different (P > 0.05) from that of wildtype (T30) strains.In addition, none of the engineered strains showed obvious antagonism to M. anisopliae, and overexpression of fusion did not affect the self-recognition of MaChit42 on M. anisopliae cell wall.

Fig. 5 Fig. 7 Fig. 6
Fig. 5 Cellular localization analysis of Sm1 FITC ., Chit42 FITC , and SCf FITC on hyphae of B. cinerea.FITC labeled protein was quantified by the Bradford method and 2.5 mΜ protein was added into the hyphae germinated from 600 µl B. cinerea mycelium pre-inoculated in a glass paper slide at 28 ℃ for 12 h.FM4-64 was used as B. cinerea cell membrane stains.The fluorescence intensity of FITC-labeled proteins and B. cinerea cell membrane were measured by ImageJ software

Fig. 8
Fig. 8 Mycoparasitic interaction model of T. afroharzianum OE:SCf with B. cinerea.The interaction was involved the chemotropism sensing, hyphae coiling, hydrophobicity modulation, cell wall adhere, virulence reduction, and killing pathogen by ROS

Table 1
Inhibitory effects of T. afroharzianum strains on phytopathogens