Abstract
In this study, we characterized the miR482 family in cotton using existing small RNA datasets and the recently released draft genome sequence of Gossypium raimondii, a diploid cotton species whose progenitor is the putative contributor of the Dt (representing the D genome of tetraploid) genome of the cultivated tetraploid cotton species G. hirsutum and G. barbadense. Of the three ghr-miR482 members reported in G. hirsutum, ghr-miR482a has no homolog in G. raimondii, ghr-miR482b and ghr-miR482c each has a single homolog in G. raimondii. Gra-miR482d has five homologous loci (gra-miR482d, f-i) in G. raimondii and also exists in G. hirsutum (ghr-miR482d). A variant, miR482.2 that is a homolog of miR2118 in other species, is produced from several GHR-MIR482 loci in G. hirsutum. Approximately 12% of the G. raimondii NBS-LRR genes were predicted targets of various members of the gra-miR482 family. Based on the rationale that the regulatory relationship between miR482 and NBS-LRR genes will be conserved in G. raimondii and G. hirsutum, we investigated this relationship using G. hirsutum miR482 and G. raimondii NBS-LRR genes, which are not currently available in G. hirsutum. Ghr-miR482/miR482.2-mediated cleavage was confirmed for three of the four NBS-LRR genes analysed. As in tomato, miR482-mediated cleavage of NBS-LRR genes triggered production of phased secondary small RNAs in cotton. In seedlings of the susceptible cultivar Sicot71 (G. hirsutum) infected with the fungal pathogen Verticillium dahliae, the expression levels of ghr-miR482b/miR482b.2, ghr-miR482c and ghr-miR482d.2 were down-regulated, and several NBS-LRR targets of ghr-miR482c and ghr-miR482d were up-regulated. These results imply that, like tomato plants infected with viruses or bacteria, cotton plants are able to induce expression of NBS-LRR defence genes by suppression of the miRNA-mediated gene silencing pathway upon fungal pathogen attack.
Highlights
MicroRNAs are 20–24 nucleotides long small noncoding RNAs and are processed from MIRNA genes that are transcribed by RNA polymerase II
By searching the published cotton small RNA datasets, we found that gra-miR482 exists in G. hirsutum; there are at least four miR482 members in the G. hirsutum genome
The miR482 reported by Wang et al [22] is a variant of ghr-miR482b because its 1st – 20th nucleotides are identical to the 3rd – 22nd nucleotides of ghr-miR482b (Figure 1)
Summary
MicroRNAs (miRNAs) are 20–24 nucleotides (nt) long small noncoding RNAs and are processed from MIRNA genes that are transcribed by RNA polymerase II. A number of miRNAs in Arabidopsis have been shown to either positively or negatively regulate elicitor flg22-induced callose deposition, a signature of the PTI defense response [8]. Arabidopsis plants elicited by flg showed induction of MIR393 transcription and down-regulation of miR393 targets, including three F-box auxin receptors TIR1 (Transport Inhibitor Response 1), AFB2 (Auxin signaling F-Box proteins 2) and AFB3, and increased resistance to Pseudomonas syringae [9]. The N gene from Nicotiana benthamiana [13], which encodes a TIR (the Toll and Interleukin-1 Receptor) type of nucleotide binding site (NBS)-leucine-rich-repeat (LRR) receptor protein that confers resistance to tobacco mosaic virus (TMV), was found to be cleaved by nta-miR6019 and nta-miR6020 [11]. Using a transient assay system, it has been shown that N. benthamiana mRNAs encoding NBS-LRR proteins could be silenced by tomato miR482 [12]
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