Exogenous Melatonin RegulatesAlternaria brassicaeReproduction and Pathogenicity in a Dose-Dependent Manner [miRNA]

The development of viroid-elimination technology is an important means to ensure the production of tomatoes. Viroid is an obligate pathogen that can infect the apical meristem cells. The combination of low-temperature therapy and meristem culture is a commonly used method for viroid eradication, but tomatoes cannot withstand long-term low-temperature treatment. This study identified a new HSVd genotype and measured the growth and physiological parameters of diseased tomato “Alisa Craig”. Five HSVd-targeting ds-sRNAs were synthesized to match different regions of the HSVd genome, and their permeability in plants was tested. By screening the types of ds-sRNA and processing duration, the optimal treatment for inhibiting HSVd in diseased tomato shoot terminal was obtained. miRNAs produced by HSVd genome were detected by using miRNA-seq and degradation-seq to verify the occurrence of the ds-sRNA induced HSVd gene silencing. Exogenous ds-sRNA can penetrate all cells including vascular bundles, thin-walled cells, and stem apical meristematic tissues and induced HSVd gene silencing in shoot terminals, leading to the accumulation of miRNAs produced by HSVd in plants. Overall design: Isolation of vd-sRNAs was strictly conducted according to Kutnjak et al. (2015). 10-50 nt small RNAs were separated. NEBNext®Small RNA Library Prep Set for Illumina kit (E7580L, NEB) was used to construct small RNA libraries according to the manufacturer's instructions. The libraries were sent for sequencing on an IlluminaHiseq2000 platform (LC-Bio., Hangzhou, China). Three biological replicates were conducted. Reads that shorter than 15 nt were removed since their nonuniquely. Readsfrom each set were mapped to HSVd genome. Thecount and type of genome mapped nonuniquely sRNA reads were thereafter compared among ddH2O and ds-sRNA-H3 36 h fed samples. A confirmatory experiment by degradomeanalysis of ddH2O and ds-sRNA-H3 36 h fed HSVd-infected tomato cuts were conducted according to Navarro et al. (2021) as well. The degradome sequencing was conducted by LC-Bio. One biological replicate for each sample were conducted.