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Background: Fusarium crown rot (FCR) is a chronic and severe disease in cereal production in semi-arid regions worldwide. A putative quantitative trait locus conferring FCR resistance, Qcrs.cpi-1H, had previously been mapped on the long arm of chromosome 1H in barley., Results: In this study, five pairs of near-isogenic lines (NILs) targeting the 1HL locus were developed. Analysing the NILs found that the resistant allele at Qcrs.cpi-1H significantly reduced FCR severity. Transcriptomic analysis was then conducted against three of the NIL pairs, which placed the Qcrs.cpi-1H locus in an interval spanning about 11 Mbp. A total of 56 expressed genes bearing single nucleotide polymorphisms (SNPs) were detected in this interval. Five of them contain non-synonymous SNPs. These results would facilitate detailed mapping as well as cloning gene(s) underlying the resistance locus., Conclusion: NILs developed in this study and the transcriptomic sequences obtained from them did not only allow the validation of the resistance locus Qcrs.cpi-1H and the identification of candidate genes underlying its resistance, they also allowed the delineation of the resistance locus and the development of SNPs markers which formed a solid base for detailed mapping as well as cloning gene(s) underlying the locus.

Key Message: This study demonstrates how identification of genes underpinning disease-resistance QTL based on differential expression and SNPs can be improved by performing transcriptomic analysis on multiple near isogenic lines. Transcriptomic analysis has been widely used to understand the genetic basis of a trait of interest by comparing genotypes with contrasting phenotypes. However, these approaches identify such large sets of differentially expressed genes that it proves difficult to isolate which genes underpin the phenotype of interest. This study tests whether using multiple near isogenic lines (NILs) can improve the resolution of RNA-seq-based approaches to identify genes underpinning disease-resistance QTL. A set of NILs for a major effect Fusarium crown rot-resistance QTL in barley on the 4HL chromosome arm were analysed under Fusarium crown rot using RNA-seq. Differential gene expression and single nucleotide polymorphism detection analyses reduced the number of putative candidates from thousands within individual NIL pairs to only one hundred and two genes, which were differentially expressed or contained SNPs in common across NIL pairs and occurred on 4HL. Our findings support the value of performing RNA-seq analysis using multiple NILs to remove genetic background effects. The enrichment analyses indicated conserved differences in the response to infection between resistant and sensitive isolines suggesting that sensitive isolines are impaired in systemic defence response to Fusarium pseudograminearum.

Fusarium pseudograminearum is an important pathogen of wheat and barley, particularly in semi-arid environments. Previous genome assemblies for this organism were based entirely on short read data and are highly fragmented. In this work, a genetic map of F. pseudograminearum has been constructed for the first time based on a mapping population of 178 individuals. The genetic map, together with long read scaffolding of a short read-based genome assembly, was used to give a near-complete assembly of the four F. pseudograminearum chromosomes. Large regions of synteny between F. pseudograminearum and F. graminearum, the related pathogen that is the primary causal agent of cereal head blight disease, were previously proposed in the core conserved genome, but the construction of a genetic map to order and orient contigs is critical to the validation of synteny and the placing of species-specific regions. Indeed, our comparative analyses of the genomes of these two related pathogens suggest that rearrangements in the F. pseudograminearum genome have occurred in the chromosome ends. One of these rearrangements includes the transposition of an entire gene cluster involved in the detoxification of the benzoxazolinone (BOA) class of plant phytoalexins. This work provides an important genomic and genetic resource for F. pseudograminearum, which is less well characterized than F. graminearum. In addition, this study provides new insights into a better understanding of the sexual reproduction process in F. pseudograminearum, which informs us of the potential of this pathogen to evolve., (© 2016 BSPP AND JOHN WILEY & SONS LTD.)

The conserved protein complex known as Mediator conveys transcriptional signals by acting as an intermediary between transcription factors and RNA polymerase II. As a result, Mediator subunits play multiple roles in regulating developmental as well as abiotic and biotic stress pathways. In this report we identify the head domain subunits MEDIATOR18 and MEDIATOR20 as important susceptibility factors for Fusarium oxysporum infection in Arabidopsis thaliana. Mutants of MED18 and MED20 display down-regulation of genes associated with jasmonate signaling and biosynthesis while up-regulation of salicylic acid associated pathogenesis related genes and reactive oxygen producing and scavenging genes. We propose that MED18 and MED20 form a sub-domain within Mediator that controls the balance of salicylic acid and jasmonate associated defense pathways.

