WAX INDUCER1 (HvWIN1) transcription factor regulates free fatty acid biosynthetic genes to reinforce cuticle to resist Fusarium head blight in barley spikelets.

Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most devastating diseases of wheat and barley. Resistance to FHB is highly complex and quantitative in nature, and is most often classified as resistance to spikelet infection and resistance to spread of pathogen through the rachis. In the present study, a resistant (CI9831) and a susceptible (H106-371) two-row barley genotypes, with contrasting levels of spikelet resistance to FHB, pathogen or mock-inoculated, were profiled for metabolites based on liquid chromatography and high resolution mass spectrometry. The key resistance-related (RR) metabolites belonging to fatty acids, phenylpropanoids, flavonoids and terpenoid biosynthetic pathways were identified. The free fatty acids (FFAs) linoleic and palmitic acids were among the highest fold change RR induced (RRI) metabolites. These FFAs are deposited as cutin monomers and oligomers to reinforce the cuticle, which acts as a barrier to pathogen entry. Quantitative real-time PCR studies revealed higher expressions of KAS2, CYP86A2, CYP89A2, LACS2 and WAX INDUCER1 (HvWIN1) transcription factor in the pathogen-inoculated resistant genotype than in the susceptible genotype. Knockdown of HvWIN1 by virus-induced genes silencing (VIGS) in resistant genotype upon pathogen inoculation increased the disease severity and fungal biomass, and decreased the abundance of FFAs like linoleic and palmitic acids. Notably, the expression of CYP86A2, CYP89A2 and LAC2 genes was also suppressed, proving the link of HvWIN1 in regulating these genes in cuticle biosynthesis as a defense response.

Identification of metabolites related to mechanisms of resistance in barley against Fusarium graminearum, based on mass spectrometry.

Fusarium head blight (FHB) is an economically important disease of the family Triticeae, as, apart from yield reduction it also causes quality deterioration by producing mycotoxins. Host resistance is the most promising way to control the disease. Metabolic profiling was applied to identify resistance related (RR) metabolites against Fusarium graminearum in five FHB-resistant genotypes ('Chevron', 'H5277-44', 'H5277-164', 'M92-513' and 'M122') relative to one FHB-susceptible genotype ('Stander'). The disease severity was assessed in greenhouse to group the genotypes based on FHB-resistance. The disease was quantified as the proportion of diseased spikelets (PSD) and the area under the disease progress curve (AUDPC). Spikelets were collected at 72 h post inoculation. Metabolites were extracted into an aqueous solution of methanol and analyzed using a LC-hybrid-MS system. Metabolite abundances were subjected to a resistant versus susceptible pair-wise analysis, using a t test. Resistance related (RR) metabolites, both constitutive (RRC) and induced (RRI), were identified amongst metabolites whose levels were significantly higher in resistant genotype than in susceptible. Among 1,430 RR metabolites, 115 were putatively identified. These RR metabolites belonged to different chemical groups: fatty acids: linolenic acid; phenylpropanoids: p-coumaric, sinapic acid; flavonoids: naringenin, kaempferol glucoside, catechol glucoside. In addition, resistance indicator metabolites, such as deoxynivalenol (DON) and DON-3-O-glucoside (D3G) were also detected. The amount of total DON synthesized converted to D3G (PDC) was the greatest in resistant genotype 'Chevron' (PDC = 0.76). The role of the resistance-related and resistance-indicator metabolites on plant defense, and their use as potential biomarkers to screen barley genotypes for FHB resistance is discussed.

Mass spectrometry based metabolomics to identify potential biomarkers for resistance in barley against fusarium head blight (Fusarium graminearum).

Resistance in Triticeae to fusarium head blight (FHB) is quantitatively inherited. Metabolomics as a tool was used to better understand the mechanisms of resistance and to identify potential FHB resistance biomarker metabolites in barley. Five FHB-resistant two-row barley genotypes (CIho 4196, Zhedar-1, Zhedar-2, Fredrickson, and Harbin-2r) and one FHB-susceptible genotype (CH 9520-30) were each inoculated with either pathogen-suspension or mock-solution. Disease severity, quantified as the proportion of spikelets diseased, varied among genotypes, being the greatest in CH 9520-30. Spikelets were sampled, metabolites extracted with aqueous methanol, and analyzed using an LC-ESI-LTQ-Orbitrap system. A pair wise, resistant vs. susceptible, t-test identified 1774 significant treatment peaks. Canonical discriminant analysis of peak abundance allowed the genotypes to be sorted into three clusters: (i) CH9520-30, (ii) Harbin-2r, (iii) the remaining four genotypes. The t-test was further used to identify resistance-related (RR) and pathogenesis-related (PR) metabolites. The pathogen-produced virulence factor deoxynivalenol (DON), and its detoxification product, DON-3-O-glucoside (D3G) were designated as resistance indicator (RI) metabolites. Metabolites (RR, PR, or RI) occurring in at least two resistant genotypes, showing a two-fold or greater abundance in resistant vs. susceptible lines, and also known to have plant defense functions were selected as potential FHB resistance biomarker metabolites. These included phenylalanine, p-coumaric acid, jasmonate, linolenic acid, total DON produced (TDP), and the proportion of DON converted to D3G (PDC). Total DON was the lowest in CIho 4196, while PDC was the highest in Zhedar-2. The application of RR, PR, and RI metabolites as potential biomarkers to enhance resistance is discussed.