| Trichoderma guizhouense NJAU 4742 has been previously isolated and identified in our laboratory,and successfully applied in the fermentation and production of bioorganic fertilizers.Application of Trichoderma-based bioorganic fertilizers have shown significant plant growth-promotion effect and biocontrol function under greenhouse and field condition.To study the direct interaction between Trichoderma and plants is helpful to understand the functional mechanism of Trichoderma-based bioorganic fertilizers,so that we can improve the application effectiveness of Trichoderma in agricultural practice.T.guizhouense NJAU 4742 showed promotion of plant growth and activation of plant immune response.By means of the plant phenotypic analysis,fungal gene knockout,fungal secreted proteome analysis and plant transcriptome analysis,we demonstrated the promotive effect of NJAU 4742 and its interaction with plants at both molecular and tissue level.In addition,we compared the corresponding modes of plant to beneficial and pathogenic fungi.The main results were as follows:1.Plant growth-promotion effect of T.guizhouense NJAU 4742 and its mechanism.Under the condition of soil culture,T.guizhouense NJAU 4742 significantly promoted the growth of maize plants compared to control plants.The height of maize seedlings inoculated with T.guizhouense NJAU 4742 were increased by 10%,and the root weight was increased by more than 30%.T.guizhouense NJAU 4742 could colonize on the root surface and root hairs of maize plants.Under hydroponics condition,T.guizhouense NJAU 4742 also showed significant induction on the growth of maize seedlings,especially on the root system.Compared with the control group,the length of primary root of maize seedlings inoculated with Trichoderma spores was increased by 30%,the number of secondary roots was doubled,and the total length of roots was increased by three times,showing that the root density of maize seedlings was significantly increased under the treatment of Trichderma.In addition T.guizhouense NJAU 4742 had significant growth-promotion effect on rice,tomato and Arabidopsis seedlings.The root length and fresh weight of rice seedlings treated with T.guizhouense NJAU 4742 were both increased by 30%,and the germination rate of tomato seeds treated with T.guizhouense NJAU 4742 spore solution was increased by 20%.Besides,T.guizhouense NJAU 4742 promoted the elongation of the primary roots and the growth of the shoots of tomato seedlings,of which the fresh weight was increased by 20%.Moreover,T.guizhouense NJAU 4742 regulated the root growth and development of Arabidopsis.After the inoculation of Trichoderma,the fresh weight of Arabidopsis plants was increased by 50%and the root configuration of Arabidopsis plants changed obviously,exhibiting inhibited elongation of the primary roots,increased formation of lateral root primordia and promoted growth of lateral roots.By using maize plants and Arabidopsis DR5::GUS mutant lines,T.guizhouense NJAU 4742 promoted the growth of plants by inducing plant endogenous auxin pathway.We detected the plant hormones in the metabolites of T.guizhouense NJAU 4742 using liquid mass spectrometry(LC-MS/MS),among which the content of ethylene precursors(ACC)was highest.Then we took advantage of the enzyme-linked immunoprecipitation reaction(ELISA)kits to confirm that T.guizhouense NJAU 4742 could synthesize ethylene.Under the condition of separate inoculation,volatile metabolites of T.guizhouense NJAU 4742 significantly induced the growth and development of the roots of maize and rice seedlings.The treatment of Trichoderma volatile metabolites and ethephon could stimulate the response of auxin signal in Arabidopsis DR5::GUS mutant seedlings.The growth-promotion effect of double deletion mutant in the ethylene synthesis pathways of T.guizhouense NJAU 4742 of which the ethylene production decreased by 80%,was significantly reduced,indicating that ethylene was involved in the growth-promotion effect of Trichoderma.2.The activation of plant immune response by T.guizhouense NJAU 4742.The Arabidopsis seedlings treated with ethephon or T.guizhouense NJAU 4742 spores showed leaf yellowness response.Moreover,the treatment of T.guizhouense NJAU 4742 triggered a series of immune response in maize plants.There was production of ROS(H2O2)in maize tissues;up-regulation of defense-related genes PR1,PR4,PR5,PR precursor,LOX,ACO,ETR,CWI;changes of resistance-related hormones salicylic acid(SA),jasmonic acid/ethylene(JA/ET)content and reinforcement of cell wall via callose deposition.By activating the immune response of maize,T.guizhouense NJAU 4742 induced the systematic resistance of maize plants to Fusarium verticillioides NJAU 1012.After pre-treatment of Trichoderma on maize roots for two weeks,the infection area of Fusarium on the leaf tissues of maize was decreased significantly.3.MAMPs of T.guizhouense NJAU 4742In order to explain the molecular mechanism of Trichoderma-plant interaction,we explored and validated the MAMPs in the volatile metabolites and secreted proteins of T.guizhouense NJAU 4742.We explored the elicitor function of ethylene synthesized by T.guizhouense NJAU 4742 by means of fungal gene knock-out.Using the hygromycin B(hyg B)and geneticin(G418)resistance selective markers,we constructed double mutant strains of which the ethylene content was decreased by 80%.Subsequently,we conducted phenotypic analysis on the interaction between Trichoderma double mutant strains and Arabidopsis seedlings(Col-0).Compared with the wild type fungal strain,the growth-promotion effect of double mutant strains on Arabidopsis leaf area and seedling fresh weight was reduced by 20%.Moreover,the yellowness response of Arabidopsis disappeared and the callose deposition in Arabidopsis leaves was decreased by 30-40%,indicating that Trichoderma-derived ethylene played an important role in the induction of yellowness response and callose deposition in Arabidopsis during Trichoderma-plant interaction.However,there were other elicitors or MAMPs in the volatile metabolites of T.guizhouense NJAU 4742.In addition,the