A triticale tapetal non-specific lipid transfer protein (nsLTP) is translocated to the pollen cell wall.

KEY MESSAGE: A Triticeae type III non-specific lipid transfer protein (nsLTP) was shown for the first time to be translocated from the anther tapetum to the pollen cell wall. Two anther-expressed non-specific lipid transfer proteins (nsLTPs) were identified in triticale (× Triticosecale Wittmack). LTPc3a and LTPc3b contain a putative signal peptide sequence and eight cysteine residues in a C-Xn-C-Xn-CC-Xn-CXC-Xn-C-Xn-C pattern. These proteins belong to the type III class of nsLTPs which are expressed exclusively in the inflorescence of angiosperms. The level of LTPc3 transcript in the anther was highest at the tetrad and uninucleate microspore stages, and absent in mature pollen. In situ hybridization showed that LTPc3 was expressed in the tapetal layer of the developing triticale anther. The expression of the LTPc3 protein peaked at the uninucleate microspore stage, but was also found to be associated with the mature pollen. Accordingly, an LTPc3a::GFP translational fusion expressed in transgenic Brachypodium distachyon first showed activity in the tapetum, then in the anther locule, and later on the mature pollen grain. Altogether, these results represent the first detailed characterization of a Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation.

Investigating Triticeae anther gene promoter activity in transgenic Brachypodium distachyon.

MAIN CONCLUSION: In this report, we demonstrate that Brachypodium distachyon could serve as a relatively high throughput in planta functional assay system for Triticeae anther-specific gene promoters. There remains a vast gap in our knowledge of the promoter cis-acting elements responsible for the transcriptional regulation of Triticeae anther-specific genes. In an attempt to identify conserved cis-elements, 14 pollen-specific and 8 tapetum-specific Triticeae putative promoter sequences were analyzed using different promoter sequence analysis tools. Several cis-elements were found to be enriched in these sequences and their possible role in gene expression regulation in the anther is discussed. Despite the fact that potential cis-acting elements can be identified within putative promoter sequence datasets, determining whether particular promoter sequences can in fact direct proper tissue-specific and developmental gene expression still needs to be confirmed via functional assays preferably performed in closely related plants. Transgenic functional assays with Triticeae species remain challenging and Brachypodium distachyon may represent a suitable alternative. The promoters of the triticale pollen-specific genes group 3 pollen allergen (PAL3) and group 4 pollen allergen (PAL4), as well as the tapetum-specific genes chalcone synthase-like 1 (CHSL1), from wheat and cysteine-rich protein 1 (CRP1) from triticale were fused to the green fluorescent protein gene (GFP) and analyzed in transgenic Brachypodium. This report demonstrates that this model species could serve to accelerate the functional analysis of Triticeae anther-specific gene promoters.

Data on the epitope mapping of soybean A2 and A3 glycinin.

The data information provided in this article relate to our research article "Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts" (Saeed et al., 2016) [1]. Here we provide western blot detection of glycinin subunits by soy-sensitive human sera, ELISA screens with overlapping synthetic peptides (epitope mapping), and various database/server epitope searches.

Protein engineering of Saccharomyces cerevisiae transporter Pdr5p identifies key residues that impact Fusarium mycotoxin export and resistance to inhibition.

Cereal infection by the broad host range fungal pathogen Fusarium graminearum is a significant global agricultural and food safety issue due to the deposition of mycotoxins within infected grains. Methods to study the intracellular effects of mycotoxins often use the baker's yeast model system (Saccharomyces cerevisiae); however, this organism has an efficient drug export network known as the pleiotropic drug resistance (PDR) network, which consists of a family of multidrug exporters. This study describes the first study that has evaluated the potential involvement of all known or putative ATP-binding cassette (ABC) transporters from the PDR network in exporting the F. graminearum trichothecene mycotoxins deoxynivalenol (DON) and 15-acetyl-deoxynivalenol (15A-DON) from living yeast cells. We found that Pdr5p appears to be the only transporter from the PDR network capable of exporting these mycotoxins. We engineered mutants of Pdr5p at two sites previously identified as important in determining substrate specificity and inhibitor susceptibility. These results indicate that it is possible to alter inhibitor insensitivity while maintaining the ability of Pdr5p to export the mycotoxins DON and 15A-DON, which may enable the development of resistance strategies to generate more Fusarium-tolerant crop plants.

