Stem-loop RT-PCR Research Articles

Transcriptional dynamics and regulatory function of milRNAs in Ascosphaera apis invading Apis mellifera larvae.

In the present study, small RNA (sRNA) data from Ascosphaera apis were filtered from sRNA-seq datasets from the gut tissues of A. apis-infected Apis mellifera ligustica worker larvae, which were combined with the previously gained sRNA-seq data from A. apis spores to screen differentially expressed milRNAs (DEmilRNAs), followed by trend analysis and investigation of the DEmilRNAs in relation to significant trends. Additionally, the interactions between the DEmilRNAs and their target mRNAs were verified using a dual-luciferase reporter assay. In total, 974 A. apis milRNAs were identified. The first base of these milRNAs was biased toward U. The expression of six milRNAs was confirmed by stem-loop RT-PCR, and the sequences of milR-3245-y and milR-10285-y were validated using Sanger sequencing. These miRNAs grouped into four significant trends, with the target mRNAs of DEmilRNAs involving 42 GO terms and 120 KEGG pathways, such as the fungal-type cell wall and biosynthesis of secondary metabolites. Further investigation demonstrated that 299 DEmilRNAs (novel-m0011-3p, milR-10048-y, bantam-y, etc.) potentially targeted nine genes encoding secondary metabolite-associated enzymes, while 258 (milR-25-y, milR-14-y, milR-932-x, etc.) and 419 (milR-4561-y, milR-10125-y, let-7-x, etc.) DEmilRNAs putatively targeted virulence factor-encoded genes and nine genes involved in the MAPK signaling pathway, respectively. Additionally, the interaction between ADM-B and milR-6882-x, as well as between PKIA and milR-7009-x were verified. Together, these results not only offer a basis for clarifying the mechanisms underlying DEmilRNA-regulated pathogenesis of A. apis and a novel insight into the interaction between A. apis and honey bee larvae, but also provide candidate DEmilRNA-gene axis for further investigation.

Mobile microRNAs (miRNAs) responsive to excess nickel in pumpkin (Cucurbita maxima L.)

Nickel (Ni) is a toxic heavy metal that inhibits plant growth, development, and reproduction. MicroRNAs (miRNA) travel from cell to cell or organ to carry messages to regulate gene expression. The objective of this study is to find mobile miRNAs that are Ni-responsive and are present in pumpkin (Cucurbita maxima L.) phloem sap. For this purpose, pumpkin seedlings were exposed to Ni (100 μM, NiCl2), and root, shoot, and phloem-sap specimens were collected at 0 (control), 24, and 48 hours of the treatment. The stem-loop RT-qPCR and stem-loop semi-quantitative RT-PCR methods were used to determine the abundance of 14 miRNAs in the phloem sap. Compared to the control, the abundance of miR160, miR167, miR393, miR397, and miR398 are suppressed in Ni-treated seedlings. The reduction was verified by grafting experiments, revealing that miR167 and miR393 are Ni-responsive and move/travel from the leaf-to-root direction. Those phloem-residential miRNAs potentially play a role in the Ni-response mechanism. This study can help to understand the early response mechanism of plants against excess Ni and lead to identifying miRNA-mediated long-distance communication of plants.

Biodistribution of Therapeutic Small Interfering RNAs Delivered with Lipid-Substituted Polyethylenimine-Based Delivery Systems.

Small interfering RNAs (siRNAs) have emerged as a powerful tool to manipulate gene expression in vitro. However, their potential therapeutic application encounters significant challenges, such as degradation in vivo, limited cellular uptake, and restricted biodistribution, among others. This study evaluates the siRNA delivery efficiency of three different lipid-substituted polyethylenimine (PEI)-based carriers, named Leu-Fect A-C, to different organs in vivo, including xenograft tumors, when injected into the bloodstream of mice. The siRNA analysis was undertaken by stem-loop RT-PCR, followed by qPCR or digital droplet PCR. Formulating siRNAs with a Leu-Fect series of carriers generated nanoparticles that effectively delivered the siRNAs into K652 and MV4-11 cells, both models of leukemia. The Leu-Fect carriers were able to successfully deliver BCR-Abl and FLT3 siRNAs into leukemia xenograft tumors in mice. All three carriers demonstrated significantly enhanced siRNA delivery into organs other than the liver, including the xenograft tumors. Preferential biodistribution of siRNAs was observed in the lungs and spleen. Among the delivery systems, Leu-Fect A exhibited the highest biodistribution into organs. In conclusion, lipid-substituted PEI-based delivery systems offer improvements in addressing pharmacokinetic challenges associated with siRNA-based therapies, thus opening avenues for their potential translation into clinical practice.

