Genome Editing of a Deoxynivalenol-Induced Transcription Factor Confers Resistance to Fusarium graminearum in Wheat.

Deoxynivalenol (DON) is a mycotoxin virulence factor that promotes growth of the Fusarium graminearum fungus in wheat floral tissues. To further our understanding of the effects of DON exposure on plant cell function, we characterized DON-induced transcriptional changes in wheat spikelets. Four hundred wheat genes were differentially expressed during infection with wild-type F. graminearum as compared with a Δtri5 mutant strain that is unable to produce DON. Most of these genes were more induced by the DON-producing strain and included genes involved in secondary metabolism, signaling, transport, and stress responses. DON induction was confirmed for a subset of the genes, including TaNFXL1, by treating tissues with DON directly. Previous work indicates that the NFXL1 ortholog represses trichothecene-induced defense responses and bacterial resistance in Arabidopsis, but the role of the NFXL family has not been studied in wheat. We observed greater DON-induced TaNFXL1 gene expression in a susceptible wheat genotype relative to the F. graminearum-resistant genotype Wuhan 1. Functional testing using both virus-induced gene silencing and CRISPR-mediated genome editing indicated that TaNFXL1 represses F. graminearum resistance. Together, this suggests that targeting the TaNFXL1 gene may help to develop disease resistance in cultivated wheat.

Characterization of QTL and eQTL controlling early Fusarium graminearum infection and deoxynivalenol levels in a Wuhan 1 x Nyubai doubled haploid wheat population.

BACKGROUND: Fusarium head blight (FHB) is a major disease of cereal crops, caused by the fungal pathogen Fusarium graminearum and related species. Breeding wheat for FHB resistance contributes to increase yields and grain quality and to reduce the use of fungicides. The identification of genes and markers for FHB resistance in different wheat genotypes has nevertheless proven challenging. RESULTS: In this study, early infection by F. graminearum was analyzed in a doubled haploid population derived from the cross of the moderately resistant wheat genotypes Wuhan 1 and Nyubai. Three quantitative trait loci (QTL) were identified: 1AL was associated with lower deoxynivalenol content, and 4BS and 5A were associated with reduced F. graminearum infection at 2 days post inoculation. Early resistance alleles were inherited from Wuhan 1 for QTL 1AL and 4BS and inherited from Nyubai for the 5A QTL. Cis and trans expression QTL (eQTL) were identified using RNA-seq data from infected head samples. Hotspots for trans eQTL were identified in the vicinity of the 1AL and 4BS QTL peaks. Among differentially expressed genes with cis eQTL within the QTL support intervals, nine genes had higher expression associated with FHB early resistance, and four genes had higher expression associated with FHB early susceptibility. CONCLUSIONS: Our analysis of genotype and gene expression data of wheat infected by F. graminearum identified three QTL associated with FHB early resistance, and linked genes with eQTL and differential expression patterns to those QTL. These findings may have applications in breeding wheat for early resistance to FHB.

Correction to: Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

Following publication of the original article [1], we have been notified that some important information was omitted by the authors in the Copyright note. The Copyright note should read as below.

Identification and mapping of expressed genes associated with the 2DL QTL for fusarium head blight resistance in the wheat line Wuhan 1.