Background and Aims: Fusarium crown rot caused by the fungal pathogen Fusarium pseudograminearum is a disease of wheat and barley, bearing significant economic cost. Efforts to develop effective resistance to this disease have been hampered by the quantitative nature of resistance and a lack of understanding of the factors associated with resistance and susceptibility. Here, we aimed to dissect transcriptional responses triggered in wheat by F. pseudograminearum infection., Methods: We used an RNA-seq approach to analyse host responses during a compatible interaction and identified >2700 wheat genes differentially regulated after inoculation with F. pseudograminearum . The production of a few key metabolites and plant hormones in the host during the interaction was also analysed., Key Results: Analysis of gene ontology enrichment showed that a disproportionate number of genes involved in primary and secondary metabolism, signalling and transport were differentially expressed in infected seedlings. A number of genes encoding pathogen-responsive uridine-diphosphate glycosyltransferases (UGTs) potentially involved in detoxification of the Fusarium mycotoxin deoxynivalenol (DON) were differentially expressed. Using a F. pseudograminearum DON-non-producing mutant, DON was shown to play an important role in virulence during Fusarium crown rot. An over-representation of genes involved in the phenylalanine, tryptophan and tyrosine biosynthesis pathways was observed. This was confirmed through metabolite analyses that demonstrated tryptamine and serotonin levels are induced after F. pseudograminearum inoculation., Conclusions: Overall, the observed host response in bread wheat to F. pseudograminearum during early infection exhibited enrichment of processes related to pathogen perception, defence signalling, transport and metabolism and deployment of chemical and enzymatic defences. Additional functional analyses of candidate genes should reveal their roles in disease resistance or susceptibility. Better understanding of host responses contributing to resistance and/or susceptibility will aid the development of future disease improvement strategies against this important plant pathogen., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company.)

Background: Fusarium crown rot (FCR) is a major cereal disease in semi-arid areas worldwide. Of the various QTL reported, the one on chromosome arm 3BL (Qcrs.cpi-3B) has the largest effect that can be consistently detected in different genetic backgrounds. Nine sets of near isogenic lines (NILs) for this locus were made available in a previous study. To identify markers that could be reliably used in tagging the Qcrs.cpi-3B locus, a NIL-derived population consisting of 774 F10 lines were generated and exploited to assess markers selected from the existing linkage map and generated from sequences of the 3B pseudomolecule., Results: This is the first report on fine mapping a QTL conferring FCR resistance in wheat. By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb. Seven markers co-segregating with the locus were developed. This interval contains a total of 63 gene-coding sequences based on the 3B pseudomolecule, and six of them were known to encode disease resistance proteins. Several of the genes in this interval were among those responsive to FCR infection detected in an earlier study., Conclusions: The accurate localization of the Qcrs.cpi-3B locus and the development of the markers co-segregating with it should facilitate the incorporation of this large-effect QTL conferring FCR resistance into breeding programs as well as the cloning of the gene(s) underlying the QTL.

Some of the most devastating agricultural diseases are caused by root-infecting pathogens, yet the majority of studies on these interactions to date have focused on the host responses of aerial tissues rather than those belowground. Fusarium oxysporum is a root-infecting pathogen that causes wilt disease on several plant species including Arabidopsis thaliana. To investigate and compare transcriptional changes triggered by F. oxysporum in different Arabidopsis tissues, we infected soil-grown plants with F. oxysporum and subjected root and leaf tissue harvested at early and late timepoints to RNA-seq analyses. At least half of the genes induced or repressed by F. oxysporum showed tissue-specific regulation. Regulators of auxin and ABA signalling, mannose binding lectins and peroxidases showed strong differential expression in root tissue. We demonstrate that ARF2 and PRX33, two genes regulated in the roots, promote susceptibility to F. oxysporum. In the leaves, defensins and genes associated with the response to auxin, cold and senescence were strongly regulated while jasmonate biosynthesis and signalling genes were induced throughout the plant.

