Identification of differentially regulated proteins in response to a compatible interaction between the pathogen Fusarium graminearum and its host, Triticum aestivum

Using proteomic analyses, a study was carried out aimed at understanding the molecular mechanism of interaction between Fusarium graminearum and Triticum aestivum. Wheat spikelets were inoculated with H2O and conidia spores of F. graminearum. Proteins were extracted from spikelets harvested at three time points: 1, 2 and 3 days post inoculation. About 1380 protein spots were displayed on 2-D gels stained with Sypro Ruby. In total, 41 proteins were detected to be differentially regulated due to F. graminearum infection, and were analyzed with LC-MS/MS for their identification. The proteins involved in the antioxidant and jasmonic acid signaling pathways, pathogenesis-related response, amino acid synthesis and nitrogen metabolism were up-regulated, while those related to photosynthesis were less abundant following F. graminearum infection. The DNA-damage inducible protein was found to be induced and glycosylated in F. graminearum-infected spikelets. Using TargetP program, seven of the identified wheat proteins were predicted to be located in the chloroplast, implying that the chloroplast is the organelle mostly affected by F. graminearum infection. Eight identified fungal proteins possess possible functions such as antioxidant and acquiring carbon from wheat through glycolysis in a compatible interaction between F. graminearum and wheat.     

Histological and molecular analysis of Rdg2a barley resistance to leaf stripe

Barley (Hordeum vulgare L.) leaf stripe is caused by the seed-borne fungus Pyrenophora graminea. We investigated microscopically and molecularly the reaction of barley embryos to leaf stripe inoculation. In the resistant genotype NIL3876-Rdg2a, fungal growth ceased at the scutellar node of the embryo, while in the susceptible near-isogenic line (NIL) Mirco-rdg2a fungal growth continued past the scutellar node and into the embryo. Pathogen-challenged embryos of resistant and susceptible NILs showed different levels of UV autofluorescence and toluidine blue staining, indicating differential accumulation of phenolic compounds. Suppression subtractive hybridization and cDNA amplified fragment-length polymorphism (AFLP) analyses of embryos identified P. graminea-induced and P. graminea-repressed barley genes. In addition, cDNA-AFLP analysis identified six pathogenicity-associated fungal genes expressed during barley infection but at low to undetectable levels during growth on artificial media. Microarrays representing the entire set of differentially expressed cDNA-AFLP fragments and 100 barley homologues of previously described defence-related genes were used to study gene expression changes at 7 and 14 days after inoculation in the resistant and susceptible NILs. A total of 171 significantly modulated barley genes were identified and assigned to four groups based on timing and genotype dependence of expression. Analysis of the changes in gene expression during the barley resistance response to leaf stripe suggests that the Rdg2a-mediated response includes cell-wall reinforcement, signal transduction, generation of reactive oxygen species, cell protection, jasmonate signalling and expression of plant effector genes. The identification of genes showing leaf stripe inoculation or resistance-dependent expression sets the stage for further dissection of the resistance response of barley embryo cells to leaf stripe.     

The transcriptome of Fusarium graminearum during the infection of wheat

Fusarium graminearum causes head blight disease in wheat and barley. To help understand the infection process on wheat, we studied global gene expression of F. graminearum in a time series from 24 to 196 h after inoculation, compared with a noninoculated control. The infection was rapid and, after 48 h, over 4,000 fungal genes were expressed. The number of genes expressed increased over time up to 96 h (>8,000 genes), and then declined at the 144- and 192-h post-inoculation time points. After subtraction of genes found expressed on complete medium, during carbon or nitrogen starvation, and on barley, only 355 were found exclusively expressed in wheat, mostly genes with unknown function (72.6%). These genes were mainly found in single-nucleotide polymorphism-enriched islands on the chromosomes, suggesting a higher evolutionary selection pressure. The annotated genes were enriched in functional groups predicted to be involved in allantoin and allantoate transport, detoxification, nitrogen, sulfur and selenium metabolism, secondary metabolism, carbohydrate metabolism, and degradation of polysaccharides and ester compounds. Several putative secreted virulence factors were also found expressed in wheat.     

