Proteinaceous inhibitors of microbial xylanases

At the end of 1990s two structurally different proteinaceous inhibitors of xylanases were discovered in the grain of wheat (Triticum aestivum). They were named TAXI (T. aestivum xylanase inhibitor) and XIP (xylanase-inhibiting protein). Later it was shown that TAXI and XIP in wheat are present in several isoforms encoded by different genes. TAXI- and XIP-like inhibitors have also been found in other cereals-barley, rye, rice, maize, etc. All these proteins can specifically inhibit activity of fungal and bacterial xylanases belonging to families 10 and 11 of glycoside hydrolases, but they do not affect endogenous enzymes produced by plants. A common viewpoint is that the presence of proteinaceous inhibitors in cereals is a response of plants to pathogenic attack by microorganisms. A few years ago, an inhibitor of a third type was discovered in wheat. It was named TLXI (thaumatin-like xylanase inhibitor) because of its similarity to the thaumatin family of plant proteins. In this review, the occurrence of proteinaceous inhibitors of xylanases in different cereals, their specificity towards fungal and bacterial enzymes, as well as structural features responsible for enzyme sensitivity to various types of inhibitors are discussed.     

The xylanase inhibitor TAXI‐III counteracts the necrotic activity of a Fusarium graminearum xylanase in vitro and in durum wheat transgenic plants

The xylanase inhibitor TAXI‐III has been proven to delay Fusarium head blight (FHB) symptoms caused by Fusarium graminearum in transgenic durum wheat plants. To elucidate the molecular mechanism underlying the capacity of the TAXI‐III transgenic plants to limit FHB symptoms, we treated wheat tissues with the xylanase FGSG_03624, hitherto shown to induce cell death and hydrogen peroxide accumulation. Experiments performed on lemmas of flowering wheat spikes and wheat cell suspension cultures demonstrated that pre‐incubation of xylanase FGSG_03624 with TAXI‐III significantly decreased cell death. Most interestingly, a reduced cell death relative to control non‐transgenic plants was also obtained by treating, with the same xylanase, lemmas of TAXI‐III transgenic plants. Molecular modelling studies predicted an interaction between the TAXI‐III residue H395 and residues E122 and E214 belonging to the active site of xylanase FGSG_03624. These results provide, for the first time, clear indications in vitro and in planta that a xylanase inhibitor can prevent the necrotic activity of a xylanase, and suggest that the reduced FHB symptoms on transgenic TAXI‐III plants may be a result not only of the direct inhibition of xylanase activity secreted by the pathogen, but also of the capacity of TAXI‐III to avoid host cell death.     

Structural and functional characterization of TRI3 trichothecene 15‐O‐acetyltransferase from Fusarium sporotrichioides

Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13‐epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15‐O‐trichothecene acetyltransferase isolated from F. sporotrichioides and the “in vivo” characterization of Δtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15‐decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15‐decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3‐O‐trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.     

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.     

A real-time qPCR assay to quantify Fusarium graminearum biomass in wheat kernels

Aims: To develop a real‐time PCR assay to quantify Fusarium graminearum biomass in blighted wheat kernels. Methods and Results: Primers designed to amplify a gene in the trichothecene biosynthetic cluster (TRI6) were evaluated for sensitivity and specificity. Primer pair Tri6_10F/Tri6_4R specifically and consistently amplified a 245‐bp DNA fragment from F. graminearum. A workflow was developed and validated to extract DNA from infested grain. The assay detected as little as 10 μg of F. graminearum mycelia in 1 g of ground wheat grain with a high correlation between fungal biomass and cycle threshold values (R² = 0·9912; P = 0·004). In field‐inoculated grain, qPCR measurements of biomass correlated closely with deoxynivalenol levels (R = 0·82, P < 0·0001) and two visual techniques to assess grain quality (R = 0·88, P < 0·0001 and R = 0·81, P < 0·0001). Conclusions: The qPCR assay provided accurate and precise assessments of the amount of F. graminearum biomass in blighted wheat kernels. This method represents a technical advance over other approaches to quantify kernel colonization and real‐time PCR detection methodologies for F. graminearum that do not correlate quantification of fungal genomic DNA to biomass. Significance and Impact of the Study: Quantifying F. graminearum biomass, especially low levels of growth associated with kernels that are visually asymptomatic, represents a new approach to screen for resistance to kernel infection, an understudied yet potentially important avenue to reduce the impact of head blight.     

