Arabidopsis thaliana, their contribution to antiviral immunity in Nicotiana benthamiana, a model plant for plant-pathogen interaction studies, is not well understood. In this study, we investigated the role of N. benthamiana NbAGO2 gene against TMV (Tomato mosaic virus) infection. Silencing of NbAGO2 by transient expression of an hpRNA construct recovered GFP (Green fluorescent protein) expression in GFP-silenced plant, demonstrating that NbAGO2 participated in RNAi process in N. benthamiana. Expression of NbAGO2 was transcriptionally induced by both MeSA  treatment and TMV infection. Down-regulation of NbAGO2 gene by amiR-NbAGO2 transient expression compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Our study provides evidence for the antiviral role of NbAGO2 against a Tobamovirus family virus TMV in N. benthamiana, and SA  mediates this by induction of NbAGO2 expression upon TMV infection. Our data also highlighted that miR403a was involved in TMV defense by regulation of target NbAGO2 gene in N. Benthamiana.RNAi (RNA interference) is an important defense response against virus infection in plants. The core machinery of the RNAi pathway in plants include DCL (Dicer Like), AGO (Argonaute) and RdRp (RNA dependent RNA polymerase). Although involvement of these RNAi components in virus infection responses was demonstrated in  Caenorhabditis elegans by injection of double-stranded RNA (dsRNA), which was therefore identified as the molecule mediating sequence specific inhibitory effects during RNAi process [Virus diseases threaten crop production and agriculture globally. Plant evolved and developed sophisticated mechanisms to defend against virus infections. RNA interference (RNAi) is an antiviral defense mechanism in plants. The history of our understanding of RNAi for viral defense can be traced back to 1928, when Wingard reported the recovery of virus infected plant from later virus infection . In 1998 process . In 1999 process . RNAi wa process ,7,8. BriArabidopsis thaliana, the functions of these two family proteins were heavily investigated. AGO1 plays key role against viruses like turnip crinckle virus  [Cucumovirus) [Potexvirus) [Potyvirus) [Geminivirus) [Plantago asiatica mosaic virus  [Caulimovirus) [Tobravirus) [Potyvirus) in maize [Tobamovirus) in tobacco [Bromovirus) and rice stripe virus , but not wheat dwarf geminivirus  [Nicotiana benthamiana , several but limited studies were reported on antiviral role of AGO and DCL family proteins. NbDCL4 plays key role against zucchini yellow mosaic virus  in N. benthamiana [Arabidopsis thaliana, but is not functional in N. benthamiana [N. benthamiana [Potyvirus) and the Y satellite of CMV but not TRV or CMV alone [NbAGO2 was broadly investigated in Arabidopsis thaliana, and its antiviral role in N. benthamiana was also reported for TBSV (Tombusvirus), PVX (Potexvirus), TuMV (Potyvirus) and TCV (Tombusvirus) [NbAGO2 in RNAi-mediated antiviral role against infection by TMV, a virus belonging to Tobamoviridae. From its first use in Arabidopsis thaliana in 1996, Artificial microRNA (amiRNA) has been applied in many model and monocot species including Oryza sativa and Brachypodium distachyon [NbAGO2 expression and investigated its role for TMV responses. Our results showed that NbAGO2 played key roles in antiviral responses against TMV in N. benthamiana, and miR403a and SA was implicated in these responses.Although both AGO and Dicer/DCL proteins are involved in RNAi process, their functions are diverged in defense responses against different viruses. Furthermore, there are many AGO and DCL members in plants and they showed specialized roles against different pathogens. In model system usvirus) , cucumbemovirus) , potato exvirus)  and turntyvirus) . AGO2 istyvirus) , TCV, CMtyvirus)  and PVX tyvirus) . AGO4 futyvirus) , CMV [18nivirus)  and Planexvirus) . DCL1 ismovirus) , while Dmovirus)  and TCV movirus) . RDR1 isravirus) , TuMV [2ravirus)  and CMV ravirus) . RDR2 coravirus)  and TuMVravirus) . In othein maize  and agai tobacco . In ricenivirus) . In virothamiana . Althougthamiana . NbRDR6 thamiana . NbRDR6 MV alone . AGO prousvirus) ,33. Therstachyon ,35,36. Vstachyon . We usedN. benthamiana plants were used. Wild type N. benthamiana was provided by Prof. Xiaohan Mo from the Yunnan Tobacco Research Institute  and the GFP16c line was provided by Prof. Feng Li from Huazhong Agricultural University . N. benthamiana seeds were cultured in sterilized nutrient soil and vermiculite (1:1) at growth temperature of 22–25 °C with photoperiod of 14:10 (light:dark) and 70% relative humidity. Three-leaf stage seedlings were used for following experiments. 0.5 mM MeSA  in water solution was sprayed on the leaves and the leaves were collected one day after treatment. Water solution was sprayed as mock treatment. For GFP visualization, TMV-GFP infected plants were illuminated with a hand-held UV lamp at 320 nm or under Nikon SMZ18 microscope .Wild type and 16c GFP (Green fluorescent protein)-transgenic (GFP16c) N. benthamiana leaves infected with TMV and mock solution were collected and frozen in liquid nitrogen after five hours. The collected leaves were ground in liquid nitrogen and soaked with 5 mL 60% ethanol, and the supernatant after centrifugation was used to detect absorbance at 310 nm, which was then converted to SA concentration according to the standard curve [The content of endogenous salicylic acid (SA) was determined by UV Vis Spectrophotometry. A set of standard solutions, containing 5, 10, 20, 30, and 40 μg/mL of SA, were prepared in 60% ethanol solution and one of the standard solution was used for measuring maximum absorption wavelength within a 280–320 nm wavelength range , which was used as a subsequent detection wavelength. The maximum absorption wavelength was 310 nm and the linear regression equation according to the absorbance of five standard solutions was deduced. rd curve .GFP gene, GFP interfering sequences were amplified by Forward primerEcoRV and then was subcloned into binary vector pCB2004B to produce hairpin construct for insert GFP fragment.For silencing of www.benthgenome.com website.For construction of viral pCV (CaLCuV)-based (cabbage leaf-curl virus) amiR vector, following oligonucleotides were designed by amiR design function in amiR1:amiR2:amiR3:Arabidopsis thaliana miR159b as template backbone and the amplicons were cloned into pCVA vector by XbaI/KpnI sites. The same amplicons were cloned into pBI121 vector by XbaI/KpnI sites to produce non-viral binary vector based amiR constructs.The above primers were used to amplify amiR fragment using NbAGO2 gene, open reading frame (ORF) region of NbAGO2 was amplified by the primersN. benthamiana and the amplicon was cloned into pBI121 vector by BamHI/SacI sites.To overexpress GTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATGGTGCTATCCCCTATCCTGAGCTTgaattc-3′; miR393a, 5′-ctcgagCCAAAGGGCTAATAGCATGATGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATCCAAAGGGCTAATAGCATGATgaattc-3′; and miR403, 5′-ctcgagAGTTTGTGCCTAGTGAATCTAAGTTGTTGTTGTTATGGTCTAATTTAAATATGGTCTAAAGAAGAAGAATAGTTTGTGCCTAGTGAATCTAAgaattc-3′. The restriction endonuclease sites are shown in lower-case letter, and the STTM spacer sequences were underlined. The oligonucleotides and complementary sequences were annealed to produce dsDNA and this dsDNA was then ligated into the pGreen-GUS-Competitor vector by XhoI and EcoRI respectively.For development of STTM (Short tandem target mimic) constructs for miR390a, miR393a and miR403a, the following sequences and their complementary sequences were designed and synthesized. miR390a, 5′-ctcgagGGTGCTATCCCCTATCCTGAGCTTAgrobacterium tumefaciens GV3101 competent cells. The transformants were grown to optical density OD600 = 1 at 28 °C, cells were collected by centrifugation and resuspended in inoculation buffer (10 mM 2-(N-Morpholino)ethanesulfonic acid (MES), 10 mM MgCl2, 200 μM acetosyringone). OD600 values of agrobacterium inoculation solution were all adjusted to 1.0 unless stated otherwise. The inoculation solution was leaf-infiltrated into three-leaf stage N. benthamiana by needleless syringe. For pCV-based silencing experiment, pCVB was simultaneously transformed and co-infiltrated with recombinant pCVA vectors. For virus infection, infectious clone of TMV expressing green fluorescent protein (TMV-GFP), pJL24, was infected as the same way. For pJL24, OD600 value of Agrobacterium inoculation solution was 0.1. In all experiments, inoculation buffer with empty vector was used as mock treatment.The above constructs were transformed into 260/OD280 using NanoDrop2000 Spectrophotometer  and agarose gel electrophoresis. One µg of total RNA was used as template and cDNA was synthesized by random hexamer using GoScript Reverse Transcription System .Leaf tissues were grounded in liquid nitrogen by using mortar and pestle. The powder was homogenized in Trizol reagent  and extracted following manufacturer’s protocols. DNase was used to remove contaminated genomic DNA. Total RNA quality was confirmed by measurement of ODNbAGO2 level by qRT-PCR. The primer sets 5′-ATGTGAAATGGTACGGGCTGAA-3′ and 5′-CAACAAGGTTCCACTGGCATTT-3′ were used for NbAGO2. As an internal control, steady state level of N. benthamiana GAPDH gene was evaluated by primer set 5′-CTGACAAGGACAAGGCTGCT-3′ and 5′-AAGCAGCTCTTCCACCTCTC-3′. The ratio of mRNA level between treatment and mock samples was determined by the 2−ΔΔCT method. At least three biological replicates with two technical replications were carried out, and the differences were evaluated by t-test.The cDNAs were subjected to PCR experiment. The PCR reaction was run on qTOWER 2.2  equipment. Fast SYBR Green Master Mix  was used for evaluation of +, positively charged)  by an electrokinetic capillary apparatus. 