The development of synthetic anion transporters is motivated by their potential application as treatment for diseases that originate from deficient anion transport by natural proteins. Transport of bicarbonate is important for crucial biological functions such as respiration and digestion. Despite this biological relevance, bicarbonate transport has not been as widely studied as chloride transport. Herein we present an overview of the synthetic receptors that have been studied as bicarbonate transporters, together with the different assays used to perform transport studies in large unilamellar vesicles. We highlight the most active transporters and comment on the nature of the functional groups present in active and inactive compounds. We also address recent mechanistic studies that have revealed different processes that can lead to net transport of bicarbonate, as well as studies reported in cells and tissues, and comment on the key challenges for the further development of bicarbonate transporters. The development of synthetic compounds for the transmembrane transport of bicarbonate is discussed in this Review, presenting the structures of the different transporters and the methods to study the transport process in liposomal membranes, together with the most recent studies on transport mechanism and biological activity in cells and tissues. As the concentration of CO2 in aqueous solutions is three orders of magnitude higher than that of H2CO3,Bicarbonate is, after chloride, the most abundant anion in extracellular fluids, with concentrations of 22–26 mM in blood serum and cerebrospinal fluid.3)2 and that the IUPAC recommends the use of ‘hydrogencarbonate’, which better describes the HCO3− anion.We note that ‘bicarbonate’ is a trivial name that originates from the carbonate to calcium stoichiometry in Ca. The HCO3− transporter AE1 then transports HCO3− out of the red blood cells, in exchange for Cl−  by AE1, after which the CO2 diffuses out of the red blood cells to get exhaled.A first example of bicarbonate transport is found in red blood cells, which take up CO3− from the urine into the blood takes place to avoid acidification of the organism. CO2 diffuses from the urine into renal cells to get converted into HCO3− and H+ by CAII, resembling the process in red blood cells. The H+ is transported back into the urine by ATPase to give a net absorption of HCO3−. The HCO3− is then transported into the blood by the HCO3−/Cl− exchanger AE1 in certain renal cells .arene scaffold, as an efficient chloride and bicarbonate transporter.Gale and co‐workers compared diaryl (thio)ureas 3 a–c and ceramide 6 are early examples of amide‐based bicarbonate transporters. While the former contain two amide groups pre‐organized by an aryl scaffold to bind the same anion, the latter combines one amide and two alcohol groups as anion binding units. T4 a–l was first studied in 2011 with the commonly used ISE assay (with LUVs suspended in Na2SO4).4 h as the best bicarbonate transporter of the series ureas I], which is indicative of the activity of the transporter at the beginning of the experiment, independent of the concentration. Decalin bis‐urea 8 a showed good Cl−/HCO3− antiport activity in the lucigenin assay at 0.004 mol %, and the [I] determined for this compound was 360 s−1. of 970 s−1 for the latter process.2 also was studied with the lucigenin assay, giving a [I] of 110 s−1, which revealed that decalins 8 a–b are among the few synthetic transporters with significantly higher efficiency than prodigiosin 2.35 d afforded a specific rate [I] of 1600 s−1.35 d showed a specific rate more than ten‐fold higher than prodigiosin 2 in the lucigenin assay, while 31 and 12 i performed only marginally better than 2 in the ISE assay, supports the conclusion that bambusuril 35 d and its analogues (such as 35 h and 35 k) are the most active HCO3− transporters reported to date.The transport curves from a lucigenin assay can be used to obtain the specific initial rate arene scaffold similar to that of squaramide 20. While these two compounds performed Cl−/NO3− antiport with similar activities, 39 is inactive as Cl−/HCO3− antiporter under the conditions used to obtain efficient transport by 20.−/NO3− antiport have no acidic NH or OH groups. We suggest that the poor response obtained for these compounds in Cl−/HCO3− antiport assays might be related to their inability to perform H+Cl− symport, a process that can lead to net Cl−/HCO3− antiport, as discussed in Section 4.2.Although transport of bicarbonate is in general more challenging than transport of nitrate, most transporters able to mediate Cl4.22, as elaborated on in Section 1.1 of this review. Using the newly developed EuL1 assay, we have recently shown that in addition to actual transport of HCO3−, CO2 diffusion can also lead to apparent transport of HCO3− into LUVsIn biological processes, the transport of bicarbonate is often linked to the diffusion of CO3− to LUVs in absence of any transporter, some CO2 can diffuse into the LUVs, where it will react with H2O into HCO3− and H+  is identical to that exterior (at pH∼7.4). In the EuL1 assay, this process leads to a minimal amount of HCO3− entering the LUVs when using a buffer solution with 5 mM HEPES at pH 7. A higher buffer concentration  led to more HCO3− inside the LUVs, as the buffer prevented the acidification of the interior of the LUVs upon diffusion of CO2.Upon addition of HCO+ Figure  leading 3−/Cl− antiporter, the addition of HCO3− triggers the HCO3−/Cl− exchange process, which equilibrates the concentrations of HCO3− inside and outside the vesicles  are identical in the interior and exterior of the LUVs. This could be achieved with a cation transporter such as monensin, which is able to efficiently perform H+/Na+ or H+/K+ transport, but also by anionophores that can perform H+X− symport or OH−/X− antiport . In the lucigenin assay, no gradients of HCO3− or CO2 are present, but the high NaHCO3 concentration in the experiments (225 mM) can serve as a buffer, thus allowing HCl influx into the LUVs. The 13C NMR assay reveals interior HCO3− and does also give a response upon apparent HCO3− transport, as demonstrated with monensin.Most assays for HCOactual and apparent transport of HCO3−. A− and NO3− did also show activity for Cl−/HCO3− antiport. Many of the compounds described as HCO3− transporters have acidic NH groups , as exemplified by transporters 2, 8 a, 8 b, 23 c–e, and 24 d.[3− transport ureas toxicity,80, 97− and HCO3− transport in the disease CF (see Section 1.1) has motivated anion transport studies in epithelial cell lines, using electrophysiological studies[−/Cl− antiport.− transporters has been tested by the YFP method and showed encouraging anion transport activity.[2), Obatoclax (9 a), and compounds 25 c and 31 were also found to cause an increase of the intracellular pH upon perfusion of cells with a solution of NaHCO3 (after a pre‐treatment with NH4Cl), indicating HCO3− transport into the cells.The deficient Cl3− and Cl− and resulting in ‘sticky mucus’.30 b and 31 was shown to increase the pH and the volume of the ASL, while decreasing the viscosity.40, Figure −, but also HCO3−.3− by 40.The disease CF has a drastic impact on the properties of airway surface liquid (ASL), originating from the impaired transport of HCO5−/NO3− and Cl−/HCO3− antiport, but also H+Cl− symport (or OH−/Cl− antiport) and have thus a limited selectivity. Recent studies have shown that the ability of these compounds to dissipate pH gradients could lead to apparent HCO3− transport, a process that relies on CO2 diffusion rather than on transport of HCO3− anions across the membrane. Efficient anion transporters, such as decalin bisurea 8 a and bisthiourea 8 b and carbazole bisamides 23 c–e and bisthioamide 24 d, were found to act via the CO2 diffusion mechanism when present at low transporter concentrations, while true HCO3−/Cl− antiport is the main mechanism at higher concentrations. To the best of our knowledge, bambusurils (35) are the only synthetic transporters that preferentially act via HCO3−/Cl− antiport and that achieve high rates of transport. However, the fluorinated bambusurils are too lipophilic to be deliverable and to have any biological activity. For therapeutic applications, it would thus be of great interest to develop true HCO3− transporters without acidic NH groups, that could be tested on cells and tissues. On the other hand, compounds such as synthetic tambjamines 30 b, 31 and Amphotericin B 40 are not selective HCO3− transporters; nevertheless, they have shown very promising results on cells, tissues and even in animal studies. Apart from biological applications in the context of cystic fibrosis or other channelopathies, HCO3− transporters could also find applications in sensing and CO2 concentration. Furthermore, the lessons learned from all these studies on synthetic HCO3− transporters have also deepened the fundamental insights in ion transport processes by ionophores in general and demonstrated how compound structures can be optimised to achieve maximal activities.In this Review we have given an overview of the different synthetic anion receptors that were demonstrated to be active as bicarbonate transporters. The majority of these compounds uses ureas, thioureas, amides, thioamides or pyrrolic groups to interact with anions via NH‐based H‐bond donor groups. These compounds can typically perform ClThe authors declare no conflict of interest.6Luis Martínez‐Crespo was born in Felanitx (Spain) in 1985. He received his MSc in 2011 and his PhD in 2015 from the University of the Balearic Islands (UIB), under the supervision of Prof. Antonio Costa and Dr. Carmen Rotger. He has worked as a post‐doctoral researcher with Prof. Pablo Ballester in the Institute of Chemical Research of Catalonia (ICIQ), with Dr. Hennie Valkenier at the Université Libre de Bruxelles (ULB) and with Prof. Simon Webb at the University of Manchester (UoM). Currently, he works as a Maria Zambrano fellow at the UIB, with Prof. Antonio Costa. His research focuses on the development of supramolecular systems for transmembrane transport and signalling.Hennie Valkenier is originally from The Netherlands, where she studied at the University of Groningen . She has worked as a post‐doc with Prof. Anthony Davis at the University of Bristol and with Profs. Kristin Bartik and Gilles Bruylants at the Université libre de Bruxelles (ULB), before getting a permanent position as FNRS researcher in the Engineering of Molecular NanoSystems laboratory at the ULB in 2018. She was awarded an ERC starting grant that same year. With her team and collaborators, she is developing synthetic transporters for ions and new methods to study transport processes in liposomes.


