Rhizoctonia solani is a soil-borne phytopathogen with a broad host range, classified into multiple anastomosis groups (AGs). While the rice sheath blight pathogen AG-1 IA is well-studied, defense mechanisms against AG-4, which infects diverse monocots and dicots, remain unclear. Here, we screened 153 Brachypodium distachyon accessions for resistance to AG-4 HG-I+II using both leaf and soil inoculation assays. The accessions exhibited a quantitative resistance spectrum in both assays. Some accessions were resistant to both methods, while others showed resistance in only one, indicating that leaf and root defenses operate independently. Unlike the salicylic acid (SA)-dependent resistance observed in several accessions against AG-1 IA, AG-4 leaf resistance appears to be largely SA-independent. Among 22 resistant accessions, only eight induced the SA marker BdWRKY38, whereas twelve induced the jasmonic acid (JA) marker BdAOS. Additionally, resistance in Bd3-1 was unaffected by expression of the SA hydroxylase NahG. These results are consistent with previous findings that exogenous application of SA, JA, or ethylene fails to confer AG-4 resistance. Instead, most AG-4-resistant accessions showed common upregulation of cell wall biosynthesis-related genes following leaf infection, suggesting that cell wall fortification may contribute to defense against AG-4. These findings establish B. distachyon as a valuable model for dissecting resistance mechanisms against R. solani AG-4.

Medicago truncatula (Mt) is a relatively drought-tolerant model legume widely cultivated in Australia. Unlike previous studies that focus on specific plant components, this work reanalyses the metabolite pattern along with transcriptome data to understand the integrated response of the entire plant system to water deficit stress. Physiological and transcriptomic analyses of the leaves, taproots, and fibrous roots were performed in response to moderate and severe drought conditions. Our findings revealed that plants prioritize water supply to aboveground organs, leading to a significant decline in the root system water content during active growth. At the whole plant level, a coordinated upregulation involving LEA proteins, proline, and ABA metabolism was observed. Furthermore, carbohydrate metabolism, essential for sustaining tissue growth, was significantly altered by drought stress. Despite the well-established link between water deficit and reduced photosynthesis, which compromises carbon availability within the plant, the activation of a complete set of sucrose- and starch-degrading and -synthesising enzymes was detected. These enzymes act in concert with hexose and sucrose transporters to remobilise carbon throughout the plant system. In addition to enhanced carbon remobilisation, a notable root-specific downregulation of ethylene synthesis was observed, shedding light on the mechanism regulating plant growth under drought stress. In conclusion, our findings reveal a strong organ-specific and coordinated molecular response across progressive drought stress levels.

Acibenzolar‐S‐methyl (ASM) is a plant protection compound known for its bacterial control, plant activation, and for enhancing heat and drought stress tolerance in turfgrass. However, the comprehensive transcriptional response to ASM treatment in creeping bentgrass (CB) has not been elucidated. To address this, we assembled RNA‐sequencing (RNA‐seq) reads from bentgrass to create its reference set of expressed genes. By conducting RNA‐seq analysis on RNA samples from ASM‐treated leaves and controls in CB, we comprehensively identified genes that responded to ASM treatment. To characterize the functional properties of the ASM‐responsive genes, we analyzed genes putatively encoding transcription factors (TFs), including WRKY TFs. We identified genes’ putatively encoding WRKY TFs in CB with conserved responses to ASM, analogous to their homologs in Brachypodium, which are known to mediate defense against sheath blight. This identified ASM‐responsive gene set will provide a valuable genomic resource for further detailed analysis of gene expression and transcriptional regulation in response to ASM treatment in CB.