Chloroplast ultrastructure regeneration with protection of photosystem II is responsible for the functional ‘stay‐green’ trait in wheat

CN17 is a functional stay‐green wheat variety that exhibits delayed leaf senescence and enhanced photosynthetic competence. To better understand these valuable traits, levels of chlorophyll a and b, soluble proteins, unsaturated fatty acids, and other components of CN17 were assayed. In addition, chloroplast ultrastructure, chloroplast number, and differences in gene expression between CN17 and a control variety, MY11, were examined. By 21 d post‐anthesis (DPA), CN17 leaves exhibited a significantly higher maximal photochemical efficiency for photosystem II (PSII) (F _v /F _m ) and a significantly higher efficiency of excitation capture by open PSII reaction centres (F_v′/F_m′). In addition, chlorophyll degradation in CN17 was delayed by approximately 14 d, and was not blocked as observed in cosmetic stay‐green phenotypes. The soluble protein content (Ps) of CN17 was higher than MY11 at all timepoints assayed, and the ratio of unsaturated to saturated fatty acids was significantly higher. CN17 also exhibited isolated granal lamellae associated with vesicles and diminished peroxidation, and between 35 and 42 DPA, a sharp decrease in chloroplast number was detected. Taken together, these results strongly support the hypothesis that chloroplast ultrastructure regeneration is responsible for the functional stay‐green trait of CN17, and gene expression data provide insight into the mechanistic details.     

Time‐resolved dissection of the molecular crosstalk driving Fusarium head blight in wheat provides new insights into host susceptibility determinism

Fungal plant diseases are controlled by a complex molecular dialogue that involves pathogen effectors able to manipulate plant susceptibility factors at the earliest stages of the interaction. By probing the wheat–Fusarium graminearum pathosystem, we profiled the coregulations of the fungal and plant proteins shaping the molecular responses of a 96‐hr‐long infection's dynamics. Although no symptoms were yet detectable, fungal biomass swiftly increased along with an extremely diverse set of secreted proteins and candidate effectors supposed to target key plant organelles. Some showed to be early accumulated during the interaction or already present in spores, otherwise stored in germinating spores and detectable in an in vitro F. graminearum exudate. Wheat responses were swiftly set up and were evidenced before any visible symptom. Significant wheat protein abundance changes co‐occurred along with the accumulation of putative secreted fungal proteins and predicted effectors. Regulated wheat proteins were closely connected to basal cellular processes occurring during spikelet ontogeny, and particular coregulation patterns were evidenced between chloroplast proteins and fungal proteins harbouring a predicted chloroplast transit peptide. The described plant and fungal coordinated responses provide a resourceful set of data and expand our understanding of the wheat–F. graminearum interaction.     

谷物中蛋白类木聚糖酶抑制剂研究进展

近年来研究发现谷物中存在能抑制木聚糖酶活性的蛋白质,降低了木聚糖酶在动物生产中的应用效果。自第一种木聚糖酶抑制剂蛋白首先在小麦中被发现以来,随后发现的木聚糖酶抑制剂分别属于三种不同的类型,即TAXI型、XIP型和TLXI型。综述了三种不同类型木聚糖酶抑制剂的研究概况,为研究木聚糖酶抑制剂的作用机理,及进一步开发具有良好抗逆性的木聚糖酶提供理论基础。     

Biochemical and structural characterization of TLXI,the Triticum aestivum L. thaumatin-like xylanase inhibitor

