Root Colonization by Trichoderma atroviride Triggers Induced Systemic Resistance Primarily Independent of the Chitin-mediated Signaling Pathway in Arabidopsis

Abstract

Beneficial root endophytic fungi induce systemic responses, growth promotion, and induced systemic resistance (ISR) in colonized host plants. The soil application of chitin, a main component of fungal cell walls, also systemically induces disease resistance. Therefore, chitin recognition and its downstream signaling pathway mediate ISR triggered by beneficial fungi colonizing the root. The present study compared systemic disease resistance and transcriptional responses induced by Trichoderma, a representative beneficial root endophytic fungus, and chitin in Arabidopsis. Significant plant growth promotion was observed under root colonization by the three beneficial fungi tested: Trichoderma atroviride, Serendipita indica, and S. vermifera. Only T. atroviride and S. indica triggered ISR against the necrotrophic fungal pathogen Alternaria brassicicola. Induced systemic resistance triggered by T. atroviride was compromised in the chitin-receptor mutant, whereas systemic resistance caused by the soil application of chitin was not. A transcriptome ana­lysis demonstrated that chitin-regulated genes were mostly shared with those regulated by T. atroviride; however, many of the latter were specific. The commonly enriched gene ontologies for these genes indicated that the T. atroviride inoculation and chitin application systemically controlled similar transcriptional responses, mainly associated with cell wall functions. Therefore, Trichoderma may trigger ISR primarily independent of the chitin-mediated signaling pathway; however, chitin and Trichoderma may systemically induce similar cellular functions aboveground.

Plants have evolved a complex immune system against microbial pathogen infection (Jones and Dangl, 2006). Regarding the surveillance of microbes in host extracellular spaces, conserved microbial elicitors called pathogen-/microbe-associated mole­cular patterns (PAMPs/MAMPs) are recognized by pattern recognition receptors (PRRs) localized on the cell surface (Dodds and Rathjen, 2010). Well-studied PAMP/MAMP-PRR combinations include the flagellin epitope flg22-FLS2 (FLAGELLIN SENSITIVE2), leucine-rich-repeat-type PRR for bacteria and chitin-CERK1 (Chitin Elicitor Receptor Kinase 1), lysin motif (LysM)-type PRR for fungi in the model plant Arabidopsis (Shu et al., 2023). The perception of PAMPs/MAMPs leads to pattern-triggered immunity (PTI), which activates a cellular defense response to restrict microbial invasion (Yuan et al., 2021). Microbial pathogens render the plant susceptible to disease by deploying virulence effectors into host cells to suppress PTI. However, plants recognize pathogen effectors via intracellular nucleotide-binding leucine-rich repeat receptors and induce a strong defense response accompanied by hypersensitive cell death, which is called effecter-triggered immunity (ETI) (Jones and Dangl, 2006).

This plant immune system comprises similar cell-autonomous events to innate immunity in animals; however, plants lack the adaptive immune system in animals (Dodds and Rathjen, 2010). Therefore, plants have developed original systemic immune systems to induce disease resistance against the next pathogen attack in distal parts from the infection site (Pieterse et al., 2014). Systemic acquired resistance (SAR) is a well-studied systemic immunity triggered by PTI and ETI upon pathogen infection. The induction of SAR depends on salicylic acid (SA) and is a long-lasting form of disease resistance against a broad spectrum of (hemi-)biotrophic pathogens (Durrant and Dong, 2004; Vlot et al., 2021). On the other hand, systemic immunity may also be triggered by beneficial or commensal microbes in the plant’s rhizosphere, and is called induced systemic resistance (ISR) (Pieterse et al., 2014; Vlot et al., 2021). Unlike SAR, ISR depends on jasmonic acid and ethylene (ET), which function antagonistically to SA and act mainly against necrotrophic pathogens. Root endophytes include plant growth-promoting rhizobacteria (PGPR), such as Pseudomonas spp. and Bacillus spp., and plant growth-promoting fungi (PGPF), including Trichoderma spp. and Serendipita spp., which are known as ISR-inducing rhizospheric microbes (Barazani et al., 2007; Ray et al., 2018; Vlot et al., 2021). As their names suggest, PGPR and PGPF promote plant growth through root colonization. Root colonization by arbuscular mycorrhizal (AM) fungi, which establish mutual symbiosis with approximately 70% of terrestrial plants by exchanging photosynthates for soil-derived mineral nutrients, also triggers ISR (Cameron et al., 2013).