Reduced susceptibility to Fusarium head blight in Brachypodium distachyon through priming with the Fusarium mycotoxin deoxynivalenol

The fungal cereal pathogen Fusarium graminearum produces deoxynivalenol (DON) during infection. The mycotoxin DON is associated with Fusarium head blight (FHB), a disease that can cause vast grain losses. Whilst investigating the suitability of Brachypodium distachyon as a model for spreading resistance to F. graminearum, we unexpectedly discovered that DON pretreatment of spikelets could reduce susceptibility to FHB in this model grass. We started to analyse the cell wall changes in spikelets after infection with F. graminearum wild‐type and defined mutants: the DON‐deficient Δtri5 mutant and the DON‐producing lipase disruption mutant Δfgl1, both infecting only directly inoculated florets, and the mitogen‐activated protein (MAP) kinase disruption mutant Δgpmk1, with strongly decreased virulence but intact DON production. At 14 days post‐inoculation, the glucose amounts in the non‐cellulosic cell wall fraction were only increased in spikelets infected with the DON‐producing strains wild‐type, Δfgl1 and Δgpmk1. Hence, we tested for DON‐induced cell wall changes in B. distachyon, which were most prominent at DON concentrations ranging from 1 to 100 ppb. To test the involvement of DON in defence priming, we pretreated spikelets with DON at a concentration of 1 ppm prior to F. graminearum wild‐type infection, which significantly reduced FHB disease symptoms. The analysis of cell wall composition and plant defence‐related gene expression after DON pretreatment and fungal infection suggested that DON‐induced priming of the spikelet tissue contributed to the reduced susceptibility to FHB.     

PEG-simulated drought stress and spike in vitro culture are used to study the impact of water stress on barley malt quality

Water deficient or drought stress is a major factor causing deterioration or instability of malt barley quality. In the studies on the influence of drought stress during grain filling on malt quality formation or metabolic changes, it is quite difficult to obtain the uniform plant individuals and water condition in pot or field experiments. In this study, we combined barley spike in vitro culture and PEG-6000 simulated drought to determine the genotypic difference in the changes of grain metabolites and the expression level of the genes encoding β-amylase and β-glucan using two Tibetan wild barley accessions and two cultivated genotypes differing in malt quality stability under drought stress. Under simulated drought, grain weight and β-glucan content were dramatically reduced and β-amylase activity was increased, and a lot of metabolites were markedly changed for all genotypes. On the whole, the changes were relatively smaller in the wild barley. Meanwhile, the expressions of Bmy1 related to β-amylase synthesis and GSL1, GSL4 and GSL7 related to β-glucan synthesis were up-regulated and down-regulated under drought stress, respectively, being consistent with the changes of β-amylase activity and β-glucan content in the four barley genotypes. The current results showed that PEG-6000 simulated drought and spike in intro culture may provide the basically similar information on grain development or metabolites as do in the field experiments, and it is suitable for use in studies on the influence of drought stress on quality traits during grain filling stage of barley or other cereal crops.     

大麦赤霉病抗扩展性鉴定与评价

利用禾谷镰刀菌单花滴注接种研究了245份大麦品种对赤霉病抗扩展性。结果表明,大麦赤霉病抗性除了抗初侵染外还存在抗扩展类型。比较了接种后7d、14d和21d的病小穗数和病小穗率以覆由不同期病小穗率获得的病程曲线面积等7个指标性状,并对其进行遗传参数分析,发现21d病小穗率指标在品种间具帔大的变幅、遗传变异系数和遗传率,21d病小穗数和病程曲线面积与病小穗率呈极显著正相关。不同大麦品种抗扩展性表现不一,供试品种中以Suyin21、乌金一号、莆846193、盐97001、96AC20-30五个品种21d病小穗率最低,为高抗品种,占全部供试品种的2．04％。     