Using patient serum to epitope map soybean glycinins reveals common epitopes shared with many legumes and tree nuts.

Soybean consumption is increasing in many Western diets; however, recent reviews suggest that the prevalence of soy allergy can be as high as 0.5% for the general population and up to 13% for children. The immunoglobulin-E (IgE) binding of sera from six soy-sensitive adult human subjects to soybean proteins separated by 2D gel electrophoresis was studied. Synthetic peptide sets spanning the mature glycinin subunit A2 and A3 primary sequences were used to map the IgE-binding regions. Putative epitopes identified in this study were also localized on glycinin hexamer models using bioinformatics software. We identified linear IgE-binding epitopes of the major storage protein Gly m 6 by screening individual soy-sensitive patient sera. These epitopes were then further analysed by 3D in silico model localization and compared to other plant storage protein epitopes. Web-based software applications were also used to study the ability to accurately predict epitopes with mixed results. A total of nine putative IgE-binding epitopes were identified in the glycinin A3 (A3.1-A3.3) and A2 (A2.1-A2.6) subunits. Most patients' sera IgE bound to only one or two epitopes, except for one patient's serum which bound to four different A2 epitopes. Two epitopes (A3.2 and A2.4) overlapped with a previously identified epitope hot spot of 11S globulins from other plant species. Most epitopes were predicted to be exposed on the surface of the 3D model of the glycinin hexamer. Amino acid sequence alignments of soybean acidic glycinins and other plant globulins revealed one dominant epitope hot spot among the four reported hot spots. This study may be helpful for future development of soy allergy immunotherapy and diagnosis.

A molecular and proteomic investigation of proteins rapidly released from triticale pollen upon hydration.

Analysis of Triticale (×Triticosecale Wittmack cv. AC Alta) mature pollen proteins quickly released upon hydration was performed using two-dimensional gel electrophoresis followed by mass spectrometry. A total of 17 distinct protein families were identified and these included expansins, profilins, and various enzymes, many of which are pollen allergens. The corresponding genes were obtained and expression studies revealed that the majority of these genes were only expressed in developing anthers and pollen. Some genes including glucanase, glutathione peroxidase, glutaredoxin, and a profilin were found to be widely expressed in different reproductive and vegetative tissues. Group 11 pollen allergens, polygalacturonase, and actin depolymerizing factor were characterized for the first time in the Triticeae. This study represents a distinctive combination of proteomic and molecular analyses of the major cereal pollen proteins released upon hydration and therefore at the forefront of pollen-stigma interactions.

Proteomic profiling of two maize inbreds during early gibberella ear rot infection.

Fusarium graminearum is the causal agent of gibberella ear rot in maize ears, resulting in yield losses due to mouldy and mycotoxin-contaminated grain. This study represents a global proteomic approach to document the early infection by F. graminearum of two maize inbreds, B73 and CO441, which differ in disease susceptibility. Mock- and F. graminearum-treated developing kernels were sampled 48 h post-inoculation over three field seasons. Infected B73 kernels consistently contained higher concentrations of the mycotoxin deoxynivalenol than the kernels of the more tolerant inbred CO441. A total of 2067 maize proteins were identified in the iTRAQ analysis of extracted kernel proteins at a 99% confidence level. A subset of 878 proteins was identified in at least two biological replicates and exhibited statistically significantly altered expression between treatments and/or the two inbred lines of which 96 proteins exhibited changes in abundance >1.5-fold in at least one of the treatments. Many proteins associated with the defense response were more abundant after infection, including PR-10 (PR, pathogenesis-related), chitinases, xylanase inhibitors, proteinase inhibitors, and a class III peroxidase. Kernels of the tolerant inbred CO441 contained higher levels of these defense-related proteins than B73 kernels even after mock treatment, suggesting that these proteins may provide a basal defense against Fusarium infection in CO441.

Intracellular expression of a single domain antibody reduces cytotoxicity of 15-acetyldeoxynivalenol in yeast.

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V(H)H) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V(H)H immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V(H)H within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 mug.ml(-1). We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 mug.ml(-1). The proof of concept established within this eukaryotic system suggests that in planta V(H)H expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.