Chitin is a β-1,4-linked linear polymer of N-acetylglucosamine and a well-known elicitor derived from fungal cell walls that induces disease resistance (Sharp, 2013). Additionally, the soil application of chitin improves plant growth in various crops, which is considered to be independent of induced disease resistance. We recently reported that supplementing soils with chitin systemically induced disease resistance against necrotrophic fungal pathogens in Arabidopsis, cabbage, strawberry, and rice (Parada et al., 2018; Takagi et al., 2022). Therefore, the application of chitin to soils and beneficial root endophytic fungi induce similar systemic responses in plants, namely, growth promotion and disease resistance. This similarity infers that ISR by beneficial fungi occurs through chitin recognition and its downstream signaling pathway. The present study compared systemic disease resistance induced by a representative PGPF, Trichoderma, and chitin against the necrotrophic fungal pathogen, Alternaria brassicicola, in Arabidopsis thaliana. The evaluation of systemic disease resistance using a chitin-receptor CERK1 mutant indicated that Trichoderma triggered ISR primarily independent of the chitin-mediated signaling pathway. The results of a comparative transcriptome ana­lysis showed that a Trichoderma inoculation and chitin application in roots systemically controlled similar transcriptional responses aboveground.

Materials and Methods

Plant and fungal materials

A. thaliana (L) Heynh. accession Col-0 and cerk1-2 (GABI_096F06) (Miya et al., 2007) were used as the wild type and chitin receptor mutant, respectively. Trichoderma atroviride ATCC 20476 and Serendipita vermifera MAFF305830 (Warcup, 1988) were maintained on potato dextrose agar (PDA) (Difco) medium at 25°C. S. indica WP2 (Sherameti et al., 2005) was maintained on 1/6-strength Czapek Dox agar medium containing 0.8‍ ‍g‍ ‍L^–1 yeast extract and 15‍ ‍g‍ ‍L^–1 agar at 25°C.

Plant growth conditions, fungal inoculation, and chitin application

Arabidopsis seeds were sown on sterilized culture soil (Bestmix No. 3; Nippon Rockwool) and grown under controlled environmental conditions with 14-h light/8-h dark cycles at 23°C for seven weeks by watering 1,000-fold diluted fertilizer (HYPONeX [N–P–K=6–10–5]; Hyponex Japan) weekly.

Regarding the fungal inoculation, pieces of agar medium plugs of maintained fungal strains were placed on YEPG medium (Yeast extract 3‍ ‍g‍ ‍L^–1, Polypeptone 3‍ ‍g‍ ‍L^–1, and D-glucose 20‍ ‍g‍ ‍L^–1) and cultured using a rotary shaker at 25°C in the dark for two weeks. After removing the liquid medium, the harvested mycelia were homogenized with a blender (Nissei) at 10,000‍ ‍rpm for 10 s. Distilled water was added to prepare mycelial suspensions at the indicated concentrations. Five milliliters of each mycelial suspension was irrigated into soils two weeks after sowing, and Arabidopsis seedlings were grown for an additional five weeks. The water dispersion of chitin nanofibers (CNF) (MARINE NANO-FIBER CN-01), which are directly produced from chitin powder by physically grinding microfibrils (nanofibrillation) and may be used like a water solution (Ifuku and Saimoto, 2012), was purchased from Marine Nano-fiber and used for the chitin treatment unless otherwise indicated. Chitin oligosaccharide (CO) mixture powder (NA-COS-Y: Yaizu Suisankagaku Industry), containing CO with a degree of polymerization of 2–6, was also used. Upon preparation, the culture soil was mixed with an equal volume of 0.1% (w/v) CNF water dispersion or 0.1% (w/v) CO water solution before sowing, based on our previous study (Kaminaka et al., 2020). Distilled water was used for control experiments.

