Findings support a negative regulatory role of novel-miR0032-5p in CAT expression and indicate that lncRNA1386.1 participates in a candidate shared miRNA-responsive regulatory relationship.
Abstract
Long non-coding RNAs (lncRNAs) participate in insect immune regulation, but their relationship with antioxidant responses during fungal infection remains unclear. Here, we examined a candidate regulatory relationship among lncRNA1386.1, novel-miR0032-5p, and the catalase gene (CAT) in Apis cerana worker larvae infected with Ascosphaera apis. Dual-luciferase reporter assays showed that M-miR0032-5p reduced the activity of reporters containing the predicted miRNA response element within lncRNA1386.1 or CAT, whereas mutation of these sequences weakened or abolished the response. In infected larvae, lncRNA1386.1 silencing and novel-miR0032-5p overexpression reduced CAT transcript abundance and CAT protein concentration, and increased dihydroethidium (DHE) fluorescence intensity. These treatments were also accompanied by alterations in the transcript abundance of the host genes Dorsal1 and Relish and the fungal genes Chit3 and STE11-like. Novel-miR0032-5p inhibition generally produced opposite changes in CAT-related measurements, DHE fluorescence intensity, and the selected host and fungal transcripts. lncRNA1386.1 silencing was associated with increased larval survival, whereas novel-miR0032-5p inhibition was associated with a higher hazard of first visible external mycelial growth without significantly affecting survival. These findings support a negative regulatory role of novel-miR0032-5p in CAT expression and indicate that lncRNA1386.1 participates in a candidate shared miRNA-responsive regulatory relationship. This candidate regulatory relationship was associated with CAT-related antioxidant regulation, superoxide-associated DHE fluorescence, immune-related transcription, selected fungal transcriptional responses, and visible external mycelial growth during A. apis infection.
ABSTRACT Chalkbrood, a destructive larval disease caused by the fungal pathogen Ascosphaera apis, causes substantial losses in apiculture. Although host–pathogen interactions in Apis mellifera larvae infected with A. apis have been investigated, immune defense mechanisms in the Asian honey bee Apis cerana remain unclear. This study examined whether the lncRNA6470/ace-miR-750-y axis modulates the response of A. cerana larvae to A. apis infection. Stem-loop RT-PCR, Sanger sequencing, dual-luciferase reporter assays, RNA interference, and RT-qPCR were used to characterize ace-miR-750-y, lncRNA6470, AcPP2A, host immune genes, and selected A. apis genes associated with signal transduction, transcriptional regulation, and RNA metabolism. Larval survival and chalkbrood incidence were also evaluated after ncRNA perturbation. ace-miR-750-y was expressed in larval guts and showed infection-associated expression changes. Computational prediction identified 214 candidate mRNA targets and 143 candidate lncRNA interactors of ace-miR-750-y, from which the lncRNA6470/ace-miR-750-y/AcPP2A axis was selected for experimental validation. Modulation of ace-miR-750-y significantly affected AcPP2A expression. ace-miR-750-y overexpression increased host immune genes associated with antimicrobial peptide production and peroxidase activity, whereas its inhibition reduced their expression. Modulation of ace-miR-750-y was also associated with altered expression of selected A. apis genes and changes in larval survival and chalkbrood incidence. These findings suggest that ace-miR-750-y may play an important regulatory role in the immune response of A. cerana worker larvae to A. apis infection, and that its interaction with lncRNA6470 may contribute to ceRNA-like regulation during fungal challenge.
Xiaoxue Fan, Kaiyao Zhang, Yun Yang et al.· Virulence· 0 citations
Microsporidia rely extensively on host resources, yet how parasite-derived microRNAs coordinate infection remains poorly understood. Here, we investigated the function of nce-miR-12220, a miRNA identified in Nosema ceranae spores, during infection of Apis mellifera workers. Target prediction, dual-luciferase assays, and fluorescence in situ hybridization were combined with RNA interference and miRNA gain- and loss-of-function experiments. nce-miR-12220 interacted sequence-specifically with binding regions in ATP-A and γ-tubulin and was detected in infected honeybee midgut epithelial cells. Silencing either target gene reduced expression of the N. ceranae virulence-associated gene NcRBL and improved worker survival relative to the scramble control. In infected workers, nce-miR-12220 overexpression increased endogenous ATP-A and γ-tubulin transcript abundance, whereas inhibition produced the opposite effect. Overexpression also reduced expression of the Toll pathway-associated genes Cactus and dorsal and the antimicrobial peptide genes Defensin and Hymenoptaecin, increased N. ceranae spore load and sucrose consumption, and decreased midgut ATP content. Inhibition of nce-miR-12220 reversed these responses and reduced parasite burden. Survival after nce-miR-12220 manipulation changed in the predicted direction but did not reach statistical significance. Together, these findings identify nce-miR-12220 as a parasite-derived regulator that promotes N. ceranae proliferation while reshaping host immune and energetic responses, and suggest that this microRNA and its target network may provide candidates for controlling bee nosemosis.