The impact of Fusarium culmorum infection on the protein fractions of raw barley and malted grains

Contaminating fungi, such as Fusarium species, produce metabolites that may interfere with normal barley grain proteolysis pattern and consequently, affect malt and beer quality. Protein compositional changes of an initial mixture of 20 % Fusarium culmorum infected and 80 % noninfected mature barley grains and respective malt are reported here. Proteolytic activity of infected barley grains (IBG) and respective malt, with controls (uninfected grains), were characterized using protease inhibitors from each class of this enzyme, including metallo-, cysteine, serine, and aspartic proteases, as well as uninhibited protease fractions. The proteins were extracted according to the Osborne fractionation and separated by size exclusion chromatography. Additionally, two-dimensional (2D) gel electrophoresis (GE) was used to analyze hydrophobic storage proteins isolated from the control and IBG. Analyses revealed that F. culmorum IBG had a twofold increase of proteolytic activity compared to the control sample, which showed an increase in all protease classes with aspartic proteases dominating. Infected and control malt grains were comparable with cysteine proteases representing almost 50 % of all proteolytic enzymes detected. Protein extractability was 31 % higher in IBG compared to the control barley. The albumin fraction showed that several metabolic proteins decreased and increased at different rates during infection and malting, thus showing a complex F. culmorum infection interdependence. Prolamin storage proteins were more hydrophobic during barley fungal infection. F. culmorum interfered with the grain hydrolytic protein profile, thereby altering the grain's protein content and quality.     

Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare

Using proteomic techniques, a study aimed at isolating and identifying proteins associated with resistance to fusarium head blight (FHB) was conducted on six barley genotypes of varying resistance. At anthesis, barley spikelets were point inoculated with Fusarium graminearum macroconidial suspensions or mock inoculum. In total, 43 acidic protein spots out of 600 were detected 3 days postinoculation to be differentially expressed due to FHB and were identified. Identification of proteins responsive to FHB included those associated with oxidative burst and oxidative stress response, such as malate dehydrogenase and peroxidases, and pathogenesis-related (PR). An increase in abundance of PR-3 or PR-5 could be associated with the resistant genotypes CI4196, Svansota, and Harbin, as well as the intermediate resistant genotype CDC Bold. On the contrary, the susceptible genotype Stander showed a decrease in abundance of these acidic PR-proteins. In the susceptible and intermediate resistant genotypes Stander and CDC Bold, as well as CI4196, the increased abundance of proteins associated with an oxidative response might have prepared the terrain for saprophytic fungal invasion. On the contrary, in the resistant sources Harbin and Svansota we did not observed change in abundance of these proteins. Not a single significant change in acidic protein abundance could be detected in Chevron. Three distinct response patterns are reported from these six barley genotypes.     

Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains

The feasibility of producing plant cell wall polysaccharide-hydrolysing feed enzymes in the endosperm of barley grain was investigated. The coding region of a modified xylanase gene (xynA) from the rumen fungus, Neocallimastix patriciarum, linked with an endosperm-specific promoter from cereal storage protein genes was introduced into barley by Agrobacterium-mediated transformation. Twenty-four independently transformed barley lines with the xylanase gene were produced and analysed. The fungal xylanase was produced in the developing endosperm under the control of either the rice glutelin B-1 (GluB-1) or barley B1 hordein (Hor2-4) promoter. The rice GluB-1 promoter provided an apparently higher expression level of recombinant proteins in barley grain than the barley Hor2-4 promoter in both transient and stable expression experiments. In particular, the mean value for the fungal xylanase activity driven by the GluB-1 promoter in the mature grains of transgenic barley was more than twice that with the Hor2-4 promoter. Expression of the xylanase transgene under these endosperm-specific promoters was not observed in the leaf, stem and root tissues. Accumulation of the fungal xylanase in the developing grains of transgenic barley followed the pattern of storage protein deposition. The xylanase was stably maintained in the grain during grain maturation and desiccation and post-harvest storage. These results indicate that the cereal grain expression system may provide an economic means for large scale production of feed enzymes in the future.     