The Ccr4-Not complex is mostly known as the major eukaryotic deadenylase. However, several studies have uncovered roles of the complex, in particular of the Not subunits, unrelated to deadenylation and relevant for translation. In particular, the existence of Not condensates that regulate translation elongation dynamics has been reported. Typical studies that evaluate translation efficiency rely on soluble extracts obtained after the disruption of cells and ribosome profiling. Yet cellular mRNAs in condensates can be actively translated and may not be present in such extracts.In this work, by analyzing soluble and insoluble mRNA decay intermediates in yeast, we determine that insoluble mRNAs are enriched for ribosomes dwelling at non-optimal codons compared to soluble mRNAs. mRNA decay is higher for soluble RNAs, but the proportion of co-translational degradation relative to the overall mRNA decay is higher for insoluble mRNAs. We show that depletion of Not1 and Not4 inversely impacts mRNA solubilities and, for soluble mRNAs, ribosome dwelling according to codon optimality. Depletion of Not4 solubilizes mRNAs with lower non-optimal codon content and higher expression that are rendered insoluble by Not1 depletion. By contrast, depletion of Not1 solubilizes mitochondrial mRNAs, which are rendered insoluble upon Not4 depletion.Our results reveal that mRNA solubility defines the dynamics of co-translation events and is oppositely regulated by Not1 and Not4, a mechanism that we additionally determine may already be set by Not1 promoter association in the nucleus.The online version contains supplementary material available at 10.1186/s13059-023-02871-7. Adequate regulation of gene expression is essential for health, fitness, and development of all living organisms. While transcription is the most immediate and focal point of gene regulation, gene expression is also importantly controlled at post-transcriptional levels –4. RepreThe conserved Ccr4-Not complex plays a key role in mRNA metabolism . First iCcr4-Not additionally regulates translation and co-translational processes. Ribosome-associated proteins, notably the nascent polypeptide ribosome-associated complex (NAC) and Rps7A , 43, areCondensate mRNAs, like other insoluble mRNAs such as membrane-associated mRNAs, are not captured by typical ribosome and polysome profiling approaches. In this work, we used the sequencing of mRNA decay intermediates to address whether soluble mRNAs and insoluble mRNAs have different translation elongation dynamics and if different mRNA classes partition differently into soluble and insoluble mRNA pools. We further investigated if elongation dynamics of the different mRNA pools was altered immediately upon depletion of Not1, Not4, and Not5. Taken together, our data indicate that ribosomes dwell at non-optimal codons in the non-soluble RNA fraction. In turn, soluble mRNAs show more mRNA 5′ to 3′ degradation, but proportionally less co-translational decay. Additionally, we determine that the depletion of Not1 and Not4 regulate mRNA partitioning between soluble and insoluble fractions in an opposing manner, which in turn correlates with the opposite impacts on ribosome dwelling at optimal and non-optimal codons in the soluble RNA pool. Our results are compatible with a model whereby mRNA solubility sets the dynamics of co-translational events and is regulated by the Not proteins.Saccharomyces cerevisiae and we proposed that this was due to defective tethering of translating ribosomes to ribonucleoprotein (RNP) condensates, in particular ribosomes paused at non-optimal codons [S. pombe. For each sample, one aliquot was subjected to RNA-Seq to determine the transcriptome and the other to 5′P-Seq to determine mRNA decay intermediates that can provide information on co-translational decay and ribosome dwelling [In a recent study, we showed that the dynamics of translation elongation were greatly affected in the absence of Not4 and Not5 in l codons . This modwelling  . mRNA levels within the soluble RNA pool and total RNA pools correlated overall Fig. A. NevertTo investigate the distribution of 5′P mRNA decay intermediates between soluble and total RNA fractions, we compared the level of mRNA degradation intermediates determined with 5′P-Seq to the overall mRNA level defined with RNA-Seq Table S. We usedThe difference in metagene profiles of 5′P decay intermediates from soluble and total RNAs could be due to differences in the processivity of 5′ to 3′ decay in the soluble and total mRNA pools, directly or indirectly linked to differences in velocities of ribosomes. Ribosome profiling data provides information on ribosome footprints for the soluble mRNA pool. No similar data can be obtained for insoluble RNAs. However, 5′P-Seq data is informative on ribosome dwelling . Indeed,mRNA turnover can occur by mechanisms other than co-translational degradation. To get some idea of this for both RNA pools, we generated metagene profiles of mRNA 5′ ends generated by RNA-Seq and compared them to the metagene profiles of 5′P-Seq. The metagene profiles of 5′end reads of the RNA-Seq were more similar between soluble and total RNA pools Fig. SD than thFor soluble and total RNAs, the 5′P-RDO changes in the second half of the CDS compared to the first half were inversely correlated with codon optimality Fig. SF. These We next investigated the direct role of Not proteins in regulating mRNA solubilities and differences in 5′P-RDOs between total and soluble RNA pools. For this, we created Not4 and Not5 auxin-inducible degron strains, along with the Not1 degron strain described previously . ExpressLibraries from soluble and total RNA pools were generated from the degron strains and their isogenic wild-type counterpart following 15 min of auxin treatment Table SA. Some mn = 914) and decreasing  solubilities in both strains, as we suspected there would be heterogeneity within the control set at these opposing extremes. We checked for specific features of all these mRNA groups, such as their expression level, length and content in non-optimal codons, and solubility in wild type cells   and Pro (CCG) striking out Fig. B.Ser levels following Not1 depletion, but the levels of two seryl-tRNASer isoacceptors markedly decreased  in wild-type cells and in cells lacking Not5. We noted an interesting significantly higher Not1 RIP in ion Fig. A. This wion Fig. F.Fig. 6NWe thus focused on Not4 whose association with RNAs has not yet been characterized. To define to which mRNAs Not4 binds in vivo, we used a photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) approach . In two We compared the mRNAs cross-linked to Not4 to the sets of mRNAs differentially solubilized described above Fig. C. mRNAs not5Δ. We thus mapped Not1 promoter binding genome-wide to determine whether Not1 association with promoters correlated with the Not1-dependent fate of mRNAs in the cytoplasm, in particular their solubility. We fused Not1 to the micrococcal nuclease (MNase) at its own genomic locus, monitored chromatin cleavage events (ChEC), and compared them to the cleavage pattern by the free MNase. In total, 4923 promoters showed cleavages by Not1-MNase above the threshold of significance, and the pattern of Not1-MNase was specific compared to the pattern obtained with free MNase  mRNAs occurs during transcription and regulates their translation in a manner dependent upon Not5 . Above we Table SD. Highere Table SE. In pare Table SF. These Several recent studies provide evidence that the Not proteins are key for codon-optimality-related changes in mRNA stability , 66. A bWe show that the solubility of mRNAs is a determining factor in regulating co-translation dynamics. Indeed, soluble mRNAs show more relative degradation but less detectable co-translational decay compared to insoluble mRNAs, namely all mRNAs that are not soluble without distinction of the different types of insoluble mRNAs. In soluble fractions ribosome movement is faster than the action of the 5′-3′ exonuclease activity. Alternatively, or in addition, mechanisms other than co-translational decay might generate decay intermediates. These could be No-Go-Decay (NGD) that can generate 5′ to 3′ decay intermediates upstream of collided ribosomes , and morSoluble and insoluble mRNAs also exhibit a different distribution of 5′P decay intermediates along CDSs most likely due to differences in ribosome dwelling and hence velocity. Indeed, we show that ribosomes dwelling at non-optimal codons are more prominent in the insoluble RNA pool. Alternatively, the delay of 5′ to 3′ decay initiation after the last trailing ribosome, indicated by higher 5′P-RDOs at non-optimal codons for the second half of CDSs compared to the first half, is less significant for soluble mRNAs, since RNA-Seq reads relative to 5′P-Seq reads in the first half compared to the second half of CDSs are higher.In previous work, we proposed that tethering of mRNAs by Not proteins to condensates, thus to insoluble RNA pools, in particular at start and at non-optimal codons, modulates translation elongation dynamics . In lineWhile these findings indicate that the Not proteins act together and in a similar manner, several observations contradict this simple interpretation. First, depletion of Not4 or Not5 results in higher, but that of Not1 lower, relative degradation for soluble mRNAs. Higher levels of 5′P decay intermediates in the second CDS half are observed most significantly upon