Double-Stranded RNA Uptake through Topical Application,Mediates Silencing of Five CYP4 Genes and Suppresses Insecticide Resistance in Diaphorina citri

Silencing of genes through RNA interference (RNAi) in insects has gained momentum during the past few years. RNAi has been used to cause insect mortality, inhibit insect growth, increase insecticide susceptibility, and prevent the development of insecticide resistance. We investigated the efficacy of topically applied dsRNA to induce RNAi for five Cytochrome P₄₅₀ genes family 4 (CYP4) in Diaphorina citri. We previously reported that these CYP4 genes are associated with the development of insecticide resistance in D. citri. We targeted five CYP4 genes that share a consensus sequence with one dsRNA construct. Quantitative PCR confirmed suppressed expression of the five CYP4 genes as a result of dsRNA topically applied to the thoracic region of D. citri when compared to the expression levels in a control group. Western blot analysis indicated a reduced signal of cytochrome P₄₅₀ proteins (45 kDa) in adult D. citri treated with the dsRNA. In addition, oxidase activity and insecticide resistance were reduced for D. citri treated with dsRNA that targeted specific CYP4 genes. Mortality was significantly higher in adults treated with dsRNA than in adults treated with water. Our results indicate that topically applied dsRNA can penetrate the cuticle of D. citri and induce RNAi. These results broaden the scope of RNAi as a mechanism to manage pests by targeting a broad range of genes. The results also support the application of RNAi as a viable tool to overcome insecticide resistance development in D. citri populations. However, further research is needed to develop grower-friendly delivery systems for the application of dsRNA under field conditions. Considering the high specificity of dsRNA, this tool can also be used for management of D. citri by targeting physiologically critical genes involved in growth and development.     

Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca²⁺) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca²⁺ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca²⁺-ATPase (SERCA) which is involved in the regulation of the intracellular Ca²⁺ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.     

The RNA-binding protein Mex3B is a coreceptor of Toll-like receptor 3 in innate antiviral response

Recognition of viral dsRNA by Toll-like receptor 3 (TLR3) leads to induction of interferons (IFNs) and proinflammatory cytokines, and innate antiviral response. Here we identified the RNA-binding protein Mex3B as a positive regulator of TLR3-mediated signaling by expression cloning screens. Cells from Mex3b^−/− mice exhibited reduced production of IFN-β in response to the dsRNA analog poly(I:C) but not infection with RNA viruses. Mex3b^−/− mice injected with poly(I:C) was more resistant to poly(I:C)-induced death. Mex3B was associated with TLR3 in the endosomes. It bound to dsRNA and increased the dsRNA-binding activity of TLR3. Mex3B also promoted the proteolytic processing of TLR3, which is critical for its activation. Mutants of Mex3B lacking its RNA-binding activity inhibited TLR3-mediated IFN-β induction. These findings suggest that Mex3B acts as a coreceptor of TLR3 in innate antiviral response.     

Isolation and amplification of cDNA from the conserved region of the nematode Heterodera schachtii 8H07 gene with a close similarity to its homolog in rape plants

An original method was developed for isolation of small regulatory RNAs (si/miRNAs) from plant cells. PCR-amplification was carried out for the cDNA fragment of the nematode Heterodera schachtii 8H07 gene. Northern-blot hybridization of plant si/miRNAs with the cDNA fragment of the conserved region from the nematode??s 8H07 gene confirmed the high degree of their homology. In the future, the amplified cDNA fragment of the nematode 8H07 gene will be used for creating a recombinant gene with an antisense dsRNA sequence for increasing the resistance of rape plants to parasitic nematodes.     

