Abstract
Citrus dwarfing viroid (CDVd) induces stunting on sweet orange trees [Citrus sinensis (L.) Osbeck], propagated on trifoliate orange rootstock [Citrus trifoliata (L.), syn. Poncirus trifoliata (L.) Raf.]. MicroRNAs (miRNAs) are a class of non-coding small RNAs (sRNAs) that play important roles in the regulation of tree gene expression. To identify miRNAs in dwarfed citrus trees, grown in high-density plantings, and their response to CDVd infection, sRNA next-generation sequencing was performed on CDVd-infected and non-infected controls. A total of 1,290 and 628 miRNAs were identified in stem and root tissues, respectively, and among those, 60 were conserved in each of these two tissue types. Three conserved miRNAs (csi-miR479, csi-miR171b, and csi-miR156) were significantly downregulated (adjusted p-value < 0.05) in the stems of CDVd-infected trees compared to the non-infected controls. The three stem downregulated miRNAs are known to be involved in various physiological and developmental processes some of which may be related to the characteristic dwarfed phenotype displayed by CDVd-infected C. sinensis on C. trifoliata rootstock field trees. Only one miRNA (csi-miR535) was significantly downregulated in CDVd-infected roots and it was predicted to target genes controlling a wide range of cellular functions. Reverse transcription quantitative polymerase chain reaction analysis performed on selected miRNA targets validated the negative correlation between the expression levels of these targets and their corresponding miRNAs in CDVd-infected trees. Our results indicate that CDVd-responsive plant miRNAs play a role in regulating important citrus growth and developmental processes that may participate in the cellular changes leading to the observed citrus dwarf phenotype.
Highlights
Small RNAs can be divided into several categories, which include small-interferingRNAs, trans-actingsiRNAs, microRNAs, natural-antisense siRNAs, and Piwi-interacting RNAs (Borges and Martienssen, 2015; Czech et al, 2018; Zhu et al, 2018; Treiber et al, 2019)
From the non-infected trees, 8.1% of the stem and 6.1% of the root were classified as miRNAs
The most common size among the total mapped miRNAs sequences ranged between 20 and 24 nt in length, with 21-nt being the predominant miRNA class across different treatments and tissue types. This is consistent with plant antiviral RNA interference (RNAi) responses and DCL-mediated processing of dsRNA producing 21 nt siRNAs
Summary
Small RNAs (sRNAs) can be divided into several categories, which include small-interfering (si)RNAs, trans-acting (ta)siRNAs, microRNAs (miRNAs), natural-antisense siRNAs (natsiRNAs), and Piwi-interacting RNAs (piwi-RNAs) (Borges and Martienssen, 2015; Czech et al, 2018; Zhu et al, 2018; Treiber et al, 2019). One of the major components of endogenous plant sRNAs are miRNAs. miRNAs are encoded by plant MIR genes and have independent transcriptional units with their own regulatory promoters. They form double stranded stem loop structures that are processed to produce single stranded transcripts, typically 21–24 nucleotides (nt) in length (Wang et al, 2019). Plant miRNAs are incorporated into the activated RNA-induced silencing complex (RISC) to target RNAs which are complementary to the miRNA guide strand. Once the activated miRNA–RISC complex finds the complementary plant mRNA, it silences the target via RNA degradation or translational repression (Wang et al, 2019) (Figure 1A)
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