interaction between Trichoderma and Arabidopsis ethylene-signaling mutant seedlings(ein3)implicated the existence of ethylene-independent mechanism of plant innate immune response activation by Trichoderma.In addition to ethylene,we explored other MAMPs in the secreted proteins of T.guizhouense NJAU 4742 and their effector function during the Trichoderma-plant interaction.The maize tissue and Arabidopsis seedlings treated with Trichoderma secreted proteins showed enhanced immune response,including ROS accumulation and callose deposition.The content of hydrogen peroxide in the tissues of Arabidopsis seedlings showed rapid increase after inoculated with Trichoderma secreted proteins for 15 min and 30 min.Compared with the control group,the content of hydrogen peroxide in tissues of Arabidopsis seedlings doubled after treated with Trichoderma secreted proteins for 24 hours.In addition,the ROS burst response in plant leaf tissues confirmed the elicitor function of Trichoderma secreted proteins.There was an enhanced response of Arabidopsis to pathogen-derived elicitor flg22 after pretreated with Trichoderma-secreted proteins,indicating that Trichoderma secreted proteins did not inhibit the recognition ability of plant immune system,in verse.the effector function of Trichoderma proteins was mainly to stimulate the plant immune response.In order to screen and identify the MAMPs components in the secreted proteins of T.guizhouense NJAU 4742,we analyzed the phenotype of T.guizhouense NJAU 4742 secreted proteins according to the classification of different molecular weights(MW).However,the results showed that Trichoderma secreted proteins in different molecular weight ranges could all induce the callose deposition in maize and Arabidopsis tissues,so the MAMPs in the secreted proteins of T.guizhouense NJAU 4742 could not be isolated and identified according to the MW classification.Nevertheless,compared with the glucose growth condition,the secreted proteins of T.guizhouense NJAU 4742 under the fermentation condition of maize root tissues showed an enhanced function in eliciting plant immune response,so that we could obtain and identify the protein components with enhanced elicitor function in priority.Next,we conducted quantitative comparison on the secreted proteins of T.guizhouense NJAU 4742 under conditions of maize root tissues or glucose,using the method of label-free proteomic analysis.We identified 125 proteins by mass spectrometry,among which 47 proteins were up-regulated under the condition of maize root tissues compared with glucose.Most of them were in the range of 30-50 KDa molecular weight,including endochitinase,lipase,subtilisin-like protease,aspartic protease,α-1,2-mannosidase,β-1,6-glucanase and other GH family proteins.In addition,there were also proteins with unknown functions acting as MAMPs during the interaction of T.guizhouense NJAU 4742 and plants.The above studies further clarified the molecular basis of Trichoderma-plant interaction.T.guizhouense NJAU 4742 stimulated the plant innate immune response by producing protein effectors(MAMPs),which might be similar to those of the pathogenic fungi during the interaction with plants.Therefore,we took maize as the research object to compare the similarities and differences of plant corresponding modes to beneficial and pathogenic fungi.4.Different corresponding modes of plant to beneficial and pathogenic fungi.Under the condition of soil culture,there was 30%growth loss of maize plants after treated with F.verticillioides,compared with the blank controls.Under hydroponic condition,F.verticillioides showed much stronger pathogenicity to maize seedlings,causing suppression of root growth.In contrast.T.guizhouense NJAU 4742 showed significant growth-promotion effect on the maize plants under the same conditions.Therefore.Trichoderma and Fusarium influenced the growth of maize plants in different ways.Interestingly,Trichoderma and Fusarium could induce similar innate immune response in maize plants.The content of hydrogen peroxide in the root tissues of maize seedlings increased significantly after both the treatment of Trichoderma and Fusarium,and there was callose deposition in the leaf tissues observed,too.Thereafter,we inoculated the maize roots with spore suspensions of T.guizhouense NJAU 4742 and F.verticillioides respectively,and then collected the root tissues at 1 day,2 days and 3 days after fungal inoculation.The results of transcriptomic analysis on maize roots corresponding to T guizhouense NJAU 4742 and F.verticillioides exhibited opposite patterns,and the differentially expressed genes showed different changing trends.The JA/ET pathway of maize in response to F.verticillioides increased dramatically at 3 days after the inoculation of F.verticillioides spores,while that of maize response to T.guizhouense NJAU 4742 was strongest at the first day inoculated with T.guizhouense NJAU 4742 spores,and then the response of JA/ET pathway in maize root tissues decreased gradually.The results of pathway anaylsis showed that the expression of genes in flavonoids synthesis pathway in maize roots generally rose corresponding to F.verticillioides,as to T.guizhouense NJAU 4742,there was no similar change exhibited in the maize transcriptome.Based on the comparative analysis on maize root transcriptome,we demonstrated the different patterns of maize roots in response to Trichoderma and Fusarium at the level of gene transcription.Moreover,we discussed on the plant growth-defense "trade-off" balanced by different fungi.In conclusion,this study proved that T.guizhouense NJAU 4742 could not only promote the growth and development of plants,but also enhance the plant induced systemic resistance.In this research,we established the experimental system of Trichoderma-plant interaction and developed the analytic methods on Trichoderma-derived MAMPs.Meanwhile,we verified the regulatory roles of ethylene in the interaction of Trichoderma and plant,and screened and identified several potent elicitors with biocontrol functions from T.guizhouense NJAU 4742 secreted proteins. |
