Fluorescence-tunable Ag-DNA biosensor with tailored cytotoxicity for live-cell applications

DNA-stabilized silver clusters (Ag-DNA) show excellent promise as a multi-functional nanoagent for molecular investigations in living cells. The unique properties of these fluorescent nanomaterials allow for intracellular optical sensors with tunable cytotoxicity based on simple modifications of the DNA sequences. Three Ag-DNA nanoagent designs are investigated, exhibiting optical responses to the intracellular environments and sensing-capability of ions, functional inside living cells. Their sequence-dependent fluorescence responses inside living cells include (1) a strong splitting of the fluorescence peak for a DNA hairpin construct, (2) an excitation and emission shift of up to 120 nm for a single-stranded DNA construct, and (3) a sequence robust in fluorescence properties. Additionally, the cytotoxicity of these Ag-DNA constructs is tunable, ranging from highly cytotoxic to biocompatible Ag-DNA, independent of their optical sensing capability. Thus, Ag-DNA represents a versatile live-cell nanoagent addressable towards anti-cancer, patient-specific and anti-bacterial applications.

Highly activated RNA silencing via strong induction of dicer by one virus can interfere with the replication of an unrelated virus

Viruses often coinfect single host organisms in nature. Depending on the combination of viruses in such coinfections, the interplay between them may be synergistic, apparently neutral with no effect on each other, or antagonistic. RNA silencing is responsible for many cases of interference or cross-protection between viruses, but such antagonistic interactions are usually restricted to closely related strains of the same viral species. In this study, we present an unprecedented example of RNA silencing-mediated one-way interference between unrelated viruses in a filamentous model fungus,Cryphonectria parasitica. The replication of Rosellinia necatrix victorivirus 1 (RnVV1;Totiviridae) was strongly impaired by coinfection with the prototypic member of the genusMycoreovirus(MyRV1) or a mutant of the prototype hypovirus (Cryphonectria hypovirus 1, CHV1) lacking the RNA silencing suppressor (CHV1-Δp69). This interference was associated with marked transcriptional induction of key genes in antiviral RNA silencing, dicer-like 2 (dcl2) and argonaute-like 2 (agl2), following MyRV1 or CHV1-Δp69 infection. Interestingly, the inhibition of RnVV1 replication was reproduced when the levels ofdcl2andagl2transcripts were elevated by transgenic expression of a hairpin construct of an endogenousC. parasiticagene. Disruption ofdcl2completely abolished the interference, whereas that ofagl2did not always lead to its abolishment, suggesting more crucial roles ofdcl2in antiviral defense. Taken altogether, these results demonstrated the susceptible nature of RnVV1 to the antiviral silencing inC. parasiticaactivated by distinct viruses or transgene-derived double-stranded RNAs and provide insight into the potential for broad-spectrum virus control mediated by RNA silencing.

A High Level of Transgenic Viral Small RNA Is Associated with Broad Potyvirus Resistance in Cucurbits

Gene-silencing has been used to develop resistance against many plant viruses but little is known about the transgenic small-interfering RNA (t-siRNA) that confers this resistance. Transgenic cucumber and melon lines harboring a hairpin construct of the Zucchini yellow mosaic potyvirus (ZYMV) HC-Pro gene accumulated different levels of t-siRNA (6 to 44% of total siRNA) and exhibited resistance to systemic ZYMV infection. Resistance to Watermelon mosaic potyvirus and Papaya ring spot potyvirus-W was also observed in a cucumber line that accumulated high levels of t-siRNA (44% of total siRNA) and displayed significantly increased levels of RNA-dependent RNA (RDR)1 and Argonaute 1, as compared with the other transgenic and nontransformed plants. The majority of the t-siRNA sequences were 21 to 22 nucleotides in length and sense strand biased. The t-siRNA were not uniformly distributed throughout the transgene but concentrated in “hot spots” in a pattern resembling that of the viral siRNA peaks observed in ZYMV-infected cucumber and melon. Mutations in ZYMV at the loci associated with the siRNA peaks did not break this resistance, indicating that hot spot t-siRNA may not be essential for resistance. This study shows that resistance based on gene-silencing can be effective against related viruses and is probably correlated with t-siRNA accumulation and increased expression of RDR1.

Cpkk1, MAPKK of Cryphonectria parasitica, Is Necessary for Virulence on Chestnut

The role of Cpkk1, a mitogen-activated protein kinase from Cryphonectria parasitica, was investigated by generating a number of mutant strains that overexpress, under the control of the cryparin promoter, both the wild-type protein and its allele with an extensive deletion in the catalytic domain. Furthermore, a hairpin construct was built and expressed to cause specific silencing of Cpkk1 mRNA transcripts. Specific mRNA silencing or overexpression was confirmed on both Northern and Western blot analysis. Selected C. parasitica strains with Cpkk1 either silenced or overexpressed were evaluated for their biological characteristics, including virulence on European chestnut, growth on different substrates, conidial sporulation, and resistance to cell-wall-degrading enzymes. Silencing of Cpkk1 and the overexpression of a defective Cpkk1 correlated with a marked reduction in virulence on 3-year-old chestnut trees, with no statistically significant effect on fungal growth in the various conditions tested.

Tomato Chlorotic Mottle Virus Is a Target of RNA Silencing but the Presence of Specific Short Interfering RNAs Does Not Guarantee Resistance in Transgenic Plants

ABSTRACT Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5′ end of AC1 and the embedded AC4, the intergenic region and 5′ end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.