Agriculture and Agri-Food Canada

About: Agriculture and Agri-Food Canada is a facility organization based out in Ottawa, Ontario, Canada. It is known for research contribution in the topics: Population & Soil water. The organization has 10921 authors who have published 21332 publications receiving 748193 citations. The organization is also known as: Department of Agriculture and Agri-Food.

Laboratory techniques used for entomopathogenic fungi: Hypocreales

Abstract: Entomopathogenic fungi in the Order Hypocreales are important pathogens of pest and beneficial insects. Entomopathogenic Hypocreales have one of the widest spectra of host ranges among entomopathogens and, as such, many have been commercialized as biopesticides. Traditionally, classical microbiological methods have been used to isolate and identify hypocrealean fungi. While classical methods are still heavily utilized, the application of molecular methods, alone or in combination with classical methods, have provided valuable insights into the biology of entomopathogenic Hypocreales. In this chapter, we provide an overview of both classical and molecular techniques used to study entomopathogenic hypocrealean fungi. Due to the diversity of entomopathogenic fungi and the very wide range of hosts that they infect, we provide information in the form of generalizations as well as via specific examples. In many cases, readers will need to adapt these techniques to their specific requirements.

Turnip Mosaic Virus RNA Replication Complex Vesicles Are Mobile, Align with Microfilaments, and Are Each Derived from a Single Viral Genome

Abstract: Nicotiana benthamiana plants were agroinoculated with an infectious cDNA clone of Turnip mosaic virus (TuMV) that was engineered to express a fluorescent protein (green fluorescent protein [GFP] or mCherry) fused to the viral 6K2 protein known to induce vesicle formation. Cytoplasmic fluorescent discrete protein structures were observed in infected cells, corresponding to the vesicles containing the viral RNA replication complex. The vesicles were motile and aligned with microfilaments. Intracellular movement of the vesicles was inhibited when cells were infiltrated with latrunculin B, an inhibitor of microfilament polymerization. It was also observed that viral accumulation in the presence of this drug was reduced. These data indicate that microfilaments are used for vesicle movement and are necessary for virus production. Biogenesis of the vesicles was further investigated by infecting cells with two recombinant TuMV strains: one expressed 6K2GFP and the other expressed 6K2mCherry. Green- and red-only vesicles were observed within the same cell, suggesting that each vesicle originated from a single viral genome. There were also vesicles that exhibited sectors of green, red, or yellow fluorescence, an indication that fusion among individual vesicles is possible. Protoplasts derived from TuMV-infected N. benthamiana leaves were isolated. Using immunofluorescence staining and confocal microscopy, viral RNA synthesis sites were visualized as punctate structures distributed throughout the cytoplasm. The viral proteins VPg-Pro, RNA-dependent RNA polymerase, and cytoplasmic inclusion protein (helicase) and host translation factors were found to be associated with these structures. A single-genome origin and presence of protein synthetic machinery components suggest that translation of viral RNA is taking place within the vesicle.

PHYTOCHROME INTERACTING FACTOR3 Associates with the Histone Deacetylase HDA15 in Repression of Chlorophyll Biosynthesis and Photosynthesis in Etiolated Arabidopsis Seedlings

Abstract: PHYTOCHROME INTERACTING FACTOR3 (PIF3) is a key basic helix-loop-helix transcription factor of Arabidopsis thaliana that negatively regulates light responses, repressing chlorophyll biosynthesis, photosynthesis, and photomorphogenesis in the dark. However, the mechanism for the PIF3-mediated transcription regulation remains largely unknown. In this study, we found that the REDUCED POTASSIUM DEPENDENCY3/HISTONE DEACETYLASE1-type histone deacetylase HDA15 directly interacted with PIF3 in vivo and in vitro. Genome-wide transcriptome analysis revealed that HDA15 acts mainly as a transcriptional repressor and negatively regulates chlorophyll biosynthesis and photosynthesis gene expression in etiolated seedlings. HDA15 and PIF3 cotarget to the genes involved in chlorophyll biosynthesis and photosynthesis in the dark and repress gene expression by decreasing the acetylation levels and RNA Polymerase II–associated transcription. The binding of HDA15 to the target genes depends on the presence of PIF3. In addition, PIF3 and HDA15 are dissociated from the target genes upon exposure to red light. Taken together, our results indicate that PIF3 associates with HDA15 to repress chlorophyll biosynthetic and photosynthetic genes in etiolated seedlings.