Identification and characterization of microRNAs responsive to fluoride toxicity in rice (Oryza sativa L.)

MicroRNAs (miRNAs) are a class of small non-coding RNAs that act as important modulators of gene expression related to several stress responses in plants. While several miRNAs have been implicated in the modulation of multiple abiotic and biotic stresses in rice, there role in response to fluoride stress is yet to be explored. In the present study, fourteen conserved rice miRNAs with proven role in multiple stress response were analysed to identify differentially expressed miRNAs in response to fluoride toxicity in two popular rice varieties- Gobindobhog (GB; F-tolerant) and IR64 (F-sensitive). Stem-Loop RT-PCR revealed that miR156, miR166, and miR171 were significantly induced in GB seedlings post treatment with fluoride. Likewise, miR160, miR319, miR396, and miR444 were prominently induced in the fluoride-sensitive IR64. Additionally, miR393 was significantly induced post-treatment with fluoride stress in both the genotypes exhibiting a basal response to fluoride toxicity. Further, we computationally predicted the miRNA targets many of which encoded transcription factors associated with stress response mechanism. The miRNA targets were experimentally validated using ligation mediated 5′ rapid amplification of cDNA ends analysis. Quantitative RT-PCR analysis of nine selected miRNA target genes (Os11g30370, Os06g47150, Os06g03670, Os04g48290, Os02g44360, Os08g34380, Os05g05800, Os04g57050, Os04g51350) revealed simultaneous reciprocal changes in the expression patterns of the miRNAs and the corresponding target genes suggesting their involvement in the modulation of fluoride stress response in rice. Analysis of proximal promoter sequences of the F-responsive miRNAs revealed that these miRNAs possess stress-responsive, elicitor and hormonal related motifs. Overall, our results suggest that multiple conserved miRNAs are involved in fluoride toxicity and a miRNA-mediated regulation of signal response is critical for rice response to fluoride stress.

Expression Profile, Regulatory Network, and Putative Role of microRNAs in the Developmental Process of Asian Honey Bee Larval Guts.

MiRNAs, as a kind of key regulators in gene expression, play vital roles in numerous life activities from cellular proliferation and differentiation to development and immunity. However, little is known about the regulatory manner of miRNAs in the development of Asian honey bee (Apis cerana) guts. Here, on basis of our previously gained high-quality transcriptome data, transcriptome-wide identification of miRNAs in the larval guts of Apis cerana cerana was conducted, followed by investigation of the miRNAs' differential expression profile during the gut development. In addition to the regulatory network, the potential function of differentially expressed miRNAs (DEmiRNAs) was further analyzed. In total, 330, 351, and 321 miRNAs were identified in the 4-, 5-, and 6-day-old larval guts, respectively; among these, 257 miRNAs were shared, while 38, 51, and 36 ones were specifically expressed. Sequences of six miRNAs were confirmed by stem-loop RT-PCR and Sanger sequencing. Additionally, in the "Ac4 vs. Ac5" comparison group, there were seven up-regulated and eight down-regulated miRNAs; these DEmiRNAs could target 5041 mRNAs, involving a series of GO terms and KEGG pathways associated with growth and development, such as cellular process, cell part, Wnt, and Hippo. Comparatively, four up-regulated and six down-regulated miRNAs detected in the "Ac5 vs. Ac6" comparison group and the targets were associated with diverse development-related terms and pathways, including cell, organelle, Notch and Wnt. Intriguingly, it was noticed that miR-6001-y presented a continuous up-regulation trend across the developmental process of larval guts, implying that miR-6001-y may be a potential essential modulator in the development process of larval guts. Further investigation indicated that 43 targets in the "Ac4 vs. Ac5" comparison group and 31 targets in the "Ac5 vs. Ac6" comparison group were engaged in several crucial development-associated signaling pathways such as Wnt, Hippo, and Notch. Ultimately, the expression trends of five randomly selected DEmiRNAs were verified using RT-qPCR. These results demonstrated that dynamic expression and structural alteration of miRNAs were accompanied by the development of A. c. cerana larval guts, and DEmiRNAs were likely to participate in the modulation of growth as well as development of larval guts by affecting several critical pathways via regulation of the expression of target genes. Our data offer a basis for elucidating the developmental mechanism underlying Asian honey bee larval guts.