Thaumatin-like xylanase inhibitors (TLXI) are recently discovered wheat proteins. They belong to the family of the thaumatin-like proteins and inhibit glycoside hydrolase family 11 endoxylanases commonly used in different cereal based (bio)technological processes. We here report on the biochemical characterisation of TLXI. Its inhibition activity is temperature- and pH-dependent and shows a maximum at approximately 40°C and pH 5.0. The TLXI structure model, generated with the crystal structure of thaumatin as template, shows the occurrence of five disulfide bridges and three β-sheets. Much as in the structures of other short-chain thaumatin-like proteins, no α-helix is present. The circular dichroism spectrum of TLXI confirms the absence of α-helices and the presence of antiparallel β-sheets. All ten cysteine residues in TLXI are involved in disulfide bridges. TLXI is stable for at least 120 min between pH 1–12 and for at least 2 hours at 100°C, making it much more stable than the other two xylanase inhibitors from wheat, i.e. Triticum aestivum xylanase inhibitor (TAXI) and xylanase inhibitor protein (XIP). This high stability can probably be ascribed to the high number of disulfide bridges, much as seen for other thaumatin-like proteins.     

An in vitro method for the analysis of infection‐related morphogenesis in Fusarium graminearum

Fusarium graminearum is a significant pathogen of many cereal crops. With its genetic tractability, ease of culture, genome sequence availability and economic significance, F. graminearum has become the subject of intensive molecular research. Although molecular tools have been developed to enhance research into virulence determinants of F. graminearum, simple assays for infection‐related development are lacking. As such, the objective of this study was to develop an in vitro protocol for the analysis of infection‐related morphogenesis in F. graminearum. We demonstrate that two morphologically distinct hyphal structures are produced by F. graminearum during the invasion of detached wheat glumes: subcuticular hyphae and bulbous infection hyphae. Specialized wheat epidermal cells (papillae) appear to act as sites of invasion by F. graminearum on the adaxial side of detached wheat glumes. In addition, the development of bulbous infection hyphae is dependent on the pathogenicity mitogen‐activated protein kinase Gpmk1, further supporting the infection‐related nature of these structures. This relatively simple assay will contribute to the tractability of the F. graminearum system and help to uncover molecular requirements for infection‐related development.     

Capping proteins regulate fungal development,DON-toxisome formation and virulence in Fusarium graminearum

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.     

Transgenic rice plants expressing trichothecene 3-<Emphasis Type="Italic">O</Emphasis>-acetyltransferase show resistance to the <Emphasis Type="Italic">Fusarium</Emphasis> phytotoxin deoxynivalenol

Fusarium head blight (FHB) is a devastating disease of small grain cereal crops caused by the necrotrophic pathogen Fusarium graminearum and Fusarium culmorum. These fungi produce the trichothecene mycotoxin deoxynivalenol (DON) and its derivatives, which enhance the disease development during their interactions with host plants. For the self-protection, the trichothecene producer Fusarium species have Tri101 encoding trichothecene 3-O-acetyltransferase. Although transgenic expression of Tri101 significantly reduced inhibitory action of DON on tobacco plants, there are several conflicting observations regarding the phytotoxicity of 3-acetyldeoxynivalenol (3-ADON) to cereal plants; 3-ADON was reported to be highly phytotoxic to wheat at low concentrations. To examine whether cereal plants show sufficient resistance to 3-ADON, we generated transgenic rice plants with stable expression and inheritance of Tri101. While root growth of wild-type rice plants was severely inhibited by DON in the medium, this fungal toxin was not phytotoxic to the transgenic lines that showed trichothecene 3-O-acetylation activity. This is the first report demonstrating the DON acetylase activity and DON-resistant phenotype of cereal plants expressing the fungal gene. S. Ohsato and T. Ochiai-Fukuda should be considered as joint first authors.     

Effects of the infection of toxigenic fungi and an antagonistic Streptomyces strain on wheat spikes

The objective of the present study was to determine the effect on infection of wheat spikes by toxigenic fungi (Aspergillus parasiticus NRRL 2999, Fusarium tricinctum NRRL 3299, Fusarium graminearum CEREMIC 136/92) and a strain of Streptomyces sp. that is antagonistic to the above-mentioned fungi. Wheat grains (variety GRANERO INTA) were sown in 8 pots containing natural soil and kept in a greenhouse chamber. In the period of the early anthesis the wheat spikes were inoculated with conidial suspensions of each of the fungi in the presence or absence of Streptomyces. Each pot was assigned a different treatment. After an incubation of 100 days and when the wheat plants had attained maturity, the spikes were separated and the following items were determined: (a) number of grains obtained with each treatment, (b) weight of the grains, (c) average weight of the grains/treatment, (d) average number and weight of the grains/spike, and (e) invasion of the caryopses by the microorganisms determined by the analysis of the caryopses in seriate histological sections.There was a significant decrease (p<0.01) in the average weight of the caryopses and in the weight and number of grains/spike in the presence F. graminearum. The wheat grains were invaded by of F. graminearum and A. parasiticus, an effect which was partially attenuated by the presence of antagonist Streptomyces sp. Nevertheless, the effect was not strong enough to prevent the degenerative consequences on the size and weight of the grains produced by F. graminearum.     