BACKGROUND: Fusarium head blight (FHB) is a problem of great concern in small grain cereals, especially wheat. A quantitative trait locus (QTL) for FHB resistance (FHB_SFI) located on the long arm of chromosome 2D in the spring wheat genotype Wuhan 1 is a resistance locus which has potential to improve the FHB resistance of bread wheat since it confers effective resistance to wheat breeding lines. Recently, differentially expressed genes (DEG) have been identified by comparing near isogenic lines (NIL) carrying the susceptible and resistant alleles for the 2DL QTL, using RNA-Seq. In the present study, we aimed to identify candidate genes located within the genetic interval for the 2DL QTL for FHB resistance, as assessed by single floret inoculation (FHB_SFI), and possibly contributing to it. RESULTS: Combining previous and additional bioinformatics analyses, 26 DEG that were located on chromosome arm 2DL were selected for further characterization of their expression profile by RT-qPCR. Seven of those DEG showed a consistent differential expression profile between either three pairs of near isogenic lines or other genotypes carrying the R and S alleles for the 2DL QTL for FHB resistance. UN25696, which was identified in previous expression work using microarray was also confirmed to have a differential expression pattern. Those eight candidate genes were further characterized in 85 lines of a double haploid mapping population derived from the cross Wuhan 1/Nyubai, the population where the 2DL QTL was originally identified. The expression QTL for gene Traes_2DL_179570792 overlapped completely with the mapping interval for the 2DL QTL for FHB_SFI while the expression QTL for UN25696 mapped near the QTL, but did not overlap with it. None of the other genes had a significant eQTL on chromosome 2DL. Higher expression of Traes_2DL_179570792 and UN25696 was associated with the resistant allele at that locus. CONCLUSIONS: Of the 26 DEG from the 2DL chromosome further characterized in this study, only two had an expression QTL located in or near the interval for the 2DL QTL. Traes_2DL_179570792 is the first expression marker identified as associated with the 2DL QTL.

Degradation of salicylic acid by Fusarium graminearum.

Fusarium head blight (FHB) is a major cereal crop disease, caused most frequently by the fungus Fusarium graminearum. We have previously demonstrated that F. graminearum can utilize SA as sole source of carbon to grow. In this current study, we further characterized selected four fungal SA-responsive genes that are predicted to encode salicylic acid (SA)-degrading enzymes and we used a gene replacement approach to characterize them further. These included two genes predicted to encode a salicylate 1-monooxygenase, FGSG_03657 and FGSG_09063, a catechol 1, 2-dioxygenase gene, FGSG_03667, and a 2, 3-dihydroxybenzoic acid decarboxylase gene, FGSG_09061. For each gene, three independent gene replacement strains were assayed for their ability to degrade salicylic acid in liquid culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were shown to be essential for SA degradation, while a loss of 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 caused only a partial reduction of SA degradation and a loss of salicylate 1-monooxygenase FGSG_09063 had no effect when compared to wild type culture. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were identified as the first two key enzyme steps of SA degradation via catechol in the ß-ketoadipate pathway. Expression profiles for all four genes were also determined in liquid culture and in planta. Salicylate 1-monooxygenase FGSG_03657 and catechol 1, 2-dioxygenase FGSG_03667 were co-expressed and their expression was substrate dependent in liquid culture; however their expression was uncoupled in planta. Disruption of the gene for catechol 1, 2-dioxygenase FGSG_03667 was shown to have no effect on fungal virulence on wheat. Our results with 2, 3-dihydroxybenzoic acid decarboxylase FGSG_09061 raise the possibility of an alternate non-oxidative decarboxylation pathway for the conversion of SA to catechol via 2, 3-dihydrozybenzoic acid and for a connection between the oxidative and the non-oxidative decarboxylation pathways for SA conversion.

Transcriptome dynamics associated with resistance and susceptibility against fusarium head blight in four wheat genotypes.

BACKGROUND: Fusarium head blight (FHB) of wheat in North America is caused mostly by the fungal pathogen Fusarium graminearum (Fg). Upon exposure to Fg, wheat initiates a series of cellular responses involving massive transcriptional reprogramming. In this study, we analyzed transcriptomics data of four wheat genotypes (Nyubai, Wuhan 1, HC374, and Shaw), at 2 and 4 days post inoculation (dpi) with Fg, using RNA-seq technology. RESULTS: A total of 37,772 differentially expressed genes (DEGs) were identified, 28,961 from wheat and 8811 from the pathogen. The susceptible genotype Shaw exhibited the highest number of host and pathogen DEGs, including 2270 DEGs associating with FHB susceptibility. Protein serine/threonine kinases and LRR-RK were associated with susceptibility at 2 dpi, while several ethylene-responsive, WRKY, Myb, bZIP and NAC-domain containing transcription factors were associated with susceptibility at 4 dpi. In the three resistant genotypes, 220 DEGs were associated with resistance. Glutathione S-transferase (GST), membrane proteins and distinct LRR-RKs were associated with FHB resistance across the three genotypes. Genes with unique, high up-regulation by Fg in Wuhan 1 were mostly transiently expressed at 2 dpi, while many defense-associated genes were up-regulated at both 2 and 4 dpi in Nyubai; the majority of unique genes up-regulated in HC374 were detected at 4 dpi only. In the pathogen, most genes showed increased expression between 2 and 4 dpi in all genotypes, with stronger levels in the susceptible host; however two pectate lyases and a hydrolase were expressed higher at 2 dpi, and acetyltransferase activity was highly enriched at 4 dpi. CONCLUSIONS: There was an early up-regulation of LRR-RKs, different between susceptible and resistant genotypes; subsequently, distinct sets of genes associated with defense response were up-regulated. Differences in expression profiles among the resistant genotypes indicate genotype-specific defense mechanisms. This study also shows a greater resemblance in transcriptomics of HC374 to Nyubai, consistent with their sharing of two FHB resistance QTLs on 3BS and 5AS, compared to Wuhan 1 which carries one QTL on 2DL in common with HC374.