Mapping of quantitative trait loci associated with protein expression variation in barley grains

Barley (Hordeum vulgare) is an important cereal crop grown for both the feed and malting industries. Hence, there is great interest to gain deeper insight into the determinants of grain nutritional quality in order to improve the assessment of new traits. Two-dimensional gel electrophoresis was employed for the characterization of the grain proteome of doubled-haploid introgression lines (IL) representing a wild barley genome (Hordeum spontaneum Hs213) within a modern cultivar background (H. vulgare cv. Brenda). Proteome maps were subjected to differential cluster analysis and revealed ILs with similar or different protein expression patterns compared to the Brenda parent. A total of 51 quantitative trait loci for protein expression (pQTL) were detected, and proteins underlying these pQTL were further examined by mass spectrometry. Identification was successful for 49 of the segregating spots and functional annotation of proteins revealed that most proteins are involved in metabolism and disease/defence-related processes. Among those, multigene families of glyceraldehyde-3-phosphate dehydrogenases, heat shock proteins, peroxidases, and serpins were identified. Overall, eight pQTL signals were discovered in two independently grown sets of plants. The mapped spots included protein disulfide isomerase, α-amylase inhibitor BDAI, NADP malic enzyme, adenosine kinase and peroxidase BP1. Specific marker information of proteins involved in developmental events and protein storage as well as in disease- and defence-related processes now allows for targeted breeding approaches to improve the grain quality in barley.     

Genetic analysis of grain and malt quality in an elite barley population

Quantitative trait loci (QTLs) associated with grain weight, grain width, kernel hardness and malting quality were mapped in a doubled haploid population derived from two elite Australian malting barley varieties, Navigator and Admiral. A total of 30 QTLs for grain weight, grain width and kernel hardness were identified in three environments, and 63 QTLs were identified for ten malting quality traits in two environments. Three malting quality traits, namely β-amylase, diastatic power and apparent attenuation limit, were mainly controlled by a QTL linked to the Bmy1 gene at the distal end of chromosome 4H encoding a β-amylase enzyme. Six other malting quality traits, namely α-amylase, soluble protein, Kolbach index, free amino-acid nitrogen, wort β-glucan and viscosity, had coincident QTL clustered on chromosomes 1HS, 4HS, 7HS and 7HL, which demonstrated the interdependence of these traits. There was a strong association between these malt quality QTL clusters on chromosomes 1HS and 7HL and the major QTL for kernel hardness, suggesting that the use of this trait to enable early selection for malting quality in breeding programs would be feasible. In contrast, the majority of QTLs for hot-water extract were not coincident with those identified for other malt quality traits, which suggested differences in the mechanism controlling this trait. Novel QTLs have been identified for kernel hardness on chromosomes 2HL and 7HL, hot-water extract on 7HL and wort β-glucan on 6HL, and the resulting markers may be useful for marker-assisted selection in breeding programs.     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.     

Divergence in properties of two closely related α-amylase inhibitors of barley

The only inhibitor of human salivary α-amylase identified so far in Hordeum has been isolated from barley cv. Bomi endosperm. This protein has the same N-terminal sequence (23 residues), molecular mass, and isoelectric point as one of the subunits of the barley tetrameric inhibitor previously characterized. However, enzymatic cleavage of both proteins with endoproteinase Glu-C revealed that they are products of different genes. The two isoforms have diverged in their aggregative and inhibitory properties. Thus, the subunit previously characterized forms, along with two other subunits, a tetramer active towards insect but not human salivary α-amylase, while the isoform reported here behaves as a homodimer effective against the human enzyme. These results are discussed in the context of the evolution of the cereal α-amylase inhibitor family.