Rhizoctonia solani is a basidiomycete phytopathogenic fungus that causes rapid necrosis in a wide range of crop species, leading to substantial agricultural losses worldwide. The species complex is divided into 13 anastomosis groups (AGs) based on hyphal fusion compatibility and further subdivided by culture morphology. While R. solani classifications were shown to be independent of host specificity, it remains unclear whether different R. solani isolates share similar virulence mechanisms. Here, we investigated the infectivity of Japanese R. solani isolates on Brachypodium distachyon and barley. Two isolates, AG-1 IA (from rice) and AG-4 HG-I+II (from cauliflower), infected leaves of both plants, but only AG-4 HG-I+II infected roots. B. distachyon accessions Bd3-1 and Gaz-4 and barley cultivar ‘Morex’ exhibited enhanced resistance to both isolates compared to B. distachyon Bd21 and barley cultivars ‘Haruna Nijo’ and ‘Golden Promise’. During AG-1 IA infection, but not AG-4 HG-I+II infection, resistant Bd3-1 and Morex induced genes for salicylic acid (SA) and N-hydroxypipecolic acid (NHP) biosynthesis. Pretreatment with SA or NHP conferred resistance to AG-1 IA, but not AG-4 HG-I+II, in susceptible B. distachyon Bd21 and barley Haruna Nijo. On the leaves of susceptible Bd21 and Haruna Nijo, AG-1 IA developed extensive mycelial networks with numerous infection cushions, which are specialized infection structures well-characterized in rice sheath blight. In contrast, AG-4 HG-I+II formed dispersed mycelial masses associated with underlying necrosis. We propose that the R. solani species complex encompasses at least two distinct infection strategies: AG-1 IA exhibits a hemibiotrophic lifestyle, while AG-4 HG-I+II follows a predominantly necrotrophic strategy.

High-temperature stress poses a significant threat to agricultural productivity and ecological diversity. Here, we show the effects of prolonged high-temperature stress on wheat (Triticum aestivum) and the model grass Brachypodium distachyon and demonstrate that heat stress induces iron deficiency in newly emerged leaves. Quantitative trait locus analysis of B. distachyon reveals a genomic region associated with heat resilience that includes the transporter of mugineic acid family phytosiderophores 1 gene (BdTOM1). Iron-deficiency-related genes including BdTOM1 are more highly expressed in a high-temperature-tolerant B. distachyon accession at high temperature than in a sensitive accession, resulting in greater secretion of deoxymugineic acid. Treatment with proline-2'-deoxymugineic acid mitigates heat-induced growth inhibition, but excess iron treatment leads to toxicity in both species. Our findings highlight the role of heat-induced nutritional stress in prolonged high-temperature stress and suggest that iron homeostasis could provide a promising target for improving crop resilience to climate extremes.

Rice seed-borne diseases caused by the bacterial pathogens Burkholderia glumae and B. plantarii pose a major threat to rice production worldwide. To manage these diseases in a sustainable manner, a biocontrol strategy is crucial. In this study, we showed that B. gladioli NB6 (NB6), a nonpathogenic bacterium, strongly protects rice from infection caused by the above-mentioned pathogens. NB6 was isolated from the indica rice cultivar Nona Bokra seedlings, which possesses genetic resistance to B. glumae. We discovered that cell suspensions of NB6 and its culture filtrate suppressed the disease symptoms caused by B. glumae and B. plantarii in rice seedlings, which indicated that NB6 secretes a plant-protective substance extracellularly. Through purification and mass spectrometry analysis of the culture filtrate, combined with transmission electron microscopy and mutant analysis, the substance was identified as a tailocin and named BglaTNB6. Tailocins are bacteriotoxic multiprotein structures morphologically similar to headless phage tails. BglaTNB6 exhibited antibacterial activity against several Burkholderia species, including B. glumae, B. plantarii, and B. gladioli, suggesting it can prevent pathogen infection. Interestingly, BglaTNB6 greatly contributed only to the biocontrol activity of NB6 cell suspensions against B. plantarii, and not against B. glumae. BglaTNB6 was shown to be encoded by a prophage locus lacking genes for phage head proteins, and a B. gladioli strain with the coded BglaTNB6-like locus equipped with phage head proteins failed to prevent rice seedlings from being infected with B. plantarii. These results suggested that BglaTNB6 may enhance the competitiveness of NB6 against a specific range of bacteria. Our study also highlights the potential of tailocin-producing endophytes for managing crop bacterial diseases.

Pantoea dispersa is a plant-associated bacterium that often has beneficial effects on plants. In this study, we demonstrated that P. dispersa BB1, a previously identified potential biocontrol agent against the seedling rot pathogen Burkholderia glumae , could effectively suppress the symptoms of seedling blight caused by Burkholderia plantarii . To facilitate the isolation and utilization of P. dispersa as a biocontrol agent, a comparative genome analysis was performed to develop novel PCR primer sets for specifically detecting P. dispersa genes. Among the developed primer sets, the Pd725 set could detect 0.3 pg of genomic DNA of P. dispersa and evaluate the colonization of P. dispersa in plants. Using this primer set, we demonstrated that the colonization of P. dispersa in rice seeds infected with B. glumae and B. plantarii was significantly higher than that in non-infected seeds. This suggested that infection by Burkholderia pathogens promoted the colonization of P. dispersa . The Pd725 primer set could serve as a valuable tool for monitoring the colonization of P. dispersa in crops and evaluating potential new biocontrol strains.

