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–
P. 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.
