MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in various biological processes. This study examined microRNA profiles of Laodelphax striatellus using the small RNA libraries derived from virus free (VF) and rice black-streaked dwarf virus (RBSDV) infected (RB) insects. A total of 59 mature miRNAs (46 miRNA families) were identified as conserved insect miRNAs in both VF and RB libraries. Among these conserved miRNAs, 24 were derived from the two arms of 12 miRNA precursors. Nine conserved L. striatellus miRNAs were up-regulated and 12 were down-regulated in response to RBSDV infection. In addition, a total of 20 potential novel miRNA candidates were predicted in the VF and RB libraries. The miRNA transcriptome profiles and the identification of L. striatellus miRNAs differentially expressed in response to RBSDV infection will contribute to future studies to elucidate the complex miRNA-mediated regulatory network activated by pathogen challenge in insect vectors.
In this study, sequences of small RNA (sRNA) libraries derived from the insect vector Laodelphax striatellus were assembled into contigs and used as queries for database searches. A large number of contigs were highly homologous to the genome sequence of an insect dicistrovirus, himetobi P virus (HiPV). Interestingly, HiPV-derived sRNAs had a wide size distribution, and were relatively abundant throughout the 18-30 nt size range with only a slight peak at 22 nt. HiPV sRNAs had a strong bias towards the sense strand, whilst the antisense sRNAs were predominantly 21 and 22 nt. HiPV sRNAs do not have the typical features of PIWI-interacting RNAs, but their 39 ends were preferentially cleaved at UA-rich sequences. Our data suggest that HiPV sRNAs may be derived both from activities of the RNA interference pathway and from cleavage of the viral genome by other host RNases.
Argonaute (AGO) proteins are essential catalytic components of the RNA-induced silencing complex and play central roles in RNA interference. Using a combination of bioinformatics and rapid amplification of cDNA ends (RACE) methods, putative AGO subfamily members, ls-AGO1 and ls-AGO2, were cloned and characterized from the small brown planthopper, Laodelphax striatellus. The open reading frame (ORF) of ls-AGO1 is 2,820 bp long, encoding a putative protein of 939 amino acid residues, and ls-AGO2 contains an ORF of 2,490 bp, encoding 829 amino acid residues. The expected conserved PAZ and PIWI domains, and the conserved Asp-Asp-His (DDH) catalytic triad motif in the PIWI domain were observed in both ls-AGO1 and ls-AGO2. Reverse transcription-qPCR (RT-qPCR) results showed that both ls-AGO1 and ls-AGO2 were expressed in all developmental stages of L. striatellus with highest mRNA abundance in eggs. Expression of ls-AGO1 and ls-AGO2 was significantly decreased in adult insects in response to acquisition of rice black-streaked dwarf virus by second instar nymphs. mRNA expression of ls-AGO1 was significantly downregulated in response to low and high temperatures, but expression of ls-AGO2 was only affected by low temperature. ls-AGO1 and ls-AGO2 were initially downregulated when insects were transferred from rice to maize and to the wild grass Brachypodium distachyon, but expression showed partial or complete recovery 7 days after transfer. These results document that AGO subfamily members of L. striatellus are ubiquitously expressed at different developmental stages and respond to various stresses. Thus, AGO subfamily may act in regulating the stress-response of L. striatellus by controlling related gene expression.
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