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 PPV\u2013P. 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