Rui Guo, He Zang, Wenhua Xu et al.· PLoS Pathogens· 0 citations
Chalkbrood disease, caused by Ascosphaera apis, threatens Apis cerana larvae. However, miRNA-mediated antifungal regulation remains poorly defined. Here, we characterized ace-miR-3759-y in A. apis-challenged A. cerana larvae. Stem-loop RT-PCR and Sanger sequencing confirmed its gut expression. Target prediction identified 146 candidate mRNAs, and dual-luciferase assays verified AcDorsal1 and AcARL4C as direct targets, significantly suppressing reporter activity (P < 0.01). During infection, ace-miR-3759-y was upregulated (P = 0.0008), whereas AcDorsal1 was downregulated (P = 0.0011). Inhibiting ace-miR-3759-y reduced its expression (P < 0.0001), relieved target repression, enhanced reactive oxygen species (ROS) accumulation (P = 0.0006), tended to increase antimicrobial peptide (AMP) gene expression, and decreased fungal ADM-B and chsD expression. Functionally, ace-miR-3759-y inhibition reduced cumulative mortality and chalkbrood incidence (both P < 0.0001), whereas AcARL4C silencing suppressed AMP genes and aggravated disease. These findings reveal a dual-target miRNA module and provide a potential RNAi-based target for chalkbrood control.
He Zang, Haimei Yue, Xiaoxue Fan et al.· Journal of Agricultural and...· 0 citations
Simple Summary Cephalopholis sonnerati is an economically new aquaculture species in China. The red-spotted grouper nervous necrosis virus (RGNNV) exhibits high pathogenicity in larval C. sonnerati, but its immune response mechanisms remain unexplored, impeding the development of aquaculture. In the present study, we profiled brain transcriptome from healthy and naturally RGNNV-infected larvae. Many differentially expressed microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and fewer circular RNAs were identified. Enrichment analysis demonstrated that these targeted genes of differentially expressed ncRNAs were markedly enriched in innate immune defense, inflammatory, and cell death related pathways, such as JAK-STAT signaling pathway, NF-κB signaling pathway, apoptosis, and necroptosis. Furthermore, a lncRNA–miRNA–mRNA regulatory network focused on miR-93 was established, which may provide a valuable candidate target for future antiviral strategies in C. sonnerati. This study offers the first ncRNA transcriptome landscape of C. sonnerati during RGNNV infection, establishing a theoretical basis for elucidating host-RGNNV interaction mechanism in groupers.
MicroRNAs (miRNAs) are small regulatory RNAs that control gene expression through sequence-specific interactions with target transcripts and play pivotal roles in plant developmental phase transitions. Among them, the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module constitutes a conserved regulatory pathway controlling flowering time in diverse species, yet its molecular function in woody perennials with extended juvenile phases remains poorly understood. Here, seven miR156 family members were identified in Liriodendron chinense by small RNA sequencing, and the Lch-miR156b-LcSPL11 regulatory module was functionally characterized. 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) and dual-luciferase assays demonstrated that Lch-miR156b directly cleaves and represses LcSPL11 transcripts. Expression profiling further associated the Lch-miR156b-LcSPL11 module with flowering-time variation in L. chinense. In heterologous assays, ectopic expression of Lch-miR156b in Arabidopsis thaliana delayed flowering, whereas overexpression of LcSPL11, including a miRNA-resistant variant (rLcSPL11), accelerated floral transition. In addition, LcSOC1 and LcFUL showed expression patterns similar to that of LcSPL11, their promoters contained conserved GTAC cis-elements, and yeast one-hybrid assays confirmed direct binding of LcSPL11 to both promoters. Together, these findings reveal a conserved miRNA-mediated regulatory cascade in which Lch-miR156b modulates flowering through LcSPL11 and its downstream targets LcSOC1 and LcFUL, providing mechanistic insight into flowering control in a woody basal angiosperm.
Jing Wang, Wenjuan Yi, Yu Zhang et al.· International Journal of Bio...· 0 citations
Potassium (K+) deficiency severely limits global wheat productivity and food security. Genetic improvement of K+ utilization efficiency (KUE) is a sustainable strategy to address this challenge; however, the underlying molecular mechanisms remain unclear. We identified a lncRNA62732‐miR9778‐TaHAK18‐5B module in wheat (Triticum aestivum) using long noncoding RNAs (lncRNA)‐sequencing combined with competing endogenous RNA prediction. Using genetic transformation and molecular biology techniques, we analyzed the role of this module in mediating the response to K+ deficiency and the mechanisms underlying its effects. miR9778 is a Triticeae‐specific miRNA that directly targets TaHAK18‐5B. Both miR9778 silencing and TaHAK18‐5B overexpression notably enhanced K+ deficiency tolerance at the seedling stage by modulating root K+ acquisition and translocation from the roots to the shoots, whereas silencing TaHAK18‐5B had the opposite effect. Additionally, lncRNA62732 was identified as an endogenous target mimic (eTM) of miR9778 that sequesters miR9778 and prevents miR9778‐mediated cleavage of TaHAK18‐5B, thereby positively regulating wheat seedlings' growth under K+ deficiency conditions. Notably, the lncRNA62732‐miR9778‐TaHAK18‐5B module improved wheat adaptation to K+ deficiency and enhanced the KUE and grain yield under field conditions. These results reveal a novel module in mediating K+ homeostasis and provide valuable genetic resources for engineering KUE in wheat.
Ke Xu, Sihang Zhao, Hao Wu et al.· New Phytologist· 0 citations
