Genome editing in wheat microspores and haploid embryos mediated by delivery of ZFN proteins and cell-penetrating peptide complexes.

Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value-added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell-penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN-treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP.

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.

Trichostatin A increases embryo and green plant regeneration in wheat.

KEY MESSAGE: Chemical agents such as trichostatin A (TSA) can assist in optimization of doubled haploidy for rapid improvements in wheat germplasm and addressing recalcitrance issues in cell culture responses. In wheat, plant regeneration through microspore culture is an integral part of doubled haploid (DH) production. However, low response to tissue culture and genotype specificity are two major constraints in the broad deployment of this breeding tool. Recently, the structure of chromatin was shown to be linked with cell transitions during tissue culture. Specifically, repression of genes that are required for cell morphogenesis, through acetylation of histones, may play an important role in this process. Reduction of histone acetylation by chemical inhibition may increase tissue culture efficiency. Here, the role of trichostatin A (TSA) in inducing microspore-derived embryos was investigated in wheat. The optimal dose of TSA was determined for wheat cultivars and subsequently validated in F1 hybrids. A significant increase in the efficiency of DH production was observed in both cultivated varieties and F1 hybrids. Thus, the inclusion of TSA in DH protocols for wheat breeding programs is advocated.

A novel method of transgene delivery into triticale plants using the Agrobacterium transferred DNA-derived nano-complex.

Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particle-mediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.

Molecular markers as a complementary tool in risk assessments: quantifying interspecific gene flow from triticale to spring wheat and durum wheat.

Triticale is being considered as a bioindustrial crop in Canada using genetic modification. Because related spring wheat (Triticum aestivum) and durum wheat (T. durum) may exhibit synchronous flowering and grow in proximity, determination of interspecific gene flow when triticale is the pollen donor is necessary to evaluate potential risk. Pollen-mediated gene flow risk assessments generally rely on phenotypic markers to detect hybridization but DNA markers could be powerful and less ambiguous in quantifying rare interspecific gene flow. Six cultivars representing four species [spring wheat, durum wheat, triticale and rye (Secale cereale)] were screened with 235 spring wheat and 27 rye SSR markers to evaluate transferability and polymorphism. Fifty-five polymorphic markers were used in conjunction with morphological characterization to quantify interspecific gene flow from a blue aleurone (BA) triticale line to two spring wheat cultivars (AC Barrie and AC Crystal) and one durum wheat cultivar (AC Avonlea). Approximately 1.9 Million seeds from small plot experiments were visually screened in comparison with known hybrid seed. In total 2031 putative hybrids were identified and 448 germinated. Morphological analysis of putative hybrid plants identified five hybrids while molecular analysis identified 11 hybrids and two were common to both. Combined, 14 hybrids were confirmed: 10 spring wheat × triticale (0.0008 % of harvested seed): seven AC Barrie × BA triticale (0.001 %) and three AC Crystal × BA triticale (0.0005 %); and four durum wheat × triticale (0.0006 %). The occurrence of rare hybrids does not present a substantial risk to the development of GM triticale.

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale.

KEY MESSAGE: Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production. Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F1 hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.

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.

Evaluation of Genomic Prediction for Fusarium Head Blight Resistance with a Multi-Parental Population.

Fusarium head blight (FHB) resistance is quantitatively inherited, controlled by multiple minor effect genes, and highly affected by the interaction of genotype and environment. This makes genomic selection (GS) that uses genome-wide molecular marker data to predict the genetic breeding value as a promising approach to select superior lines with better resistance. However, various factors can affect accuracies of GS and better understanding how these factors affect GS accuracies could ensure the success of applying GS to improve FHB resistance in wheat. In this study, we performed a comprehensive evaluation of factors that affect GS accuracies with a multi-parental population designed for FHB resistance. We found larger sample sizes could get better accuracies. Training population designed by CDmean based optimization algorithms significantly increased accuracies than random sampling approach, while mean of predictor error variance (PEVmean) had the poorest performance. Different genomic selection models performed similarly for accuracies. Including prior known large effect quantitative trait loci (QTL) as fixed effect into the GS model considerably improved the predictability. Multi-traits models had almost no effects, while the multi-environment model outperformed the single environment model for prediction across different environments. By comparing within and across family prediction, better accuracies were obtained with the training population more closely related to the testing population. However, achieving good accuracies for GS prediction across populations is still a challenging issue for GS application.