Fluorescent staining of fungal hyphae in Arabidopsis roots

Harvested Arabidopsis roots were fixed in 70% ethanol overnight. After removing ethanol, 5% KOH was added, and samples were heated at 90°C for 30‍ ‍min. Root samples were transferred into 1% HCl for neutralization for 5‍ ‍min, washed with PBS buffer, and incubated with 5‍ ‍μg mL^–1 of WGA-Alexa Fluor 488 (Thermo Fisher Scientific) in the dark for 10‍ ‍min. Roots were washed with PBS again, and 20% TOMEI (Tokyo Chemical Industry) was added to clear tissues. Stained roots were observed under a fluorescent microscope (DM2500; Leica) with the excitation filter L5, and photo images were obtained with the equipped digital camera (DFC310; Leica).

Detection of root-colonized beneficial fungi by PCR

Harvested Arabidopsis roots were subjected to the extraction of genomic DNA (gDNA) using RBC Genomic DNA Extraction Kit Mini (Plant) (RBC Bioscience). The concentration of extracted gDNA was measured using the DS-11 spectrophotometer (DeNovix). Each PCR reaction mixture was prepared in a final volume of 25‍ ‍μL containing 5.5‍ ‍ng gDNA, 12.5‍ ‍μL of KOD One PCR Master Mix (TOYOBO), and 1.5‍ ‍μL of 5‍ ‍μM of each primer. PCR was conducted on the T100 Thermal Cycler (BIO-RAD). Detailed information on the PCR reaction, including the target genes, the sequences of the primers used, the amplicon size, and PCR reaction conditions, was provided in Table S1. PCR products were separated by 1.5% agarose gel electrophoresis with the FastGene 100 bp DNA Maker (NIPPON GENE), and images of ethidium bromide-stained gels were taken using the GelDoc GO Imaging system (BIO-RAD).

Disease resistance assay

Spores of the fungal pathogen A. brassicicola strain O-264, a causal agent of black leaf spot on Brassica plants, were prepared and inoculated on Arabidopsis leaves as previously reported (Parada et al., 2018). Ten microliters of a conidial suspension (5.0×10⁵ spores mL^–1) was inoculated onto each leaf. The diameter of the lesions that emerged on leaves was measured four d after the inoculation using ImageJ ver.1.53a.

RNA-sequencing and data ana­lysis

Approximately 100‍ ‍mg of randomly selected leaves from at least three individual seven-week-old Arabidopsis seedlings inoculated with T. atroviride or treated with chitin were used to prepare the sequencing library. Total RNA preparation was conducted according to Tominaga et al. (2021). The preparation of sequencing libraries and sequencing with strand-specific and paired-end reads (150 bp) by DNBSEQ-T7RS was performed by Genome-Lead.

Low-quality reads (<QV30) and adapter sequences of the raw reads obtained were removed by fastp (Chen et al., 2018) and mapped onto the sequence of the Arabidopsis reference genome TAIR10.43 (https://www.arabidopsis.org/) by the RNA-sequencing aligner STAR ver.2.6.1d (Dobin et al., 2013) (Table S2). Data were processed with featureCounts ver.2.0.1 (Liao et al., 2014) to obtain gene expression count data. Count data in different library sizes were normalized by the trimmed mean of the M-values normalization method, and differentially expressed genes (DEGs) were identified by comparing control and T. atroviride-inoculated or chitin-treated plants using edgeR ver.4.2.1 (Robinson et al., 2010). The list of DEGs was extracted by a false discovery rate (FDR) cut-off <0.05. Venn diagrams were generated using the Venn diagram website (https://bioinformatics.psb.ugent.be/webtools/Venn). The gene ontology (GO) enrichment ana­lysis was conducted using Shiny GO 0.77 (Ge et al., 2020), and dot plots were drawn using “ggplot2” and “ggpubr” packages in R (ver.4.3.1).

RNA-sequencing data accession number

The raw read data obtained by RNA-sequencing were deposited in the DNA Data Bank of Japan under the BioProject accession number PRJDB17932.

