Host-induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea.

Host-induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross-kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a-insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a-mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double-stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross-kingdom RNAi in the apple-B. dothidea interaction and establish HIGS as a potential disease control strategy in apple.

Genome-Wide Identification and Functional Analysis of Salvia miltiorrhiza MicroRNAs Reveal the Negative Regulatory Role of Smi-miR159a in Phenolic Acid Biosynthesis.

MicroRNAs (miRNAs) are a group of endogenous small non-coding RNAs in plants. They play critical functions in various biological processes during plant growth and development. Salvia miltiorrhiza is a well-known traditional Chinese medicinal plant with significant medicinal, economic, and academic values. In order to elucidate the role of miRNAs in S. miltiorrhiza, six small RNA libraries from mature roots, young roots, stems, mature leaves, young leaves and flowers of S. miltiorrhiza and one degradome library from mixed tissues were constructed. A total of 184 miRNA precursors, generating 137 known and 49 novel miRNAs, were genome-widely identified. The identified miRNAs were predicted to play diversified regulatory roles in plants through regulating 891 genes. qRT-PCR and 5' RLM-RACE assays validated the negative regulatory role of smi-miR159a in SmMYB62, SmMYB78, and SmMYB80. To elucidate the function of smi-miR159a in bioactive compound biosynthesis, smi-miR159a transgenic hairy roots were generated and analyzed. The results showed that overexpression of smi-miR159a caused a significant decrease in rosmarinic acid and salvianolic acid B contents. qRT-PCR analysis showed that the targets of smi-miR159a, including SmMYB62, SmMYB78, and SmMYB80, were significantly down-regulated, accompanied by the down-regulation of SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmTAT3, SmHPPR1, SmRAS, and SmCYP98A14 genes involved in phenolic acid biosynthesis. It suggests that smi-miR159a is a significant negative regulator of phenolic acid biosynthesis in S. miltiorrhiza.

Soybean-derived miRNAs specifically inhibit proliferation and stimulate apoptosis of human colonic Caco-2 cancer cells but not normal mucosal cells in culture.

MicroRNAs (miRNAs) are important regulators of gene expression in eukaryotes. Studies have shown that plant-derived miRNAs can be absorbed through diets and regulate gene expression in mammals. Although soybean-derived miRNAs have been reported, their biological functions are still unclear. In this study, we found that soybean-derived small RNAs (sRNAs) significantly inhibited the proliferation and stimulated the apoptosis of Caco-2 cells. Bioinformatics analysis indicated that the target gene set of soybean miRNAs was extensively enriched in cancer pathways. Besides, we obtained 8 target genes, including Transcription factor 7 (TCF7), associated with colon cancer through prediction. Further studies showed that gma-miR159a inhibited the proliferation of Caco-2 cells and played an important role in the inhibitory effect of sRNAs by inhibiting TCF7 protein, which are upregulated in colon cancer cells but not normal mucosal cells in culture. These findings provide a novel molecular mechanism of soybean-derived miRNAs for potential application in tumor prevention.

ACC oxidase and miRNA 159a, and their involvement in fresh fruit bunch yield (FFB) via sex ratio determination in oil palm.

Oil palm (Elaeis guineesis Jacq.) is the most productive oil-bearing crop, yielding more oil per area than any other oil-bearing crops. However, there are still efforts to improve oil palm yield, in order to serve consumer and manufacturer demand. Oil palm produces female and male inflorescences in an alternating cycle. So, high sex ratio (SR), the ratio of female inflorescences to the total inflorescences, is a favorable trait in term of increasing yields in oil palm. This study aims to understand the genetic control for SR related traits, such as fresh fruit bunch yield (FFB), by characterizing genes at FFB quantitative trait loci (QTLs) on linkage 10 (chromosome 6) and linkage 15 (chromosome 10). Published oil palm sequences at the FFB QTLs were used to develop gene-based and simple sequence repeat (SSR) markers. We used the multiple QTL analysis model (MQM) to characterize the relationship of new markers with the SR traits in the oil palm population. The RNA expression of the most linked QTL genes was also evaluated in various tissues of oil palm. We identified EgACCO1 (encoding aminocyclopropane carboxylate (ACC) oxidase) at chromosome 10 and EgmiR159a (microRNA 159a) at chromosome 6 to be the most linked QTL genes or determinants for FFB yield and/or female inflorescence number with a phenotype variance explained (PVE) from 10.4 to 15 % and suggest that these play the important roles in sex determination and differentiation in oil palm. The strongest expression of EgACCO1 and the predicted precursor of EgmiR159a was found in ovaries and, to a lesser extent, fruit development. In addition, highly normalized expression of EgmiR159a was found in female flowers. In summary, the QTL analysis and the RNA expression reveal that EgACCO1 and EgmiR159a are the potential genetic factors involved in female flower determination and hence would affect yield in oil palm. However, to clarify how these genetic factors regulate female flower determination, more investigation of their down regulation or target may be essential. Additionally, if more sex determination genes controlled by plant hormones are identified, it may possible to reveal a crosstalk of sex determination genes with hormones and environment factors.

miR159a modulates poplar resistance against different fungi and bacteria.

