ABSTRACT
Background: Small ribonucleic acids represent an important repertoire of mobile molecules that exert key roles in several cell processes including antiviral defense. Small RNA based repertoire includes both small interfering RNA (siRNA) and microRNA (miRNA) molecules. In the Prunus genus, sharka disease, caused by the Plum pox virus (PPV), first occurred on European plum (Prunus domestica) and then spread over among all species in this genus and thus classified as quarantine pathogen. Next-generation sequencing (NGS) was used for the study of siRNA/miRNA molecules; however, NGS relies on adequate extraction protocols. Currently, knowledge of PPV-Prunus interactions in terms of siRNA populations and miRNA species is still scarce, and siRNA/miRNA extraction protocols are limited to species such as peach, almond, and sweet cherry. Results: We describe a reliable procedure for siRNA/miRNA purification from Prunus salicina trees, in which previously used protocols did not allow adequate purification. The procedure was based on a combination of commercially available RNA purification kits and specific steps that yielded high quality purifications. The resulting molecules were adequate for library construction and NGS, leading to the development of a pipeline for analysis of both siRNAs and miRNAs in the PPVP. salicina interactions. Results showed that PPV infection led to altered siRNA profiles in Japanese plum as characterized by decreased 24-nt and increased 21- and 22-nt siRNAs. Infections showed miR164 and miR160 generation and increased miR166, miR171, miR168, miR319, miR157, and miR159. Conclusion: We propose this protocol as a reliable and reproducible small RNA isolation procedure for P. salicina and other Prunus species.
Subject(s)
RNA, Plant/isolation & purification , MicroRNAs/isolation & purification , RNA, Small Interfering/isolation & purification , Prunus domestica/genetics , Plant Diseases/virology , Plum Pox Virus/physiology , Host-Pathogen Interactions , High-Throughput Nucleotide Sequencing , Real-Time Polymerase Chain Reaction , Prunus domestica/immunology , Prunus domestica/virologyABSTRACT
Cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that plays an important role in innate immunity. Transfection of DNA or DNA virus infection results in the induction of type I interferon production in fibroblasts, macrophages, and dendritic cells which is dependent on cGAS. Recently, cGas (-/-) mice have been reported to be more vulnerable to fatal infection with herpes simplex virus 1 (HSV1) as compared to wild-type mice.