Statistical ana­lysis

Lesion diameters caused by the A. brassicicola inoculation were compared to control plants, and the significance of differences in the results obtained was analyzed using the Student’s t-test and Microsoft Excel (ver. 2312). All pathogen inoculation tests were conducted at least three times with more than three different plants for each treatment and genotype.

Results

Growth promotion and ISR by the beneficial fungal colonization of Arabidopsis

Root endophytes promote plant growth and systemically induce disease resistance, and are called PGPR and PGPF (Pieterse et al., 2014; Vlot et al., 2021). In Arabidopsis, Trichoderma and Serendipita species are known as PGPF (Lahrmann and Zuccaro, 2012; González-Pérez et al., 2018). To compare the effects of beneficial fungi inoculated on Arabidopsis seedlings, T. atroviride, S. indica, and S. vermifera were inoculated by irrigating the soil with mycelial suspensions. The growth of Arabidopsis seedlings inoculated with these three fungi was significantly better than that of non-inoculated seedlings at five weeks post-inoculation (Fig. 1A). Fungal colonization was confirmed by hyphal staining and PCR only in the roots of fungus-inoculated seedlings (Fig. 1B and C). These results indicate that root colonization by these beneficial fungi promoted plant growth.

Fig. 1.

Growth promotion of Arabidopsis seedlings colonized by beneficial fungi. (A) Photos of Arabidopsis seedlings grown for seven weeks on soil irrigated with hyphal homogenates (50‍ ‍mg fresh weight [FW] mL^–1) of Serendipita vermifera, S. indica, and Trichoderma atroviride. (B) Fluorescent images of inoculated fungal hyphae stained with WGA-Alexa Fluor 488 in Arabidopsis roots. The white arrowheads indicate fungal hyphae. (C) Detection of colonized beneficial fungi in Arabidopsis roots by PCR using gene-specific primers of colonized fungi and Arabidopsis: S. vermifera 5.8S coding sequence and highly variable ITS2 region of ribosomal DNA (SvITS), S. indica translation elongation factor 1 alpha (SiTEF), T. atroviride translation elongation factor 1 alpha (TaTEF), and Arabidopsis thaliana SHAGGY-related kinase 11 (iASK). Arrowheads indicate the bands corresponding to the amplicon size. M: Molecular marker, C: Control, Sv: S. vermifera, Si: S. indica, Ta: T. atroviride.

Since beneficial fungi cause ISR mainly against necrotrophic pathogens (Pieterse et al., 2014; Vlot et al., 2021), we exami­ned disease resistance against the necrotrophic fungal pathogen A. brassicicola, a causal agent of black leaf spot on Brassica plants, in the leaves of beneficial fungus-inoculated seedlings. Root colonization by T. atroviride and S. indica significantly reduced lesion sizes. In contrast, no significant differences in lesion formation were observed between S. vermifera-colonized seedlings and control seedlings (Fig. 2A). Since T. atroviride displayed significantly more ISR than S. indica, it was selected for the subsequent experiment to optimize the fungal inoculum concentration. Only the concentration used in the previous experiment (50‍ ‍mg FW mL^–1), not lower concentrations, led to significantly smaller lesions caused by the A. brassicicola infection (Fig. 2B). Therefore, the concentration of the T. atroviride inoculum of 50‍ ‍mg FW mL^–1 was used in further experiments.

Fig. 2.

Induced systemic resistance against a necrotrophic pathogen after an inoculation with beneficial fungi. (A) Lesion size on Arabidopsis seedling leaves (grown as in Fig. 1) inoculated with 10‍ ‍μL of the Alternaria brassicicola suspension (5.0×10⁵ spores mL^–1). The lesion was measured four d after the inoculation. (B) The effects of the inoculum concentration (mg fresh weight [FW] mL^–1) for the Trichoderma atroviride inoculation, conducted as in (A). Bars and error bars indicate means and standard errors, and asterisks indicate significant differences (the Student’s t-test: *P<0.05, ***P<0.001, ****P<0.0001; n≥8).

