HslO ameliorates arrested ΔrecA polA cell growth and reduces DNA damage and oxidative stress responses.

Chromosome damage combined with defective recombinase activity has been widely considered to render cells inviable, owing to deficient double-strand break repair. However, temperature-sensitive recAts polA cells grow well upon induction of DNA damage and supplementation with catalase at restrictive temperatures. These treatments reduce intracellular reactive oxygen species (ROS) levels, which suggests that recAts polA cells are susceptible to ROS, but not chronic chromosome damage. Therefore, we investigated whether polA cells can tolerate a complete lack of recombinase function. We introduced a ΔrecA allele in polA cells in the presence or absence of the hslO-encoding redox molecular chaperon Hsp33 expression plasmid. Induction of the hslO gene with IPTG resulted in increased cell viability in ΔrecA polA cells with the hslO expression plasmid. ΔrecA polA cells in the absence of the hslO expression plasmid showed rich medium sensitivity with increasing ROS levels. Adding catalase to the culture medium considerably rescued growth arrest and decreased ROS. These results suggest that hslO expression manages oxidative stress to an acceptable level in cells with oxidative damage and rescues cell growth. Overall, ROS may regulate several processes, from damage response to cell division, via ROS-sensitive cell metabolism.

Recombinase Polymerase Amplification Assay with and without Nuclease-Dependent-Labeled Oligonucleotide Probe.

The combination of recombinase polymerase amplification (RPA) and lateral flow test (LFT) is a strong diagnostic tool for rapid pathogen detection in resource-limited conditions. Here, we compared two methods generating labeled RPA amplicons following their detection by LFT: (1) the basic one with primers modified with different tags at the terminals and (2) the nuclease-dependent one with the primers and labeled oligonucleotide probe for nuclease digestion that was recommended for the high specificity of the assay. Using both methods, we developed an RPA-LFT assay for the detection of worldwide distributed phytopathogen-alfalfa mosaic virus (AMV). A forward primer modified with fluorescein and a reverse primer with biotin and fluorescein-labeled oligonucleotide probe were designed and verified by RPA. Both labeling approaches and their related assays were characterized using the in vitro-transcribed mRNA of AMV and reverse transcription reaction. The results demonstrated that the RPA-LFT assay based on primers-labeling detected 103 copies of RNA in reaction during 30 min and had a half-maximal binding concentration 22 times lower than probe-dependent RPA-LFT. The developed RPA-LFT was successfully applied for the detection of AMV-infected plants. The results can be the main reason for choosing simple labeling with primers for RPA-LFT for the detection of other pathogens.

Rapid detection of peach latent mosaic viroid by reverse transcription recombinase polymerase amplification.

Reverse transcription recombinase polymerase amplification (RT-RPA), an isothermal nucleic acid amplification and detection method, was developed to detect peach latent mosaic viroid (PLMVd) in pollen and peach leaves. Results showed that this RT-RPA detection method can be performed at 42 °C and completed in approximately 5 min, and there was no cross-reactivity with other common peach viruses. A sensitivity assay showed that the RT-RPA assay was 1000-fold more sensitive than a regular RT-PCR. Moreover, the method was successfully applied to test field-collected samples. The newly developed RT-RPA assay is rapid, sensitive, and reliable method for detection of PLMVd in peach pollen and leaves and can be utilized as an effective technique in quarantine and viroid-free certification processes.

Development of lateral flow assay combined with recombinase polymerase amplification for highly sensitive detection of Dickeya solani.

Dickeya solani, one of the most significant bacterial pathogens, infects potato plants, resulting in severe economic damage. In this study, a lateral flow assay (LFA) combined with isothermal DNA amplification was developed for rapid, specific, and sensitive diagnosis of the potato blackleg disease caused by D. solani. Recombinase polymerase amplification (RPA) was chosen for this purpose. Five primer pairs specific to different regions of the D. solani genome were designed and screened. A primer pair providing correct recognition of the target sequence was aligned with the SOL-C region specific to D. solani and flanked by fluorescein (forward primer) and biotin (reverse primer). Lateral flow test strips were constructed to detect DNA amplicons. The RPA-LFA demonstrated a detection limit equal to 14,000 D. solani colony-forming units per gram of potato tuber. This assay provided sensitivity corresponding to the polymerase chain reaction (PCR) but was implemented at a fixed temperature (39 °C) over 30 min. No unspecific reactions with Pectobacterium, Clavibacter, and other Dickeya species were observed. Detection of latent infection of D. solani in the potato tubers by the developed RPA-LFA was verified by PCR. The obtained results confirmed that RPA-LFA has great potential for highly sensitive detection of latent infection.

Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato.

In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6-98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

Food Yellow4 reprotoxicity in relation to localization of DMC1 and apoptosis in rat testes: Roles of royal jelly and cod liver oil.