Salicylic acid (SA) is known to be involved in the immunity against Clavibacter michiganensis ssp. michiganensis (Cmm) that causes bacterial canker in tomato. To identify the candidate genes associated with SA-inducible Cmm resistance, transcriptome analysis was conducted via RNA sequencing in tomato plants treated with SA. SA treatment upregulated various defense-associated genes, such as PR and GST genes, in tomato cotyledons. A comparison of SA- and Cmm-responsive genes revealed that both SA treatment and Cmm infection commonly upregulated a large number of genes. Gene Ontology (GO) analysis indicated that the GO terms associated with plant immunity were over-represented in both SA- and Cmm-induced genes. The genes commonly downregulated by both SA treatment and Cmm infection were associated with the cell cycle and may be involved in growth and immunity trade-off through cell division. After SA treatment, several proteins that were predicted to play a role in immune signaling, such as resistance gene analogs, Ca2+ sensors, and WRKY transcription factors, were transcriptionally upregulated. The W-box element, which was targeted by WRKYs, was over-represented in the promoter regions of genes upregulated by both SA treatment and Cmm infection, supporting the speculation that WRKYs are important for the SA-mediated immunity against Cmm. Prediction of protein-protein interactions suggested that genes encoding receptor-like kinases and EF-hand proteins play an important role in immune signaling. Thus, various candidate genes involved in SA-inducible Cmm resistance were identified.

Rhizoctonia solani is a necrotrophic plant pathogen with a wide host range. R. solani is a species complex consisting of thirteen anastomosis groups (AGs) defined by compatibility of hyphal fusion reaction and subgroups based on cultural morphology. The relationship between such classifications and host specificity remains elusive. Here, we investigated the pathogenicity of seventeen R. solani isolates (AG-1 to 7) in Japan towards Arabidopsis thaliana using leaf and soil inoculations. The tested AGs, except AG-3 and AG-6, induced symptoms in both methods with variations in pathogenicity. The virulence levels differed even within the same AG and subgroup. Some isolates showed tissue-specific infection behavior. Thus, the AGs and their subgroups are suggested to be not enough to define the virulence (host and tissue specificity) of R. solani. We also evaluated the virulence of the isolates on Arabidopsis plants pretreated with salicylic acid, jasmonic acid, and ethylene. No obvious effects were detected on the symptom formation by the virulence isolates, but ethylene and salicylic acid slightly enhanced the susceptibility to the weak and nonvirulent isolates. R. solani seems to be able to overcome the induced defense by these phytohormones in the infection to Arabidopsis.

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot.

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction.

Tomato brown rugose fruit virus (ToBRFV) is an emerging virus of the genus Tobamovirus. ToBRFV overcomes the tobamovirus resistance gene Tm-22 and is rapidly spreading worldwide. Genetic resources for ToBRFV resistance are urgently needed. Here, we show that clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9)-mediated targeted mutagenesis of four tomato (Solanum lycopersicum) homologs of TOBAMOVIRUS MULTIPLICATION1 (TOM1), an Arabidopsis (Arabidopsis thaliana) gene essential for tobamovirus multiplication, confers resistance to ToBRFV in tomato plants. Quadruple-mutant plants did not show detectable ToBRFV coat protein (CP) accumulation or obvious defects in growth or fruit production. When any three of the four TOM1 homologs were disrupted, ToBRFV CP accumulation was detectable but greatly reduced. In the triple mutant, in which ToBRFV CP accumulation was most strongly suppressed, mutant viruses capable of more efficient multiplication in the mutant plants emerged. However, these mutant viruses did not infect the quadruple-mutant plants, suggesting that the resistance of the quadruple-mutant plants is highly durable. The quadruple-mutant plants also showed resistance to three other tobamovirus species. Therefore, tomato plants with strong resistance to tobamoviruses, including ToBRFV, can be generated by CRISPR/Cas9-mediated multiplexed genome editing. The genome-edited plants could facilitate ToBRFV-resistant tomato breeding.

Additional file 8: Table S8. The primers used in this study.