Immunosuppression induced by entomopathogens is rescued by addition of apolipophorin III in the diamondback moth, Plutella xylostella

Apolipophorin III (ApoLpIII) has been known to play critical roles in lipid transport and immune activation in insects. This study reports a partial ApoLpIII gene cloned from the diamondback moth, Plutella xylostella. It showed that the gene was expressed in all developmental stages of P. xylostella. In larval stage, it was expressed in all tested tissues of hemocyte, fat body, gut, and epidermis. In response to bacterial challenge, the larvae showed an enhanced level of ApoLpIII expression by a quantitative real-time RT-PCR. RNA interference of ApoLpIII by its specific double stranded RNA (dsRNA) caused significant knockdown of its expression level and resulted in significant suppression in hemocyte nodule formation in response to bacterial challenge. However, larvae treated with the dsRNA exhibited a significant recovery in the cellular immune response by addition of a recombinant ApoLpIII. Parasitization by an endoparasitoid wasp, Cotesia plutellae, suppressed expression of ApoLpIII and resulted in a significant suppression in the hemocyte nodule formation. The addition of the recombinant ApoLpIII to the parasitized larvae significantly restored the hemocyte activity. Infection of an entomopathogenic bacterium, Xenorhabdus nematophila, caused potent pathogenicity of P. xylostella. However, the addition of the recombinant ApoLpIII to the infected larvae significantly prevented the lethal pathogenicity. This study suggests that ApoLpIII limits pathogenicity induced by parasitization or bacterial infection in P. xylostella.     

Predictive modelling of Lactobacillus casei KN291 survival in fermented soy beverage

The aim of the study was to construct and verify predictive growth and survival models of a potentially probiotic bacteria in fermented soy beverage. The research material included natural soy beverage (Polgrunt, Poland) and the strain of lactic acid bacteria (LAB) — Lactobacillus casei KN291. To construct predictive models for the growth and survival of L. casei KN291 bacteria in the fermented soy beverage we design an experiment which allowed the collection of CFU data. Fermented soy beverage samples were stored at various temperature conditions (5, 10, 15, and 20°C) for 28 days. On the basis of obtained data concerning the survival of L. casei KN291 bacteria in soy beverage at different temperature and time conditions, two non-linear models (r ²= 0.68–0.93) and two surface models (r ²=0.76–0.79) were constructed; these models described the behaviour of the bacteria in the product to a satisfactory extent. Verification of the surface models was carried out utilizing the validation data — at 7°C during 28 days. It was found that applied models were well fitted and charged with small systematic errors, which is evidenced by accuracy factor — Af, bias factor — Bf and mean squared error — MSE. The constructed microbiological growth and survival models of L. casei KN291 in fermented soy beverage enable the estimation of products shelf life period, which in this case is defined by the requirement for the level of the bacteria to be above 10⁶ CFU/cm³. The constructed models may be useful as a tool for the manufacture of probiotic foods to estimate of their shelf life period.     

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSs^TYRV was just as strong as those by NSs^TSWV and NSs^GRSV, even though NSs^TYRV was expressed in lower amounts. Using the system established, a set of selected NSs^TSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSs^TSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.     

High efficient expression of cellobiase gene from Aspergillus niger in the cells of Trichoderma reesei

The cellobiase gene from Aspergillus niger was cloned and connected with the strong promoter Pcbh1 from Trichoderma reesei to construct a recombinant plasmid pHB9 with the hygromycin B resistance marker. The plasmid was transformed into conidia of T. reesei using the modified PEG-CaCl₂ method. Main factors effecting the transformation were discussed and about 99-113 transformants/μg DNA could be obtained under optimal conditions. It was found that the molecular mass of the recombinant cellobiase was about 120 kDa by SDS-PAGE analysis. The activity of cellobiase could reach 5.3 IU/ml after 48 h fermentation, which was as high as 106 times compared with that of the host strain. Meanwhile, the filter paper activity of recombinant T. reesei was 1.44-fold of the host strain. Saccharification of corncob residue with the crude enzyme showed that the hydrolysis yield (84.2%) of recombinant T. reesei was 21% higher than that (69.5%) of the host strain.     

Towards an understanding of the molecular basis of effective RNAi against a global insect pest,the whitefly Bemisia tabaci