depletion of Not1 for soluble mRNAs, but upon depletion of Not4 for total mRNAs. Importantly, mRNA solubility was mildly altered by depletion of Not5, but inversely impacted by the depletions of Not1 and Not4  and PCR amplification of a 9Myc-NATMX4 cassette with Not4- and Not5-specific primers using plasmid pE641. The Not1 degron strain (13517) has already been described [MATa leu2Δ20 ura3Δ met15Δ his3Δ1) with Not4-specific primers by PCR using pE557, the strains expressing the Not1-MNase fusion (MY12601) was generated with Not1-specific primers by PCR using pE611 respectively. The free MNase strain was MY13783 (MATa tor1-1 fpr1::loxP-LEU2-loxP RPL13A-FKBP12::loxP ura3::MNAse-URA3).Plasmids and strains are listed in Table Strain 135 has alreSchizosaccharomyces pombe, before the RNA samples were then split in 2, one aliquot subjected to RNA-Seq to determine the transcriptome and the other to 5′P-Seq to determine mRNA decay.Total RNA was prepared either by the hot acid phenol method  or cellsTotal protein was prepared by post-alkaline lysis and analyzed by western blotting with antibodies to Not1, Not4, and Not5 that were our own polyclonal antibodies previously described , 73. Ant5′P-Seq.Schizosaccharomyces pombe as spike-in, was used. Each sample was split in two. One part was used for preparing conventional HT-5PSeq libraries and the other part was random fragmented prior to the preparation of HT-5PSeq libraries (negative control).RNA was prepared from 50 ml of exponentially growing cells in YPD and treated or not with auxin. HT-5PSeq libraries were generated as reported  with minFor HT-5PSeq Libraries: 7.5 μg RNA was ligated over night at 16 °C to r5P_RNA_MPX oligo (CrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU rXrXrXrXrXrX rNrNrNrNrNrNrNrN) carrying a sample barcode (rX) and unique molecular identifiers (rN). Ligase was deactivated using 5 mM EDTA and heat at 65 °C for 10 min  and subsequent purified using 1.8 × volumes of RNAClean XP beads (Beckman Coulter). Ligated RNA was then reverse transcribed using random hexamer  and oligo-dT  oligos to prime. After, the remaining RNA was degraded using NaOH. Ribosomal RNA was removed using previously described rRNA DNA oligo depletion mixes, following a duplex-specific nuclease  digestion. rRNA-depleted cDNA was amplified by PCR (17 cycles) and final product was enriched for fragments with the range of 300–500 nt using Ampure XP.Size selected HT-5P Libraries were quantified by fluorescence , size estimated using an Agilent Bioanalyzer and sequenced using a NextSeq500 Illumina sequencer (75 cycles High output kit).Cells expressing Not4 tagged at the C-terminus with the HTB tag  from itsNOT1 or NOT5 genes generated using PCR-amplification of tagged alleles and homologous recombination according to standard techniques [REB1 promoter were used as a control [600 = 0.7. Cells were washed twice with buffer A  and resuspended in 200 μl of buffer A with 0.1% digitonin. The cells were incubated for 5 min at 30 °C. Then, MNase action was induced by the addition of 5 mM CaCl2 and stopped at 150 s for Not1 and Not5 ChEC-seq and 20 min for Mnase under the control of REB1 promoter by adding EGTA to a final concentration of 50 mM. DNA was purified using MasterPure Yeast DNA purification Kit (Epicentre) according to the manufacturer’s instruction. Large DNA fragments were removed by a 5-min incubation with 2.5 × volume of AMPure beads (Agencourt) after which the supernatant was kept, and MNase-digested DNA was precipitated using isopropanol. Libraries were prepared using NEBNext kit (New England Biolabs) according to the manufacturer’s instructions. Before the PCR amplification of the libraries small DNA fragments were selected by a 5-min incubation with 0.9 × volume of the AMPure beads after which the supernatant was kept and incubated with the same volume of beads as before for another 5 min. After washing the beads with 80% ethanol the DNA was eluted with 0.1 × TE and PCR was performed. Adaptor dimers were removed by a 5-min incubation with 0.8 × volume of the AMPure beads after which the supernatant was kept and incubated with 0.3 × volume of the beads. The beads were then washed twice with 80% ethanol and DNA was eluted using 0.1 × TE. The quality of the libraries was verified by running an aliquot on a 2% agarose gel. Libraries were sequenced using a HiSeq 2500 machine in single-end mode. To analyze the Not1- and Not5-MNase binding pattern, read ends were considered to be MNase cuts and were mapped to the genome (sacCer3 assembly) using HTSstation .Not1 ChEC-Seq experiments were essentially performed as previously described  with thechniques  were use control . One samTo determine the fraction of aminoacyl-tRNAs, we followed the procedure described in . For thiFor tRNA abundance, total RNA was isolated at alkaline pH to simultaneously deacylate all tRNAs. tRNAs isolated from Not1 depleted cells were labeled with Cy3-labeled RNA/DNA stem-loop oligonucleotide and were hybridized on the same microarray with tRNAs isolated from the wild-type strain and labeled with Att647-labeled RNA/DNA stem-loop oligonucleotide. The arrays were normalized to spike-in standards, processed, and quantified with in-house python scripts.S. cerevisiae (R64-1–1) and S. pombe (ASM294v2) using STAR.Sequencing files were demultiplexed using bcl2fastq v2.20.0.422 , and adapters were trimmed using cutadapt 2.3.  at defauS. cerevisiae (R64-1–1). Counts in S. cerevisiae were calculated by aggregating RNA-Seq reads and 5′P-Seq 5′-ends, overlapping CDS positions. Differential expression was performed using DESeq2 [Second read enables to split reads between oligo-dT or random primer. That information was not used in the current analysis. CDS positions were defined with Ensembl gff version 94 for g DESeq2 .We define this as the log fold change produced by DESeq2, dividing RNA-Seq counts for the soluble fraction in a given sample by the corresponding counts for the total fraction of the same sample.S. pombe data was used in the calculation of relative degradation. We define it as the log fold change produced by DESeq2, comparing counts in 5′P-Seq to their corresponding RNA-Seq sample using estimateSizeFactors on counts mapping to S. pombe to adjust for spike-in. Enrichment is calculated using a hypergeometric test for over-representation of hits in defined gene set for GO SLIM categories for S. cerevisiae.Spike-in To calculate 5′P-Seq pausing scores, equivalent to A site ribosome dwelling occupancy, the mean depth was calculated 17nt upstream of each codon type for each strain in the regions or transcripts of interest. In all cases, the values are normalized to the mean depth over all codons for the regions or transcripts included in the calculation. Where two conditions are compared, the differential RDO is calculated as the log2 fold change of these normalized values for each codon.For 5′P-Seq, metagenes at start and stop are calculated by aggregating the depth of 5′ ends at each position relative to start or stop for every CDS and normalizing each by the total depth per million genome-wide. These values are shifted 17nt downstream for equivalency with the A-site position, in the case of co-translational decay.For scaled metagenes, every CDS was split into 100 equal bins and the mean depth of 5′ ends of RNA-Seq and 5′P-Seq was calculated for each bin. This was averaged over all transcripts of interest and normalized to the mean depth over all nucleotides in this transcript group.FASTQ files were adapter stripped, using cutadapt  and then mapped using bowtie  to sacCeFASTQ files were adapter stripped, using cutadapt  and then mapped using bowtie2  to sacCeValues for Ribo-Seq RPKMs were calculated as in our previous paper .p-value by t-test. All correlations and correlation tests were performed on groups of at least 30 in size. Enrichment of gene sets is defined via FDR after Benjamini–Hochberg adjustment from p-values generated using a hypergeometric test. All t-tests were performed on sample sizes of 30 or higher, where the central limit theorem applies regarding the normality assumption. We use Welch’s t-test in all cases, rather than Student’s t-test, resulting in more conservative p-value, which is more reliable where variances and sample sizes are unequal. In one case, we used a Wilcoxon rank sum test [t-test, so as to avoid any possible breach of the normality distribution assumption, since both samples had a size of 15 .Reported correlations were the Pearson’s product-moment correlation coefficient and were used as test statistics to generate the associated sum test  to compaAdditional file 1: Additional figures and figure legends in a single pdf file with: Figure S1. Comparison of mRNAs that are more or less soluble. Figure S2. mRNAs that are more or less soluble upon Not1 and Not4 depletion are enriched for different GO-terms. Figure S3. Comparison of RDO changes between mutants and RNA pools. Figure S4. Quality control of the Not4 PAR-CLIP. Table S5. List of strains and plasmids.Additional file 2: Table S1. 5’P-Seq and RNA-Seq of soluble and total RNA pools before and after depletion of Not1, Not4 and Not5. Additional file 3: Table S2. Changes in solubility upon Not1, Not4 and Not5 depletion.Additional file 4: Table S3. Not4 and RNAPII PAR-CLIP. Additional file 5: Table S4. Not1-ChEC-Seq.Additional file 6. A separate pdf file entitled: Uncropped blots. Related to Figure S2A.Additional file 7. Review history.