Quantitative analysis of Epstein-Barr virus DNA in plasma and stomach biopsies of patients with gastric cancer.

Epstein-Barr virus (EBV) associated gastric carcinoma (EBVaGC) is a subtype of gastric cancer with distinct histological and molecular features. The study aimed to assess the EBV DNA copy number and the prevalence of EBVaGC in gastric cancer samples taken from Iranian patients. The next aim was to assess whether the DNA and microRNAs EBV are present in plasma. EBV load was analyzed in 68 gastric cancer biopsies and compared with the results of EBV-encoded small RNA in situ hybridization (EBER-ISH) test in these patients. After the detection of 6 EBV miRNAs in gastric tissue by stem-loop RT-PCR, plasma samples were evaluated for the viral load and EBV miRNAs. Four gastric cancer cases were EBER -ISH positive (5.8%), with a significantly higher viral load than the remaining cases, 47,781 vs. 1909 copies/μg of tissue DNA. Here, was also found a significant difference in plasma EBV load between EBER-positive and EBER-negative cases. Although EBV miRNAs were detectable in all the EBER-positive tumors, the test did not detect any of these miRNAs among the plasma samples tested. Our data indicate that the prevalence of EBVaGC among Iranian patients with gastric cancer is lower than the global prevalence and although none of the EBV miRNAs were detected in plasma, evaluation of EBV microRNAs in tumor tissue, especially miR-BART7-3p, may constitute useful biomarkers for diagnosis of EBVaGC.

Ame-miR-34 Modulates the Larval Body Weight and Immune Response of Apis mellifera Workers to Ascosphara apis Invasion.

MiRNAs are critical regulators of numerous physiological and pathological processes. Ascosphaera apis exclusively infects bee larvae and causes chalkbrood disease. However, the function and mechanism of miRNAs in the bee larval response to A. apis infection is poorly understood. Here, ame-miR-34, a previously predicted miRNA involved in the response of Apis mellifera larvae to A. apis invasion, was subjected to molecular validation, and overexpression and knockdown were then conducted to explore the regulatory functions of ame-miR-34 in larval body weight and immune response. Stem-loop RT-PCR and Sanger sequencing confirmed the authenticity of ame-miR-34 in the larval gut of A. mellifera. RT-qPCR results demonstrated that compared with that in the uninfected larval guts, the expression level of ame-miR-34 was significantly downregulated (p < 0.001) in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae, indicative of the remarkable suppression of host ame-miR-34 due to A. apis infection. In comparison with the corresponding negative control (NC) groups, the expression level of ame-miR-34 in the larval guts in the mimic-miR-34 group was significantly upregulated (p < 0.001), while that in the inhibitor-miR-34 group was significantly downregulated (p < 0.01). Similarly, effective overexpression and knockdown of ame-miR-34 were achieved. In addition, the body weights of 5- and 6-day-old larvae were significantly increased compared with those in the mimic-NC group; the weights of 5-day-old larvae in the inhibitor-miR-34 group were significantly decreased in comparison with those in the inhibitor-NC group, while the weights of 4- and 6-day-old larvae in the inhibitor-miR-34 group were significantly increased, indicating the involvement of ame-miR-34 in modulating larval body weight. Furthermore, the expression levels of both hsp and abct in the guts of A. apis-infected 4-, 5-, and 6-day-old larvae were significantly upregulated after ame-miR-34 overexpression. In contrast, after ame-miR-34 knockdown, the expression levels of the aforementioned two key genes in the A. apis-infected 4-, 5-, and 6-day-old larval guts were significantly downregulated. Together, the results demonstrated that effective overexpression and knockdown of ame-miR-34 in both noninfected and A. apis-infected A. mellifera larval guts could be achieved by the feeding method, and ame-miR-34 exerted a regulatory function in the host immune response to A. apis invasion through positive regulation of the expression of hsp and abct. Our findings not only provide a valuable reference for the functional investigation of bee larval miRNAs but also reveal the regulatory role of ame-miR-34 in A. mellifera larval weight and immune response. Additionally, the results of this study may provide a promising molecular target for the treatment of chalkbrood disease.