Cell-penetrating peptides: Nanocarrier for macromolecule delivery in living cells.

Novel classes and applications of cell-penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP-mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP-macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP-cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.

Genetic Characterization of Multiple Components Contributing to Fusarium Head Blight Resistance of FL62R1, a Canadian Bread Wheat Developed Using Systemic Breeding.

Fusarium head blight (FHB) is a devastating fungal disease of small-grain cereals that results in severe yield and quality losses. FHB resistance is controlled by resistance components including incidence, field severity, visual rating index, Fusarium damaged kernels (FDKs), and the accumulation of the mycotoxin deoxynivalenol (DON). Resistance conferred by each of these components is partial and must be combined to achieve resistance sufficient to protect wheat from yield losses. In this study, two biparental mapping populations were analyzed in Canadian FHB nurseries and quantitative trait loci (QTL) mapped for the traits listed above. Nine genomic loci, on 2AS, 2BS, 3BS, 4AS, 4AL, 4BS, 5AS, 5AL, and 5BL, were enriched for the majority of the QTL controlling FHB resistance. The previously validated FHB resistance QTL on 3BS and 5AS affected resistance to severity, FDK, and DON in these populations. The remaining seven genomic loci colocalize with flowering time and/or plant height QTL. The QTL on 4B was a major contributor to all field resistance traits and plant height in the field. QTL on 4AL showed contrasting effects for FHB resistance between Eastern and Western Canada, indicating a local adapted resistance to FHB. In addition, we also found that the 2AS QTL contributed a major effect for DON, and the 2BS for FDK, while the 5AL conferred mainly effect for both FDK/DON. Results presented here provide insight into the genetic architecture underlying these resistant components and insight into how FHB resistance in wheat is controlled by a complex network of interactions between genes controlling flowering time, plant height, local adaption, and FHB resistance components.

Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores.

Microspore culture is contributing significantly in the field of plant breeding for crop improvement in general and cereals, in particular. In the present study, we investigated the uptake of fluorescently labeled cell-penetrating peptides (CPP; Tat, Tat(2), M-Tat, peptide vascular endothelial-cadherin, transportan) in the freshly isolated triticale microspores (mid-late uninucleate stage). We demonstrated that Tat (RKKRRQRRR) and Tat(2) (RKKRRQRRRRKKRRQRRR) are able to efficiently transduce GUS enzyme (272 kDa) in its functional form in 5 and 14% of the microspores, respectively, in a noncovalent manner. Pep-1, a synthetic CPP, was able to transduce GUS enzyme in its active form in 31% of the microspores. The effect of various endocytic and macropinocytic inhibitors on Tat(2)-mediated GUS enzyme delivery was studied and revealed a preferred micropinocytosis entry. DNase I protection assay and confocal laser microscopy was carried out to recommend a ratio of 4:1 Tat(2)-linear plasmid DNA (pActGUS) in complex preparation for microspore transfection. We further show that Tat(2) can successfully deliver GUS gene in near to 2% triticale microspores. The negative control mutated Tat (M-Tat: AKKRRQRRR) failed to transducer the GUS protein and transfect the GUS gene in microspore nucleus. The ability of CPPs to deliver macromolecules (protein as well as linear plasmid DNA) noncovalently has been demonstrated in triticale isolated microspores. It further confirms potential applications of CPPs in developing simple, time saving, cost effective plant genetic engineering technologies.

Trichothecenes in cereal grains.

Trichothecenes are sesquiterpenoid mycotoxins associated with fusarium head blight (FHB) of cereals, with worldwide economic and health impacts. While various management strategies have been proposed to reduce the mycotoxin risk, breeding towards FHB-resistance appears to be the most effective means to manage the disease, and reduce trichothecene contamination of cereal-based food products. This review provides a brief summary of the trichothecene synthesis in Fusarium species, their toxicity in plants and humans, followed by the current methods of screening and breeding for resistance to FHB and trichothecene accumulation.