To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR.P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency.Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity.Our results indicated that the In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.The online version contains supplementary material available at 10.1186/s12985-022-01956-2. GFP gene, showed a lower viral titer than the severe strain TuGR (a wild-type TuMV carrying a GFP gene) , and the PAGE gel was exposed to chemiluminescence film (Cytiva) to visualize the RNA. The band with a strong signal was excised and mashed in 300 μL extraction buffer  and passed through centrifuge tube filters (Costar). The supernatant was further cleaned up using the TRIzol method. RNA was diluted with 5 μL water to obtain a radioactive strength of 300 to 500 CPM. The original data of RISC assay are provided in Additional file cDNA fragments, including 32P-labeled substrate RNA (60 CPM/ μL) in 25 μL 2 × cleavage buffer  incubated with AGO1-IP beads that contain 5, 10, or 20 ng AGO1, which quantified with standard concentrations of recombinant AGO1, at 25 °C for 1 h under rotation. The RNA fragment was purified by the TRIzol method. The RNA was solubilized in 10 μL water with 10 μL RNA Gel Loading Dye (Invitrogen) and denatured at 85 °C for 2 min. The RNA was separated on 6% denaturing PAGE gel and exposed to chemiluminescence film for 5 days to obtain the image.The IP reaction for RISC activity was performed with 15 μL 1 mg/mL IgG, 30 μL beads, 500 μL lysate and ultimately resuspended in 50 μL 1 × PBS. For the slicer assay, 5 μL The signals for full-length substrate, 5’- and 3'-cleaved fragments were detected using ImageJ. For calculation of the RISC cleavage efficiency, the product signal (5'-cleaved fragment + 3'-cleaved fragment) was divided by the total signal (full-length substrate + 5'-cleaved fragment + 3'-cleaved fragment), and the percentages were calculated. Each cleavage efficiency was normalized to the corresponding AGO1-IP signal, which was detected using ImageJ.For the detection of AGO1 protein in tissue lysates, 20 μL supernatant was diluted with 20 μL 1 × PBS buffer, and the lysate was denatured in 40 μL 2 × protein sample buffer. For IP-AGO1 detection, 90 μL 1 × PBS buffer and 100 μL 2 × protein sample buffer were added to 10 μL IP beads. The protein samples were boiled at 100 °C for 10 min.The protein samples were separated and analyzed by polyacrylamide-SDS-PAGE. After electrophoresis, the separated proteins were transferred to a PVDF membrane by electroblotting. Polyclonal AGO1 , coat protein , and three HC-Pro  IgGs were produced in our lab and used as the primary antibody. Monoclonal β-tubulin antibody  was used for control. The signal was visualized by enhanced chemiluminescence detection with a Western Lightning™ Pro kit (PerkinElmer). The original images of western blot are provided in Additional file Additional file 1: Table S1. The primer list for this study.Additional file 2. Supplementary Figures.


In this review, we explore recombination in two very different virus families that have become major threats to human health. The Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Coronaviridae are positive-strand RNA viruses (CoVs) that have also become major threats to global health and economic stability, especially in the last two decades. Despite many differences, such as the make-up of their genetic material (DNA vs. RNA) and overall mechanisms of genome replication, both human herpes viruses (HHVs) and CoVs have evolved to rely heavily on recombination for viral genome replication, adaptation to new hosts and evasion of host immune regulation. In this review, we will focus on the roles of three viral exonucleases: two HHV exonucleases  and one CoV exonuclease (ExoN). We will review the roles of these three nucleases in their respective life cycles and discuss the state of drug discovery efforts against these targets. It has long been recognized that genetic recombination plays many important roles in the biology of all living organisms, including the repair of double-strand breaks (DSBs) and the generation of diversity during evolution. Recombination not only allows cells to maintain chromosomal stability and prevent genetic loss but also enables organisms to adapt and evolve. Extensive research on the mechanisms of recombination in both prokaryotic and eukaryotic cellular organisms has revealed a complex set of pathways by which genetic information is exchanged and repaired. Given the importance of these processes, it is perhaps not surprising that viruses have also evolved to rely heavily on recombination for genome replication and repair and to promote viral diversity ,3,4,5,6.When double-strand breaks arise in cellular organisms, two major pathways of homology-driven DNA repair exist to repair broken ends and prevent genetic loss. Homologous recombination (HR) utilizes a classical recombinase that can carry out strand invasion (RecA for bacteria and Rad51 for eukaryotes) . In the Herpesviridae are a large family of pathogenic double-stranded DNA viruses involved in a range of diseases affecting both people and animals. Over 90% of the human population is infected with one or more of the nine human HHVs [The man HHVs . The HHVPrimary infection with Herpes simplex virus (HSV 1/2) is associated with painful blisters affecting the skin, mouth, lips, eyes and genitals, as well as life-threatening diseases such as encephalitis, meningitis and neonatal herpes. Primary infection with VZV causes varicella (chickenpox), which can be severe in immunocompromised individuals. Infections with CMV (a β-HHV) are often asymptomatic in immunocompetent hosts, although pregnant women can transmit the virus to a developing fetus, resulting in congenital infections that can lead to permanent hearing loss, loss of vision and/or mental impairment in infants and children ,24. DuriAll HHVs establish life-long, latent infections that undergo periodic reactivation in the host . ReactivHHVs are known to induce inflammation, especially in neuronal tissues, leading to serious neurological diseases such as encephalitis, meningitis and epilepsy. HSV, CMV and the roseolaviruses have been shown to infect the central nervous system (CNS), possibly due to the ability of these viruses to alter the permeability of the blood–brain barrier. A growing body of evidence suggests that human HHVs are linked to degenerative central nervous system conditions such as Alzheimer’s disease (AD) and multiple sclerosis (MS) ,43,44,45Although the α-, β- and γ-HHVs differ in their tissue tropisms and associated pathologies, most of the replication machinery is conserved across all three subfamilies, suggesting that the mechanisms by which they replicate their DNA genomes are similar . StudiesSeveral lines of evidence support the notion that HSV performs recombination-dependent replication. Evidence for high rates of recombination between coinfecting HSV genomes comes from cell culture and animal infection models, as well as from analysis of viruses circulating in patient populations ,62,63,64Currently, HHV infections are treated primarily with agents that target viral DNA polymerases. These include nucleoside analogues acyclovir/ganciclovir (ACV/GCV), nucleotide analogues (cidofovir) and pyrophosphate mimetics (foscarnet). ACV/Val-ACV is used for first line HSV therapy and prophylaxis, although long-term treatment can lead to the development of drug resistance, especially in immunocompromised patients ,70. GCV HCoV-229E, HCoV-OC43, HCoV-NL63 and HKU1) are responsible for ~15–30% of cases of the seasonal “common cold” in humans and are largely associated with relatively mild symptoms [SARS-CoV-1 (2002–2004) [MERS-CoV (2012–present) [SARS-CoV-2 (2019–present) [Coronaviruses (CoVs) are enveloped viruses containing a single-strand, positive-sense RNA genome of approximately 26–32 kilobases, an unusually large size for an RNA virus . CoVs casymptoms ,84. Othesymptoms . Over th02–2004) , Middle present) ,88 and Spresent) . Such sppresent) ,92,93,94present) , discusspresent) ,103,104.SARS-CoV-2 in patient samples [It has been recognized since the early 1960s that RNA–RNA genome recombination can occur in RNA viruses such as poliovirus ,106. Rec samples  also relORF1a and ORF1b) that are subsequently, proteolytically processed to generate 16 non-structural proteins (nsps) (nsps1–16) [After fusion of the enveloped CoV virus