First Identification and Investigation of piRNAs in the Larval Gut of the Asian Honeybee, Apis cerana.

Piwi-interacting RNAs (piRNAs), a class of small non-coding RNAs (ncRNAs), play pivotal roles in maintaining the genomic stability and modulating biological processes such as growth and development via the regulation of gene expression. However, the piRNAs in the Asian honeybee (Apis cerana) are still largely unknown at present. In this current work, on the basis of previously gained high-quality small RNA-seq datasets, piRNAs in the larval gut of Apis cerana cerana, the nominated species of A. cerana, were identified for the first time, followed by an in-depth investigation of the regulatory roles of differentially expressed piRNAs (DEpiRNAs) in the developmental process of the A. c. cerana. Here, a total of 621 piRNAs were identified in A. c. cerana larval guts, among which 499 piRNAs were shared by 4-(Ac4 group), 5-(Ac5 group), and 6-day-old (Ac6 group) larval guts, while the numbers of unique ones equaled 79, 37, and 11, respectively. The piRNAs in each group ranged from 24 nucleotides (nt) to 33 nt in length, and the first base of the piRNAs had a cytosine (C) bias. Additionally, five up-regulated and five down-regulated piRNAs were identified in the Ac4 vs. Ac5 comparison group, nine of which could target 9011 mRNAs; these targets were involved in 41 GO terms and 137 pathways. Comparatively, 22 up-regulated piRNAs were detected in the Ac5 vs. Ac6 comparison group, 21 of which could target 28,969 mRNAs; these targets were engaged in 46 functional terms and 164 pathways. The results suggested an overall alteration of the expression pattern of piRNAs during the developmental process of A. c. cerana larvae. The regulatory network analysis showed that piR-ace-748815 and piR-ace-512574 in the Ac4 vs. Ac5 comparison group as well as piR-ace-716466 and piR-ace-828146 in the Ac5 vs. Ac6 comparison group were linked to the highest number of targets. Further investigation indicated that targets of DEpiRNAs in the abovementioned two comparison groups could be annotated to several growth and development-associated pathways, such as the Jak/STAT, TGF-β, and Wnt signaling pathways, indicating the involvement of DEpiRNAs in modulating larval gut development via these crucial pathways. Moreover, the expression trends of six randomly selected DEpiRNAs were verified using a combination of stem-loop RT-PCR and RT-qPCR. These results not only provide a novel insight into the development of the A. c. cerana larval gut, but also lay a foundation for uncovering the epigenetic mechanism underlying larval gut development.

Functional conservation and divergence of miR156 and miR529 during rice development

MicroRNAs (miRNAs) are important regulatory elements involved in the regulation of various plant developmental and physiological processes by blocking the expression of target genes. MiR156 and miR529 are two combinatorial regulators, which cooperatively target the SQUAMOSA PROMOTER BINDING-LIKE (SPL) family genes. However, there has been no report about the functional conservation and divergence of miR156 and miR529 during plant development to date. In this study, the biological function and relationship of miR156, miR529 and their target OsSPL14 in rice were explored. Overexpression of miR156e or miR529a (miR156e-OE and miR529a-OE) increased the grain size and tiller number but decreased the plant height and panicle length, while an opposite phenotype was observed for their target mimicry (miR156-MIMIC and miR529a-MIMIC) transgenic plants. Stem-loop RT-PCR results revealed ubiquitous expression of miR156 in roots, axillary buds and leaves, while miR529 was preferentially expressed in the panicle. Accordingly, OsSPL14 could be preferentially and precisely cleaved by miR529a in young panicle but by miR156 in vegetative tissues. Transgenic plants generated by the target immune strategy exhibited obvious growth defects upon the blocking of miR156 and/or miR529 function in rice, confirming that both miR156 and miR529 play important roles in controlling rice growth and development. Moreover, the miR156/miR529-OsSPL14 module negatively controlled grain size by regulating the genes associated with grain size and cell cycling, and controlled plant height through a more complicated mechanism. Taken together, our results demonstrate that miR156 and miR529 respectively function dominantly in the vegetative stage and reproductive stage to control rice growth and development by regulating the accumulation of OsSPL14. These findings facilitate a better understanding of the functional conservation and divergence of miR156 and miR529 family in the miRNA combinatorial regulatory network of plants.