32P or digoxigenin-labelled probes were prepared by the methods described as previously, and hybridization and signal visualization were performed as per the literatures [N. benthamiana NbPR1a gene, a contig sequence with transcript ID of JN247448 from N. benthamiana sequence database (www.benthgenome.com) was used and the primer pairs 5′-GGATGCCCATAACACAGCTC-3′ and 5′-CCTAGCACATCCAACACGAA-3′ were used for amplification of 313bp probe sequence. For N. benthamiana β-actin gene, a partial cDNA sequence (JQ256516.1) was used and the primer pairs 5′-CCCAAAGGCTAATCGTGAAA-3′ and 5′-GCAGCTTCCATTCCAATCAT-3′ were used for amplification of 483bp probe sequence. For TMV detection, nucleotide encoding capsid protein was used and the primer pairs 5′-CAAGCTCGAACTGTCGTTCA-3′ and 5′-GACCAGAGGTCCAAACCAAA-3′ were used for amplification of 352bp probe sequence.Ten µg of total RNA was resolved by agarose gel electrophoresis. RNAs were transferred from the gel to nylon membrane  -based vector pCVA under 35S promoter [NbAGO2 was transformed into Agrobacterium tumefaciens strain GV3101 and agro-infiltrated into N. benthamiana leaves together with pCVB. We used three different regions of NbAGO2 for amiR targeting, and named them amiR-NbAGO2-1, amiR-NbAGO2-2 and amiR-NbAGO2-3 respectively  was used. First, we used pCB2004B-hpGFP to transiently silence GFP expression in GFP16c plant. The result showed that GFP expression was largely silenced by transient overexpression of hpGFP by agroinfiltration, and both GFP fluorescence and GFP mRNA levels decreased by hpGFP expression. One should note that GFP signal in GFP16c plant was mostly localized in vascular tissue. Next, we used this system to investigate the involvement of NbAGO2 in RNA silencing. Down-regulation of NbAGO2 by amiR-NbAGO2 recovered both the GFP fluorescence and GFP mRNA levels in the hpGFP expressed GFP16c plant, while in the control plant GFP was significantly silenced  is reported to be broadly engaged in virus infection and defense response. We also investigated the involvement of SA in the TMV infection process in  by 24 h B. We the 1–4 DPI A. In tob 1–4 DPI B, and Nbthamiana A.NbAGO2 was responsive to TMV infection and MeSA treatment. Involvement of NbAGO2 in TMV infection was further investigated as follows. NbAGO2 was first silenced by pBI121-amiR-NbAGO2 agroinfiltration, and after one day the same leaf region was agroinfiltrated with TMV-GFP, an infectious TMV engineered to express green fluorescent protein, which was retained in the infiltrated area for at least 6 days. The inoculated leaves were monitored daily for the appearance of GFP fluorescence. As shown in NbAGO2 caused stronger accumulation of virus (GFP fluorescence) as compared to control empty vector (pBI121) inoculated leaves. In accordance with this, overexpression of NbAGO2 (Ox-NbAGO2) resulted in weaker virus accumulation, and this effect was abolished by simultaneous amiR-NbAGO2 expression. In addition, virus spread was reduced by treatment with MeSA , TCV, CaMV [OsRDR genes but not OsAGO genes [NbAGO2 was induced by TMV infection in virological model species N. benthamiana. NbAGO2 was shown to be involved in both RNAi and virus defense responses here. NbAGO2 was reported to mediate defense against tomato bushy stunt virus  [NbAGO2 compromise defenses against PVX, TuMV and TCV, but not Tombusvirus and CMV infections [Potexvirus) [AGO2 is a broad spectrum antiviral gene whose activity is achieved by RNAi mechanism. Artificial miRNA was shown to be effective to silence variety of genes in a diversity of plant species including N. benthamiana [Arabidopsis thaliana [N. tabacum [Oryza sativa [Glycine max [Solanum melongena [Medicago truncutula [Triticum aestivum [Vitis vinifera [Solanum tuberosum [Solanum lycopersicum [N. benthamiana AGO family genes via transient expression from both virus vector and binary vector. As infection of virus vector (pCV) hindered subsequent TMV infection, we used pBI121 for expression of amiRNA and its effects on TMV infection. It is interesting to note that AGO2 itself is targeted by miR403a in plants [NbAGO2 by STTM technology influenced TMV infection. As for the miR390a and miR393a, both of them were reported to bind to NbAGO2 protein competitively [NbAGO2 expression was induced by tomato ringspot virus  isolates ToRSV-Rasp1 or ToRSV-GYV, and silencing of NbAGO2 elevated ToRSV accumulation [NbAGO2 was involved in plant responses toward different families of viruses. Induction of RNAi pathway component genes by corresponding pathogens were reported in several manuscripts. In proteins ,53,64. Rnfection . ExpressCV, CaMV  and TCV CV, CaMV ,67. In rGO genes . Here, wusvirus) . CRISPR/fections . Transgeexvirus) . Our datthamiana , Arabidothaliana , N. taba tabacum , Oryza sa sativa , Glycinecine max , Solanumelongena , Medicaguncutula , Triticuaestivum , Vitis vvinifera , Solanumuberosum  and Solapersicum . Here, wn plants , and hertitively , therefomulation . This inNbAGO2 gene against TMV infection was investigated in N. benthamiana. NbAGO2 was demonstrated to be involved in RNAi process in N. benthamiana. NbAGO2 mRNA level was elevated by both TMV infection and MeSA treatment. Suppression of NbAGO2 gene by amiR technology compromised plant resistance against TMV infection. Inhibition of endogenous miR403a, a predicted regulatory microRNA of NbAGO2, reduced TMV infection. Taken all this together, we conclude that SA participates in NbAGO2 mediated TMV response and miR403a is also involved in this response by post-transcriptionally regulating NbAGO2 expression (The role of pression .


Potential utilization of proteins for early detection and diagnosis of various diseases has drawn considerable interest in the development of protein-based multiplex detection techniques. Among the various techniques for high-throughput protein screening, optically-encoded beads combined with fluorescence-based target monitoring have great advantages over the planar array-based multiplexing assays. This review discusses recent developments of analytical methods of screening protein molecules on microbead-based platforms. These include various strategies such as barcoded microbeads, molecular beacon-based techniques, and surface-enhanced Raman scattering-based techniques. Their applications for label-free protein detection are also addressed. Especially, the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads are successful for generating a large number of coding. High-throughput screening (HTS) of biomarkers has great potential for clinical and genetic analysis, and medical diagnostics. Because proteins can indicate the state of disease progression and the functions of normal biological processes within the human body, HTS techniques that identify proteins and their expression levels are very important for early detection, diagnosis, and therapy ,4,5,6,7.The most widely used method for protein analysis in basic research and clinical diagnostics is the enzyme-linked immunosorbent assay (ELISA) ,9,10. Ma6 fold), and hence a large dynamic range of detection level is recommended for protein detection. In this regard, small sample volume on microarray spots may reduce the dynamic range of detection in some cases [Planar microarrays (protein chips) and bead-based microarrays (suspension arrays) are widely used to date for multiplex protein detection. The microarray chip-based screening has many advantages over ELISA such as assay miniaturization, multiplexing, low consumption of samples (less than a nanoliter), and high-throughput screening ,18,19,20me cases ,22,23.TM claiming ~106 capture molecules per bead. This means that more capture biomolecules can be immobilized on the bead, and thus bead-based arrays are more probable to detect a wide range of target proteins; (2) detection is faster and sensitivity is equal to or higher than that of ELISAs because the interaction between beads and target molecules can be nearly comparable with solution-phase kinetics; (3) target molecules can be collected by using flow cytometry such as fluorescence-activated cell sorting (FACS); (4) Large-scale fabrication and surface modification is possible, and the prepared beads can be stored. Thus, customization is possible by selective mixing of antibody-conjugated microbeads; (5) beads can be used with combination of microfluidic devices to detect trace amounts of molecules in a manner of automation. As one of widely used methods for the multiplex detection of biomolecules, bar-coded (encoded) micro-sized beads (microbeads) have been used in bead-based arrays (suspension or liquid arrays) ,24,25. TIn the microbead system, capture molecules that specifically bind to target analytes are immobilized to corresponding unique bar-coded micro-sized beads. By decoding the beads, the identity of captured analytes can be determined. Thus, the system needs two readouts as shown in Until now, a number of encoding strategies such as chemical encoding, electronic encoding, graphical encoding and spectrometric encoding have been proposed and demonstrated ,31,32. AThere are various approaches for coding and decoding of beads, and for identification of the binding event of a protein with a capture molecule on beads. Fluorescence-based detection has been widely used to detect binding event owing to several major advantages: easy visualization, quantification of target molecules, selective excitation of fluorophores , rhodamine isothiocyanate (RITC), Cy series dyes or Alexa Fluor dyes) and fast readout ,40.Recently, our group has introduced several kinds of optically-encoded bead and together with strategies for protein binding event to solve some of the biggest challenges with bead-based arrays such as generating a large number of coding and label-free protein detection. This review is focused on fluorescence-based multiplex detection systems of proteins with particular emphasis on optically-encoded beads.Among the optical encoding methods, fluorescence-encoding has been most widely used in biological applications owing to the simple encoding process, easy detection of large-scale samples, and compatibility with a variety of biological chemistries ,36,41. TThe commercially available Luminex protein detection system is a representative example of fluorescence-encoded bead-based assays ,42. In tMany researchers and several companies have developed microbead-based assay systems based on the Luminex system . Very reThe other approach to overcome the limited number of fluorescence-encoded beads is the use of localized fluorescence-encoded beads. One of the examples is fluorescence dye-doped NP-embedded bead ,52,53,54Recently, our group has developed and used layer-by-layer (multilayer) fluorescence-encoded beads for protein detection as illustrated in In order to apply the bead-encoding system to bio-detections, an additional labeling step is generally required to monitor protein-binding event. Moreover, the number of matched-pair antibodies in sandwich immunoassays is limited for multiplex detection.Direct labeling method which is conjugating of fluorophores  to target can be used a. Howeveet al. have introduced label-free analyses on inverse-opaline photonic beads [Label-free techniques that monitors inherent property changes of the capture molecule by target binding can be used to avoid the above-mentioned problems, and many research groups are currently developing label-free planar chip assays based on various tools such as surface plasmon resonance (SPR), carbon nanotubes (CNTs), nanowires, nanohole arrays and interferometry ,57. The ic beads . In theiic beads .Recently, we combined aptamer-based molecular beacons (MBs)  or polydUsing a sandwich immunoassay format, up to a maximum of 30 targets can be analyzed in multiplex within the same sample, which is circumvented by the other method such as direct labeling method or label-free detection. MBs have a hairpin structure that can undergo spontaneous conformational changes upon hybridization to complementary nucleic acids or protein targets, activating fluorescence resonance energy transfer (FRET) ,62. For MBs are attached to beads by electrostatic or biotin-streptavidin interactions to detect unlabeled nucleic acids in solution for multiplex detection . Using bAs a different approach, MBs were directly coupled to multilayer fluorescence-encoded beads by covalent bonding . In thisPDA-based biosensors have attracted considerable attention due to their unique color change from blue to red in response to a variety of stimuli such as applied stress, changes in temperature or pH, and ligand-receptor binding. Thus, PDA-based biosensors have been applied to a wide range of analytes, including proteins, viruses, antibacterial peptides, antibodies, and pharmacologically active compounds.Most PDA-based biosensors are prepared in the form of free-floating vesicles of 100–200 nm or planar chips ,65,66. PAlthough these studies are at an early stage, the combination of optically-encoded beads with fluorescence-based methods could evolve as a powerful label-free detection method in such fields as separation using direct detection of ligand-target binding events, flow cytometry, multiplexing ability, and easy and real-time recognition of ligand type and binding event by using CLSM.Nanostructures of noble metal such as gold and silver exhibit an optical phenomenon known as surface-enhanced Raman scattering (SERS), which enhances Raman scattering of molecules adsorbed thereon. When SERS is used as a coding method, it has advantages for bioassays over other optical tools: (1) A large number of different Raman signatures can be obtained using different reporter molecules. Since SERS peaks are narrow (less than 0.5 nm), spectral overlap is minimized, and thus a large number of coding can be created by the combination of chemicals; (2) Choice of photoexcitation line is very flexible covering UV to NIR region; (3) There is no photobleaching in Raman scattering; (4) They can afford non-invasive analysis of biomolecules and thus are applicable to high-throughput screening of various biomolecules ,70,71,72So far, a large number of SERS-coded materials and readout techniques have been reported ,77,78,79Monodisperse micro-sized polystyrene beads prepared by seed polymerization were used as stable templates for SERS encoding by our group. Silver NPs were embedded on sulfonated micro-beads polystyrene (PS) beads and then Raman-labeled organic compounds were adsorbed on the silver NPs. Then, the beads were coated with a silica shell using tetraethoxyorthosilicate (TEOS) for easy surface modification and chemical stability. The SERS-encoded beads had uniform size and produced highly intense and reproducible Raman signatures. Moreover, the size of PS beads could be controlled by changing backbone size, and additional function such as magnetic property can be incorporated to the SERS-encoded beads. The protein p53 which is tumor suppressor protein was chosen as a model to show that the SERS-encoded beads could be used for protein detection. By using p53 antibody-conjugated SERS-encoded beads, the p53 tumor suppressor protein in a protein mixture was successfully detected by sandwich-type bioassays.The key advantage of this system comes from combination of flow cytometry with optically-encoded beads ,80. SERSTo take the advantage of the combination of fluorescence-based immunoassays with optically-encoded beads, the choice of fluorescent dye requires special considerations for avoiding spectral overlap. Because fluorescent dyes have narrow excitation wavelengths, they can be selectively excited by laser sources and spectral emission overlap can be avoided. When combining QD-encoded beads and fluorescence-based detection, the overlap can be generally avoided by using emitting spectra, although QDs can be excited by broad wavelengths. Because fluorescence-based coding is based on different emitting wavelengths, combination of fluorescence-based immunoassays with optically-encoded beads could limit coding numbers, and consequently multiplexing ability.One of main advantages of combining fluorescence-based protein detection with SERS-encoded beads is that the target binding event and the type of ligand can be simultaneously recognized by fluorescence and SERS, respectively, with single laser-line excitation and without interference by coding number.Fluorescence is quenched by the interaction between metal surfaces and fluorescent dye molecules, and thus, fluorescent dyes are widely used as Raman label compounds to produce resonance Raman signals. However, in the case of using fluorescence and SERS together, fluorescence part is physically separated from silver NPs as SERS substrate, and this prevents quenching of fluorescence.Another point to be considered is that fluorescence can overlap the SERS spectrum. The best approach is to avoid overlap. For example, when we used FITC (530 nm) or Cy5.5 (670 nm) as fluorescent dyes for target detection in the case of silver-based SERS coding (514-nm laser source), the FITC spectra covered the SERS signal but the Cy5.5 spectra did not, as shown in On the other hand, in SERS decoding of fluorescence with SERS beads, broad fluorescence background of FITC at 530 nm almost covered the SERS peaks. This overlap could be avoided by fluorescence photobleaching. Because SERS peaks are not photobleachable, only the intensity of fluorescence was gradually decreased by laser illumination. After about 100 s, fluorescence was almost completely photobleached, and the 4-MT SERS peaks were obtained as shown in Currently, the analysis of multiple analytes in a single biological sample is required for diagnostic applications. These demands can be met by using multiplex platforms such as planar and bead-based arrays. Bead-based arrays have many advantages in sensitivity, flexibility, and the requirement of small sample volume over planar arrays. In particular, the combination of fluorescence-based detection with optically-encoded beads can provide a robust and efficient approach for setting up multiplexed assays. In this review, we have briefly summarized recent developments in the area of optically-encoded beads based screening of protein molecules. We have also focused on the optically-encoded beads such as multilayer fluorescence beads and SERS-encoded beads which have potential to generate a large number of coding for multiplexing detection. Combination of several strategies like molecular beacon-based techniques or PDA techniques with those beads is also discussed to show the potential for label-free protein.Even though plenty of success has been, in order for bead-based assays to be more practically achieved in this field, several issues still need to be resolved, including a large number of optical codes, rapid readout method, safety, cost, sensitivity, and ease of use for bioapplications such as multiple protein detection in clinical diagnostics. (1) With regard to coding materials, unlimited coding number has not been fully accomplished. Fluorescence-based beads can be limited in their number and/or toxicity. Although the coding number of SERS beads has great potential in respect of coding numbers, they have not been completely established. So far, only several to dozens of Raman dyes are widely used, and different signal intensity and signal complexity sometimes limit their practical coding number.(2) For detection of protein binding, on-beads label-free detection is still at the beginning stage. Development of more smart and practical detection method is necessary to multiplex, fast and sensitive detection.(3) The great advantages of optically-encoded beads came from well developed decoding and sorting system. So far decoding and sorting with flow cytometry seem to give best performance and can be immediately applicable and promising way.The combination of fluorescence-based detection and SERS materials could make bead-based assays more attractive in the medical and diagnostic fields. We also expect that the recently developed fluorescence-based label-free method will significantly contribute to the expanded use of bead-based assays.