particle at the cell membrane, the capped positive-strand genome is deposited into the cytoplasm where it can be directly translated to produce two large overlapping ORFs (sps1–16) ,120. Nspsps1–16) ,122,123.sps1–16) .Several of the CoV replicase proteins are universal among positive-strand RNA viruses, such as RNA-dependent RNA polymerase  and helicase (nsp13); however, in addition, CoVs and the other Nidoviruses encode proteins that are unique and play roles not typically associated with RNA viruses ,127,128.The second evolutionary adaptation made by CoVs to improve replication fidelity is the acquisition of a novel RNA exoribonuclease (ExoN) with proofreading ability . ProofreAn interesting result of the acquisition of ExoN is its effect on the sensitivity of CoVs to nucleoside analogue inhibitors. Although many RNA viruses can be treated with ribavirin, this drug has minimal activity against the CoVs. Ribavirin is thought to be misincorporated into the RNA of sensitive viruses, such as RSV and HCV, resulting in lethal mutagenesis in the viral progeny . ExoN isAs previously noted, ExoN has been shown to be essential for recombination in three different strains of CoV, including murine hepatitis virus (MHV) and the β-coronaviruses SARS-CoV-2 and MERS-CoV, as demonstrated by RNA next-generation sequencing technology . All thrSARS-CoV-2, evasive variants quickly appeared, underscoring the fact that vaccines alone are not sufficient to combat the current crisis and should not be solely relied upon to prepare for future emergences. Currently, there are limited treatment options for CoV infections beyond the aforementioned mRNA vaccines. Several monoclonal antibody cocktails have proven effective; however, resistance of the newer variants to the mAb therapies is of concern. Historically, nucleoside and nucleotide analogues that target viral polymerases have provided an effective first-line therapy for RNA and DNA viruses alike. However, as described above, ribavirin and other nucleoside analogues are largely ineffective against CoVs because of ExoN [SARS-CoV, SARS-CoV-2 and MERS-CoV and has been approved for SARS-CoV-2 use [While multiple coronavirus vaccines were rapidly developed in an effort to reduce the uncontrolled spread of  of ExoN . The nucoV-2 use . HoweveroV-2 use . Other toV-2 use ,143. UnfAlthough HHVs and CoVs differ in many respects, we are struck by some remarkable similarities. Recombination plays a major role in the replication strategies of both virus families, and the HHV and CoV nucleases involved in resection  are essential for viral replication. Both viruses encode proofreading exonucleases (PolExo and ExoN) that have been shown to be essential for ensuring replication fidelity. The evidence reviewed so far thus suggests that the three viral nucleases discussed herein provide promising new targets for antiviral therapy.vaccinia virus, another family of large DNA viruses, employs an unusual recombination system in which the 3′-to-5′ exo of the viral polymerase is proposed to resect broken DNAs, generating 5′ ssDNA tails that anneal with complementary ssDNA [While a detailed discussion is beyond the scope of this review, it is worth noting that other viruses also stimulate recombination-dependent replication (RDR). Like the herpesviruses, baculoviruses encode a 5′-to-3′ exonuclease that interacts with an ssDNA annealing protein to make a complex (Exo/SSAP) that can stimulate SSA ,144. Intry ssDNA ,146. TheAs previously discussed, antiviral drug discovery has centered mainly around the misincorporation of toxic nucleotide/side analogues into the viral genome via the polymerase. The complex viral replication machinery of HHVs and CoVs offers a wealth of underexplored, essential targets involved in the synthesis and processing of genomic material. In addition to potential standalone antiviral agents, exploiting these essential targets would open the door for powerful combination therapies that could reduce the onset of resistance, improve efficacy and also aid in reducing dose-limiting toxicity.The wide array of nucleic acid processing enzymes (nucleases), characterized by their ability to cleave the phosphodiester bonds of either DNA or RNA, has been extensively reviewed in . This brSeveral notable efforts aimed at inhibiting enzymes of this type have produced novel DAAs. For example, compounds such as raltegravir and dolutegravir target the HIV integrase, a TMID nuclease that catalyzes an essential strand transfer reaction ,149,150.Exonucleases non-specifically cleave the terminal phosphodiester bond of either DNA or RNA, whereas endonucleases catalyze internal cleavage often in a sequence-selective manner. It is important to note that the exonucleases operate with a preferred polarity, excising specifically at either the 5′ or 3′ terminus. Mechanistically, TMID exonucleases employ the magnesium ions to both position and activate the phosphodiester bond for cleavage . Bond scHuman herpes viruses encode several TMID nuclease enzymes from different superfamilies: the alkaline nuclease (DEK family) , the terAll HHVs encode a well-conserved version of this important protein ,157,158.Crystal structures of the alkaline nucleases from both EBV  ,165,166 2+ ions required for exonuclease activity [The polymerase activity is a well-established antiviral target, primarily using nucleoside/tide inhibitors that are misincorporated during DNA synthesis ,171,172.activity ,57, the activity ,175,176.The genetic experiments described above support the hypothesis that AN is an excellent target for anti-HHV drug discovery. This hypothesis is also supported by previous efforts to inhibit ANs from HSV and CMV with small molecules ,178,179 In our laboratory, we conducted high-throughput screening of a small library of compounds against the alkaline nucleases derived from SOX, EBV, CMV and HSV. This screen identified several hits in the low micromolar range, such as purpurogallin and compound 2 ; howeverWe also tested integrase inhibitors, BXA and two 8-hydroxyquinolines (HQs), from our library for inhibition of HSV/CMV in cell culture, as well as inhibition of purified TMID proteins . The HIVThe ExoN domain of nsp14 is a magnesium-dependent exoribonuclease that acts in a 3′→5′ fashion on both ss- and dsRNA ,96. Nsp1SARS-CoV-2 and treated with ATA (500 mM) and PV6R (200 mM), leading to 66.6- and 25-fold reductions in the viral genome copy number, respectively.Biochemical and computational screenings have been performed to identify small molecule inhibitors of the proofreading activity. Not surprisingly, many of these leads possess well-recognized metal-binding pharmacophores . A fluor50 17.43–21.99 μM) and were further investigated for antiviral activity against a related seasonal coronavirus, HCoV-OC43 [Another more recent study used a computer-generated model of ExoN in complex with RNA to evaluate potential ExoN inhibitors . This inCoV-OC43 . InteresThe scale of the current pandemic has underscored the need for more effective and safe antiviral agents for both recurring and emerging viral pathogens. The success of targeting viral nucleases characterized by a two-metal-ion-dependent active site, such as HIV integrase and influenza PA endonuclease, with potent and selective small molecule inhibitors suggests that similar viral enzymes could also be exploited for new antiviral drug discovery. In this review, we evaluated the potential of targeting viral exonucleases from both human herpesvirus and coronavirus pathogens and reviewed the underlying contribution these enzymes make in the viral life cycle. PolExo and ExoN are critical for proofreading and ensuring replication fidelity. Targeting this vital function is anticipated to induce lethal mutagenesis through catastrophic levels of mutations in progeny genomes and to provide potential synergy with mutagenic nucleoside analogues. From the standpoint of drug discovery, exonucleases have received almost no attention compared to the endonucleases, such as integrase and PA. Although attempts have been made to identify early lead matter through screening, the majority of identified hits are compounds that indiscriminately bind metals. We are focused on the use of structure-based methods to optimize drug-like compounds that can engage the metal-binding centers of the exonuclease active sites with high selectivity. We believe this approach holds significant value in developing next-generation antiviral drugs.


