Exploring natural variation for rice sheath blight resistance in Brachypodium distachyon.
ABSTRACT: Sheath blight caused by the soil borne fungus Rhizoctonia solani AG1-IA is one of the major diseases of rice in the world. Genetic resistance in rice against this disease has not been very successful. Brachypodium distachyon is considered as a model species for several cereal crops and it has been studied in the past to identify novel sources of disease resistance against cereal crop diseases. Therefore, the current study was designed to explore nonhost disease resistance in Brachypodium accessions against sheath blight pathogen of rice, Rhizoctonia solani. A total of 19 Brachypodium distachyon accessions were screened for resistance against Rhizoctonia solani AG1-IA. Different levels of resistance reactions were observed among accessions. Quantification of jasmonic acid (JA) and salicylic acid (SA) concentration in selected resistant (Bd3-1), moderately susceptible (Bd21), and susceptible (Bd30-1) inbred accessions revealed that Bd3-1 accumulated more JA upon pathogen infection compared to Bd21 or Bd30-1. In contrary, no differences were observed for SA accumulation in tested accessions suggesting that the resistance to R. solani in Brachypodium is due to an SA-independent defense pathway. Our study provides a new foundation to explore this area for more durable resistance against this disease.
Project description: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.
Project description: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.
Project description: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.
Project description:<i>Brachypodium distachyon</i> is an established model for monocotyledonous plants. Numerous markers intended for gene discovery and population genetics have been designed. However to date, very few indel markers with larger and easily scored length polymorphism differences, that distinguish between the two morphologically similar and highly utilized <i>B. distachyon</i> accessions, Bd21, the reference genome accession, and Bd21-3, the transformation-optimal accession, are publically available. In this study, 22 indel markers were designed and utilized to produce length polymorphism differences of 150 bp or more, for easy discrimination between Bd21 and Bd21-3. When tested on four other <i>B. distachyon</i> accessions, one case of multiallelism was observed. It was also shown that the markers could be used to determine homozygosity and heterozygosity at specific loci in a Bd21 x Bd3-1 F2 population. The work done in this study allows researchers to maintain the fidelity of Bd21 and Bd21-3 stocks for both transgenic and nontransgenic studies. It also provides markers that can be utilized in conjunction with others already available for further research on population genetics, gene discovery and gene characterization, all of which are necessary for the relevance of <i>B. distachyon</i> as a model species.
Project description:The ND18 strain of Barley stripe mosaic virus (BSMV) infects several lines of Brachypodium distachyon, a recently developed model system for genomics research in cereals. Among the inbred lines tested, Bd3-1 is highly resistant at 20 to 25 °C, whereas Bd21 is susceptible and infection results in an intense mosaic phenotype accompanied by high levels of replicating virus. We generated an F(6:7) recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21 and used the RILs, and an F(2) population of a second Bd21 × Bd3-1 cross to evaluate the inheritance of resistance. The results indicate that resistance segregates as expected for a single dominant gene, which we have designated Barley stripe mosaic virus resistance 1 (Bsr1). We constructed a genetic linkage map of the RIL population using SNP markers to map this gene to within 705 Kb of the distal end of the top of chromosome 3. Additional CAPS and Indel markers were used to fine map Bsr1 to a 23 Kb interval containing five putative genes. Our study demonstrates the power of using RILs to rapidly map the genetic determinants of BSMV resistance in Brachypodium. Moreover, the RILs and their associated genetic map, when combined with the complete genomic sequence of Brachypodium, provide new resources for genetic analyses of many other traits.
Project description:Next-generation sequencing (NGS) technologies have enabled genome re-sequencing for exploring genome-wide polymorphisms among individuals, as well as targeted re-sequencing for the rapid and simultaneous detection of polymorphisms in genes associated with various biological functions. Therefore, a simple and robust method for targeted re-sequencing should facilitate genotyping in a wide range of biological fields. In this study, we developed a simple, custom, targeted re-sequencing method, designated "multiplex PCR targeted amplicon sequencing (MTA-seq)," and applied it to the genotyping of the model grass Brachypodium distachyon. To assess the practical usability of MTA-seq, we applied it to the genotyping of genome-wide single-nucleotide polymorphisms (SNPs) identified in natural accessions (Bd1-1, Bd3-1, Bd21-3, Bd30-1, Koz-1, Koz-3, and Koz-4) by comparing the re-sequencing data with that of reference accession Bd21. Examination of SNP-genotyping accuracy in 443 amplicons from eight parental accessions and an F1 progeny derived by crossing of Bd21 and Bd3-1 revealed that ~95% of the SNPs were correctly called. The assessment suggested that the method provided an efficient framework for accurate and robust SNP genotyping. The method described here enables easy design of custom target SNP-marker panels in various organisms, facilitating a wide range of high-throughput genetic applications, such as genetic mapping, population analysis, molecular breeding, and genomic diagnostics.