FgFim,a key protein regulating resistance to the fungicide JS399‐19, asexual and sexual development,stress responses and virulence in Fusarium graminearum

Fimbrin is an actin‐bundling protein found in intestinal microvilli, hair cell stereocilia and fibroblast filopodia. Its homologue Sac6p has been shown to play a critical role in endocytosis and diverse cellular processes in Saccharomyces cerevisiae. FgFim from the wheat scab pathogenic fungus Fusarium graminearum strain Y2021A, which is highly resistant to the fungicide JS399‐19, was identified by screening a mutant library generated by HPH‐HSV‐tk cassette‐mediated integration. The functions of FgFim were evaluated by constructing a deletion mutant of FgFim, designated ΔFgFim‐15. The deletion mutant exhibited a reduced rate of mycelial growth, reduced conidiation, delayed conidium germination, irregularly shaped hyphae, a lack of sexual reproduction on autoclaved wheat kernels and a dramatic decrease in resistance to JS399‐19. ΔFgFim‐15 also exhibited increased sensitivity to diverse metal cations, to agents that induce osmotic stress and oxidative stress, and to agents that damage the cell membrane and cell wall. Pathogenicity assays showed that the virulence of the FgFim deletion mutant on flowering wheat heads was impaired, which was consistent with its reduced production of the toxin deoxynivalenol in host tissue. All of these defects were restored by genetic complementation of the mutant with the parental FgFim gene. Quantitative real‐time polymerase chain reaction (PCR) assays showed that the basal expression of three Cyp51 genes, which encode sterol 14α‐demethylase, was significantly lower in the mutant than in the parental strain. The results of this study indicate that FgFim plays a critical role in the regulation of resistance to JS399‐19 and in various cellular processes in F. graminearum.     

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.     

Metabolic pathways of the wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm amyloplast revealed by proteomics

Background  

By definition, amyloplasts are plastids specialized for starch production. However, a proteomic study of amyloplasts isolated from wheat (Triticum aestivum Butte 86) endosperm at 10 days after anthesis (DPA) detected enzymes from many other metabolic and biosynthetic pathways. To better understand the role of amyloplasts in food production, the data from that study were evaluated in detail and an amyloplast metabolic map was outlined.     

Arbuscular mycorrhiza influences carbon‐use efficiency and grain yield of wheat grown under pre‐ and post‐anthesis salinity stress

• Soil salinity severely affects and constrains crop production worldwide. Salinity causes osmotic and ionic stress, inhibiting gas exchange and photosynthesis, ultimately impairing plant growth and development. Arbuscular mycorrhiza (AM) have been shown to maintain light and carbon use efficiency under stress, possibly providing a tool to improve salinity tolerance of the host plants. Thus, it was hypothesized that AM will contribute to improved growth and yield under stress conditions.
• Wheat plants (Triticum aestivum L.) were grown with (AMF+) or without (AMF?) arbuscular mycorrhizal fungi (AMF) inoculation. Plants were subjected to salinity stress (200 mm NaCl) either at pre‐ or post‐anthesis or at both stages. Growth and yield components, leaf chlorophyll content as well as gas exchange parameters and AMF colonization were analysed.
• AM plants exhibited a higher rate of net photosynthesis and stomatal conductance and lower intrinsic water use efficiency. Furthermore, AM wheat plants subjected to salinity stress at both pre‐anthesis and post‐anthesis maintained higher grain yield than non‐AM salinity‐stressed plants.
• These results suggest that AMF inoculation mitigates the negative effects of salinity stress by influencing carbon use efficiency and maintaining higher grain yield under stress.