Blackleg, caused by Leptosphaeria maculans, is one of the most important diseases of oilseed and vegetable crucifiers worldwide. The present study describes (1) the construction of a genetic linkage map, comprising 255 markers, based upon simple sequence repeats (SSR), sequence-related amplified polymorphism, sequence tagged sites, and EST-SSRs and (2) the localization of qualitative (race-specific) and quantitative (race non-specific) trait loci controlling blackleg resistance in a doubled-haploid population derived from the Australian canola (Brassica napus L.) cultivars Skipton and Ag-Spectrum using the whole-genome average interval mapping approach. Marker regression analyses revealed that at least 14 genomic regions with LOD ≥ 2.0 were associated with qualitative and quantitative blackleg resistance, explaining 4.6-88.9 % of genotypic variation. A major qualitative locus, designated RlmSkipton (Rlm4), was mapped on chromosome A7, within 0.8 cM of the SSR marker Xbrms075. Alignment of the molecular markers underlying this QTL region with the genome sequence data of B. rapa L. suggests that RlmSkipton is located approximately 80 kb from the Xbrms075 locus. Molecular marker-RlmSkipton linkage was further validated in an F(2) population from Skipton/Ag-Spectrum. Our results show that SSR markers linked to consistent genomic regions are suitable for enrichment of favourable alleles for blackleg resistance in canola breeding programs.

Fusarium crown rot (FCR) of wheat and barley, predominantly caused by the fungal pathogen Fusarium pseudograminearum, is a disease of economic significance. The quantitative nature of FCR resistance within cultivated wheat germplasm has significantly limited breeding efforts to enhanced FCR resistance in wheat. In this study, we characterized the molecular responses of Brachypodium distachyon (Brachypodium hereafter) to F. pseudograminearum infection using RNA-seq to determine whether Brachypodium can be exploited as a model system towards better understanding of F. pseudograminearum-wheat interaction. The transcriptional response to infection in Brachypodium was strikingly similar to that previously reported in wheat, both in shared expression patterns of wheat homologs of Brachypodium genes and functional overlap revealed through comparative gene ontology analysis in both species. Metabolites produced by various biosynthetic pathways induced in both wheat and Brachypodium were quantified, revealing a high degree of overlap between these two species in metabolic response to infection but also showed Brachypodium does not produce certain defence-related metabolites found in wheat. Functional analyses of candidate genes identified in this study will improve our understanding of resistance mechanisms and may lead to the development of new strategies to protect cereal crops from pathogen infection.

Plants produce a variety of secondary metabolites to defend themselves from pathogen attack, while pathogens have evolved to overcome plant defences by producing enzymes that degrade or modify these defence compounds. However, many compounds targeted by pathogen enzymes currently remain enigmatic. Identifying host compounds targeted by pathogen enzymes would enable us to understand the potential importance of such compounds in plant defence and modify them to make them insensitive to pathogen enzymes., Here, a proof of concept metabolomics-based method was developed to discover plant defence compounds modified by pathogens using two pathogen enzymes with known targets in wheat and tomato. Plant extracts treated with purified pathogen enzymes were subjected to LC-MS, and the relative abundance of metabolites before and after treatment were comparatively analysed., Using two enzymes from different pathogens the in planta targets could be found by combining relatively simple enzymology with the power of untargeted metabolomics. Key to the method is dataset simplification based on natural isotope occurrence and statistical filtering, which can be scripted., The method presented here will aid in our understanding of plant-pathogen interactions and may lead to the development of new plant protection strategies. [ABSTRACT FROM AUTHOR]

Background: Fusarium crown rot (FCR) is a major cereal disease in semi-arid areas worldwide. Of the various QTL reported, the one on chromosome arm 3BL (Qcrs.cpi-3B) has the largest effect that can be consistently detected in different genetic backgrounds. Nine sets of near isogenic lines (NILs) for this locus were made available in a previous study. To identify markers that could be reliably used in tagging the Qcrs.cpi-3B locus, a NIL-derived population consisting of 774 F10 lines were generated and exploited to assess markers selected from the existing linkage map and generated from sequences of the 3B pseudomolecule. Results: This is the first report on fine mapping a QTL conferring FCR resistance in wheat. By three rounds of linkage mapping using the NILs and the NIL-derived population, the Qcrs.cpi-3B locus was mapped to an interval of 0.7 cM covering a physical distance of about 1.5 Mb. Seven markers co-segregating with the locus were developed. This interval contains a total of 63 gene-coding sequences based on the 3B pseudomolecule, and six of them were known to encode disease resistance proteins. Several of the genes in this interval were among those responsive to FCR infection detected in an earlier study. Conclusions: The accurate localization of the Qcrs.cpi-3B locus and the development of the markers co-segregating with it should facilitate the incorporation of this large-effect QTL conferring FCR resistance into breeding programs as well as the cloning of the gene(s) underlying the QTL. [ABSTRACT FROM AUTHOR]