Translocation and nuclear accumulation of monomer and dimer of HIV-1 Tat basic domain in triticale mesophyll protoplasts.

Cellular internalization of cell-penetrating peptide HIV-1 Tat basic domain (RKKRRQRRR) was studied in Triticale cv AC Alta mesophyll protoplasts. Fluorescently labeled monomer (Tat) and dimer (Tat(2)) of Tat basic domain efficiently translocated through the plasma membrane of mesophyll protoplast and showed distinct nuclear accumulation within 10 min of incubation. Substitution of first arginine residue with alanine in Tat basic domain (M-Tat) severely reduced cellular uptake of the peptide (3.8 times less than Tat). Tat(2) showed greater cellular internalization than Tat (1.6 times higher). However, characteristics of cellular uptake remained same for Tat and Tat(2). Cellular internalization of Tat and Tat(2) was concentration dependent and non-saturable whereas no significant change in cellular uptake was observed even at higher concentrations of M-Tat. Low temperature (4 degrees C) remarkably increased cellular internalization of Tat as well as Tat(2) but M-Tat showed no enhanced uptake. Viability test showed that peptide treatment had no cytotoxic effect on protoplasts further indicating involvement of a common mechanism of peptide uptake at all the temperatures. Endocytic inhibitors nocodazole (10 muM), chloroquine (100 muM) and sodium azide (5 mM) did not show any significant inhibitory effect on cellular internalization of either Tat or Tat(2). These results along with stimulated cellular uptake at low temperature indicate that Tat peptide is internalized in the plant protoplasts in a non-endocytic and energy-independent manner. Competition experiments showed that non-labeled peptide did not inhibit or alter nuclear accumulation of fluorescent Tat or Tat(2) suggesting active transport to the nucleus was not involved. Studies in mesophyll protoplasts show that internalization pattern of Tat peptide is apparently similar to that observed in mammalian cell lines.

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos.

The uptake of five fluorescein labeled cell-penetrating peptides (Tat, Tat(2), mutated-Tat, peptide vascular endothelial-cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell-penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell-penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell-penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2-fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated-Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active beta-glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat(2)-mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat(2)-mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat(2)-GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell-penetrating peptide-cargo uptake in wheat immature embryos. We also studied Tat(2)-plasmid DNA (carrying Act-1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat(2)-plasmid DNA complex showed 3.3-fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine 2000 to the Tat(2)-plasmid DNA complex resulted in 1.5-fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell-penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.

High Density Single Nucleotide Polymorphism (SNP) Mapping and Quantitative Trait Loci (QTL) Analysis in a Biparental Spring Triticale Population Localized Major and Minor Effect Fusarium Head Blight Resistance and Associated Traits QTL.

Triticale (xTriticosecale Wittmack) is an important feed crop which suffers severe yield, grade and end-use quality losses due to Fusarium head blight (FHB). Development of resistant triticale cultivars is hindered by lack of effective genetic resistance sources. To dissect FHB resistance, a doubled haploid spring triticale population produced from the cross TMP16315/AC Ultima using a microspore culture method, was phenotyped for FHB incidence, severity, visual rating index (VRI), deoxynivalenol (DON) and some associated traits (ergot, grain protein content, test weight, yield, plant height and lodging) followed by single nucleotide polymorphism (SNP) genotyping. A high-density map consisting of 5274 SNPs, mapped on all 21 chromosomes with a map density of 0.48 cM/SNP, was constructed. Together, 17 major quantitative trait loci were identified for FHB on chromosomes 1A, 2B, 3A, 4A, 4R, 5A, 5R and 6B; two of incidence loci (on 2B and 5R) also co-located with loci for severity and VRI, and two other loci of VRI (on 1A and 4R) with DON accumulation. Major and minor loci were also identified for all other traits in addition to many epistasis loci. This study provides new insight into the genetic basis of FHB resistance and their association with other traits in triticale.

