Viral Small-RNA Analysis of Bombyx mori Larval Midgut during Persistent and Pathogenic Cytoplasmic Polyhedrosis Virus Infection.

UNLABELLED: The lepidopteran innate immune response against RNA viruses remains poorly understood, while in other insects several studies have highlighted an essential role for the exo-RNAi pathway in combating viral infection. Here, by using deep-sequencing technology for viral small-RNA (vsRNA) assessment, we provide evidence that exo-RNAi is operative in the silkworm Bombyx mori against both persistent and pathogenic infection of B. mori cytoplasmic polyhedrosis virus (BmCPV) which is characterized by a segmented double-stranded RNA (dsRNA) genome. Further, we show that Dicer-2 predominantly targets viral dsRNA and produces 20-nucleotide (nt) vsRNAs, whereas an additional pathway is responsive to viral mRNA derived from segment 10. Importantly, vsRNA distributions, which define specific hot and cold spot profiles for each viral segment, to a considerable degree overlap between Dicer-2-related (19 to 21 nt) and Dicer-2-unrelated vsRNAs, suggesting a common origin for these profiles. We found a degenerate motif significantly enriched at the cut sites of vsRNAs of various lengths which link an unknown RNase to the origins of vsRNAs biogenesis and distribution. Accordingly, the indicated RNase activity may be an important early factor for the host's antiviral defense in Lepidoptera. IMPORTANCE: This work contributes to the elucidation of the lepidopteran antiviral response against infection of segmented double-stranded RNA (dsRNA) virus (CPV; Reoviridae) and highlights the importance of viral small-RNA (vsRNA) analysis for getting insights into host-pathogen interactions. Three vsRNA pathways are implicated in antiviral defense. For dsRNA, two pathways are proposed, either based on Dicer-2 cleavage to generate 20-nucleotide vsRNAs or based on the activity of an uncharacterized endo-RNase that cleaves the viral RNA substrate at a degenerate motif. The analysis also indicates the existence of a degradation pathway that targets the positive strand of segment 10.

Phytochemical Profile and Evaluation of the Antioxidant, Cyto-Genotoxic, and Antigenotoxic Potential of Salvia verticillata Hydromethanolic Extract.

This study comprises the phytochemical characterization, the evaluation of the total phenolic content (TPC) and antioxidant activity (AA), and the investigation of the cyto-genotoxic and antigenotoxic potential of hydromethanolic extract derived from Salvia verticillata L. leaves. HPLC-DAD-ESI-MS and HPLC-DAD were used for the characterization of the extract and determination of the major ingredients. Afterwards, the TPC and AA were determined. The cytotoxic and genotoxic effect of the extract on cultured human lymphocytes at concentrations of 10, 25, and 50 µg mL-1 was investigated via the Cytokinesis Block MicroNucleus (CBMN) assay. Moreover, its antigenotoxic potential against the mutagenic agent mitomycin C (MMC) was assessed using the same assay. The hydromethanolic extract comprises numerous metabolites, with rosmarinic acid being the major compound. It had a high value of TPC and exerted significant AA as shown by the results of the Ferric Reducing Antioxidant Power (FRAP) and Radical Scavenging Activity by DPPH• assays. A dose-dependent cytotoxic potential was recorded, with the highest dose (50 µg mL-1) exhibiting statistically significant cytotoxicity. None of the tested concentrations induced significant micronuclei (MN) frequencies, indicating a lack of genotoxicity. All tested concentrations reduced the MMC-mediated genotoxic effects, with the two lowest showing statistically significant antigenotoxic potential.

Two new infraspecific taxa of Verbascum delphicum (Scrophulariaceae, Scrophularieae) from mainland Greece and the island of Evvia.

Verbascum delphicum Boiss. & Heldr. subsp. cervi Zografidis (Scrophulariaceae, Scrophularieae) is described as a subspecies new to science and illustrated. It is narrowly distributed in the Greek National Park of Mt Parnitha (Attica, Greece) with a very small population size. The new subspecies is a seldom-collected taxon, previously overlooked and misidentified as consubspecific with the autonymous subspecies, an endemic of the island of Evvia (Greece). Also described in this study is a new variety of subsp. delphicum from Mt Ochi of southern Evvia.

APRF1 promotes flowering under long days in Arabidopsis thaliana.

Arabidopsis thaliana flowering time mutants revealed the function of numerous genes that regulate the transition from vegetative to reproductive growth. Analyses of their loci have shown that many of them act as chromatin modifiers. In this study, a combination of molecular and genetic approaches have been implemented, to characterize the function of APRF1 (ANTHESIS POMOTING FACTOR 1) gene in A. thaliana and to investigate its role in plant development. APRF1 encodes for a low molecular weight nuclear WDR protein which displays functional homology to the Swd2 protein, an essential subunit of the yeast histone methylation COMPASS complex. Compared to WT plants, total loss-of-function aprf1 mutants exhibited shoot apical meristem (SAM) alterations and increased growth rates. However, the vegetative phase of aprf1 plants was prolonged and bolting was delayed, indicating an impairment in flowering under long days (LD). On the contrary, overexpression of APRF1 accelerates flowering. Consistent with the late flowering phenotype, the molecular data confirmed that FLC and SOC1 expression were significantly altered in the aprf1 mutants. Our data suggest that APRF1 acts upstream of FLC and promotes flowering under LD.

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery.

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

Transcriptional regulation and functional involvement of the Arabidopsis pescadillo ortholog AtPES in root development.

The Pescadillo gene is highly conserved from yeasts to human and has been shown to impact on both the cell cycle and on ribosome biogenesis. However, the biological function and transcriptional regulation of the plant orthologs remain unclear. In the present study, we have implemented a combination of molecular and genetic approaches, in order to characterize the Arabidopsis thaliana pescadillo ortholog (AtPES) and its role in root development. The RNAi transgenic lines displayed severely compromised meristem structures and a reduction of the primary root length of up to 70%. The correct pattern of the cell files is distorted, whereas in the root elongation and differentiation zone the epidermal and cortex cells appear abnormally enlarged. Yeast two hybrid and BiFC experiments confirmed that AtPES interacts physically with AtPEIP1 and AtPEIP2, the orthologs of the murine Bop1 and WDR12. Promoter deletion analysis revealed that AtPES expression depends on a number of transcription factor binding sites, with the TELO-box being a crucial site for regulating its accurate tissue-specific manifestation. Our results indicate that AtPES is firmly regulated at the transcriptional level and that the corresponding protein plays a role in root developmental processes.