Here, we report the detection of a novel alphavirus in Australian mosquitoes, provisionally named Yada Yada virus (YYV). Phylogenetic analysis indicated that YYV belongs to the mosquito-specific alphavirus complex. The assembled genome is 11,612 nucleotides in length and encodes two open reading frames. Here, we report the detection of a novel alphavirus in Australian mosquitoes, provisionally named Yada Yada virus (YYV). Phylogenetic analysis indicated that YYV belongs to the mosquito-specific alphavirus complex. The assembled genome is 11,612 nucleotides in length and encodes two open reading frames. Alphavirus, family Togaviridae) are small (10- to 12-kb) single-stranded positive-sense RNA viruses and include species important to human and animal health, such as Chikungunya virus and Eastern equine encephalitis virus , supporting previous studies that have detected mosquito-specific alphaviruses from only Anopheles and Culex species .To investigate the vector of YYV, the assembled contigs were compared to a cytochrome oxidase I (COI) database of Australian mosquito species  using BLspecies 25. Due toThe discovery of YYV expands the diversity and geographic range of the mosquito-specific alphavirus complex and in doing so will help reveal the virus origin and evolution of host switching . In addiMN733821. The sequencing reads are available in the SRA database via BioProject accession number PRJNA594295.The YYV genome sequence has been deposited in GenBank under the accession number


The effect of the experimental parameters, such as pH, contacting time, adsorbent dosage, agitation type, and rotating speed were investigated and optimized for an efficient removal of Cd(II) ions at an initial concentration of 250 mg/L. The amount of adsorbed Cd(II) by MAAC beads increased at a pH of 6 with a removal efficiency over 90%. The maximum adsorption capacity reached was 70 mg/g of adsorbent at an initial Cd(II) concentration of 150 mg/L, whereas at 250 mg/L the adsorption capacity lowered until 60 mg/g. Sorption isotherms were calculated using Langmuir, Freundlich, Temkin, and Dubinin–Radushkevich equations, and were better described by the Freundlich and Temkin models. These results proved the removal efficiency and the potential use under real environmental conditions of the MAAC beads, due to their easy recovery from contaminated aqueous solutions.This study presents an effective magnetic separation method for cadmium removal, based on the use of a novel nanostructured material as an adsorbent. This adsorbent involves the incorporation of magnetite nanoparticles (Fe Removing toxic heavy metals from wastewater is becoming one of the greatest matters of interest due to their large toxicity in the environment and all living beings ,2. HeavyTherefore, new and more effective technologies are required to reduce heavy metals to environmentally acceptable levels at a reasonable cost. In this regard, one of the most promising methods is metal biosorption, which consists in the use of natural materials from biological sources to uptake metals from solution. The main challenge consists in the development of biosorbents with high adsorption capacities and uptake velocities. Different plants, raw materials from microbiological origin, or biopolymers such as chitin, alginate, pectin, and agarose, show great affinity towards metal ions and have been tested in water originated from industrial effluents ,14,15,16d-mannuronic acid and α-L-guluronic acid residues, is a promising candidate since it is very cheap, non-toxic [Among them, alginate, a natural polysaccharide extracted from brown algae and composed of β-on-toxic , and preon-toxic ,22,23,24When alginate is mixed with divalent cations, such as calcium, a spontaneous cross-linking reaction occurs, and a three-dimensional hydrogel is formed as a consequence of interchain associations, allowing in this way the preparation of gel beads of different sizes by tailoring the reaction conditions. This is the main process behind the development of metal biosorbents with a large surface area  that canHowever, the separation or extraction of metal loaded biomaterial beads from the treated aqueous media is a matter of concern that is being addressed by means of magnetic separation strategies. This technique is also used in conventional industries, like mineral separation in the steel industry or biomarker separation in biomedical detection kits. Magnetic adsorbents, also called magsorbents, are receiving considerable attention  since th3O4-NPs) into the alginate beads as the active magnetic part of the absorbent to allow for an easy separation and recovery of the beads from treated waters [The magsorbents have enormous advantages with different applications, like the addition of passivating or flocculating agents with no additional pollution being produced. One of the most used strategies consists of incorporating magnetite nanoparticles  was obtained from Alfa Aesar , cadmium chloride hemi(pentahydrate) 99% (CdCl2·2.5H2O) was obtained from Acros Organics , commercial activated carbon powder (MW = 12.01 g) was obtained from PANREAC , ferrous sulfate (FeSO4·7H2O), calcium chloride, ammonium hydroxide, and sodium alginate were purchased from Sigma . All chemicals used were of analytical grade and without purification. Ferric chloride  at 60 °C and stirred mechanically for 30 min. The NPs were washed several times with distilled water, and re-dispersed in 150 mL of water.Fen method . In this3O4-NPs solution. Finally, 3.0 g of activated carbon powder was added. The obtained suspension was mixed for 4 h, and then added dropwise into a bath of 0.27 M calcium chloride under a continuous constant speed of 450 rpm using a New Era NE-300 syringe pump . Magnetic alginate activated carbon (MAAC) beads were instantly formed and left in the solution bath for hardening during 30 min. The beads were then cleaned with distilled water. Afterwards, cleaned MAAC beads were reserved in distilled water (pH = 6.5) and dried overnight at 60 °C for the adsorption experiments. Magnetic alginate beads were prepared using calcium chloride as a cross-linking agent. Firstly, 2.0 g of sodium alginate was weighted using a Mettler AT100 analytical balance, which was subsequently added to the previously prepared Fe3O4-NPs and MAAC beads was characterized by transmission electron microscopy (TEM) using a JEOL JEM-1011 microscope  operating at 100 kV and by scanning electron microscopy (SEM) analysis using a ZEISS FE-SEM ULTRA Plus (30 kV) microscope . The morphology of the Feθ). The Fourier transform infrared spectra (FT-IR) were recorded on a Varian FT-IR 670  spectrophotometer in the range 400–4000 cm−1. Thermogravimetric analyses (TGA) were performed on heating, from 50 to 840 °C, 10 mg of sample at 10 °C/min under nitrogen flow (20 mL/min) using a TGA Perkin Elmer Pyris 7 . Magnetic properties were assessed by measuring the magnetization curve using a vibrating sample magnetometer (VSM)  with an applied field between −10,000 and 10,000 Oe at room temperature.Structural studies were performed by X-ray diffraction (XRD) using a Philips diffractometer  with Cu Kα radiation (λ = 1.5406Å), with a step size of 0.02° and a counting time of 2 s per step from 10° to 80° (2The concentration of Cd(II) ions was measured by inductively coupled plasma optical emission spectrophotometry (ICP-OES) using an emission spectrometer Perkin Elmer Model Optima 3300 DV .Several adsorption experiments were carried out, in this study, in order to investigate the optimum conditions to achieve the highest amount of Cd(II) removal. To optimize the process of removing Cd(II), experimental parameters, such as adsorbent dosage, contacting time, pH, rotating speed, and agitation type  and magnetic stirring agitation, where the solution is moved in a vertical circular motion as result of a magnetic stirring bar). The effect of the agitation type was investigated while varying one of the following parameters: the adsorbent dosage (2.5–15 mg), the contacting time , and the agitation speed  using a cadmium concentration of 10 mg/L at room temperature. These optimized parameters were used to study the pH effect on cadmium adsorption by the MAAC beads. The pH range studied was varied from 3.0 to 11 and adjusted with 0.1 M HCl and 0.05 M NaCl using a pH meter Milwaukee pH51 waterproof . The effect of initial concentrations of Cd(II) was studied from 10 to 250 mg/L while keeping all the other optimized parameters unchanged.R is the removal efficiency; C0 (mg/L) is the initial concentration; and tC (mg/L) is the concentration at any time t.The cadmium removal efficiency was calculated using the following equation: q value shows the metal amount adsorbed per specific adsorbent amount (mg/g). The adsorption capacity at time t, tq (mg/g), was obtained as follows:V (L) is the volume and M (g) is the dry weight of adsorbent.The eq, was determined using the equation:eC (mg/L) is the equilibrium metal concentration present in solution.The adsorption amount at equilibrium, 3O4-NPs was studied by TEM and the different surface views of the MAAC beads were analyzed by SEM.The morphology of the Fe3O4-NPs used to synthesize the MAAC beads. The nanoparticles revealed a nanosphere morphology with an average particle size of 11 nm. Also, the average crystallite size was determined with the Scherrer equation [L is the mean size of the ordered  domains; K is a constant related to crystallite shape normally taken as 0.9; λ is the X-ray wavelength in nanometers; β is the peak width of the diffraction peak profile at half the maximum intensity (FWHM); and θ is the Bragg angle. equation :(4)L=KλβThe crystallite size calculated from the X-ray diffractogram, as shown in 3O4-NPs. In In θ = 30.1, 35.8, 43.1, 53.9, 57.2, and 63.1° can be indexed to (200), (311), (400), (422), (440), and (511) planes corresponding to the magnetite crystalline