A chalcone synthase-like gene is highly expressed in the tapetum of both wheat (Triticum aestivum L.) and triticale (xTriticosecale Wittmack).

A novel anther-specific chalcone synthase-like gene, TaCHSL1, was isolated and characterized. The TaCHSL1 transcript was detected only within the tapetum during the "free" and early vacuolated microspore stages in both wheat and triticale. Sequence analysis indicated that the 41.8 kDa TaCHSL1 deduced protein belongs to a small distinct group of type III polyketide synthases that includes angiosperm and gymnosperm orthologs shown to be anther-specific. TaCHSL1 sequence characteristics and conservation, as well as its restricted expression pattern, point to a distinct and important biochemical role in developing anthers.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.

A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

Proteomic analyses of Fusarium graminearum grown under mycotoxin-inducing conditions.

Non-gel-based quantitative proteomics technology was used to profile protein expression differences when Fusarium graminearum was induced to produce trichothecenes in vitro. As F. graminearum synthesizes and secretes trichothecenes early in the cereal host invasion process, we hypothesized that proteins contributing to infection would also be induced under conditions favouring mycotoxin synthesis. Protein samples were extracted from three biological replicates of a time course study and subjected to iTRAQ (isobaric tags for relative and absolute quantification) analysis. Statistical analysis of a filtered dataset of 435 proteins revealed 130 F. graminearum proteins that exhibited significant changes in expression, of which 72 were upregulated relative to their level at the initial phase of the time course. There was good agreement between upregulated proteins identified by 2-D PAGE/MS/MS and iTRAQ. RT-PCR and northern hybridization confirmed that genes encoding proteins which were upregulated based on iTRAQ were also transcriptionally active under mycotoxin producing conditions. Numerous candidate pathogenicity proteins were identified using this technique. These will provide leads in the search for mechanisms and markers of host invasion and novel antifungal targets.

Use of phage display to select novel cystatins specific for Acanthoscelides obtectus cysteine proteinases.

Cysteine proteinases from larvae of the common bean weevil, Acanthoscelides obtectus (Coleoptera: Bruchidae), were isolated by ion exchange affinity chromatography on a CM-Cellulose column and used to select mutant cystatins from a library made with the filamentous M13 phage display system. The library contained variant cystatins derived from the nematode Onchocerca volvulus cystatin through mutagenesis of loop 1, which contains the QVVAG motif that is involved in binding to proteinases. After three rounds of selection, the activity of variant cystatins against papain and cysteine proteinases from A. obtectus was assayed by ELISA. Two different variant cystatins (presenting amino acids DVVSA and NTSSA at positions 65-69) bound to A. obtectus cysteine proteinases more tightly than to papain. In contrast, the wild type had similar affinity for A. obtectus proteinases and for papain. These two selected variants cystatins have greater specificity towards A. obtectus cysteine proteinases than the original sequence and could represent good candidate genes for the production of transgenic plants resistant to this insect pest.

Midgut proteases from Mamestra configurata (Lepidoptera: Noctuidae) larvae: characterization, cDNA cloning, and expressed sequence tag analysis.

The activities of digestive protease within the midgut of Mamestra configurata (bertha armyworm) larvae were examined using specific substrates and protease inhibitors. The bulk of the activity was associated with serine proteases comprising trypsin-, chymotrypsin-, and elastase-like enzymes. At least 10-15 serine protease isozymes were detected using one-dimension gelatin gel electrophoresis. Cysteine or aspartic protease activities were not present; however, amino- and carboxypeptidase activities were associated with the midgut extract. Midgut proteases were active in the pH range of 5.0-12.0 with peaks at pH 7.5 and 11.0. In general, the middle region of the midgut exhibited a higher pH (approximately 8.0) than either the posterior or anterior regions (approximately 7.3-7.7). Moulting larvae possessed a neutral gut pH that was 0.5-1.5 units below that of feeding larvae. Degenerate PCR and expressed sequence tag (EST)-based approaches were used to isolate 30 distinct serine protease encoding cDNAs from a midgut-specific cDNA library including 8 putative trypsins, 9 chymotrypsins, 1 elastase, and 12 whose potential activities could not be determined. cDNAs encoding three amino- and two carboxypeptidases were also identified. Larvae feeding upon artificial diet containing 0.2% soybean trypsin inhibitor experienced a significant delay in development.