Chitin receptor CERK1 functions in disease resistance systemically induced by T. atroviride and chitin

Chitin is a PAMP/MAMP used by plants to sense the presence of fungi in intracellular spaces (Shu et al., 2023). Similar to beneficial fungal colonization, the application of chitin to soils has been shown to promote plant growth and induce systemic resistance (Parada et al., 2018; Kaminaka et al., 2020; Takagi et al., 2022). Therefore, the involvement of chitin in ISR triggered by T. atroviride root colonization was investigated using the chitin receptor LysM-type PRR CERK1-deficient mutant cerk1-2 (Miya et al., 2007). ISR against A. brassicicola observed in wild-type plants inoculated with T. atroviride was compromised in cerk1-2 (Fig. 3A). In the present study, chitin nanofibers were used as chitin because they induce a more robust plant response than other chitins through the rapid and massive production of CO by chitinase (Egusa et al., 2015; Kaminaka et al., 2020). Systemic disease resistance against A. brassicicola was significantly induced, even in cerk1-2, by the application of chitin to soils (Fig. 3B). The application of CO to soils also showed a similar result (Fig. S1), which indicated that resistance systemically induced by any form of chitin is independent of CERK1 functions. These results reveal that ISR triggered by T. atroviride root colonization may be regulated differently from, and potentially independent of, chitin-triggered systemic disease resistance in Arabidopsis.

Fig. 3.

Effects of chitin receptor deficiency on induced systemic resistance against a necrotrophic pathogen. Disease resistance against Alternaria brassicicola on the leaves of wild-type (Col-0) and cerk1-2 seedlings (A) inoculated with Trichoderma atroviride and (B) treated with chitin, as conducted in Fig. 2. Bars and error bars indicate means and standard errors, and asterisks indicate significant differences (the Student’s t-test: *P<0.05, ***P<0.001; n≥8).

Comparative transcriptome ana­lysis of T. atroviride-inoculated or chitin-treated Arabidopsis seedlings

To elucidate the mole­cular mechanisms underlying ISR caused by the root colonization of T. atroviride, Arabidopsis roots were inoculated with T. atroviride or treated with chitin, and seedling leaves were used for RNA-sequencing to elucidate the mole­cular mechanisms responsible for ISR caused by T. atroviride. Compared with control seedlings, 1,724 DEGs were identified in T. atroviride-inoculated seedlings, including 617 up-regulated and 1,107 down-regulated DEGs (Fig. 4A, B and Table S3). We identified 95 DEGs in chitin-treated seedlings, including 24 up-regulated and 71 down-regulated DEGs (Fig. 4A, B and Table S4). More than 95% of DEGs in chitin-treated seedlings were shared with those in T. atroviride-inoculated seedlings (Fig. 4A and B).

Fig. 4.

Transcriptome ana­lysis of leaves of Arabidopsis seedlings inoculated with Trichoderma atroviride and treated with chitin. Venn diagrams of up-regulated (A) and down-regulated (B) differentially expressed genes (DEGs) identified by a false discovery rate (FDR) cut-off <0.05. The top 20 enriched biological process gene ontology (GO) terms with the lowest FDR values for up-regulated (C) and down-regulated (D) DEGs upon the T. atroviride inoculation. The circle size indicates the FDR value. The complete list of enriched GO terms is presented in Table S5.

A GO enrichment ana­lysis of DEGs identified in the leaves of T. atroviride-inoculated seedlings was then performed. Regarding up-regulated DEGs, GO terms associated with cell wall functions (e.g., “Cellulose biosynthesis/metabolic process”, “Plant-type cell wall organization or biogenesis”, and “Cell wall polysaccharide/macromolecule metabolic process”) were dominantly overrepresented in the biological process category (Fig. 4C and Table S5) and were also identified as enriched GO terms for up-regulated DEGs in chitin-treated seedling leaves (Fig. S2A and Table S6). The enrichment for categories related to the negative regulation of the phosphorus metabolic process, kinase inhibitor activity, and endocytic pathway (e.g., endosome, Golgi, or vesicle) was also indicated (Fig. 4C and Table S5). Regarding down-regulated DEGs, the GO term “Response to chitin” was highly enriched, and overrepresented GO terms associated with cellular responses to oxygen levels, transcription factors, and NAD/NAD(P)+ nucleoside activity were also found (Fig. 4D and Table S5). These GO terms were also overrepresented for down-regulated DEGs in chitin-treated seedlings (Fig. S2B and Table S6).