Indole-3-acetic acid in Fusarium graminearum: Identification of biosynthetic pathways and characterization of physiological effects.

Fusarium graminearum is a devastating pathogenic fungus causing fusarium head blight (FHB) of wheat. This fungus can produce indole-3-acetic acid (IAA) and a very large amount of IAA accumulates in wheat head tissues during the first few days of infection by F. graminearum. Using liquid culture conditions, we have determined that F. graminearum can use tryptamine (TAM) and indole-3-acetonitrile (IAN) as biosynthetic intermediates to produce IAA. It is the first time that F. graminearum is shown to use the l-tryptophan-dependent TAM and IAN pathways rather than the indole-3-acetamide or indole-3-pyruvic acid pathways to produce IAA. Our experiments also showed that exogenous IAA was metabolized by F. graminearum. Exogenous IAA, TAM, and IAN inhibited mycelial growth; IAA and IAN also affected the hyphae branching pattern and delayed macroconidium germination. IAA and TAM had a small positive effect on the production of the mycotoxin 15-ADON while IAN inhibited its production. Our results showed that IAA and biosynthetic intermediates had a significant effect on F. graminearum physiology and suggested a new area of exploration for fungicidal compounds.

Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia.

The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m-3 for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m-3 to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.

Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

Components of priming-induced resistance to Fusarium head blight in wheat revealed by two distinct mutants of Fusarium graminearum.

Two mutants (tri6Δ and noxABΔ) of the fungal pathogen Fusarium graminearum were assessed for their ability to prime immune responses in wheat (cv. Roblin) against challenge with pathogenic F. graminearum. Priming treatments generated Fusarium head blight (FHB)-resistant wheat phenotypes and reduced the accumulation of fungal mycotoxins in infected tissues. Microarray analysis identified 260 transcripts that were differentially expressed during the priming period. Expression changes were observed in genes associated with immune surveillance systems, signalling cascades, antimicrobial compound production, oxidative burst, secondary metabolism, and detoxification and transport. Specifically, genes related to jasmonate, gibberellin and ethylene biosynthesis exhibited differential expression during priming. In addition, the induction of the phenylpropanoid pathways that lead to flavonoid, coumarin and hydroxycinnamic acid amide accumulation was also observed. This study highlights the utility of nonpathogenic mutants to both elicit and delineate stages of defence responses in wheat.

Phylogeny of the genus Synchytrium and the development of TaqMan PCR assay for sensitive detection of Synchytrium endobioticum in soil.