phase (JCPDS 79-0417). The relative intensity and locations of the peaks, similar to the theoretical pattern of magnetite, clearly evidence that the crystallinity nature of Fe3O4-NPs embedded in the MAAC beads structure is preserved during all the synthesis procedures.The X-ray diffraction pattern of the dried beads is shown in −1 corresponds to the –OH stretching vibration of alginate; the peaks at 2918 and 2630 cm−1 belong to symmetric and asymmetric C–H stretching, respectively. The bands which appeared at 1585 and 1409 cm−1 are assigned to asymmetric and symmetric stretching vibrations of carboxyl groups of alginate, respectively. The band at 1076 cm−1 is attributed to C–O–C groups [−1 which corresponds to the stretching vibration of Fe–O bonds in Fe3O4-NPs [−1 [Chemical topography of the MAAC beads is analyzed by FT-IR spectrometry as shown in C groups . Anothere3O4-NPs . It can -NPs [−1 . Magnetization of the beads was measured with a vibrating sample magnetometer working from −10 kOe to 10 kOe at room temperature. The results were normalized to the amount of magnetic mass content of the sample, which was obtained by TGA analysis. The percentage of magnetite in the sample was 22.84%. In S) of the MAAC beads shows a value of 48 emu/g Fe3O4, which is smaller than bulk magnetite (MS-bulk = 92 emu/g). This is in accordance to what can be expected for small nanoparticles where magnetization becomes reduced by the presence of a dead magnetic layer. Nevertheless, the net magnetization of the MAAC beads was higher when exposed to an external magnetic field due to the contribution of the large number of Fe3O4-NPs contained in each bead. This fact ensures their strong drag to an external applied permanent magnet and facilitates an almost instantaneous separation from the aqueous medium, as shown in the inset of Saturation magnetization  solution (10 mg/L) and 8 mg of adsorbent, at pH = 7, were kept under orbital or magnetic agitation for distinct times to measure subsequent aliquots of metal removal at an agitation speed of 250 rpm. The effect of the contacting time on the removal efficiency of Cd(II) by the MAAC beads at different times is shown in The influence of the rotating speed on the adsorption process was also studied by analyzing the metal removal for magnetic and orbital agitation from 150 to 500 rpm. The results, as shown in Magnetic agitation of the MAAC beads has shown to be generally more efficient than orbital agitation, as shown in In addition, the influence between adsorbent and adsorbed materials on the removal procedure was assessed by keeping constant all the experimental parameters  while varying the mass of MAAC beads between 2 and 15 mg. This adsorbent dose range was selected as a criterion for successful employment of this material to industrial applications by reducing costs using minimum adsorbent dosages. In The solution pH is one of the most important parameters able to govern metal adsorption, since it acts on the protonation of the binding sites of the adsorbent and consequently strongly affects the adsorption capacity . The inf2 and Cd(OH)3 [As observed in  Cd(OH)3 .+ and Cd(II) ions [The adsorption capacity of Cd(II) by MAAC beads at a pH value of 3–4 attains removal efficiency up to 78% which can be attributed to an insufficient number of deprotonated sites, arising from the competition of available binding sites between HII) ions . By incrII) ions . This reThe effect of the Cd(II) initial concentration was studied from 10 to 250 mg/L at optimized conditions studied previously . In Conversely, the removal efficiency of Cd(II) by the MAAC beads decreased with increasing initial cadmium concentration in solution. This may be due to the saturation adsorption sites of the MAAC beads throughout the adsorption process, leading to a reduction uptake with increasing Cd(II) initial concentration. In general, the adsorption isotherm describes the mobility of a substance from an aqueous media to a solid-phase at constant conditions of temperature and pH. The equilibrium is only established when a dynamic balance is reached between the adsorbed substance onto the adsorbent surface and the remaining one in solution. This phenomenon can be described by several isotherm models, depending on the specific details of the adsorption process, the adsorbent properties, and the adsorbed species, like Langmuir, Freundlich, Temkin, or Dubinin–Radushkevich isotherms.The Langmuir isotherm assumes monolayer and homogeneous adsorption onto a surface containing a finite number of adsorption sites that possess equal affinity for the adsorbate ,48. Onceeq (mg/g) is the amount adsorbed; eC (mg/L) is the adsorbate concentration in solution, both at equilibrium; LK (L/mg) is the Langmuir adsorption constant; and mq (mg/g) is the maximum sorption capacity for monolayer formation on the adsorbent.The linear form of the Langmuir isotherm is given by :(6)Ceqe=LR, defined by Weber and Chakraborti in 1974, can be represented as [C0 (mg/mL) is the adsorbate initial concentration.Hereby, a fundamental characteristic of the Langmuir isotherm regarding a dimensionless constant, known as separation factor ented as :(7)RL=11The Freundlich isotherm assumes that the adsorbent has an energetically heterogeneous surface and has a non-uniform distribution for adsorption. The isotherm is represented by the non-linear equation :(8)qe= KF and 𝑛 are empirical constants, being indicative of the adsorption extent and the f nonlinearity degree between solution and concentration, respectively. The linear form of the Freundlich isotherm is given by :(9)logqeTK (L/g) is the Temkin isotherm equilibrium binding constant. The Temkin isotherm model is assumed by the ratio between the adsorption heat and adsorbent surface coverage . The adsR (8.314 J/mol·K) is the universal gas constant; T (K) is the absolute temperature; and Tb is the Temkin isotherm constant.However, the rearranged linear form of the Temkin isotherm equation is commonly used, represented by :(11)qe=Rmq (mg/g) is the adsorption capacity; β is the activity coefficient pertinent to mean adsorption energy; and ε is the Polanyi potential, which can be calculated through Equation (13) [The Dubinin–Radushkevich (D–R) isotherm model is usually applied to describe the characteristic porosity of the adsorbent and adsorption energy. The non-linear form is expressed as :(12)qe= ion (13) :(13)ε=RTThe linear form of this model is described as : (14)lnqE, per molecule of adsorbate can be calculated by the relationship (kJ/mol) [However, it also specifies the nature of the adsorption process through physisorption or chemisorption of metal ions, with its mean free energy, (kJ/mol) : (15)E=1The adsorption equilibrium was studied using the following optimized parameters: magnetic agitation with a rotating speed of 300 rpm, 14 mg of MAAC beads, contacting time of 1 h, and pH = 6. The Langmuir, Freundlich, Temkin, and D-R parameters of Cd(II) adsorption onto MAAC beads were obtained and listed in 2, represented in B index and TK constant were 18.9 J/mol and 0.169 L/g, which is an indication of the adsorption heat and physical adsorption process. According to the Freundlich model, the value of 1/n = 0.794 (n = 1.26), indicates a physical and favorable adsorption of Cd(II) onto the MAAC beads with an adsorption capacity of 1.27 mg/g [E, was 1.581 KJ/mol, suggesting that the uptake of Cd(II) by MAAC beads was by physisorption with a maximum monolayer adsorption capacity of 47.68 mg/g. The comparison of R.27 mg/g ,54. The LR, with a value of 0.333, indicates a favorable equilibrium, but the very low value of R2 of 0.753 reveals that the sorption cannot be described by the Langmuir isotherm model. Moreover, the mq calculated was 94.34 mg/g, revealing a high Cd(II) adsorption.Parameter Metal ion adsorption by an adsorbent is influenced by several factors, such as the metal ion size, the number of active groups and pore size present on the surface of the adsorbent, and the experimental conditions in the adsorption process .In our case, Cd(II) adsorption is better described by the Freundlich and Temkin isotherms, which is in accordance with the multi-layer structure adsorption head, and heterogenous surface with different affinity sites on the MAAC beads. These results suggest that the adsorption follows a physical process, although to further understand the underlying mechanism, more studies are needed. 3O4-NPs with activated carbon in alginate-based beads. The magnetic bead structure observed by SEM analysis showed a high number of inter-connected pores and a large specific surface area, which is the reason for fast and efficient adsorption of Cd(II) ions from solution. The prepared beads showed superparamagnetic behavior, with well distributed Fe3O4-NPs and activated carbon on the beads’ structure. The effects of contacting time, initial Cd(II) concentration, pH, adsorbent dose, agitation type, and rotating speed on the adsorption process were discussed. All procedures showed a higher removal efficiency of Cd(II) when beads were under magnetic agitation. At pH 6, the adsorption isotherm that better described the study equilibrium was the Freundlich and Temkin isotherms, with R2 > 0.90 and R2 = 0.89, respectively.The present work presents a cost-effective method for Cd(II) removal from aqueous medium by developing a novel magnetic hybrid nanostructured material through the encapsulation of FeMagnetic hybrid beads are good candidates for efficient magnetic removal of Cd(II) and other heavy metals present in wastewaters, being useful for industrial applications since they combine a set of advantageous features: biocompatibility, cost-effective synthetic production, easy handling, and fast separation from any aqueous solution under an external magnetic force. Future work is intended to use this material in real environmental conditions and at an industrial scale.