Transcriptome-Wide Characterization of piRNAs during the Developmental Process of European Honey-Bee Larval Guts.

piRNAs play pivotal roles in maintaining genome stability, regulating gene expression, and modulating development and immunity. However, there are few piRNA-associated studies on honey-bees, and the regulatory role of piRNAs in the development of bee guts is largely unknown. Here, the differential expression pattern of piRNAs during the developmental process of the European honey-bee (Apis mellifera) larval guts was analyzed, followed by investigation of the regulatory network and the potential function of differentially expressed piRNAs (DEpiRNAs) in regulating gut development. A total of 843 piRNAs were identified in the larval guts of A. mellifera; among these, 764 piRNAs were shared by 4- (Am4 group), 5- (Am5 group), and 6-day-old (Am6 group) larval guts, while 11, 67, and one, respectively, were unique. The first base of piRNAs in each group had a cytosine (C) bias. Additionally, 61 up-regulated and 17 down-regulated piRNAs were identified in the “Am4 vs. Am5” comparison group, further targeting 9, 983 genes, which were involved in 50 GO terms and 142 pathways, while two up-regulated and five down-regulated piRNAs were detected in the “Am5 vs. Am6” comparison group, further targeting 1, 936 genes, which were engaged in 41 functional terms and 101 pathways. piR-ame-742536 and piR-ame-856650 in the “Am4 vs. Am5” comparison group as well as piR-ame-592661 and piR-ame-31653 in the “Am5 vs. Am6” comparison group were found to link to the highest number of targets. Further analysis indicated that targets of DEpiRNAs in these two comparison groups putatively regulate seven development-associated signaling pathways, seven immune-associated pathways, and three energy metabolism pathways. Moreover, the expression trends of five randomly selected DEpiRNAs were verified based on stem-loop RT-PCR and RT-qPCR. These results were suggestive of the overall alteration of piRNAs during the larval developmental process and demonstrated that DEpiRNAs potentially modulate development-, immune-, and energy metabolism-associated pathways by regulating the expression of corresponding genes via target binding, further affecting the development of A. mellifera larval guts. Our data offer a novel insight into the development of bee larval guts and lay a basis for clarifying the underlying mechanisms.

Abstract 2212: Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis

Abstract Breast cancer (BC) is the most common non-cutaneous malignancy and the second leading cause of cancer-related death in American women. Health disparities in incidence and clinical outcome are also reported among different racial groups, most notably African American (AA) women, who are often diagnosed at a young age with aggressive BC and exhibit greater mortality than Caucasian American (CA) women. Since socioeconomic difficulties can have tremendous impact on psychophysiology besides limiting the access to optimal healthcare, we examined the serum levels of stress (cortisol) and satiety (leptin) hormones as well as inflammatory cytokines (resistin and interleukin-6/IL-6) in AA and CA women. To observe a potential epigenomic connection, we also performed profiling of a targeted set of exosomal microRNAs in serum samples. The study was conducted under an Institutional Review Board (IRB) approved protocol. All subjects participated voluntarily, and their consents were obtained. Serum levels of resistin, IL-6, leptin, and cortisol were quantified by Enzyme linked Immunosorbent Assay (ELISA) using commercial kits. Exosomes were isolated using precipitation method and recovered by standard centrifugation. Total RNA was isolated from exosomal preps and subjected to stem-loop RT-PCR for quantitation of a set of inflammation-associated microRNAs. We found that the levels of resistin, leptin, IL-6 and cortisol were higher in women with a BC diagnosis than non-BC subjects. Moreover, AA women with or without BC showed significantly higher levels of these cytokines and hormones in their serum as compared to the CA women with or without a BC diagnosis, respectively. We also observed differential expression of several microRNAs in serum of BC women as compared to their normal counterparts, of which five (miR511, miR33a, miR27a, miR6794, miR143-3p) exhibited highest presence in serum exosomes of AA women with BC. Together, these findings suggest that relatively greater exposure of minority women to social stressors may have epigenomic consequences and may potentially be linked to the observed BC racial health disparities. Citation Format: Sarabjeet Kour Sudan, Kunwar Somesh Vikramdeo, Amod Sharma, Sachin Kumar Deshmukh, Sanjeev Kumar Srivastava, Teja Poosarla, Nicolette P. Holliday, Donna L. Dyess, Ajay P. Singh, Seema Singh. Racially disparate serum levels of inflammatory cytokines, satiety and stress hormones, and exosomal microRNAs in women with or without a breast cancer diagnosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2212.