Potato wart, caused by the fungal pathogen Synchytrium endobioticum, is a serious disease with the potential to cause significant economic damage. The small subunit (SSU) and internal transcribed spacer (ITS) ribosomal DNA (rDNA) were sequenced for several Synchytrium spp., showing a high rate of variability for both of these markers among the different species and monophyly of the genus within phylum Chytridiomycota. The intergenic nontranscribed spacer (IGS) of rDNA was sequenced for different pathotypes and showed no intraspecific variation within S. endobioticum, similar to the other rDNA markers from this study. To facilitate screening for the pathogen in soil, three TaqMan polymerase chain reaction (PCR) assays were developed from SSU, ITS, and IGS rDNA sequences to detect S. endobioticum sporangia in the chloroform-flotation fraction of sieved soil extracts. In the screening portion of the method, a first TaqMan assay targeting the SSU rDNA was developed with positive results that were further confirmed with amplicon melt analysis. A synthetic reaction control cloned into a plasmid was incorporated into the procedure, facilitating the validation of negative results. The presence of the reaction control did not adversely affect the efficiency of the SSU target amplification. A second TaqMan assay targeting the ITS-1 region was developed as a confirmatory test. There was 100% accordance between the SSU and ITS-1 TaqMan assays. Utilizing these two assays in tandem achieved good specificity for S. endobioticum, generating negative results with the cloned SSU and ITS-1 regions from all 14 other Synchytrium spp. considered. Spike recovery experiments indicated that these assays, targeting the SSU and ITS-1 rDNA regions, developed from a phylogeny dataset of the genus, could reliably detect a single sporangium in the chloroform flotation fraction of a soil extract. Good correlation between microscopic detection of sporangia and PCR results in both positive and negative soil samples was dually demonstrated for both the SSU and ITS-1 assays.

Effect of salicylic acid on Fusarium graminearum, the major causal agent of fusarium head blight in wheat.

Salicylic acid (SA) is one of the key signal molecules in regulating plant resistance to diverse pathogens. In Arabidopsis thaliana, it is predominantly associated with resistance against biotrophic and hemibiotrophic pathogens, and triggering systemic acquired resistance. In contrast, the effect of SA on the defence efficiency of wheat against fusarium head blight (FHB) and its causal agent, Fusarium graminearum, is still poorly understood. Here we show that the F. graminearum mycelial growth and conidia germination were significantly inhibited, and eventually halted in the presence of increasing concentration of SA in both liquid and solid media. Addition of SA also significantly reduced the production of the mycotoxin deoxynivalenol (DON). However the inhibitory effect of SA required acidic growth conditions to be observed while basic conditions allowed F. graminearum to use SA as a carbon source. High performance liquid chromatography (HPLC) analysis confirmed the capacity of F. graminearum to metabolize SA. To better understand the effect of SA on F. graminearum mycelial growth, we have compared the expression profiles of SA-treated and untreated F. graminearum liquid cultures after 8 and 24 h of treatment, using an F. graminearum custom-commercial microarray. The microarray analysis suggested that F. graminearum can metabolize SA through either the catechol or gentisate pathways that are present in some fungal species. Inoculation of F. graminearum conidia in a SA-containing solution has led to reduced FHB symptoms in the very susceptible Triticum aestivum cv. Roblin. In contrast, no inhibition was observed when SA and conidia were inoculated sequentially. The expression patterns for the wheat PR1, NPR1, Pdf1.2, and PR4 genes, a group of indicator genes for the defence response, suggested that SA-induced resistance contributed little to the reduction of symptoms in our assay conditions. Our results demonstrate that, although F. graminearum has the capacity to metabolize SA, SA has a significant and direct impact on F. graminearum through a reduction in efficiency of germination and growth at higher concentrations.

High-dose supplemental selenite to male Syrian hamsters fed hypercholesterolaemic diets alters Ldlr, Abcg8 and Npc1l1 mRNA expression and lowers plasma cholesterol concentrations.