In planta RNAi against essential insect genes offers a promising route to control insect crop pests, but is constrained for many insect groups, notably phloem sap-feeding hemipterans, by poor RNAi efficacy. This study conducted on the phloem-feeding whitefly Bemisia tabaci reared on tomato plants investigated the causes of low RNAi efficacy and routes to ameliorate the problem. Experiments using tomato transgenic lines containing ds-GFP (green fluorescent protein) revealed that full-length dsRNA is phloem-mobile, ingested by the insects, and degraded in the insect. We identified B. tabaci homologs of nuclease genes (dsRNases) in other insects that degrade dsRNA, and demonstrated that degradation of ds-GFP in B. tabaci is suppressed by administration of dsRNA against these genes. dsRNA against the nuclease genes was co-administered with dsRNA against two insect genes, an aquaporin AQP1 and sucrase SUC1, that are predicted to protect B. tabaci against osmotic collapse. When dsRNA constructs for AQP1, SUC1, dsRNase1 and dsRNase2 were stacked, insect mortality was significantly elevated to 50% over 6 days on artificial diets. This effect was accompanied by significant reduction in gene expression of the target genes in surviving diet-fed insects. This study offers proof-of-principle that the efficacy of RNAi against insect pests can be enhanced by using dsRNA to suppress the activity of RNAi-suppressing nuclease genes, especially where multiple genes with related physiological function but different molecular function are targeted.     

Development of a functional assay for Ca2+ release activity of IP3R and expression of an IP3R gene fragment in the baculovirus-insect cell system

Receptor-stimulated phosphoinositide (PI) hydrolysis is an important and ubiquitous mechanism of intracellular signaling. Inositol 1,4,5-trisphosphate (IP₃), generated by phosphoinositide (PI) hydrolysis, binds to and gates an intracellular Ca²⁺ channel, the IP₃ receptor (IP₃R), which is therefore a central component of this signaling cascade. Here we describe the development of a baculovirus (BV)/Sf(S. frugiperda) cell system that can be used to look at IP₃R function. Agonist-evoked changes in intracellular Ca²⁺ levels [Ca²⁺]_i were measured (using Fura2) in Sf cells expressing the gene encoding the muscarinic acetylcholine receptor (vmlAchR). Furthermore, we have constructed a recombinant BV (vlP₃R), with the core of the IP₃R ligand-binding domain from the Drosophila IP₃R, under the polyhedrin promoter. The recombinant protein from such a virus was expected to act as a large ligand sink for IP3, generated by stimulation of vmlAchR. Cells coinfected with recombinant BV carrying the potential dominant-negative vIP₃R construct and vmlAchR have been used to assay the modulation of IP₃R-mediated Ca²⁺ release, by the ligand sink.     

Identification of quantitative trait loci involved in the response of common bean to Pseudomonas syringae pv. phaseolicola

Halo blight, caused by Pseudomonas syringae pv. phaseolicola (Burkn.) Downs (Psp), is an important disease in common bean (Phaseolus vulgaris L.). This study investigated the genetic control of the resistance to two local isolates of Psp (ITA-812 and ITA-684) in a recombinant inbred line (RIL) population derived from the cross between the bean genotypes Xana and Cornell 49242. The cultivar Cornell 49242 exhibited moderate resistance to these isolates, whereas cultivar Xana was susceptible. The RIL population showed a continuous variation in response to the two isolates. Analysis revealed four significant quantitative trait loci (QTLs): Psp4^812XC and Psp6.1^812XC located on linkage groups Pv04 and Pv06 (for the response to isolate ITA-812), and Psp6.1^684XC and Psp6.2^684XC located on Pv06 (for the response to isolate ITA-684). The QTLs Psp6.1^812XC and Psp6.1^684XC were located in the same genetic region (Psp6.1), close to the Psp6.2 region in which the QTL Psp6.2^684XC was mapped. A genetic dissection was undertaken to verify the consistency of these three QTLs located on the end of Pv06. Four sets of RILs were established according to the genotypes (Xana and Cornell 49242) of the underlying markers for the regions Psp6.1 and Psp6.2. Re-evaluation of these sets of lines revealed significant differences relative only to isolate ITA-684. The set of lines with the Cornell genotype in both regions was significantly more resistant than the other three sets of lines. This suggested that both regions were necessary to detect a significant effect in the response to isolate ITA-684. In the physical positions corresponding to these two genetic regions, in silico analysis revealed 16 candidate genes (putative orthologous genes) that carried sequences homologous to the resistance genes RPM1, FLS2, RPG1/RPG1-B, and Pto—all of which confer resistance to P. syringae in different species. The results confirm that, apart from the major genes implicated in resistance to Psp, specific bean genotypes exhibit a quantitative mode of inheritance of resistance to Psp.     