Arabidopsis thaliana as model systems, we have identified the within-host speed of virus colonization and multiplication in the reproductive structures as the main determinants of the efficiency of seed transmission. These results contribute to shedding light on the mechanisms by which plant viruses disperse and optimize their fitness and may help in the design of more-efficient strategies to prevent seed infection.One of the major factors contributing to plant virus long-distance dispersal is the global trade of seeds. This is because more than 25% of plant viruses can infect seeds, which are the main mode of germplasm exchange/storage, and start new epidemics in areas where they were not previously present. Despite the relevance of this process for virus epidemiology and disease emergence, the infection traits associated with the efficiency of virus seed transmission are largely unknown. Using turnip mosaic and cucumber mosaic viruses and their natural host  Arabidopsis thaliana accessions with Turnip mosaic virus and Cucumber mosaic virus. Using these plant-virus interactions, we analyzed the relationship between the effect of virus infection on rosette and inflorescence weights; short-, medium-, and long-term seed survival; virulence; the number of seeds produced per plant; virus within-host speed of movement; virus accumulation in the rosette and inflorescence; and efficiency of seed transmission measured as a percentage and as the total number of infected seeds. Our results indicate that the best estimators of percent seed transmission are the within-host speed of movement and multiplication in the inflorescence. Together with these two infection traits, virulence and the number of seeds produced per infected plant were also associated with the number of infected seeds. Our results provide support for theoretical predictions and contribute to an understanding of the determinants of a process central to plant-virus interactions.Although vertical transmission from parents to offspring through seeds is an important fitness component of many plant viruses, very little is known about the factors affecting this process. Viruses reach the seed by direct invasion of the embryo and/or by infection of the ovules or the pollen. Thus, it can be expected that the efficiency of seed transmission would be determined by (i) virus within-host multiplication and movement, (ii) the ability of the virus to invade gametic tissues, (iii) plant seed production upon infection, and (iv) seed survival in the presence of the virus. However, these predictions have seldom been experimentally tested. To address this question, we challenged 18 IMPORTANCE One of the major factors contributing to plant virus long-distance dispersal is the global trade of seeds. This is because more than 25% of plant viruses can infect seeds, which are the main mode of germplasm exchange/storage, and start new epidemics in areas where they were not previously present. Despite the relevance of this process for virus epidemiology and disease emergence, the infection traits associated with the efficiency of virus seed transmission are largely unknown. Using turnip mosaic and cucumber mosaic viruses and their natural host Arabidopsis thaliana as model systems, we have identified the within-host speed of virus colonization and multiplication in the reproductive structures as the main determinants of the efficiency of seed transmission. These results contribute to shedding light on the mechanisms by which plant viruses disperse and optimize their fitness and may help in the design of more-efficient strategies to prevent seed infection. The ability to be transmitted is arguably the most important determinant of parasite fitness. Indeed, most theoretical models of the evolution of parasites consider infection traits, such as virulence, i.e., the effect of infection on host fitness , or with2–Vertical transmission is particularly frequent in plant viruses, as more than 25% of all known species are vertically transmitted through seeds , 12. See11–In general, plant viruses achieve seed transmission in two ways according to the distribution of the virus in the seed. The first is through contamination of the seed coat. In this case, during germination, the virus infects the seedling through abrasions caused by soil particles , 20. ThiSoybean mosaic virus seed transmission is controlled by plant gene homologs of Arabidopsis thalianaDCL3 and RDR6, which are involved in small RNA-mediated gene silencing (Barley stripe mosaic virus (BSMV), Cucumber mosaic virus (CMV), and Pea seed-borne mosaic virus genes encoding the replicase and movement proteins has been associated with the efficiency of seed transmission  (Potyviridae), CMV (Bromoviridae), and Arabidopsis thaliana (Brassicaceae) (referred to here as Arabidopsis). Both viruses are commonly found in natural populations of Arabidopsis at a prevalence of up to 80%  suggests that this virus may also be seed transmitted  TuMV and CMV speed of within-host movement and multiplication, as proxies of the ability of the virus to reach and invade gametic tissues, respectively; (ii) the effect of virus infection on plant growth and progeny production, as a measure of virulence; and (iii) short-, medium-, and long-term survival of infected seeds. Using multivariate mixed models, we investigated which of these infection traits are associated with the efficiency of virus seed transmission, quantified both as a percentage and as the total number of infected seeds, and their relative importance. We constructed global models that considered data for both viruses together as well as virus-specific models, such that we could differentiate traits broadly associated with seed transmission from virus species-specific determinants of this process. We validated the resulting models by measuring, in a larger set of 18 Arabidopsis accessions, seed transmission and the most relevant estimators of these traits, and we compared the values predicted by our models with experimental quantifications of seed transmission.To address these questions, we utilized p to 80% , indicatnsmitted , 27, andnsmitted , 33. In virulent . Thus, oST) and as the total number (IS) of TuMV- and CMV-infected seeds in 18 Arabidopsis accessions . Similar results were obtained when seed transmission was quantified as the number of infected seeds  . Both measures of seed transmission depended on the Arabidopsis accession  and on the interaction between accession and virus species . Thus, we analyzed both ST and IS for each virus separately.We quantified seed transmission as a percentage  but not to the virus isolate  . The interaction between these factors was significant . Similar trends were observed when the number of TuMV-infected seeds was analyzed (TuMV isolate UK1 (UK1-TuMV) was seed transmitted in 6/18 accessions, and JPN1-TuMV was transmitted in 12/18  and B, wanalyzed  and B.Arabidopsis accessions  and between virus isolates , with similar values for Fny-CMV (2.59% ± 0.46%) and LS-CMV (2.37% ± 0.34%) and lower values for De72-CMV (1.83% ± 0.30%). The interaction between these factors was significant . Again, similar results were obtained when CMV seed transmission was quantified as the number of infected seeds  and 2 virus isolates (Fny-CMV and JPN1-TuMV) were selected to analyze the relationship between 9 traits proposed to be linked to virus seed transmission and ST and IS. Specifically, we calculated the effects of virus infection on rosette weight (RW) (ratio of RW of infected plants/RW of mock-inoculated plants [iRW/mRW]), inflorescence weight (IW) (iIW/mIW), and short-term (seed germination percentage at time zero [G0]) (G0i/G0m), medium-term (G24i/G24m), and long-term (G48i/G48m) seed survival; virulence (V); the number of seeds produced per infected plant (iSN); virus within-host speed of movement (SM); and virus accumulation in rosette (RVA) and inflorescence (IVA) leaves, plus ST and IS .Six out of the 18 RW, IW, and G24 plus V, iSN, SM, RVA, and IVA differed according to the virus isolate , the Arabidopsis accession , and the accession-per-isolate interaction , whereas the effects of virus infection on G0 and 48G did not  . These results prompted us to analyze the variation in these infection traits for each virus separately. In JPN1-TuMV-infected plants, the effect of infection on RW, IW, and G24 plus V, iSN, RVA, and IVA varied according to the Arabidopsis accession , and the effects of infection on G0 and G48 did not  , except that in this case, only the effect of virus infection on G0 did not vary between accessions   . Note th≥ 0.078) . Similar= 0.472) . Note thArabidopsis accessions. In this subset of accessions, ST and IS significantly varied according to the virus isolate , the Arabidopsis accession , and the interaction between them . In agreement, virus-specific analyses showed that for both viruses, ST and IS varied significantly between Arabidopsis accessions . ST and IS in the six Arabidopsis accessions showed similar trends between experiments .The seed transmission percentage and number of infected seeds were also estimated in the six Arabidopsis accession, and/or both factors.Thus, all the analyzed infection traits, except for the effect of infection on short-term seed survival, showed variability according to the virus isolate, the Since the efficiency of seed transmission depended on the plant-virus genotype-per-genotype interaction, and we showed a similar variation for most infection traits proposed to be associated with this mode of transmission, we considered these infection traits as potential estimators of seed transmission. Thus, we performed more-detailed analyses of the association between infection traits and virus seed transmission utilizing multiple-regression model selection analyses .SM, V, RVA, IVA, and the effect of infection on IW, G0, and G24 . IVA and SM had the highest relative importance , explaining most of the variance in ST . Again, IVA and SM were the most important estimators , with virulence playing a less relevant role  , with SM and IVA being the most important estimators (both having a relative importance of 37%) and G48i/G48m having lower relative importance (18%). Thus, our results indicated that IVA and SM were chief estimators of ST, with V and G48i/G48m playing secondary roles in a virus-specific manner  and IVA  (ST and the virus-specific secondary estimators (V and G48i/G48m) was also detected  . The best-ranked model contained ce in ST . Becausece, 14%) . In addic manner . Regardl< 0.001) . A weake≤ 0.002) .IS contained ST, iSN, V, IVA, and the effect of infection on RW and G48 . In this case, ST, iSN, and V had the highest relative importance  . Again, ST, iSN, and V were the most important estimators . The best-ranked model explaining Fny-CMV IS included ST, V, iSN, SM, IVA, and the effect of infection on RW and IW , with ST, V, and iSN being the most important estimators   and with iSN  and negatively associated with V  . In agreement with ST-explaining models, a weak negative association between virulence and ST was detected in the virus-specific models  but not in the global model   . The besctively) . Bivaria≤ 0.001) . These re traits . ST was = 0.138) .Arabidopsis accessions and one isolate per virus inoculated at a given host phenological stage, and therefore, the derived results