The aim of the present study was to elucidate possible cholesterol-lowering mechanism(s) of high-dose supplemental Se in the form of selenite, a known hypocholesterolaemic agent. Male Syrian hamsters (four groups, ten per group) were fed semi-purified diets for 4 weeks containing 0.1 % cholesterol and 15 % saturated fat with selenite corresponding to varying levels of Se: (1) Se 0.15 parts per million (ppm), control diet; (2) Se 0.85 ppm; (3) Se 1.7 ppm; (4) Se 3.4 ppm. Lipids were measured in the bile, faeces, liver and plasma. The mRNA expression of several known regulators of cholesterol homeostasis (ATP-binding cassette transporters g5 (Abcg5) and g8 (Abcg8), 7-hydroxylase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor (LdLr) and Nieman-Pick C1-like 1 protein (Npc1l1)) were measured in the liver and/or jejunum. Oxysterols including 24-(S)-hydroxycholesterol, 25-hydroxycholesterol and 27-hydroxycholesterol (27-OHC) were measured in the liver. Significantly lower total plasma cholesterol concentrations were observed in hamsters consuming the low (0.85 ppm) and high (3.4 ppm) Se doses. The two highest doses of Se resulted in decreased plasma LDL-cholesterol concentrations and increased mRNA levels of hepatic Abcg8, Ldlr and jejunal Ldlr. Higher hepatic 27-OHC and TAG concentrations and lower levels of jejunal Npc1l1 mRNA expression were noted in the 1.7 and 3.4 ppm Se-treated hamsters. Overall, Se-induced tissue changes in mRNA expression including increased hepatic Abcg8 and Ldlr, increased jejunal Ldlr and decreased jejunal Npc1l1, provide further elucidation regarding the hypocholesterolaemic mechanisms of action of Se in the form of selenite.

Genome-wide identification and evaluation of novel internal control genes for Q-PCR based transcript normalization in wheat.

To accurately quantify gene expression using quantitative PCR amplification, it is vital that one or more ideal internal control genes are used to normalize the samples to be compared. Ideally, the expression level of those internal control genes should vary as little as possible between tissues, developmental stages and environmental conditions. In this study, 32 candidate genes for internal control were obtained from the analysis of nine independent experiments which included 333 Affymetrix GeneChip Wheat Genome arrays. Expression levels of the selected genes were then evaluated by quantitative real-time PCR with cDNA samples from different tissues, stages of development and environmental conditions. Finally, fifteen novel internal control genes were selected and their respective expression profiles were compared using NormFinder, geNorm, Pearson correlation coefficients and the twofold-change method. The novel internal control genes from this study were compared with thirteen traditional ones for their expression stability. It was observed that seven of the novel internal control genes were better than the traditional ones in expression stability under all the tested cDNA samples. Among the traditional internal control genes, the elongation factor 1-alpha exhibited strong expression stability, whereas the 18S rRNA, Alpha-tubulin, Actin and GAPDH genes had very poor expression stability in the range of wheat samples tested. Therefore, the use of the novel internal control genes for normalization should improve the accuracy and validity of gene expression analysis.

Antioxidant Supplements Improve Profiles of Hepatic Oxysterols and Plasma Lipids in Butter-fed Hamsters.

Hypercholesterolemic diets are associated with oxidative stress that may contribute to hypercholesterolemia by adversely affecting enzymatically-generated oxysterols involved in cholesterol homeostasis. An experiment was conducted to examine whether the cholesterol-lowering effects of the antioxidants selenium and α-tocopherol were related to hepatic oxysterol concentrations. Four groups of male Syrian hamsters (n = 7-8) were fed high cholesterol and saturated fat (0.46% cholesterol, 14.3% fat) hypercholesterolemic semi-purified diets: 1) Control; 2) Control + α-tocopherol (67 IU all-racemic-α-tocopheryl-acetate/kg diet); 3) Control + selenium (3.4 mg selenate/kg diet); and 4) Control + α-tocopherol + selenium. Antioxidant supplementation was associated with lowered plasma cholesterol concentrations, decreased tissue lipid peroxidation and higher hepatic oxysterol concentrations. A second experiment examined the effect of graded selenium doses (0.15, 0.85, 1.7 and 3.4 mg selenate/kg diet) on mRNA expression of the oxysterol-generating enzyme, hepatic 27-hydroxylase (CYP27A1, EC 1.14.13.15), in hamsters (n = 8-9) fed the hypercholesterolemic diets. Supplementation of selenium at 3.4 mg selenate/kg diet was not associated with increased hepatic 27-hydroxylase mRNA. In conclusion, the cholesterol lowering effects of selenium and α-tocopherol were associated with increased hepatic enzymatically generated oxysterol concentrations, which appears to be mediated via improved antioxidant status rather than increased enzymatic production.