François Eudes

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.

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.

Effect of Trichothecenes Produced by Fusarium graminearum during Fusarium Head Blight Development in Six Cereal Species

Fusarium head blight (FHB) is a complex cereal disease associated with trichothecene production; these mycotoxins are factors of aggressiveness in wheat. Six species (bread and durum wheat, triticale, rye, barley and oats) were submitted to point inoculations with two isogenic strains of Fusarium graminearum; a wild strain (Tri5 +) produced trichothecenes and the mutated strain (Tri5 −) did not. The trichothecene-producing strain was generally more aggressive than the non-producing strain, but this varied according to crop species. The difference in aggressiveness was less pronounced in rye, a very resistant species. High resistance levels were observed in oats due to the large spacing between florets. In six-row barley, despite the existence of a moderate Type II resistance, the fungus was often observed to move externally from one floret to another within the dense spike, without penetrating the rachis. Bread wheat had low resistance to the trichothecene-producing strain and good resistance to the non-producing strain. Triticale responded to the strains in a similar way but was somewhat more resistant to both: symptoms on the spikelets and rachis of the triticales were restricted to below the point of inoculation. Durum wheat was susceptible to the trichothecene-producing strain and only moderately resistant to the non-producing strain, which was able to cause serious damage only to this species. Our study confirmed that the role of trichothecenes in FHB pathogenesis differs among species. The failure of the trichothecene non-producing F. graminearum strain to spread within the inflorescence of wheat, triticale, rye and barley, and the significant reduction of spread in the durum wheat spike strongly suggested that trichothecenes are a major determinant of fungal spread and disease development in Triticeae.

A novel method to induce direct somatic embryogenesis, secondary embryogenesis and regeneration of fertile green cereal plants

A direct somatic embryogenesis and secondary embryogenesis protocol was developed for seven cereal species, thus providing a new vista for in vitro plant genetic transformation or propagation. This paper describes a novel process that has been successfully developed for efficient regeneration of a wide range of cereal species and genotypes. This tissue culture and regeneration system does not require formation of callus tissues and takes approximately 2 months to complete, shorter than any of the currently available systems requiring 3-4 months. Rapid induction of direct somatic embryogenesis in barley (Hordeum vulgare), common wheat (Triticum aestivum), durum wheat (T. durum) and derived amphiploids, wild wheat (T. monococcum and T. urartu), rye (Secale cereale) and oats (Avena sativa) was induced from excised immature scutellum on DSEM medium. Newly developed globular embryos were cultured on SEM medium for a second cycle of embryogenesis followed by germination (GEM medium) and regeneration of embryos into normally growing green and fertile plants. In vitro techniques to induce direct somatic embryogenesis, secondary embryogenesis and plant regeneration from these cereals require a specific sequence of defined media and controlled environments. The sequence and the timing of the media used, as well as their hormonal composition and balance are critical aspects of this process. The organic and mineral compositions of these media are not new but are important for supporting and sustaining rapid growth of the tissues.

Phytotoxicity of eight mycotoxins associated with the fusariosis of wheat spikelets.