Discussion

Trichoderma is widely used as a biocontrol agent mainly against soil-borne diseases in various crops through its mycoparasitism and secretomes, including volatile organic compounds (VOCs), cell wall-degrading enzymes (CDWEs), reactive oxygen species, and antimicrobial secondary metabolites (Alfiky and Weisskopf, 2021; Yao et al., 2023). Additionally, Trichoderma induces systemic responses in host plants, including growth promotion and disease resistance known as ISR. The combination of these functions may cause the biocontrol effects of Trichoderma; however, available knowledge on each function needs to be improved. To obtain novel insights into plant–Trichoderma interactions, we investigated the mole­cular mechanisms underlying Trichoderma-ISR by focusing on the involvement of chitin—recognition and signaling—in Arabidopsis. The ana­lysis of systemic disease resistance in chitin receptor-deficient mutants revealed that Trichoderma triggered systemic resistance against necrotrophic pathogens primarily independent of the chitin-mediated signaling pathway.

The root colonization of all exami­ned endophytic fungi, T. atroviride, S. indica, and S. vermifera, promoted plant growth, which is consistent with previous findings from various plants (Lahrmann and Zuccaro, 2012; Ray et al., 2018; Alfiky and Weisskopf, 2021). In contrast, only T. atroviride and S. indica significantly increased disease resistance against the necrotrophic pathogen, A. brassicicola, in the leaves of root-colonized Arabidopsis seedlings. ISR by the root colonization of T. atroviride and S. indicia in Arabidopsis has been reported (Salas-Marina et al., 2011; Lahrmann and Zuccaro, 2012), but not for S. vermifera. Sarkar et al. (2019) revealed the mycoparasitism of S. vermifera against Bipolaris sorokiniana, a causal agent of spot blotch and common root rot diseases, by mainly reducing the pathogen’s root infection in barley. They also suggested disease resistance systemically induced by S. vermifera in roots. Therefore, S. vermifera may be able to cause weak ISR, which may not be sufficient for detection using our Arabidopsis pathosystem.

ISR in Arabidopsis has been extensively exami­ned using PGPR, such as Pseudomonas and Bacillus species (Pieterse et al., 2014; Vlot et al., 2021). However, knowledge of ISR by beneficial fungi is limited due to AM fungi being a non-host. The present study demonstrated that the function of the chitin-receptor CERK1 was required for ISR by Trichoderma in Arabidopsis. The ectomycorrhizal fungus Laccaria bicolor triggered ISR against the cabbage looper Trichoplusia ni and induced systemic susceptibility against the hemi-biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 in non-host Arabidopsis plants in a CERK1-dependent manner (Vishwanathan et al., 2020). Since the treatments of heat-killed L. bicolor and chitin also systemically induced disease resistance, the authors proposed that ISR without a symbiotic association may be triggered by the root perception of PAMPs/MAMPs, which was supported by our previous findings (Takagi et al., 2022). In rice, supplementing soils with chitin systemically induced disease resistance against the necrotrophic pathogen Bipolaris oryzae through the function of LysM-type PRRs, OsCERK1, and OsCEBiP. In contrast, chitin-ISR in Arabidopsis was not mediated by CERK1 in this study, which indicated that the general model for chitin perception mediated by CERK1 function does not apply to chitin-induced systemic disease resistance in Arabidopsis. Although the mechanism for chitin perception by LysM-type PRRs in Arabidopsis is similar to that in rice, CERK1 functions differ in terms of its binding ability to chitin; unlike rice CERK1 (OsCERK1), Arabidopsis CERK1 binds to CO (Liu et al., 2012; Shinya et al., 2012; Yang et al., 2022). Therefore, the difference in the chitin perception mechanism may be explained by the different requirements of CERK1 functions for chitin-ISR between Arabidopsis and rice. To address this point, Arabidopsis LysM-type PRR(s) involved in chitin-ISR and Trichoderma-induced ISR need to be characterized using loss-of-function mutants, which will be conducted in a subsequent study.