are specific only for these particular plant-virus genotype-per-genotype interactions and inoculation conditions. We analyzed the applicability of our models to other Arabidopsis accessions and TuMV/CMV isolates by using them to estimate the efficiency of seed transmission in the set of 18 Arabidopsis accessions and 5 virus isolates inoculated at the same phenological stage as the 6 accessions used for model construction. To do so, we quantified the relevant infection traits identified by the models , and we incorporated these values into the best-ranked global and virus-specific models to predict percentages and numbers of virus-infected seeds. The resulting values were compared with those obtained by experimental testing of seed infection  (x coefficients near 1) (ST (x coefficient = 3.63) and overestimated CMV ST (x coefficient = 0.10), particularly at seed transmission values of >10% , although R2 values were generally lower than those for percent seed transmission (x coefficient near 1) than the global one, but under- and overestimates of TuMV and CMV IS, respectively, were smaller than in the case of ST-estimating models   to D. Ho near 1)  and C. S near 1) . On the  of >10%  and D. V of >10% . Experimsmission  to H. Agg models . Note thparately .Arabidopsis accessions, i.e., high versus low seed transmission, and virus-specific models infer seed transmission values more accurately than the global ones.Hence, for the host phenological stage at inoculation used in our experiments, our multivariate models fairly estimate trends of TuMV and CMV seed transmission across The ability to be transmitted is a key component of parasite fitness , 9, 10. Arabidopsis accession-virus species/isolate interaction, indicating that traits controlled by the host and/or the parasite determine this process. Global multivariate models identified the virus speed of within-host movement and its level of multiplication in the plant inflorescence as the best estimators of percent seed transmission. These variables can be considered proxies for the ability of the virus to reach and invade gametic tissues, respectively  in other Arabidopsis, TuMV is transmitted through seeds and in some accessions with high efficiency, adding this to the list of vertically transmitted plant viruses. As mentioned above, in Arabidopsis, TuMV is considered a sterilizing virus . This reduces the resources available for virus multiplication, which in turn prevents maximization of horizontal transmission . These results highlight the complexity of the interactions between different infection traits in determining the contribution of vertical transmission to virus fitness.Not surprisingly, models generally included the speed of within-host movement of the virus and its multiplication in the inflorescence as secondary determinants of the number of infected seeds, the two traits associated with percent seed transmission. This is likely the consequence of the major contribution of the percentage of seed transmission to the number of infected seeds. Indeed, equivalent models for In summary, by using a multivariate approach, this work provides a highly detailed analysis of the infection traits linked to the efficiency of plant virus seed transmission and identify virus within-host speed of movement and multiplication in the plant reproductive organs as major determinants of this process. We also show that a greater contribution of vertical transmission to virus fitness is associated with lower virus virulence. These results support theoretical predictions and contribute to shedding light on the mechanisms by which plant viruses achieve vertical transmission and optimize their fitness.AB194802), JPN1-TuMV (GenBank accession number KM094174), Fny-CMV , LS-CMV , and De72-CMV (not sequenced) were used. JPN1-TuMV was obtained from a field-infected Raphanus sativus (Brassicaceae) plant (Diplotaxis erucoides L. plant (Brassicaceae) , and transcripts were used to infect N. benthamiana plants for virus multiplication.Virus isolates UK1-TuMV (GenBank accession number e) plant , and De7icaceae) ; both vie clones –56 by inArabidopsis accessions were used (Arabidopsis (2). Plants were mechanically inoculated, either with N. benthamiana TuMV- and CMV-infected tissue ground in a solution containing 0.1 M Na2HPO4, 0.5 M NaH2PO4, and 0.02% DIECA  or with inoculation buffer for mock-inoculated plants. Inoculations were done when plants were at developmental stages 1.05 to 1.06  such that (i) accessions represented a range of TuMV and CMV seed transmissions (The 18 missions  and (ii)missions . We alsoArabidopsis accessions and the two virus isolates, we conducted a time course experiment of viral infection. For each Arabidopsis accession, 85 plants per virus were inoculated with JPN1-TuMV and Fny-CMV each, and the other 10 were mock inoculated. Five infected plants per accession were harvested at regular intervals. Because each accession has a different developmental schedule  was quantified from three disks with a diameter of 4 mm collected from different systemically infected leaves, and virus accumulation in the inflorescence (IVA) was quantified from the collected 1-cm pieces. For plants that were allowed to complete their life cycle, RVA and IVA were quantified at the end of the flowering period to ensure maximum viral multiplication in plant structures. Form these plant samples, total RNA extracts were obtained using TRIzol reagent , and 10 ng of total RNA was added to Brilliant III Ultra-Fast SYBR green qRT-PCR master mix  according to the manufacturer’s recommendations. Specific primers were used to amplify a 70-nucleotide (nt) fragment of the TuMV coat protein (CP) gene and a 106-nt fragment of the CMV CP gene  for each individual plant. For plants included in the time course experiment, at each time point, virus accumulation in the rosette  in the plant inflorescence was quantified as centimeters per day from flower meristem formation to silique ripening, thus covering the time interval during which the virus can enter the seed. Following methods described previously , inflorescence (IW), and seeds (SW) were obtained. RW was used as an estimate of plant resources dedicated to growth, and IW and SW were taken as estimates of plant resources dedicated to reproduction . Virulence (V) was estimated as 1 minus the ratio of the total seed weight of infected (iSW) to the total seed weight of mock-inoculated (mSW) plants. The total numbers of seeds produced per mock-inoculated (mSN) and infected (iSN) plant were also quantified. To do so, we obtained the weight of 200 seeds for each replicate and derived the weight of a single seed. Using this value and the total seed weight of the corresponding plant, we calculated SN. Virus infection did not affect the weight of 200 seeds .Aboveground plant structures were harvested at complete senescence. The weights of the rosette (oduction . The eff2). According to methods described previously (ST) was then estimated using an expression reported previously (p = 1 − (1 − y/n)1/k, where p is the probability of virus transmission by a single seed, y is the number of positive samples, n is the total number of samples assayed (n  =  50), and k is the number of seedlings per sample (k  =  2). To calculate the total number of infected seeds per plant (IS), we multiplied iSN by ST.The efficiency of CMV and TuMV seed transmission was estimated both as a percentage of infected seeds and as the total number of infected seeds that gave rise to infected progeny per plant in grow-out tests. For each virus, 100 seeds per replicate were washed in a 10% bleach solution to ensure that any viral infection that occurred was not simply the result of the presence of virus on the seed coat but rather was the result of embryonic infection. Next, seeds were placed into petri dishes containing Murashige-Skoog medium, stratified for 3 days at 4°C, and germinated in a growth chamber at 22°C, under 16 h of light , medium-term (G24), and long-term (G48) seed survival were measured as the ratio of infected to mock-inoculated seed germination percentages. We quantified these three measures of seed survival because they can yield different information on the processes determining the efficiency of seed transmission: short-term survival occurs prior to our measures of seed transmission, and it can be considered a predictor of this trait. On the other hand, seed aging is posterior to our measures of seed transmission, and modifications of medium- and long-term survival can be considered a consequence of seed infection that modify the efficiency of seed transmission in the long run. In any case, the three seed survival measures can be considered potential estimators of virus efficiency of seed transmission.We measured short-, medium-, and long-term seed survival as surrogates of the effect of infection on seed viability. Short-term seed survival was measured as percent germination of seeds derived from infected and mock-inoculated plants 4 months after harvest to avoid biases due to seed dormancy, according to the protocol described above. To estimate medium- and long-term seed survival, seeds were artificially aged, and their germination percentage was measured. The artificial aging process was a modification of the “basal thermotolerance assay” described previously . BrieflyiSN and IS, which were normalized using a log transformation. Therefore, differences between virus species and plant accessions were analyzed by generalized linear models (GzLMs), applying the corresponding linked function and considering virus isolate nested to virus species and Arabidopsis accession as random factors. Virus species-specific analyses considered virus isolate and Arabidopsis accession as random factors .Arabidopsis accession as random effects, and TuMV- and CMV-specific models included only the latter random effect. Models for percent seed transmission were constructed using a Poisson function and a log-linked function, as this function best reflected the distribution of the data according to Akaike’s information criterion (AIC) (R package rriskDistributions [i) as the difference between the AIC of a given model and that of the best-ranked model (i) of each model was calculated according to the expression ωi = exp(−0.5Δi)/Σexp(−0.5Δi). The relative importance of a given estimator included in the best-ranked model was calculated by decomposing the R2 value of the model into components corresponding to each estimator using the R package relaimpo . Modelsed model . Finallyrelaimpo . BivariaArabidopsis accessions and the 5 virus isolates for which the efficiency of seed transmission was initially quantified. For this set of accessions, we quantified all the variables considered in the best-ranked models except for SM, G24, and G48. We estimated SM using values from the six-accession experiment. To do so, we calculated bivariate correlations between SM and all other variables. Because SM was an accession-specific trait, we used average values per accession. Based on these analyses, we identified the variable with the highest association with SM, and we used the equation of the linear relationship to estimate SM in the 18 Arabidopsis accessions. We could not follow the same approach for G24 and G48 as they were quantified for each individual plant, and no other trait significantly correlated with these two variables. Because these variables generally had very low relative importance in the best-ranked models, we considered them a constant in our simulations. ST and IS for the 18 Arabidopsis accessions were simulated using the best-ranked models , and th