Polyploidy is a widespread phenomenon in eukaryotes that can lead to phenotypic novelty and has important implications for evolution and diversification. The modification of phenotypes in polyploids relative to their diploid progenitors may be associated with altered gene expression. However, it is largely unknown how interactions between duplicated genes affect their diurnal expression in allopolyploid species. In this study, we explored parental legacy and hybrid novelty in the transcriptomes of an allopolyploid species and its diploid progenitors. We compared the diurnal transcriptomes of representative Brachypodium cytotypes, including the allotetraploid Brachypodium hybridum and its diploid progenitors Brachypodium distachyon and Brachypodium stacei. We also artificially induced an autotetraploid B. distachyon. We identified patterns of homoeolog expression bias (HEB) across Brachypodium cytotypes and time-dependent gain and loss of HEB in B. hybridum. Furthermore, we established that many genes with diurnal expression experienced HEB, while their expression patterns and peak times were correlated between homoeologs in B. hybridum relative to B. distachyon and B. stacei, suggesting diurnal synchronization of homoeolog expression in B. hybridum. Our findings provide insight into the parental legacy and hybrid novelty associated with polyploidy in Brachypodium, and highlight the evolutionary consequences of diurnal transcriptional regulation that accompanied allopolyploidy.

SUMMARY Rhizoctonia solani is a soil‐borne necrotrophic fungus that causes sheath blight in grasses. The basal resistance of compatible interactions between R. solani and rice is known to be modulated by some WRKY transcription factors (TFs). However, genes and defense responses involved in incompatible interaction with R. solani remain unexplored, because no such interactions are known in any host plants. Recently, we demonstrated that Bd3‐1, an accession of the model grass Brachypodium distachyon, is resistant to R. solani and, upon inoculation with the fungus, undergoes rapid induction of genes responsive to the phytohormone salicylic acid (SA) that encode the WRKY TFs BdWRKY38 and BdWRKY44. Here, we show that endogenous SA and these WRKY TFs positively regulate this accession‐specific R. solani resistance. In contrast to a susceptible accession (Bd21), the infection process in the resistant accessions Bd3‐1 and Tek‐3 was suppressed at early stages before the development of fungal biomass and infection machinery. A comparative transcriptome analysis during pathogen infection revealed that putative WRKY‐dependent defense genes were induced faster in the resistant accessions than in Bd21. A gene regulatory network (GRN) analysis based on the transcriptome dataset demonstrated that BdWRKY38 was a GRN hub connected to many target genes specifically in resistant accessions, whereas BdWRKY44 was shared in the GRNs of all three accessions. Moreover, overexpression of BdWRKY38 increased R. solani resistance in Bd21. Our findings demonstrate that these resistant accessions can activate an incompatible host response to R. solani, and BdWRKY38 regulates this response by mediating SA signaling.

Rhizoctonia solani is a necrotrophic phytopathogen belonging to basidiomycetes. It causes rice sheath blight which inflicts serious damage in rice production. The infection strategy of this pathogen remains unclear. We previously demonstrated that salicylic acid-induced immunity could block R. solani AG-1 IA infection in both rice and Brachypodium distachyon . R. solani may undergo biotrophic process using effector proteins to suppress host immunity before necrotrophic stage. To identify pathogen genes expressed at the early infection process, here we developed an inoculation method using B. distachyon which enables to sample an increased amount of semi-synchronous infection hyphae. Sixty-one R. solani secretory effector-like protein genes ( RsSEPGs ) were identified using in silico approach with the publicly available gene annotation of R. solani AG-1 IA genome and our RNA-sequencing results obtained from hyphae grown on agar medium. Expression of RsSEPGs was analyzed at 6, 10, 16, 24, and 32 h after inoculation by a quantitative reverse transcription-polymerase chain reaction and 52 genes could be detected at least on a single time point tested. Their expressions showed phase-specific patterns which were classified into 6 clusters. The 23 RsSEPGs in the cluster 1–3 and 29 RsSEPGs in the cluster 4–6 are expected to be involved in biotrophic and necrotrophic interactions, respectively.