The transcriptome ana­lysis revealed that most DEGs identified in chitin-treated seedlings were shared with those in Trichoderma-inoculated seedlings, indicating the minor or no contribution of chitin-triggered functions in Trichoderma-induced systemic responses. Additionally, 94% of DEGs identified in Trichoderma-inoculated seedlings were specific; therefore, Trichoderma-specific transcriptional responses may contribute to systemic responses, including ISR and growth promotion. The GO terms involved in cell wall functions were mainly overrepresented by the GO enrichment ana­lysis of up-regulated DEGs in Trichoderma-inoculated seedlings. Recent studies revealed the involvement and importance of cell wall functions (e.g., cell wall biogenesis, composition, and integrity) in the induction of disease resistance (Bacete et al., 2018; Molina et al., 2021; Baez et al., 2022). Therefore, the modulation of cell wall conditions by transcriptional changes may be a major cellular event inducing disease resistance aboveground in Trichoderma-induced systemic responses. In our previous study, the GO terms associated with cell wall functions were also enriched in the leaves of rice seedlings grown on chitin-supplemented soils (Takagi et al., 2022). However, unlike in Arabidopsis, the genes involved in cell wall functions were down-regulated in rice. These opposite aboveground transcriptional responses may also describe the different requirements of CERK1 functions for chitin-ISR between Arabidopsis and rice.

The enriched GO terms for DEGs were similar between the Trichoderma inoculation and chitin treatment. Therefore, this inoculation/treatment may induce systemic responses by modulating similar cellular functions mainly associated with aboveground cell wall functions, even if the requirement of LysM-type PRRs is different (Fig. 5). In the present study, we identified a specific signaling pathway mediated by CERK1 in ISR triggered by Trichoderma, which is primarily independent of the chitin-mediated signaling pathway. Plant hormones and secretomes, including effectors, VOCs, and CDWEs, also participate in ISR by Trichoderma (Alfiky and Weisskopf, 2021). Therefore, we plan to conduct further studies with a focus on the involvement of these molecules in order to elucidate the mechanisms underlying the Trichoderma-specific signaling pathway systemically inducing disease resistance in Arabidopsis.

Fig. 5.

Proposed model for chitin- and Trichoderma-induced systemic resistance (ISR) in Arabidopsis. Chitin supplementation into soils and root colonization by Trichoderma atroviride systemically up-regulate cell wall-related genes in leaves and induce disease resistance against the necrotrophic fungal pathogen Alternaria brassicicola. The function of Chitin Elicitor Receptor Kinase 1 (CERK1), the lysin motif (LysM)-type pattern recognition receptor required for chitin perception (Miya et al., 2007), is required for ISR by Trichoderma, whereas CERK1 is not involved in ISR by chitin. Therefore, chitin and Trichoderma may systemically modulate similar cellular functions for aboveground ISR. However, Trichoderma induces systemic responses primarily independent of the chitin-mediated signaling pathway.

Citation

Sakai, A., Yamagata, H., Naito, K., Yoshioka, M., Tominaga, T., Ifuku, S., and Kaminaka, H. (2024) Root Colonization by Trichoderma atroviride Triggers Induced Systemic Resistance Primarily Independent of the Chitin-mediated Signaling Pathway in Arabidopsis. Microbes Environ 39: ME24038.

https://doi.org/10.1264/jsme2.ME24038

Acknowledgements

We thank Dr. Hirofumi Nakagami (Max-Planck Research Institute), Dr. Hiroshi Otani (Tottori University), and Dr. Patrick Schäfer (Justus Liebig University) for providing cerk1-2 seeds, Alternaria brassicicola, and Serendipita indica, respectively. This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI grant number 22K19182.

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