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The fungus, Fusarium oxysporum f. sp. cubense (Foc), is the causal agent of Fusarium wilt disease, which is the most serious disease affecting the whole banana industry. Although extensive studies have characterized many Foc-responsive genes in banana, the molecular mechanisms on microRNA level underlying both banana defense and Foc pathogenesis are not yet fully understood. In this study, we aimed to reveal the role of miRNA during banana-Foc TR4 interactions. Illumina sequencing was used to reveal the changes in small RNAome profiles in roots of Foc TR4-inoculated ‘Tianbaojiao’ banana (Musa acuminata cv. Tianbaojiao) in the early stages (i.e. 5 h, 10 h and 25 h post Foc TR4 inoculation, respectively). The expression of some differentially expressed (DE) miRNAs and their predicted target genes was studied by using quantitative real time PCR (qRT-PCR). Totally, 254 known miRNAs from 31 miRNA families and 28 novel miRNAs were identified. Differential expression analysis identified 84, 77 and 74 DE miRNAs at the three respective Foc TR4 infection time points compared with control healthy banana (CK). GO and KEGG analysis revealed that most of the predicted target genes of DE miRNAs (DET) were implicated in peroxisome, fatty acid metabolism, auxin-activated signaling pathway, sulfur metabolism, lignin metabolism and so on, and many known stress responsive genes were identified to be DETs. Moreover, expected inverse correlations were confirmed between some miRNA and their corresponding target genes by using qRT-PCR analysis. Our study revealed that miRNA play important regulatory roles during the banana-Foc TR4 interaction by regulating peroxidase, fatty acid metabolism, auxin signaling, sulfur metabolism, lignin metabolism related genes and many known stress responsive genes.

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Introns exist not only in coding sequences (CDSs) but also in untranslated regions (UTRs) of a gene. Recent studies in animals and model plants such as Arabidopsis have revealed that the UTR-introns (UIs) are widely presented in most genomes and involved in regulation of gene expression or RNA stability. In the present study, we identified introns at both 5′UTRs (5UIs) and 3′UTRs (3UIs) of sweet orange genes, investigated their size and nucleotide distribution characteristics, and explored the distribution of cis-elements in the UI sequences. Functional category of genes with predicted UIs were further analyzed using GO, KEGG, and PageMan enrichment. In addition, the organ-dependent splicing and abundance of selected UI-containing genes in root, leaf, and stem were experimentally determined. Totally, we identified 825 UI- and 570 3UI-containing transcripts, corresponding to 617 and 469 genes, respectively. Among them, 74 genes contain both 5UI and 3UI. Nucleotide distribution analysis showed that 5UI distribution is biased at both ends of 5′UTR whiles 3UI distribution is biased close to the start site of 3′UTR. Cis- elements analysis revealed that 5UI and 3UI sequences were rich of promoter-enhancing related elements, indicating that they might function in regulating the expression through them. Function enrichment analysis revealed that genes containing 5UI are significantly enriched in the RNA transport pathway. While, genes containing 3UI are significantly enriched in splicesome. Notably, many pentatricopeptide repeat-containing protein genes and the disease resistance genes were identified to be 3UI-containing. RT-PCR result confirmed the existence of UIs in the eight selected gene transcripts whereas alternative splicing events were found in some of them. Meanwhile, qRT-PCR result showed that UIs were differentially expressed among organs, and significant correlation was found between some genes and their UIs, for example: The expression of VPS28 and its 3UI was significantly negative correlated. This is the first report about the UIs in sweet orange from genome-wide level, which could provide evidence for further understanding of the role of UIs in gene expression regulation.