Food Yellow 4 (FY4) is a lemon-yellow-colored synthetic organic azo dye, which is used widely for imparting pleasant and attractive appearance to foods and cosmetics. The present study aimed at evaluating the possible mechanism underlying the FY4-induced reprotoxicity in rats, and the potential supportive role of royal jelly (RJ) or cod liver oil (CLO), which is a natural remedy with several pharmacological benefits, against induced toxicity. Forty-eight male rats were divided into different groups-the control group, the CLO group (0.4 mL/kg), the RJ group (300 mg/kg), the FY4 group (500 mg/kg b.w.), and the co-treated groups (FY4 + CLO or FY4 + RJ). Semen analysis, serum hormones, and enzyme activities were estimated. Immunohistochemical staining was performed using anti-PCNA, anti-Sox 9, anti-STRA8, anti-DMC1, and anti-ssDNA antibody. The FY4 group exhibited a significant decrease in sperm concentration and motility percentage (%) and a substantial reduction in the TES and LH levels. Testicular LDH, ACP, and SDH were observed to be inhibited. Furthermore, co-localization of DMC1 and ssDNA, which reflected apoptotic induction in the leptotene and zygotene spermatocytes, respectively, was observed to have markedly elevated in the FY4 treated rats, with fewer PCNA-positive and SOX9-positive cells and higher ssDNA-positive cells in the seminiferous epithelium in comparison to the control groups. Interestingly, co-treatment with CLO or RJ exhibited healthy sperms and restored their features, activated the enzyme production, and raised the levels of sexual hormones. In addition, both RJ and CLO restored the features of the testicular tissue as observed under a light microscope, and limited the apoptosis as observed through antibody staining. Collectively, the results of the present study revealed that the co-administration of RJ or CLO with FY4 improved the biochemical, hormonal, and structural aspects of the testicular tissue in rats. Therefore, CLO and RJ may be considered promising agents that would be able to improve the testicular structure and function in the FY4-exposed individuals.

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Rapid Authentication of Ginkgo biloba Herbal Products Using the Recombinase Polymerase Amplification Assay.

Species adulteration in herbal products (HPs) exposes consumers to health risks. Chemical and morphological methods have their own deficiencies when dealing with the detection of species containing the same active compounds in HPs. In this study, we developed a rapid identification method using the recombinase polymerase amplification (RPA) assay to detect two species, Ginkgo biloba and Sophora japonica (as adulteration), in Ginkgo biloba HPs. Among 36 Ginkgo biloba HP samples, 34 were found to have Ginkgo biloba sequences, and 9 were found to have Sophora japonica sequences. During the authentication process, the RPA-LFS assay showed a higher specificity, sensitivity and efficiency than PCR-based methods. We initially applied the RPA-LSF technique to detect plant species in HPs, demonstrating that this assay can be developed into an efficient tool for the rapid on-site authentication of plant species in Ginkgo biloba HPs.

Rapid detection of potyviruses from crude plant extracts.

Potyviruses (genus Potyvirus; family Potyviridae) are widely distributed and represent one of the most economically important genera of plant viruses. Therefore, their accurate detection is a key factor in developing efficient control strategies. However, this can sometimes be problematic particularly in plant species containing high amounts of polysaccharides and polyphenols such as yam (Dioscorea spp.). Here, we report the development of a reliable, rapid and cost-effective detection method for the two most important potyviruses infecting yam based on reverse transcription-recombinase polymerase amplification (RT-RPA). The developed method, named 'Direct RT-RPA', detects each target virus directly from plant leaf extracts prepared with a simple and inexpensive extraction method avoiding laborious extraction of high-quality RNA. Direct RT-RPA enables the detection of virus-positive samples in under 30 min at a single low operation temperature (37 °C) without the need for any expensive instrumentation. The Direct RT-RPA tests constitute robust, accurate, sensitive and quick methods for detection of potyviruses from recalcitrant plant species. The minimal sample preparation requirements and the possibility of storing RPA reagents without cold chain storage, allow Direct RT-RPA to be adopted in minimally equipped laboratories and with potential use in plant clinic laboratories and seed certification facilities worldwide.

Resolvase OsGEN1 Mediates DNA Repair by Homologous Recombination.

Yen1/GEN1 are canonical Holliday junction resolvases that belong to the RAD2/XPG family. In eukaryotes, such as budding yeast, mice, worms, and humans, Yen1/GEN1 work together with Mus81-Mms4/MUS81-EME1 and Slx1-Slx4/SLX1-SLX4 in DNA repair by homologous recombination to maintain genome stability. In plants, the biological function of Yen1/GEN1 remains largely unclear. In this study, we characterized the loss of function mutants of OsGEN1 and OsSEND1, a pair of paralogs of Yen1/GEN1 in rice (Oryza sativa). We first investigated the role of OsGEN1 during meiosis and found a reduction in chiasma frequency by ∼6% in osgen1 mutants, compared to the wild type, suggesting a possible involvement of OsGEN1 in the formation of crossovers. Postmeiosis, OsGEN1 foci were detected in wild-type microspore nuclei, but not in the osgen1 mutant concomitant with an increase in double-strand breaks. Persistent double-strand breaks led to programmed cell death of the male gametes and complete male sterility. In contrast, depletion of OsSEND1 had no effects on plant development and did not enhance osgen1 defects. Our results indicate that OsGEN1 is essential for homologous recombinational DNA repair at two stages of microsporogenesis in rice.

Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.

Yin Yang 1 (YY1) is a multifunctional protein with regulatory potential in tumorigenesis. Ample studies demonstrated the activities of YY1 in regulating gene expression and mediating differential protein modifications. However, the mechanisms underlying YY1 gene expression are relatively understudied. G-quadruplexes (G4s) are four-stranded structures or motifs formed by guanine-rich DNA or RNA domains. The presence of G4 structures in a gene promoter or the 5'-UTR of its mRNA can markedly affect its expression. In this report, we provide strong evidence showing the presence of G4 structures in the promoter and the 5'-UTR of YY1. In reporter assays, mutations in these G4 structure forming sequences increased the expression of Gaussia luciferase (Gluc) downstream of either YY1 promoter or 5'-UTR. We also discovered that G4 Resolvase 1 (G4R1) enhanced the Gluc expression mediated by the YY1 promoter, but not the YY1 5'-UTR. Consistently, G4R1 binds the G4 motif of the YY1 promoter in vitro and ectopically expressed G4R1 increased endogenous YY1 levels. In addition, the analysis of a gene array data consisting of the breast cancer samples of 258 patients also indicates a significant, positive correlation between G4R1 and YY1 expression.

Method for Bxb1-mediated site-specific integration in planta.

Gene targeting in plants through homologous recombination has been sparsely reported, although notable breakthroughs have been achieved in recent years. In particular, the use of zinc finger nucleases to promote homologous end joining has revived the promise that homologous gene targeting could someday become practical for plant genetic engineering. An alternative and complementary approach that has progressed steadily over the years has been recombinase-mediated site-specific integration. In this approach, a first recombination site is introduced into the genome to serve as a target for inserting subsequent DNA. Here, we describe the method for generating the chromosomal target and the subsequent insertion of new DNA into the chromosomal target by Bxb1-mediated site-specific integration. This method would permit the comparison of different molecular constructs at the same genomic locations.

The histidine utilization (hut) genes of Pseudomonas fluorescens SBW25 are active on plant surfaces, but are not required for competitive colonization of sugar beet seedlings.

The ability to monitor the spatial and temporal distribution of signals in complex environments is necessary for an understanding of the function of bacteria in the wild. To this end, an existing recombinase-based transcriptional reporter strategy (recombinase-based in vivo expression technology, RIVET) has been extended and applied to the plant-colonizing bacterium Pseudomonas fluorescens SBW25. Central to the project was a rhizosphere-inducible locus, rhi14, which functional analyses show is hutT, a histidine-inducible gene that is required for histidine utilization. A transcriptional fusion between hutT and a promoterless site-specific recombinase (tnpR(mut168)) results in excision of a chromosomally integrated tetracycline-resistance cassette in a histidine-dependent manner. The dose- and time-responsiveness of the promoterless recombinase to histidine closely mirrored the histidine responsiveness of an identical hutT fusion to promoterless lacZ. To demonstrate the effectiveness of the strategy, the activity of hutT was monitored on sugar beet seedlings. Low levels of transcriptional activity were detected in the phyllosphere, rhizosphere and in plant extract, but not in vermiculite devoid of seedlings. The histidine concentration in the rhizosphere was estimated to be 0.6 microg ml(-1). The ecological significance of the hut locus was examined by competing a hutT deletion mutant against the wild-type during colonization of sugar beet seedlings. No impact on competitive fitness was detected, suggesting that the ability to utilize plant-derived histidine is not essential for bacterial colonization.

Visualization of the annealing of complementary single-stranded DNA catalyzed by the herpes simplex virus type 1 ICP8 SSB/recombinase.

The rate of annealing of long linear complementary single-stranded (ss) DNAs can be increased greatly by certain DNA-binding proteins including the herpes simplex virus type 1 ICP8 SSB/recombinase. Using electron microscopy, we have investigated the DNA-protein structures involved in ICP8-mediated DNA annealing. We show that the formation of superhelical ICP8-ssDNA filaments is required for annealing. Two superhelices interact with each other to form a coiled-coil, which is the intermediate in annealing. In this process, the superhelices likely rotate and translocate relative to each other. Psoralen/UV photocrosslinking studies revealed that meta-stable contacts form at sites of limited sequence homology during the annealing. Partial proteolysis of ICP8 in the protein-ssDNA complexes showed that Mg2+ induces conformational changes in the N-terminal region (amino acid residues 1-305) of ICP8. In addition to Mg2+, Ca2+ and, to a significantly lesser extent, Cu2+ and Mn2+, were found to induce superhelix formation of the ICP8-ssDNA filament and to facilitate annealing. Mechanisms for how the coiled-coil structures facilitate annealing are discussed.