Adsorption of cell-penetrating peptide Tat2 and polycation luviquat FC-370 to triticale microspore exine.

Microspores are the pre-gametophyte stage of pollen, and have proven to be a successful tissue culture material for the production of doubled haploid plants. Microspore culture has also been used as a platform for the production of transgenic plants. The use of cell-penetrating peptides (CPPs) as transfection agents in microspores has been previously demonstrated, but at low efficiencies. Here, the pH dependent adsorption of the cationic CPP Tat2 to the sporopollenin surface of the microspore (the exine) has been explored using electrophoretic light scattering (ELS). Furthermore the adsorption of a commercially available polycationic polymer; Luviquat FC-370 (polyquaternium D-16, PQ-D16) was similarly measured using ELS. It was found that a suspension media with a pH less than 7.0 showed an approximately ten fold decrease in the amount of Tat2 that was required before apparent surface neutralization. This data suggests that a change in the surface chemistry of the microspore occurs in acidic pH conditions, that modulates the binding affinity of the CPP Tat2 in a non-trivially complex manner.

Tetrabutylphosphonium Bromide Reduces Size and Polydispersity Index of Tat2:siRNA Nano-Complexes for Triticale RNAi.

Cell-penetrating peptides (CPPs) are short 8-30 amino-acid oligopeptides that act as effective transducers of macromolecular cargo, particularly nucleic acids. They have been implemented in delivering dsDNA, ssDNA, and dsRNA into animal and plant cells. CPPs and nucleic acids form nano-complexes that are often 100-300 nm in size but still effectively transit the cell membrane of animal cells, but are less effective with plant cells due to the plant cell wall. To overcome this obstacle, nano-complexes of the CPP Tat2 and various lengths of nucleic acid (21-mer siRNA duplex (dsRNA) to ~5.5 kb circular plasmid) were evaluated for size using dynamic light scattering (DLS), under conditions of increasing ionic strength (Ic) and addition of phase transfer catalyst salts (tetrabutylammonium bromide-TBAB and tetrabutylphosphonium bromide-TBPB) and sugars (maltose-mannitol solution). It was found that the combination of 21-mer siRNA:Tat2 complexes with TBPB produced small 10-20 nm diameter nano-complexes with a polydispersity index (PDI) of ~0.1. Furthermore, it was found that for each length of nucleic acid that a linear mathematical relationship existed between the theoretical volume of the nano-complex and the nucleic acid length. Next, nano-complex formulation was tested for its ability to carry small interfering RNA molecules into plant cells and to trigger silencing of phytoene desaturase (PDS) in Triticale leaves. RT-qPCR showed 75% suppression of PDS, demonstrating that TBPB acts as an adjuvant in effecting the entry and efficacy of siRNA in young Triticale plants.

Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores.

Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity.

Solvent and Water Mediated Structural Variations in Deoxynivalenol and Their Potential Implications on the Disruption of Ribosomal Function.

Fusarium head blight (FHB) is a disease of cereal crops caused by trichothecene producing Fusarium species. Trichothecenes, macrocylicic fungal metabolites composed of three fused rings (A-C) with one epoxide functionality, are a class of mycotoxins known to inhibit protein synthesis in eukaryotic ribosomes. These toxins accumulate in the kernels of infected plants rendering them unsuitable for human and animal consumption. Among the four classes of trichothecenes (A-D) A and B are associated with FHB, where the type B trichothecene deoxynivalenol (DON) is most relevant. While it is known that these toxins inhibit protein synthesis by disrupting peptidyl transferase activity, the exact mechanism of this inhibition is poorly understood. The three-dimensional structures and H-bonding behavior of DON were evaluated using one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy techniques. Comparisons of the NMR structure presented here with the recently reported crystal structure of DON bound in the yeast ribosome reveal insights into the possible toxicity mechanism of this compound. The work described herein identifies a water binding pocket in the core structure of DON, where the 3OH plays an important role in this interaction. These results provide preliminary insights into how substitution at C3 reduces trichothecene toxicity. Further investigations along these lines will provide opportunities to develop trichothecene remediation strategies based on the disruption of water binding interactions with 3OH.