RNAi-mediated down-regulation of fasciclin-like proteins (FoFLPs) in Fusarium oxysporum f. sp. lycopersici results in reduced pathogenicity and virulence

Fusarium oxysporum f. sp. lycopersici (Fol) is majorly responsible for causing vascular wilt disease in tomato by blocking transpirational pull, thereby interfering and suppressing overall host immune response. This is mainly achieved by fungal invasion and colonization within the host, resulting in hyphal formation that instigates secondary infection response inside the plant. Earlier reports show the role of fasciclin-like proteins (FLPs) in cell-to-cell adhesions and signaling cascade. Moreover, deletion mutant of FLPs explained its role in development stages of Magnaporthe oryzae and Lentula edodes. Therefore, in present study, based on bioinformatic analysis, we have identified putative FoFLP genes encoding Fusarium-specific fasciclin like proteins. We have exploited the RNAi technology to analyse and understand role of these FoFLPs in virulence during wilt disease in tomato. Interestingly, the morphogenesis of fungal RNAi transformants of FoFLP1, FoFLP3, FoFLP4 and FoFLP5 showed significant reduction in spore count and spore germination frequency, thereby suggesting role of FoFLPs in conidiation. Furthermore, we have detected FoFLP1, FoFLP3, FoFLP4 and FoFLP5 specific siRNAs in the respective fungal transformants, using stem-loop RT-PCR and northern hybridization suggesting targeting of cognate FoFLPs transcripts. Moreover, the fruit invasion and plant infection assays using FoFLP fungal transformants showed late onset of disease with significant reduction in disease symptoms in the infected plants. Although the FoFLP-RNAi fungal transformants entered the host plant by penetrating root cortex, the infected plants showed minimum fungal colonization as a result of siRNA-mediated targeting of endogenously expressed FoFLPs. This further inhibited the propagation of fungal RNAi transformants within root cortex and affected its ability to cause secondary infection response, which led to reduced disease incidence and disease severity index. Altogether, our results show that FoFLPs might play an important role in conidiation and pathogenicity, and can serve as potent RNAi targets. Therefore, Host-Induced Gene Silencing (HIGS) can effectively be used to target FoFLPs in order to raise tomato transgenics resistant to Fusarium wilt.

Set of miRNAs Involved in Sulfur Uptake and the Assimilation Pathway of Indian Mustard (B. juncea) in Response to Sulfur Treatments.

MicroRNAs (miRNAs) play an important role in the regulation of gene expression. They play a regulatory role in various nutrient assimilatory pathways of plants; however, their role in the regulation of sulfur uptake and assimilatory pathways in mustard cultivars under high/low sulfur conditions is not elucidated. Sulfur is essential for plant growth and development, and its deficiency can cause a decline in oil seed content and thus lower the economic yield in Brassica juncea. In this study, different miRNAs involved in the regulation of sulfur uptake and assimilation pathways in B. juncea were identified using a psRNA target analyzer and miRanda database tools. The predicted miRNAs that belong to 10 highly conserved families were validated using stem-loop RT-PCR. The B. juncea cultivars Pusa Jaikisan, Pusa Bold, and Varuna were kept in sulfur-excessive (high) and -deficient (insufficient) conditions, and expression studies of miRNAs and their target mRNAs were carried out using qRT-PCR. The correlation between the expression pattern of miRNAs and their target genes showed their potential role in sulfur uptake and assimilation. Analysis with 5′ RACE revealed the authentic target of miRNAs. The influence of S treatments on metabolites and sulfur content was also studied using GC-MS and a CHNS analyzer. Our study showed the potential role of miRNAs in the regulation of sulfur uptake and assimilation and put forward the implications of these molecules to enhance the sulfur content of B. juncea.