The study of eight mycotoxins from Fusarium spp. at concentrations ranging from 0 to 4.3 mg/L for deoxynivalenol, 3-acetyldeoxynivalenol, nivalnol, and a culture filtrate, and from 0 to 14.3 mg/L for T2 toxin, HT2 toxin, diacetoxyscirpenol, and zearalenone, demonstrate that trichothecenes are phytotoxic. The inhibition of coleoptile elongation by each trichothecene is well described by a linear equation and these compounds show differences in phytotoxicity. Deoxynivalenol and 3-acetyldeoxynivalenol are 2.5 times more toxic than T2 toxin, 8 times more toxic than HT2 toxin, and 13 times more toxic than diacetoxyscirpénol. The culture filtrate, which contains deoxynivalenol and 3-acetyldeoxynivalenol, is the most toxic to the coleoptile. A mixture of four purified trichothecenes, deoxynivalenol, 3-acetyldeoxynivalenol, T2 toxin, and nivalénol, revealed differences in trichothecene susceptibility among 11 wheat cultivars. Moreover, reduction of coleoptile elongation of these cultivars was correlated with scabsusceptibility (r = 0.74; P = 0.0068).The study of eight mycotoxins from Fusarium spp. at concentrations ranging from 0 to 4.3 mg/L for deoxynivalenol, 3-acetyldeoxynivalenol, nivalnol, and a culture filtrate, and from 0 to 14.3 mg/L for T2 toxin, HT2 toxin, diacetoxyscirpenol, and zearalenone, demonstrate that trichothecenes are phytotoxic. The inhibition of coleoptile elongation by each trichothecene is well described by a linear equation and these compounds show differences in phytotoxicity. Deoxynivalenol and 3-acetyldeoxynivalenol are 2.5 times more toxic than T2 toxin, 8 times more toxic than HT2 toxin, and 13 times more toxic than diacetoxyscirpenol. The culture filtrate, which contains deoxynivalenol and 3-acetyldeoxynivalenol, is the most toxic to the coleoptile. A mixture of four purified trichothecenes, deoxynivalenol, 3-acetyldeoxynivalenol, T2 toxin, and nivalenol, revealed differences in trichothecene susceptibility among 11 wheat cultivars. Moreover, reduction of coleoptile elongation of these cultivars was correlated with scab...

Impact of trichothecenes on Fusarium head blight [Fusarium graminearum] development in spring wheat (Triticum aestivum)

>Fusarium head blight pathogens (Fusarium spp.) produce trichothecenes that have been demonstrated to play a role in the pathogenesis. To test the impact of trichothecenes on a broad range of genotypes, 18 spring wheat lines (Triticum aestivum) were inoculated with two Fusarium graminearum strains, the genetically modified GzT40 strain, which could not produce trichothecene, and the wild parental Gz3639 strain. During 3 weeks of observation, the two fungal strains showed extreme differences in aggressiveness in all but three of wheat genotypes tested. While the GzT40 mutant did not spread into the rachis, the wild-type strain quickly spread in the spike. This work confirms earlier findings that trichothecenes are a principal determinant of F. graminearum aggressiveness on most spring wheat cultivars. Therefore, trichothecenes may serve as a useful screening tool in programs breeding for resistance to Fusarium head blight of wheat.Key words: trichothecenes, deoxynivalenol, mycotoxins, Fusarium head blight, Fusarium graminearum, spring wheat, Triticum aestivum.

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, Tat2, 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 β‐glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat2‐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 Tat2‐mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat2–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 Tat2‐plasmid DNA (carrying Act‐1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat2–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 Tat2–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.

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.

The Stripe Rust Resistance Gene Yr10 Encodes an Evolutionary-Conserved and Unique CC–NBS–LRR Sequence in Wheat

The first seedling or all-stage resistance (R) R gene against stripe rust isolated from Moro wheat (Triticum aestivum L.) using a map-based cloning approach was identified as Yr10. Clone 4B of this gene encodes a highly evolutionary-conserved and unique CC-NBS-LRR sequence. Clone 4E, a homolog of Yr10, but lacking transcription start site (TSS) and putative TATA-box and CAAT-box, is likely a non-expressed pseudogene. Clones 4B and 4E are 84% identical and divergent in the intron and the LRR domain. Gene silencing and transgenesis were used in conjunction with inoculation with differentially avirulent and virulent stripe rust strains to demonstrate Yr10 functionality. The Yr10 CC-NBS-LRR sequence is unique among known CC-NBS-LRR R genes in wheat but highly conserved homologs (E = 0.0) were identified in Aegilops tauschii and other monocots including Hordeum vulgare and Brachypodium distachyon. Related sequences were also identified in genomic databases of maize, rice, and in sorghum. This is the first report of a CC-NBS-LRR resistance gene in plants with limited homologies in its native host, but with numerous homologous R genes in related monocots that are either host or non-hosts for stripe rust. These results represent a unique example of gene evolution and dispersion across species.

Development and assessment of DArT markers in triticale

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.