Project description:Metabolite composition and concentrations in seed grains are important traits of cereals. To identify the variation in the seed metabolotypes of a model grass, namely Brachypodium distachyon, we applied a widely targeted metabolome analysis to forty inbred lines of B. distachyon and examined the accumulation patterns of 183 compounds in the seeds. By comparing the metabolotypes with the population structure of these lines, we found signature metabolites that represent different accumulation patterns for each of the three B. distachyon subpopulations. Moreover, we found that thirty-seven metabolites exhibited significant differences in their accumulation between the lines Bd21 and Bd3-1. Using a recombinant inbred line (RIL) population from a cross between Bd3-1 and Bd21, we identified the quantitative trait loci (QTLs) linked with this variation in the accumulation of thirteen metabolites. Our metabolite QTL analysis illustrated that different genetic factors may presumably regulate the accumulation of 4-pyridoxate and pyridoxamine in vitamin B6 metabolism. Moreover, we found two QTLs on chromosomes 1 and 4 that affect the accumulation of an anthocyanin, chrysanthemin. These QTLs genetically interacted to regulate the accumulation of this compound. This study demonstrates the potential for metabolite QTL mapping in B. distachyon and provides new insights into the genetic dissection of metabolomic traits in temperate grasses.
Project description:Brachypodium distachyon is an emerging model plant for studying biological phenomena in temperate grasses. Study of the growth scale is essential to analyse spatio-temporal changes in molecular factors throughout the life cycle. For sensitive and robust staging based on morphology in B. distachyon, we demonstrated the utility of the BBCH (Biologische Bundesanstalt, Bundessortenamt and CHemical industry) scale, which is comparable to the Zadoks scale conventionally used for Triticeae crops. We compared the chronological progression of B. distachyon accessions Bd21 and Bd3-1, in addition to the progression of Chinese Spring wheat. The comparison of growth stages illustrates the morphological similarities and differences in the timing of life cycle events. Furthermore, we compared metabolite accumulation patterns across different growth stages and across different stress conditions using a widely targeted metabolome analysis. Metabolic profiling determined commonalities and specificities in chemical properties that were dependent on organisms, growth stages and/or stress conditions. Most metabolites accumulated equivalently in B. distachyon and wheat. This qualitative similarity indicated the superiority of B. distachyon as a model for Triticeae crops. The growth scale of B. distachyon should provide a conceptual framework for comparative analysis and for knowledge integration between this model grass and crops in the Pooideae subfamily.
Project description:We constructed seven small RNA libraries of Rhizoctonia solani AG1 IA and sequenced using Illumina GA II. The seven samples include mycelium cultured on PDA without rice incubated, 6 different stages at 10 hours (10h), 18h, 24h, 32h, 48h and 72h spanning the Rhizoctonia solani AG1 strains infection rice. We identified miRNA-like small RNAs (milRNAs) using MIREAP and mirdeep2. The milRNAs were used for further analysis of interactions between milRNA and mRNA that may involve in plant-infection. Overall design: Identification of milRNAs in R. solani AG1.
Project description:<h4>Background and aims</h4>Cold is a major constraint for cereal cultivation under temperate climates. Winter-hardy plants interpret seasonal changes and can acquire the ability to resist sub-zero temperatures. This cold acclimation process is associated with physiological, biochemical and molecular alterations in cereals. Brachypodium distachyon is considered a powerful model system to study the response of temperate cereals to adverse environmental conditions. To date, little is known about the cold acclimation and freezing tolerance capacities of Brachypodium. The main objective of this study was to evaluate the cold hardiness of seven diploid Brachypodium accessions.<h4>Methods</h4>An integrated approach, involving monitoring of phenological indicators along with expression profiling of the major vernalization regulator VRN1 orthologue, was followed. In parallel, soluble sugars and proline contents were determined along with expression profiles of two COR genes in plants exposed to low temperatures. Finally, whole-plant freezing tests were performed to evaluate the freezing tolerance capacity of Brachypodium.<h4>Key results</h4>Cold treatment accelerated the transition from the vegetative to the reproductive phase in all diploid Brachypodium accessions tested. In addition, low temperature exposure triggered the gradual accumulation of BradiVRN1 transcripts in all accessions tested. These accessions exhibited a clear cold acclimation response by progressively accumulating proline, sugars and COR gene transcripts. However, whole-plant freezing tests revealed that these seven diploid accessions only have a limited capacity to develop freezing tolerance when compared with winter varieties of temperate cereals such as wheat and barley. Furthermore, little difference in terms of survival was observed among the accessions tested despite their previous classification as either spring or winter genotypes.<h4>Conclusions</h4>This study is the first to characterize the freezing tolerance capacities of B. distachyon and provides strong evidence that some diploid accessions such as Bd21 have a facultative growth habit.