A cypovirus encoded microRNA negatively regulates the NF-κB pathway to enhance viral multiplication in Silkworm, Bombyx mori

MicroRNAs (miRNAs) are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level. Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes, little is known about RNA virus-encoded miRNAs. Bombyx mori cypovirus (BmCPV) is a double-stranded RNA virus with a segmented genome that causes cytoplasmic polyhedrosis disease in silkworm larvae. To date, the interaction between BmCPV and silkworm remains largely unclear. 22 candidate BmCPV-encoded miRNAs were identified in this study through small RNA sequencing, stem-loop RT-PCR and qRT-PCR. Then, generation and function analyses were conducted on one of the candidate miRNAs, BmCPV-miR-1, in the BmN cells and the silkworm larvae by RNA interference, quantitative PCR, dual-luciferase assay. Our results revealed that BmCPV-miR-1 was encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome. Its generation depended on Dicer-1 and might also be correlated with Dicer-2, Argonaute-1 and Argonaute-2. Moreover, BmCPV-miR-1 could suppress the expression of the target gene, B. mori inhibitor of nuclear factor kappa-B kinase subunit beta (BmIKKβ), via binding to the target mRNA 3′-untranslated region, which fine-tuned the host NF-κB signaling pathway and consequently enhanced viral replication. Our results provide new evidence supporting the hypothesis that RNA viruses could generate miRNAs to modulate antiviral host defense.

Detection and Quantification of Small Noncoding RNAs in Marine Diatoms.

Endogenous small noncoding RNAs (sRNAs) are a large family of essential regulators of gene expression in both eukaryotes and prokaryotes. Various types of sRNAs with different size and mapping to different genome locations have been recently identified in diatoms, a successful group of phytoplankton in the marine environment. However, their biogenesis and regulatory function are still largely unknown and unexplored in these microalgae, also due to the lack of methods for their experimental analysis. Herein, we present a point-by-point description of the protocols for detection and quantification of sRNAs by Northern-blot analysis and quantitative stem-loop RT-PCR, established in the diatom molecular model specie Phaeodactylum tricornutum.

Host-mediated attenuation of gut sucrase in mustard aphid Lipaphis erysimi impaired its parthenogenetic reproduction on Indian mustard Brassica juncea.

The nefarious hemipteran mustard aphid (Lipaphis erysimi) inflicts colossal yield losses in Brassica crops including Indian mustard (Brassica juncea). Lack of an accessible resistance source has been the primary impediment in breeding varietal resistance against aphids. In recent years, in planta RNAi-mediated resistance has been demonstrated in model plants as a potential tool for protection against insect pests. However, translational application in crop species is imperative for critical assessment of this technology in breeding effective resistance. The essential role of sucrase 1 (SUC1) in mitigating osmotic pressure imposed by sucrose-rich phloem sap inside the insect gut is corroborated by its expression pattern in L.erysimi. Transgenic lines of Indian mustard were developed expressing SUC1 hairpin RNA for its host-mediated delivery into the infesting aphids. The expression of the dsRNA encoding cassette, and generation of siRNA molecules in transgenic B.juncea lines were verified by quantitative reverse transcription (RT)-PCR, stem-loop RT-PCR and Northern hybridization. Rearing of L.erysimi on the transgenic lines resulted in 22-40% reduction in aphid fecundity. The observed retardation in aphid reproduction was coherent with the detection of SUC1-specific siRNA molecules and attenuation of the SUC1 transcript level in L.erysimi fed on the transgenic lines. Augmenting varietal resistance can substantially reduce usage of toxic agrochemicals in crop protection. This attempt was the first successful demonstration of host-mediated RNAi of an aphid gene in any Brassica crop. It paves the way for more rigorous attempt of engineering RNAi-based resistance against aphids in Brassica crops. © 2021 Society of Chemical Industry.

RNAi-mediated silencing of PEX6 and GAS1 genes of Fusarium oxysporum f. sp. lycopersici confers resistance against Fusarium wilt in tomato.

The online version contains supplementary material available at 10.1007/s13205-021-02973-8.