Pteropus medius bats sampled in Bangladesh during 2013 and 2014.Despite molecular and serologic evidence of Nipah virus in bats from various locations, attempts to isolate live virus have been largely unsuccessful. We report isolation and full-genome characterization of 10 Nipah virus isolates from  Pteropus hypomelanus bats (P. vapmyrus bats (P. lylei bats in Cambodia (Nipah virus (NiV) is an emerging zoonotic virus carried by bats. It is considered a global health priority by the World Health Organization and has pandemic potential because of its zoonotic nature, human-to-human transmissibility, wide geographic distribution of bat reservoir species, high case-fatality rate in humans, and lack of available vaccine or therapeutic agents (https://www.searo.who.int/entity/emerging_diseases/links/nipah_virus_outbreaks_sear/en/).Bangladesh has reported seasonal outbreaks of infectious NiV almost annually since 2001, and India has reported 2 outbreaks in neighboring West Bengal, the last in 2007 .We report isolation of NiVs from P. medius and Rousettus leschenaultia) for NiV. Samples were collected with permission from the Government of Bangladesh Forestry Office and under Institutional Animal Care and Use Committees .During January 2011–April 2014, we collected 2,749 bat samples from various ongoing projects in the region. We collected samples nondestructively from individual bats as described  that had no reported cases . Sampleshttp://www.biosciences.ie/gibco). We added 50 μL of each sample to 2 wells, incubated each sample for 90 min, remove the inoculum, and added 200 μL EMEM to each well. Plates were incubated at 37°C, checked at 7 days postinfection for a cytopathic effect (CPE), and frozen at −80°C.We performed virus isolation under BioSafety Level 4 containment. For virus isolation from PCR-positive samples, we prepared Vero cells in 96-well plates in Eagle minimum essential medium (EMEM) containing 10% fetal bovine serum and 1× antibiotic–antimycotic mixture  for RNA extraction according to the manufacturer’s protocol.To confirm the identity of isolated viruses, we subjected each sample to electron microscopy and PCR. All isolates had morphology consistent with NiV and were positive by NiV-specific PCR. Virus stocks were amplified in Vero cells, and supernatants were harvested and clarified by centrifugation at 10,000 × https://www.neb.com/) according to the manufacturer’s instructions. DNA libraries were subjected to a liquid-based target capture method to separate and enrich for specific NiV sequences. NiV-specific 120-mer biotinylated DNA baits were designed to capture the entire genome. Hybridized probes and captured NiV genetic material were immobilized on magnetic beads, and contaminating host material was washed away, which increased the number of virus-specific reads. We mapped all reads to NiV sequence JN808863 by using the Map to Reference Function in Geneious version 7.1.6 (https://www.geneious.com/previous-versions/). The consensus nucleotide sequence was selected by majority rules at each site.We used a virus enrichment strategy (To ascertain 5′ and 3′ ends because of low coverage and PCR errors and chimeras, we downloaded full-genome NiV sequences from GenBank and aligned them by using MAFFT in Geneious. A 90% similarity threshold was used to generate consensus 5′ and 3′ sequences of 120 nt. Final consensus full genomes had 10× coverage. We downloaded full-genome henipaviruses from GenBank, generated alignment by using the MAFFT plugin in Geneious, and tested node robustness by using 1,000 bootstrap replicates under the general time reversible plus gamma model in PHYML (Full-length genome sequences were obtained from 10 bat NiV isolates. We constructed a phylogenetic tree  that shoP. medius bats, a natural virus reservoir in Bangladesh. With an improved enrichment-based NGS strategy, we generated complete genome sequences for 10 bat NiV isolates with higher efficiency than for traditional PCR-based sequencing methods. NiV has been difficult to isolate from bats and, similar to results of previous studies of Hendra virus (We report isolation of NiV from None of the bat NiV isolate sequences were identical with any previously detected human NiV isolate sequences, suggesting that NiV spillover into humans is a rare event. However, the genetic diversity of bat NiV isolates needs to be fully identified.


MAT1) locus, which typically comprises two idiomorphic genes. The presence of one or both allelic variants at the locus determines the reproductive strategy in fungi—homothallism versus heterothallism. It has been hypothesized that self-fertility via homothallism is widespread in lichen-forming fungi. To test this hypothesis, we characterized the MAT1 locus of 41 genomes of lichen-forming fungi representing a wide range of growth forms and reproductive strategies in the class Lecanoromycetes, the largest group of lichen-forming fungi. Our results show the complete lack of genetic homothallism suggesting that lichens evolved from a heterothallic ancestor. We argue that this may be related to the symbiotic lifestyle of these fungi, and may be a key innovation that has contributed to the accelerated diversification rates in this fungal group.Fungal reproduction is regulated by the mating-type ( MAT1) -box characterizes the core MAT1-2 gene  . This loiomorphs . These v1-2 gene . The tra1-2 gene . The mol1-2 gene .MAT1 locus as, in general, sexual reproduction requires the expression of genes from both MAT1 idiomorphs  uni-/bidirectional mating-type switching when individuals of one or both mating types are able to reversibly (or irreversibly) switch to the opposite mating type forming a mixed, functionally heterothallic colony; and 2) unisexuality when individuals of the same mating type are able to undergo sexual reproduction regardless of the absence of a compatible mating partner. Self-fertility can also be achieved via pseudohomothallism, when opposite mating-type nuclei are packed within a single spore which produces, upon germination, a heterokaryotic, self-fertile mycelium     to predoriaceae) , 2015. Iriaceae) . It is sMAT1 locus of a set of genomes of lichen-forming fungi representing a wide range of growth forms and reproductive strategies, with particular focus on the Lecanoromycetes, the largest clade of lichenized fungi. Our findings will contribute to the understanding of the regulation of reproductive processes and the evolution of the mating locus in the Lecanoromycetes. This will further contribute to our understanding of the mechanisms behind the accelerated diversification of this important and diverse group of symbiotic fungi.In this study, we tested the hypothesis of widespread, ancestral primary homothallism in lichen-forming fungi using genomic data. For this purpose, we gathered whole-genome sequence data and characterized the MAT1 core gene, MAT1-1 or MAT1-2, flanked by the highly conserved cytoskeleton protein (SLA2) and DNA lyase (APN2) genes , the genetic basis of sexual reproduction in lichens remain largely unknown. Here, we analyzed the mating-type locus from 41 genomes representing 4 classes and 9 orders of lichenized fungi. Our results show the loss of primary homothallism in the Lecanoromycetes, the largest group of lichenized fungi . In all 2) genes  online. s abrupt  online. strophus . We founspecific  online: iliensis ; similar species  online. ustulata , the MATMAT1 locus across the wide taxonomic diversity of the Lecanoromycetes under study, including several supposedly asexual lichens. Furthermore, our results indicate a highly conserved organization and synteny of the MAT1 locus in lichens. By and large, our results strongly support the hypothesis of an ancestral heterothallic state in lichens. This scenario is thus similar to what has been described for the evolution of breeding systems in other ascomycete genera such as Cochliobolus  and de facto self-fertile. This would also mean that lichen thalli would comprise multiple mycobiont genomes, thus rendering metagenomic assemblies further challenging . Noteworthy, we showed widespread heterothallism in the largest, phenotypically most heterogeneous group of lichen-forming fungi. We hypothesize that this is related to the symbiotic lifestyle of this fungal group. Furthermore, the consistency of this character allows us to speculate that this may be implicated in the accelerated diversification rates found in the Lecanoromycetes . As such, our study sets the stage for further exploration of the reproductive strategy of lichens, as well as of its evolutionary outcomes.This is the first broad scale study dissecting the genetic architecture of the mating locus in lichen-forming fungi. We characterized the Xanthoria parietina and Gyalolechia flavorubescens), Umbilicariales (Umbilicaria pustulata [Umbilicaria hispanica [Umbilicaria muehlenbergii), Ostropales (Graphis scripta), Peltigerales (Leptogium austroamericanun), and Lecanorales  and Arthoniomycetes (Arthonia rubrocinta).A total of 41 lichen-forming fungal species were included in this study  online. ustulata , Umbilicispanica  and UmbiTotal genomic DNA of 27 specimens of Parmeliaceae were extracted from apothecia or thalli using the Quick-DNA Fungal/Bacterial Miniprep Kit  following the manufacturers’ instructions. DNA concentration was measured using the Qubit dsDNA BR Assay kit . Paired-end libraries (250 bp) were built either using TrueSeq or Nextera XT DNA library preparation kits . Sequencing of Nextera XT libraries was carried out by the University of Illinois at Chicago Research Resource Center  on Illumina NextSeq platform while TruSeq libraries were sequenced on Illumina MiSeq platform at the Pritzker Laboratory for Molecular Systematics and Evolution at The Field Museum, Chicago, IL.http://ww.usadellab.org/cm/? page=trimmomatic) Properly working, 18 February 2019 access (Arthonia rubrocincta (PRJNA256244) and Graphis scripta (PRJNA256475), and the metagenomes of Leptogium austroamericanum (PRJNA256476) and Dibaeis baeomyces (PRJNA256246).Raw sequences were downloaded from an Illumina BaseSpace application and quality-trimmed and filtered using Trimmomatic-0.36  and Arthonia rubrocinta (Arthoniomycetes) were used as outgroup.We followed a similar procedure to infer the phylogenetic relationships among 39 lichen-forming fungi belonging to the class Lecanoromycetes. For this purpose, we extended the orthologs gene set to 3,156 single-copy genes of Pezizomycotina as implemented in BUSCO v3 , resultiSLA2 of Xanthoria polycarpa (CAI59767.1), APN2 of Xanthoria parietina (CAI59775.1), Alpha-domain of MAT1-1 of Xanthoria polycarpa (CAI59771.1), and HMG-domain of MAT1-2 of Dufourea flammea (CAI59780.2) from SLA2, APN2) were included in further analyses.In order to identify the mating-type locus in every genome, we first selected protein sequences of Umbilicaria pustulata (Two loci of opposite mating type from ustulata  were aliustulata . Sequencustulata . Regionsustulata . IntergeGenome Biology and Evolution online.Supplementary DataClick here for additional data file.