Plant defense inducers that mimic functions of the plant immune hormone salicylic acid (SA) often affect plant growth. Although benzothiadiazole (BTH), a synthetic analog of SA, has been widely used to protect crops from diseases by inducing plant defense responses, we recently demonstrated that SA, but not BTH, confers resistance against Rhizoctonia solani, the causal agent of sheath blight disease, in Brachypodium distachyon. Here, we demonstrated that BTH compromised the resistance of Bd3-1 and Gaz4, the two sheath blight-resistant accessions of B. distachyon, which activate SA-dependent signaling following challenge by R. solani. Moreover, upon analyzing our published RNA-seq data from B. distachyon treated with SA or BTH, we found that BTH specifically induces expression of genes related to chloroplast function and jasmonic acid (JA) signaling, suggesting that BTH attenuates R. solani resistance by perturbing growth-defense trade-offs and/or by inducing a JA response that may increase susceptibility to R. solani. Our findings demonstrated that BTH does not work as a simple mimic of SA in B. distachyon, and consequently may presumably cause unfavorable side effects through the transcriptional alteration, particularly with respect to R. solani resistance.

Plant cell surface receptor-like kinases (RLKs) mediate the signals from microbe-associated molecular patterns (MAMPs) that induce immune responses. Lipopolysaccharide (LPS), the major constituent of the outer membrane of gram-negative bacteria, is a common MAMP perceived by animals and plants; however, the plant receptors/co-receptors are unknown except for LORE, a bulb-type lectin S-domain RLK (B-lectin SD1-RLK) in Arabidopsis. OsCERK1 is a multifunctional RLK in rice that contains lysin motifs (LysMs) and is essential for the perception of chitin, a fungal MAMP, and peptidoglycan, a bacterial MAMP. Here, we analyzed the relevance of OsCERK1 to LPS perception in rice. Using OsCERK1-knockout mutants (oscerk1), we evaluated hydrogen peroxide (H2O2) production and gene expression after LPS treatment. We also examined the LPS response in knockout mutants for the B-lectin SD1-RLK genes in rice and for all LysM-protein genes in Arabidopsis. Compared with wild-type rice cells, LPS responses in oscerk1 cells were mostly diminished. By contrast, rice lines mutated in either of three B-lectin SD1-RLK genes and Arabidopsis lines mutated in the LysM-protein genes responded normally to LPS. From these results, we conclude that OsCERK1 is an LPS receptor/co-receptor and that the LPS perception systems of rice and Arabidopsis are significantly different.

Summary Rhizoctonia solani is a soil‐borne fungus causing sheath blight. In consistent with its necrotrophic life style, no rice cultivars fully resistant to R. solani are known, and agrochemical plant defense activators used for rice blast, which upregulate a phytohormonal salicylic acid (SA)‐dependent pathway, are ineffective towards this pathogen. As a result of the unavailability of genetics, the infection process of R. solani remains unclear. We used the model monocotyledonous plants Brachypodium distachyon and rice, and evaluated the effects of phytohormone‐induced resistance to R. solani by pharmacological, genetic and microscopic approaches to understand fungal pathogenicity. Pretreatment with SA, but not with plant defense activators used in agriculture, can unexpectedly induce sheath blight resistance in plants. SA treatment inhibits the advancement of R. solani to the point in the infection process in which fungal biomass shows remarkable expansion and specific infection machinery is developed. The involvement of SA in R. solani resistance is demonstrated by SA‐deficient NahG transgenic rice and the sheath blight‐resistant B. distachyon accessions, Bd3‐1 and Gaz‐4, which activate SA‐dependent signaling on inoculation. Our findings suggest a hemi‐biotrophic nature of R. solani, which can be targeted by SA‐dependent plant immunity. Furthermore, B. distachyon provides a genetic resource that can confer disease resistance against R. solani to plants.

Chitin is widely distributed in fungal cell walls, and its fragments act as potent elicitors in several plant systems. Indeed, pretreatment of rice seedlings with the chitin elicitor results in increased resistance to rice blast. Our histochemical and reverse genetic analyses suggest that the rice blast fungus possesses a mechanism to avoid generation of the chitin elicitor in the first-invaded cell, but none of the genes encoding chitin-binding domains that were tested was responsible for the mechanism. The release of the chitin elicitor during the infection process is thus limited, and the chitin elicitor induces basal resistance, which is nonspecific but not strong enough to avoid infection completely. Therefore, we manipulated cellular responses upon sensing the chitin to evoke an HR-like response by modifying CEBiP, a receptor for the chitin elicitor, with the intracellular region of XA21. Rice plants constitutively expressing the chimeric receptor displayed enhanced resistance to rice blast. In future studies using molecular breeding for disease resistance, a combination approach in which both the chimeric receptor and chitinase are overexpressed simultaneously, or using another receptor-like protein kinase that induces more effective HR, would be helpful.