Identification of microRNA-like RNAs from Trichoderma asperellum DQ-1 during its interaction with tomato roots using bioinformatic analysis and high-throughput sequencing

MicroRNA-like small RNAs (milRNAs) and their regulatory roles in the interaction between plant and fungus have recently aroused keen interest of plant pathologists. Trichoderma spp., one of the widespread biocontrol fungi, can promote plant growth and induce plant disease resistance. To investigate milRNAs potentially involved in the interaction between Trichoderma and tomato roots, a small RNA (sRNA) library expressed during the interaction of T. asperellum DQ-1 and tomato roots was constructed and sequenced using the Illumina HiSeqTM 2500 sequencing platform. From 13,464,142 sRNA reads, we identified 21 milRNA candidates that were similar to other known microRNAs in the miRBase database and 22 novel milRNA candidates that possessed a stable microRNA precursor hairpin structure. Among them, three milRNA candidates showed different expression level in the interaction according to the result of stem-loop RT-PCR indicating that these milRNAs may play a distinct regulatory role in the interaction between Trichoderma and tomato roots. The potential transboundary milRNAs from T. asperellum and their target genes in tomato were predicted by bioinformatics analysis. The results revealed that several interesting proteins involved in plant growth and development, disease resistance, seed maturation, and osmotic stress signal transduction might be regulated by the transboundary milRNAs. To our knowledge, this is the first report of milRNAs taking part in the process of interaction of T. asperellum and tomato roots and associated with plant promotion and disease resistance. The results might be useful to unravel the mechanism of interaction between Trichoderma and tomato.

Trans-species small RNAs move long distances in a parasitic plant complex.

Parasitic plants exchange various types of RNAs with their host plants, including mRNA, and small non-coding RNA. Among small non-coding RNAs, miRNA production is known to be induced at the haustorial interface. The induced miRNAs transfer to the host plant and activate secondary siRNA production to silence target genes in the host. In addition to interfacial transfer, long-distance movement of the small RNAs has also been known to mediate signaling and regulate biological processes. In this study, we tested the long-distance movement of trans-species small RNAs in a parasitic-plant complex. Small RNA-Seq was performed using a complex of a stem parasitic plant, Cuscuta campestris, and a host, Arabidopsis thaliana. In the host plant's parasitized stem, genes involved in the production of secondary siRNA, AtSGS3 and AtRDR6, were upregulated, and 22-nt small RNA was enriched concomitantly, suggesting the activation of secondary siRNA production. Stem-loop RT-PCR and subsequent sequencing experimentally confirmed the mobility of the small RNAs. Trans-species mobile small RNAs were detected in the parasitic interface and also in distant organs. To clarify the mode of long-distance translocation, we examined whether C. campestris-derived small RNA moves long distances in A. thaliana sgs3 and rdr6 mutants or not. Mobility of C. campestris-derived small RNA in sgs3 and rdr6 mutants suggested the occurrence of direct long-distance transport without secondary siRNA production in the recipient plant.

Topical application of double-stranded RNA molecules deriving from Tomato yellow leaf curl virus reduces cognate virus infection in tomato

Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted single-stranded DNA (ssDNA) virus, causes the most important viral disease of tomato worldwide. TYLCV-mediated disease is mainly controlled via extensive insecticide sprays aiming at the whitefly vector. RNA-based vaccination was proven to be a non-transgenic approach leading to efficient plant virus control. In this work, double-stranded RNA (dsRNA) molecules deriving from sequences of the C4 and V2 genes of TYLCV-Mild were produced in vitro and topically applied onto tomato plants along with the virus (via agroinfiltration). DsC4 and dsV2 application reduced disease incidence to 23 and 46 %, respectively, while TYLCV positive control reached 64 %. Bioinformatics analysis of the virus-specific small interfering RNAs (vsiRNAs) from TYLCV-infected tomato revealed 'hot' and 'cold' spots in the TYLCV-Mild genome. Interestingly, the viral C-strand had twofold siRNA reads when compared to that of the V-strand. Overall, vsiRNAs of negative and positive polarity were almost equal (53.5 vs. 46.6 %); vsiRNAs of negative polarity prevailed at the V-strand. Stem-loop RT-PCR validated the presence of six vsiRNAs (hot or cold spots) in TYLCV-Mild-infected and dsRNA-treated tomato. The exogenously applied dsRNA was found to rapidly move systemically in tomato and was detected for 54 days post treatment (dpt). The applied dsRNA molecules were successfully processed by the Dicer-like proteins (DCLs) in tomato since small interfering RNAs (siRNAs) deriving from the dsRNA were detected for at least 54 dpt. This consists the first report of dsRNA-based vaccination applied against a monopartite geminivirus.