Functional and Key Gene Expression Analyses of Chicken Monocyte-derived Dendritic Cells with Recombinant Interleukin 4.

The central role of dendritic cells (DCs) as bridging innate and adaptive immunity leads to the expanding use of these cells in the poultry vaccine studies. The most effective way to produce enough DCs is monocyte transformation by combined induction of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). In this study full length of chicken IL-4 (cIL-4) cDNA was cloned, characterized and expressed in Escherichia coli. Subsequently, the expressed IL-4 was used to induce monocytes- derived DCs (MDDC). Typical features of DCs such as long membrane protrusions, apparently was dominant only four days after cytokine induction. Analyses of selected key genes' expression also confirmed that most of the monocytes shifted to DCs. The findings of the present study strongly suggest that the cloning and expression of cIL-4 in the bacterial host without any codon optimization or other modifications could produce mature MDDC in six to seven days.

Assessment of Ustilago maydis as a fungal model for root infection studies.

Ustilago maydis is a fungus infecting aerial parts of maize to form smutted galls. Due to its interest as a genetic tool in plant pathology, we evaluated its ability to penetrate into plant roots. The fungus can penetrate between epidermic root cells, forming inter and intracellular pseudohyphae. Root infection didn't provoke gall formation on the maize lines tested, and targeted PCR detection showed that U. maydis, unlike the other maize smut fungus Sporisorium reilianum, has a weak aptitude to grow from the roots up to the aerial part of maize. We also observed that U. maydis can infect Medicago truncatula hairy roots as an alternative host. This plant species is a model host to study root symbiosis, and this pathosystem can provide new insights on root-microbe interactions. Considering that U. maydis could be a soil fungus, we tested its responsiveness to GR24, a strigolactone analogue. Strigolactones are root exuded molecules which activate mitochondrial metabolism of arbuscular mycorrhizal (AM) fungi. Physiologic and molecular analysis revealed that GR24 also increases cell respiration of U. maydis. This result points out that strigolactones could have an incidence on several rhizospheric microbes. These data provide evidences that the biotrophic pathogen U. maydis has to be considered for studying root infection.

The life cycle of the smut fungus Moesziomyces penicillariae is adapted to the short-cycle of the host, Pennisetum glaucum.

Moesziomyces penicillariae (Brefield) Vànky is a basidiomycete fungus responsible for smut disease on pearl millet, an important staple food in the sub-Sahelian zone. We revisited the life cycle of this fungus. Unlike other Ustilaginales, mating of sporidia was never observed and monoclonal cultures of monokaryotic sporidia were infectious in the absence of mating with compatible partner. These data argued for an atypical monokaryotic diploid cell cycle of M. penicillariae, where teliospores only form solopathogenic sporidia. After inoculation of monoclonal solopathogenic strains on spikelets, the fungus infects the ovaries and induces the folding of the micropilar lips, as observed during early pollination steps. The infected embryo then becomes disorganized and the fungus invades peripheral ovary tissues before sporulating. We evaluated the systemic growth abilities of the fungus. After root inoculation, mycelium was observed around and inside the roots. As argued by transmission electron microscopy (TEM) observations and polymerase chain reaction (PCR) detection using specific primers for M. penicillariae, the fungus can grow from roots to the caulinar meristems. In spite of this systemic growth, no sori were formed on the varieties of pearl millet tested after root inoculation. All together, these data suggest that the reduced life cycle of M. penicillariae--i.e. dispersal of 'ready to infect' solopathogenic sporidia, floral infection--is an adaptation to the aetiology of this disease to short-cycle pearl millet varieties from the sub-Sahel.

Solopathogenic strain formation strongly differs among Ustilaginaceae species.

The pathogenicity of smut fungi is initiated by the fusion of two compatible saprotrophic yeasts that give rise to the formation of dikaryotic pathogenic hyphae. It has been described in the literature that complementation assays of auxotrophic yeasts of Ustilago maydis have allowed the isolation of diploid strains that are solopathogenic, i.e. pathogenic in the absence of mating. The occurrence of such strains from germinating teliospores was not investigated. We evaluated the ability of teliospores to generate solopathogenic strains in three species of smut fungi: Sporisorium reilianum f.sp. zeae, U. maydis and Moesziomyces penicillariae. Using an approach based on the stability of pseudohyphae of solopathogenic strains, we isolated the strain SRZS1 from teliospores of S. reilianum. Microscopic observations and analyses of mating-type alleles showed that SRZS1 is monokaryotic and diploid. Inoculation tests on maize plantlets indicated that SRZS1 is infectious. The same protocol was applied to polyteliosporal isolates from M. penicillariae, U. maydis and S. reilianum of diverse geographic origin. Surprisingly, all strains from teliospores of M. penicillariae were solopathogenic, whereas only few solopathogenic strains were obtained from the other two species. The possible incidence of solopathogenic strain production in the biology of these species is discussed.