Trees are inevitably attacked by different kinds of pathogens in their life. However, little is known about the regulatory factors in poplar response to different pathogen infections. MicroRNA159 (miR159) is a highly conserved microRNA (miRNA) in plants and regulates plant development and stress responses. Here, transgenic poplar overexpressing pto-miR159a (OX-159) showed antagonistic regulation mode to poplar stem disease caused by fungi Cytospora chrysosperma and bacteria Lonsdalea populi. OX-159 lines exhibited a higher susceptibility after inoculation with bacterium L. populi, whereas enhanced disease resistance to necrotrophic fungi C. chrysosperma compared with wild-type (WT) poplars. Intriguingly, further disease assay found that OX159 line rendered the poplar susceptible to hemi-biotrophic fungi Colletotrichum gloeosporioide, exhibiting larger necrosis and lower ROS accumulation than WT lines. Transcriptome analyses revealed that more down-regulated differentially expressed genes with disease-resistant domains in OX-159 line compared with WT line. Moreover, the central mediator NPR1 of salicylic acid (SA) pathway showed a decrease in expression level, while jasmonic acid/ethylene (JA/ET) signal pathway marker genes ERF, as well as PR3, MPK3, and MPK6 genes showed an increase level in OX159-2 and OX159-5 compared with WT lines. Further spatio-temporal expression analysis revealed JA/ET signaling was involved in the dynamic response process to C. gloeosporioides in WT and OX159 lines. These results demonstrate that overexpression of pto-miR159a resulted in the crosstalk changes of the downstream hub genes, thereby controlling the disease resistance of poplars, which provides clues for understanding pto-miR159a role in coordinating poplar-pathogen interactions.

Nested miRNA Secondary Structure Is a Unique Determinant of miR159 Efficacy in Arabidopsis.

MicroRNAs (miRNAs) are 20- to 24-nucleotide small RNAs, and whenever a pri-miRNA precursor includes another miRNA precursor, and both of these precursors may generate independent non overlapping mature miRNAs, we called them nested miRNAs. However, the functional and regulatory roles of nested miRNA structures in plants are still unknown. In this study, the Arabidopsis nested miR159a structure, which consists of two nested miRNAs, miR159a.1, and miR159a.2, was used as a model to determine miRNA-mediated gene silencing in plants. Complementation analysis of nested miR159a structures revealed that the miR159a structure can differentially complement the mir159ab phenotype, and a duplex nested structure in the tail end region of the pre-miR159a fold back may have a possible dominant function, indicating the importance of the flanking sequence of the stem in the cleavage of the mature miRNA. Furthermore, continuously higher expression of the miR159a.2 duplex in the severe leaf curl phenotype indicates that miR159a.2 is functional in Arabidopsis and suggests that in plants, a miRNA precursor may encode multiple regulatory small RNAs. Taken together, our study demonstrates that the nested miR159a structure regulated by duplex mutations of miR159a has a unique pattern and provides novel insight into silencing efficacy of Arabidopsis miR159a.

Soybean-derived gma-miR159a alleviates colon tumorigenesis by suppressing TCF7/MYC in mice.

Previous reports have shown that plant-derived microRNAs (miRNAs) regulate mammalian gene expression through dietary intake. Our prior study found that gma-miR159a, which is abundant in soybean, significantly inhibited the proliferation of colon cancer cells. In the current study, dietary gma-miR159a was utilized to study its anti-colon cancer function in azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon cancer mice. Under processing conditions, gma-miR159a exhibited excellent stability in cooked soybean. In vitro, gma-miR159a suppressed the expression of the oncogene MYC downstream of the Wnt signaling pathway by targeting the TCF7 gene, significantly inhibiting the growth of colon cancer cells. The in vivo experiments showed that gma-miR159a and soybean RNA (total RNA extracted from soybean) significantly reduced tumor growth in AOM/DSS-induced colon cancer mice by gavage. This effect disappeared when anti-miR159a was present. In addition, gma-miR159a and soybean RNA significantly attenuated inflammation in colon cancer mice. These results showed that long-term dietary intake of soybean-derived gma-miR159a effectively prevented the occurrence of colon cancer and colitis, which provides novel evidence for the prevention function of soybean.

Fine-Tuning Roles of Osa-miR159a in Rice Immunity Against Magnaporthe oryzae and Development.

BACKGROUND: Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases of rice. An increasing number of microRNAs (miRNAs) have been reported to fine-tune rice immunity against M. oryzae and coordinate with growth and development. RESULTS: Here, we showed that rice microRNA159a (Osa-miR159a) played a positive role in rice resistance to M. oryzae. The expression of Osa-miR159a was suppressed in a susceptible accession at 12, 24, and 48 h post-inoculation (hpi); it was upregulated in a resistant accession of M. oryzae at 24 hpi. The transgenic rice lines overexpressing Osa-miR159a were highly resistant to M. oryzae. In contrast, the transgenic lines expressing a short tandem target mimic (STTM) to block Osa-miR159a showed enhanced susceptibility. Knockout mutations of the target genes of Osa-miR159a, including OsGAMYB, OsGAMYBL, and OsZF, led to resistance to M. oryzae. Alteration of the expression of Osa-miR159a impacted yield traits including pollen and grain development. CONCLUSIONS: Our results indicated that Osa-miR159a positively regulated rice immunity against M. oryzae by downregulating its target genes. Proper expression of Osa-miR159a was critical for coordinating rice blast resistance with grain development.