The segmented negative-sense RNA genome of influenza A virus is assembled into ribonucleoprotein complexes (RNP) with viral RNA-dependent RNA polymerase and nucleoprotein (NP). It is in the context of these RNPs that the polymerase transcribes and replicates viral RNA (vRNA). Host acidic nuclear phosphoprotein 32 (ANP32) family proteins play an essential role in vRNA replication by mediating the dimerization of the viral polymerase via their N-terminal leucine-rich repeat (LRR) domain. However, whether the C-terminal low-complexity acidic region (LCAR) plays a role in RNA synthesis remains unknown. Here, we report that the LCAR is required for viral genome replication during infection. Specifically, we show that the LCAR directly interacts with NP and this interaction is mutually exclusive with RNA. Furthermore, we show that the replication of a short vRNA-like template that can be replicated in the absence of NP is less sensitive to LCAR truncations compared with the replication of full-length vRNA segments which is NP-dependent. We propose a model in which the LCAR interacts with NP to promote NP recruitment to nascent RNA during influenza virus replication, ensuring the co-replicative assembly of RNA into RNPs. The influenza A virus genome is composed of negative-sense single-stranded viral RNA (vRNA) segments, which are assembled into separate viral ribonucleoprotein (vRNP) complexes with a heterotrimeric RNA-dependent RNA polymerase and multiple copies of viral nucleoprotein (NP) . The virlignment . Furtherlignment . It has lignment ,10,11. Tlignment . HoweverAcidic nuclear phosphoprotein 32 (ANP32) family proteins are known as important host factors of influenza viruses specifically supporting viral genome replication ,17. ThesIn this paper, we investigate the role of the ANP32 LCAR in influenza A virus genome replication. We show that the ANP32 LCAR is required for RNA synthesis during virus infection and interacts directly with NP. Two previously identified RNA binding grooves of NP contribute to the interaction. We also find that the LCAR is important for polymerase function in a minigenome assay using a full-length neuraminidase (NA) genome segment as template RNA. However, the LCAR is dispensable for RNA production from a 47-nucleotide(nt)-long template which can be replicated in the absence of NP . We propl-glutamine (Gibco). All cells were maintained at 37°C with 5% CO2. Influenza A/WSN/33 (H1N1) virus was generated using the pHW2000 eight-plasmid system (Human embryonic kidney (HEK) 293T cells (293T) and the previously described ANP32A/ANP32B double knockout human embryonic kidney 293T cells (293T-DKO)  were culd system . Plasmidd system  expressid system , pPOLI-Nd system  expressid system . Plasmidd system . All con293T-DKO cells in six-well plates were transiently transfected with 2.5 μg of plasmids expressing the indicated ANP32 proteins or their truncated versions. Twenty-four hours post-transfection, cells were infected with influenza A/WSN/33 virus at a multiplicity of infection (MOI) of 5 in DMEM containing 0.5% FBS. Eight hours post-infection, cells were harvested for the extraction of total RNA, and RNA levels were analysed by primer extension.32P-labeled NA or NP segment-specific primers. A primer targeting cellular 5S rRNA was included as an internal control. Transcription products were resolved by 6% or 12% denaturing PAGE with 7 M urea in TBE buffer and detected by phosphorimaging on an FLA-5000 scanner (Fuji). ImageJ was used to quantify cDNAs and values were normalized to the cDNA derived from the 5S rRNA control. The values for the ‘vector’ control were subtracted from the sample values. Data were analysed using Prism 8 (GraphPad).Total RNA was extracted from 293T-DKO cells in six-well plate using 500 μl TRI reagent (Sigma) according to the manufacturer's instructions and 2 μg of RNA were subjected to primer extension analysis as previously described . BrieflyApproximately 80% confluent monolayers of 293T-DKO cells in six-well plates were transfected with plasmids pcDNA-PB1 (1 μg), pcDNA-PB2 (1 μg), pcDNA-PA (0.5 μg), and pcDNA-NP (2 μg) together with pPOLI-NA (0.2 μg) and plasmids encoding the indicated ANP32 proteins (2.5 μg) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For the NP-independent replication assay pcDNA-NP was omitted and the pPOLI-NA plasmid expressing NA vRNA was replaced with pPOLI-NP47 expressing a 47-nt short vRNA-like template derived from the NP segment. Total RNA from transfected cells was isolated using TRI reagent at the indicated time points post transfection. The extracted RNA was analysed by primer extension assay as previously described .huANP32A, huANP32B, chANP32A and their truncated versions were probed with rabbit anti-huANP32A , rabbit anti-huANP32B , and rabbit anti-chANP32A  antibodies. Beta-actin was probed with a mouse anti-beta-actin antibody . Goat anti-rabbit and anti-mouse antibodies conjugated to horseradish peroxidase (HRP) were used as secondary antibodies. Detection was carried out using Amersham ECL Western blotting detection reagents .Escherichia coli. Proteins were purified on Glutathione Sepharose . GST-tagged or untagged NP were released with 25 mM reduced glutathione or cleavage with PreScission protease overnight, respectively, and further purified on a Superdex 200 Increase 10/300 GL column  using 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl and 5% (v/v) glycerol. Full-length and truncated GST-tagged huANP32A, huANP32B and chANP32A were cloned into pGEX-6P-1 as described above for NP. After purification on Glutathione Sepharose, GST-tagged ANP32 proteins were eluted using 25 mM reduced glutathione. Proteins were buffer exchanged in 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl and 5% (v/v) glycerol to remove glutathione using Amicon concentrators (Millipore). An empty pGEX-6P-1 vector was used to produce a GST tag as negative control. Untagged chANP32A (1–220) was produced following the same protocol as described above for NP.Wildtype and mutant NP from influenza A/NT/60/1968 (H3N2) virus were cloned into pGEX-6P-1 vector  with an N-terminal GST tag followed by a PreScission protease site for expression in 2, 1 mM PMSF, 1× complete EDTA-free protease inhibitor cocktail tablet (Roche)) in the presence or absence of 250 U of Benzonase (Sigma). 400 μl of cell lysate was applied to the beads with bait bound as described above. The beads were then incubated for another 3–5 h and washed 3 times before eluting overnight with 25 mM reduced glutathione. Samples were analysed by western blotting.All pull-down assays were performed in 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol at 4°C. Approximately 200 μg bait  was incubated with 50 μl Glutathione Sepharose  for 2–3 h. The beads were then washed once with the same buffer as specified above before being loaded with 100 μg analyte (wildtype or mutant NP). If indicated, a 1.5× or 4.5× molar excess (over NP) of a 29-nt RNA  was added together with NP. The beads were then incubated for another 3–5 h and washed 3 times with the same buffer as above. GST tag was cleaved overnight with 50 μg PreScission protease in the presence of 1 mM DTT to release un-tagged bait from the beads. Protein samples were analysed by SDS-PAGE. To analyse binding of huANP32A and NP from cell lysates to purified GST-NP and GST-huANP32A, respectively, 293T cells in six-well plates were transfected with 3 μg of pCAGGS-huANP32A or pcDNA-NP. Forty-eight hours post transfection cells were lysed for 1 h at 37°C in 500 μl of cell lysis buffer  glycerol, 0.5% NP-40, 1 mM β-mercaptoethanol, 2 mM MgClAnalytical size exclusion chromatography (SEC) experiments were performed on a Superdex 200 Increase 10/300 GL column  in 25 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 5% (v/v) glycerol at 4°C. In the analytical SEC of NP (R416A) and chANP32A (1–220), either a mixture of purified NP (R416A) and chANP32A (1–220), or a complex of the two from a GST pull-down, was loaded on the column. The mixture of purified NP (R416A) and chANP32A (1–220) (1:2 molar ratio of NP:ANP32A) was incubated on ice for 3 h before injection onto the column.The assay was performed as described previously  with minWe have previously reported the structure of a complex between the influenza virus polymerase and ANP32A . In the ). However, deletion of most of the LCAR (construct 1–188) or complete LCAR together with part of the LRR (construct 1–149) diminished RNA accumulation to basal levels (vector). Similar results were observed for huANP32B although construct 1–188 was able to support some activity while construct 1–208 showed some reduction, as compared to wildtype  . All ANPe Figure . These re Figure ,28,29.We hypothesized that the highly acidic flexible LCAR could act as a molecular whip recruiting the basic NP to nascent RNA during influenza virus RNA genome replication. To address this, we performed assays using purified recombinant ANP32 proteins and NP. The NP we used is derived from influenza A/NT/60/1968 (H3N2) virus . Since pTo further investigate the ANP32-NP interaction, we used a series of LCAR-truncated chANP32A constructs Figure . The amoThe interaction of chANP32A with NP was further tested by size exclusion chromatography. Complexes were prepared by either GST pull-down or pre-mixing individually purified proteins. Both samples resulted in an earlier elution peak on a Superdex 200 column, compared with either chANP32A or NP alone Figure . This inTo characterize the ANP32A binding site on NP, and to gain a better understanding of the ANP32A-NP complex, we made several NP mutants based on its structure Figure  and perfNext we set out to test whether oligomeric NP could bind to chANP32A, using wildtype and a G1(4) mutant NP (R74A/R75A/R174A/R175A) in the assay. We found that wildtype NP bound to chANP32A but showed very little binding to the control GST tag Figure . JudgingIn addition to the G1 RNA-binding groove, NP possesses a second RNA-binding groove referred to as G2 . AvailabTo test this hypothesis, we generated several mutants of NP with either the tail loop (ΔT) or the C-terminal tail (ΔC) deleted. Removal of the C-terminal tail (residues 491–498) rescued chANP32A binding affinity of the monomeric G1(4) mutant, independent of whether the R416A point mutation or whole tail loop (402–428) deletion was used to prevent NP oligomerization Figure . These dGaussia luciferase (gluc1) fused to the C terminus of NP and the C-terminal half (gluc2) fused to the C terminus of huANP32A, huANP32B and chANP32A. We used huANP32A lacking the complete LCAR as negative control (huANP32A 1–149). We detected significant bioluminescence in lysates containing any of the three full-length ANP32 proteins and NP, compared with truncated ANP32A  or wildtype ANP32 proteins. Replication of the full-length template was substantially reduced in the presence of truncated huANP32A (1–188) compared to wildtype huANP32A at all time points tested. In contrast, replication of the short vRNA-like template only showed reduction at 12 h but by 24 h post transfection reached levels similar to those observed in the presence of wildtype huANP32A Figure . ChangesRecently there has been considerable interest in the ANP32 family of proteins, essential host factors in influenza virus replication that work in concert with the viral RNA polymerase to mediate the replication of the viral RNA genome ,29,36–45Segmented NSVs do not encode an equivalent to the P protein expressed by non-segmented NSVs, therefore how free NP is recruited to the nascent RNA strand to form RNPs remains elusive. From data shown in this paper, we propose that influenza virus uses the LCAR of ANP32 proteins to substitute for the function of P protein in RNP assembly. The interactions between ANP32 proteins and NP are likely to be electrostatic due to the involvement of the highly acidic LCAR and basic grooves of NP. This is different from interactions between N and P proteins from non-segmented NSVs, which are mostly hydrophobic . In addiWe propose a model in which the LCAR of ANP32 proteins is specifically involved in NP recruitment to nascent viral RNA. After polymerase dimerization mediated by the N-terminal LRR and replication initiation, the LCAR acts to capture monomeric RNA-free NP molecules to bring them spatially closer to the nascent RNA strand. As the nascent RNA strand extends, due to its higher affinity to RNA, NP dissociates from the LCAR of ANP32 proteins and binds to RNA through its RNA binding grooves, resulting in the assembly of progeny RNPs as this process repeats Figure . In the Source data as well as plasmids are available upon request.


