Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants

In plants, RNA silencing provides an adaptive immune system that inactivates pathogenic nucleic acids, guided by 21–24-mer RNAs of pathogen origin. The characterization of pathogen-related small RNAs (sRNAs) is relevant to uncovering the strategies used by pathogens to evade host defense responses. Several groups have reported the detection of viroid-derived sRNAs during infections, although the origin of these sRNAs and their chemical characteristics were poorly understood. Here, we describe the in vivo cleavage of Citrus exocortis viroid (CEVd) RNA into sRNAs of 21–22 nt, that are phosphorylated at their 5′-end and methylated at 3′. Our studies suggested that the CEVd genomic RNA might be the predominant in vivo substrate for cleavage by Dicer-like enzyme(s), which preferentially targeted residues mainly located within the right-end domain of the viroid. Further analysis on the accumulation levels of specific miRNAs controlling major regulators of leaf development and the miRNA pathway and the levels of their target mRNAs provided evidence that the endogenous tomato miRNA pathway was not affected by CEVd infection.