Production and secretion of a biologically active Closterium sex pheromone by Saccharomyces cerevisiae

The cDNA fragment coding for the Closterium sex pheromone (protoplast-release-inducing protein inducer, PR-IP inducer) was inserted into a Saccharomyces cerevisiae–Escherichia coli shuttle vector, under the control of the glucose-repressible alcohol dehydrogenase (ADH2) promoter of yeast. The yeast cells transformed by this construct produced and secreted recombinant PR-IP inducer into the surrounding culture medium. The release of PR-IP from mt⁺ cells was induced by the recombinant pheromone, whereas amino- and carboxy-terminal truncated forms did not show any biological activity. High levels of asparagine-linked glycosylation in the recombinant pheromone were also confirmed after the treatment by deglycosylation enzymes.     

Transient silencing of the grapevine gene VvPGIP1 by agroinfiltration with a construct for RNA interference

Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants.     

Improved Yield of High Molecular Weight Hyaluronic Acid Production in a Stable Strain of <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis> via the Elimination of the Hyaluronidase-Encoding Gene

Despite the significant potential of Streptococcus zooepidemicus for hyaluronic acid (HA) production with high molecular weight (MW), the HA degrading properties of hyaluronidase prevents the bacteria to achieve enhanced HA yield with high MW. In the present study, we aim to knockout the hyaluronidase enzyme and assess its effects on the yield and MW of the produced HA. The kanamycin resistance gene between the left and right arms of hyaluronidase gene was inserted into pUC18 plasmid to construct pUC18-L-kana^r-R as a recombinant suicide plasmid. The construct was then transferred into S. zooepidemicus to induce the homologous recombination between the hyaluronidase gene and the kanamycin resistance gene. Gene deletion was confirmed by PCR and enzyme assay. The product was cultured on selectable medium in which the MW of HA was increased from 1.5 to 3.8 MDa. The yield of HA production using the mutant strain was higher in all different concentrations of glucose from 40 to 120 g/l. Moreover, glucose increase results in higher HA production within both wild-type and recombinant strains. However, the growth rate of HA concentration (the slope of the plot), as a consequence of increased glucose concentration, is always higher for the recombinant strain. Unlike the wild-type strain, there was no sharp HA production drop approaching the 6 g/l HA concentration. In conclusion, hyaluronidase activity and HA concentration and MW exhibited a mutual control on each other. Based on our results, deletion of the hyaluronidase gene positively affects the yield and MW of HA.     

A “defeated” rice resistance gene acts as a QTL against a virulent strain of Xanthomonas oryzae pv. oryzae

The genetic components responsible for qualitative and quantitative resistance of rice plants to three strains (CR4, CXO8, and CR6) of Xanthomonas oryzae pv. oryzae (Xoo) were investigated using a set of 315 recombinant inbred lines (RILs) from the cross Lemont (japonica) × Teqing (indica) and a complete linkage map with 182 well distributed RFLP markers. We mapped a major gene (Xa4) and ten quantitative trait loci (QTLs) which were largely responsible for segregation of the resistance phenotype in the RILs. The Teqing allele at the Xa4 locus, Xa4 ^T , acted as a dominant resistance gene against CR4 and CXO8. The breakdown of Xa4 ^T -associated resistance mediated by the mutant allele at the avrXa4 locus in the virulent strain CR6 results from significant changes in both gene action (lose of dominance) and the magnitude of gene effect (≈50% reduction). Nevertheless, Xa4 ^T still acted as a recessive QTL with a significant residual effect against CR6. The mutant alleles at the avrXa4 locus in CXO8 and CR6 that lead to a reduction in effect, or “breakdown”, of Xa4 ^T were apparently accompanied by corresponding penalties for their fitness. The quantitative component of resistance to Xoo in the RILs was largely due to a number of resistance QTLs. Most resistance QTLs mapped to genomic locations where major resistance genes and/or QTLs for resistance to Xoo, blast and sheath blight were identified in the same cross. Most QTLs showed consistent levels of resistance against all three Xoo strains. Our results suggest that a high level of durable resistance to Xoo may be achieved by the cumulative effects of multiple QTLs, including the residual effects of “defeated” major resistance genes.