The excitation led to the dissociation of I or Br atoms within 300 fs, producing the CF2BrCF2 or CF2ICF2 radicals, respectively. All nascent CF2ICF2 underwent further dissociation of I, producing CF2CF2 with a time constant of 56 ± 5 ns. All nascent g-CF2BrCF2 isomerized into the more stable a-CF2BrCF2 with a time constant of 47 ± 5 ps. Furthermore, a-CF2BrCF2 underwent a bimolecular reaction with either itself (producing CF2BrCF2Br and CF2CF2) or Br in the CCl4 solution (producing CF2BrCF2Br) at a diffusion-limited rate. The secondary dissociation of Br from a-CF2BrCF2 was significantly slow to compete with the bimolecular reactions. Overall, approximately half of the excited CF2BrCF2I at 267 nm produced CF2BrCF2Br, whereas the other half produced CF2CF2. The excess energies in the nascent radicals were thermalized much faster than the secondary dissociation of I from CF2ICF2 and the observed bimolecular reactions, implying that the secondary reactions proceeded under thermal conditions. This study further demonstrates that structure-sensitive time-resolved infrared spectroscopy can be used to study various reaction dynamics in solution in real time.The photodissociation dynamics of CF Vicinal dihaloethanes are dissociated using ultraviolet (UV) energy to form haloethyl radicals that undergo various secondary reactions ,10,11,122ICH2I, CF2ICH2I, and CF2ICF2I, were mainly investigated because the iodoethyl radicals produced upon UV excitation contain weak C−I bonds that readily proceed with secondary dissociation . C. C2CF2 a2BrCF2I is excited by UV light, it dissociates I or Br atoms, producing CF2BrCF2 or CF2ICF2 radicals, respectively [2BrCF2I in CCl4 at 267 nm is promoted to the antibonding of the C−I (C−Br) bond, the majority of the produced radicals are expected to be CF2BrCF2. As shown in our previous TRIR spectroscopic experiment on CF2BrCF2I in the time range of 0.3−320 ps, the TRIR spectra consisted of the spectra of three nascent radicals  and the inverted spectrum of CF2BrCF2I for the bleach signal [2CF2 and 50 ± 3% CF2BrCF2Br, which were produced via secondary reactions of the reaction intermediates. Therefore, the TRIR spectra should include the spectra of the reactant (CF2BrCF2I), final products (CF2CF2 and CF2BrCF2Br), and reaction intermediates . The TRIR spectra were globally fitted using the basis spectra of CF2BrCF2I, CF2CF2, CF2BrCF2Br, a-CF2BrCF2, g-CF2BrCF2, and a-CF2ICF2, as shown in the upper panel of 2BrCF2I, CF2CF2, and CF2BrCF2Br were obtained from equilibrium FTIR measurements, and those of a-CF2ICF2, a-CF2BrCF2, and g-CF2BrCF2 were obtained from our previous experiments [2BrCF2I and CF2BrCF2Br into their conformer-specific spectra was not necessary for the fitting because their basis spectra have contributions from both conformers, and these contributions were maintained throughout the experiment. As shown in 2BrCF2I, CF2CF2, CF2BrCF2Br, a-CF2BrCF2, g-CF2BrCF2, and a-CF2ICF2 shown in the upper panel of 2BrCF2I in CCl4 at 280 K.When CFectively ,9,10,15.eriments ,15. The amp) is related to the population of the corresponding compound by ε and n represent the integrated extinction coefficient and population of the compound, respectively. The integrated extinction coefficient (ε) of a-CF2BrCF2, g-CF2BrCF2, and a-CF2ICF2 were obtained from our previous measurements [2BrCF2I, CF2CF2, and CF2BrCF2Br were determined from equilibrium FTIR measurements. Time-dependent fractional population changes of CF2BrCF2I, CF2CF2, CF2BrCF2Br, a-CF2BrCF2, g-CF2BrCF2, and a-CF2ICF2 were derived from time-dependent amplitude changes for the basis spectra of the corresponding compounds using the relation, a-CF2BrCF2, g-CF2BrCF2, and a-CF2ICF2) appeared immediately, implying that the photodissociation of CF2BrCF2I occurred within 0.3 ps [g-CF2BrCF2 correlated with the growth of a-CF2BrCF2 because g-CF2BrCF2 isomerizes into a-CF2BrCF2 at a time constant of 47 ± 5 ps [a-CF2ICF2 and g-CF2BrCF2 correlated with the growth of CF2CF2 and CF2BrCF2Br, implying that these products were formed by the secondary reactions of a-CF2ICF2 and a-CF2BrCF2.The amplitude of a basis spectrum  [g-CF2BrCF2 isomerizes into a-CF2BrCF2 with a time constant of 47 ± 5 ps [a-CF2ICF2 undergoes secondary dissociation to produce CF2CF2 + I with a time constant of 56 ± 5 ns. The nascent a-CF2ICF2 obtained from CF2ICF2I in CCl4 undergoes secondary dissociation with a time constant of 5.5 ns [a-CF2ICF2 is less than that observed previously, it clearly demonstrates that the secondary dissociation of the C−I bond is feasible and can occur on the nanosecond time scale in solution.F2BrCF2) . Almost 7 ± 5 ps . All of a-CF2BrCF2 was correlated with the growth of the final products, CF2CF2 and CF2BrCF2Br. The possible reactions of a-CF2BrCF2 to produce the final products are as follows.The decay of (a)2BrCF2Br and CF2CF2. Reaction (2), which produces CF2BrCF2Br, should proceed with a rate constant of (1.6 ± 0.3) × 1011 M−1s−1, and reaction (3), which produces CF2CF2, with a time constant of 130 ± 5 ns.If reaction (1) does not occur, reactions (2) and (3) should proceed, resulting in the formation of the two products, CF(b)a-CF2BrCF2 (reaction (3)) is too high to be realistic (vide infra), reaction (1) should occur to produce CF2CF2.As the time constant of 130 ns for the secondary dissociation of (c)2CF2 and CF2BrCF2Br, and the secondary dissociation of a-CF2ICF2 produces CF2CF2 (18% of the reactant), resulting in the final products CF2BrCF2Br (41%) and CF2CF2 (59%) if reaction (1) is the only one to proceed. Reaction (2) should proceed in addition to reaction (1) to give the observed final products (50 ± 3% CF2CF2 and 50 ± 3% CF2BrCF2Br).Reaction (1) produces equal amounts of CF(d)a-CF2BrCF2 reacts bimolecularly with itself (reaction (1)), it can also react with Br atoms in solution (reaction (2)).Furthermore, if a-CF2BrCF2, CF2CF2, and CF2BrCF2Br, resulting in rate constants for the BR of a-CF2BrCF2 with itself (reaction (1)) and the Br atoms (reaction (2)), which were approximately the same, (6.5 ± 0.3) × 1010 M−1s−1. The recovered BR rate constant is closer to the calculated diffusion-limited rate constant of 7 × 109 M−1s−1 than the obtained value of (1.6 ± 0.3) × 1011 M−1s−1, assuming that only reaction (2) produces CF2BrCF2Br.Therefore, reactions (1) and (2) were allowed to fit the fractional population changes of a-CF2ICF2, a-CF2BrCF2, and g-CF2BrCF2) produced during the photodecomposition of CF2BrCF2I was thermalized with a time constant of 15 ± 3 ps [2BrCF2I in CCl4, indicating that the observed reactions became thermal within tens of picoseconds after the photodecomposition. Therefore, the radicals should gain the energy required for the secondary bond dissociation that occurs within nanoseconds or longer. The bond dissociation energy is supplied by the solvent, indicating that the solvent becomes an energy source as well as an energy sink. As the solvent can act as an energy sink and an energy source, secondary dissociation at a longer duration than thermalization is a characteristic of the reaction in solution. For the secondary dissociation of the C−X bond, the activation energy should be as high as the C−X bond energy. As the C−Br bond energy of 22.3 ± 2.5 kcal/mol is 15.2 kcal/mol higher than the C−I bond energy of 7.1 ± 2.5 kcal/mol [12 times longer than that of the C−I bond, based on the Arrhenius equation for the rate constant at room temperature. Considering that the secondary dissociation time of thermalized a-CF2ICF2 is 56 ns, it is unlikely that the secondary dissociation of Br from thermalized a-CF2BrCF2 will take 130 ns. The excess energy in the nascent radicals  the CF2BrCF2 radicals are a-CF2BrCF2. Although most CF2BrCF2Br is produced by the BR of a-CF2BrCF2, the produced CF2BrCF2Br is in both anti and gauche conformers in the equilibrium distribution. Based on the rotational isomerization time of 47 ps for CF2BrCF2 and the calculated rotational barriers [2BrCF2Br in CCl4 at 280 K was estimated [gauche-to-anti (anti-to-gauche) transition by the DFT method using ωB97X-D/aug-cc-pVTZ. As shown in 2BrCF2Br was produced with a time constant of 83 ns, which is longer than the estimated rotational isomerization time. Thus, CF2BrCF2Br was produced in the equilibrium distribution, even though the reactant was mainly the a-CF2BrCF2 radical.The Gibbs free energy of barriers , that ofstimated  to be 12−1 spectral width, and 1 μJ of energy. The sample was excited by a portion of the 267 nm pulse and probed by a small fraction (~10 nJ) of the mid-IR pulse. The 267 nm excitation pulses were optically delayed up to 1 ns relative to the mid-IR probe pulses by a computer-controlled translation stage . The translation stage was impractical for optical delays beyond 1 ns because of the long physical distances for the stage. Thus, the excitation pulses were replaced with the nanosecond pulses  produced by a commercial nanosecond tunable laser  based on an optical parametric oscillator. These nanosecond excitation pulses were synchronized with the femtosecond probe pulses using an electronic digital delay generator , which produced an optical delay beyond 1 ns. The angle between the linearly polarized probe and the excitation pulses was set as the magic angle (54.7°) to obtain an isotropic absorbance proportional to the population of the corresponding compounds free from molecular rotation. The energy of the excitation pulse was reduced to 1–2 μJ to ensure linear absorbance and to minimize thermal lensing, which causes high background absorption on the nanosecond time scale due to solvent heating [−1) of the probe pulses, the experiment was repeated with probe pulses centered at 1300, 1230, 1160, 1080, or 950 cm−1, and the data were combined to obtain a broad spectrum. The probe pulses were passed through the sample and were detected by a 1 × 128-pixel MCT array detector  for wavenumbers > 1000 cm−1 or a 1 × 64-pixel MCT array detector  for wavenumbers < 1000 cm−1 in a 320 mm monochromator  with a grating of 100, 75, or 50 lines/mm. The 1 × 64-pixel MCT array detector was custom-made to enhance spectral responsivity at longer wavelengths (<1000 cm−1). The resulting spectra have a spectral resolution of 0.9–1.2 cm−1 for the spectral regions > 1000 cm−1 and 2.7 cm−1 for spectral regions < 1000 cm−1. The instrument response function was determined by the transient absorption of the Si wafer and was 0.2 ps in the subnanosecond experiment (0.3 ps–1 ns) and 2.5 ns in the nanosecond experiment (1 ns–10 μs). The spectra were collected at pump-probe delay times ≥ 0.3 ps to avoid signal complications due to pump-probe overlap [The TRIR spectrometer is based on a commercial Ti:sapphire oscillator/amplifier system  that generates 800 nm, 110 fs pulses with a repetition rate of 2 kHz . Half of heating ,23,24,25 overlap ,28. 2BrCF2I was purchased from Alfa Aesar, and CCl4 was obtained from Sigma-Aldrich and used without further purification. A flowing sample cell consisting of two 2-mm-thick BaF2 windows and a 100-μm-thick Teflon spacer was connected to a peristaltic tubing pump , refreshed with 50 mM CF2BrCF2I in CCl4 fast enough for each probe pulse at a repetition rate of 2 kHz. Concentration and path length were set to obtain the maximum transient absorption signal in the spectral region of interest. A sufficiently large volume of the sample was used to maintain the decrease in the sample concentration as <2% due to the photoreaction of CF2BrCF2I by 267 nm excitation. The temperature of the sample was maintained at 280 ± 2 K by passing the coolant through the sample mount block. The low temperature was beneficial in minimizing the noise in the TRIR spectra caused by the temperature gradient in the probe spot of the sample induced by the pump energy  appearing on the nano- to microsecond time scale [CFme scale ,23,24,25All quantum-chemical calculations were performed using the Gaussian 09 software (Revision D.01). The DFT method with the ωB97X-D  functional was used for molecular geometry optimization and the energies of the optimized compounds. ωB97X-D has been reported to yield satisfactory accuracy in thermochemistry, kinetics, and noncovalent interactions ,30. The 2BrCF2I in a CCl4 solution were determined over a broad time span, encompassing the entire reaction, after excitation at 267 nm using TRIR spectroscopy. UV excitation led to ultrafast dissociation of I or Br atoms, resulting in CF2BrCF2 and CF2ICF2 radicals, respectively, which further proceeded with conformer-specific reactions. CF2BrCF2 was the major photofragment with both conformers of a-CF2BrCF2 (33 ± 3%) and g-CF2BrCF2 (49 ± 3%), while a-CF2ICF2 (18 ± 3%) was the minor one. The less stable g-CF2BrCF2 was isomerized with a time constant of 47 ± 5 ps into a-CF2BrCF2. a-CF2BrCF2 further reacted bimolecularly with itself at a diffusion-limited rate to produce CF2BrCF2Br and CF2CF2, or with the dissociated Br atom to form CF2BrCF2Br. However, no secondary dissociation of C−Br in a-CF2BrCF2 was observed. In contrast, a-CF2ICF2 underwent simple secondary dissociation of the I atom with a time constant of 56 ± 5 ns to produce CF2CF2 + I. The final products contained 50 ± 3% CF2BrCF2Br and 50 ± 3% CF2CF2. Structure-sensitive TRIR spectroscopy allowed us to determine the entire reaction dynamics of photoexcited CF2BrCF2I in a CCl4 solution, including the conformer-specific dynamics of the CF2BrCF2 radicals.In this study, the photodissociation dynamics of CF


Cryphonectria parasitica, the causal agent of chestnut blight, has set an ideal model of management through the release of hypovirulent strains. However, mycovirus-based management of plant diseases is still restricted by limited approaches to search for viruses causing hypovirulence and the lack of protocols allowing effective and systemic virus infection in pathogens. RNA interference (RNAi), the eukaryotic cell system that recognizes RNA sequences and specifically degrades them, represents a promising. RNA-based disease management method. The natural occurrence of cross-kingdom RNAi provides a basis for host-induced gene silencing, while the ability of most pathogens to uptake exogenous small RNAs enables the use of spray-induced gene silencing techniques. This review describes the mechanisms behind and the potential of two RNA-based strategies, mycoviruses and RNAi, for plant disease management. Successful applications are discussed, as well as the research gaps and limitations that remain to be addressed.RNA-based strategies for plant disease management offer an attractive alternative to agrochemicals that negatively impact human and ecosystem health and lead to pathogen resistance. There has been recent interest in using mycoviruses for fungal disease control after it was discovered that some cause hypovirulence in fungal pathogens, which refers to a decline in the ability of a pathogen to cause disease.  Plant species, including agricultural, horticultural, and forestry crops, are constantly threatened by pests and pathogens. It is estimated that around 30% of crops worldwide are lost due to these harmful organisms . ChemicaMycoviruses are viruses, mostly harboring RNA genomes, that infect fungi. Some of them have been reported to decrease the virulence of their fungal host, a phenomenon called hypovirulence. This has opened up the possibility of using hypovirulence-inducing viruses to control plant pathogenic fungi. Mycoviruses commonly occur in fungi; however, only a few of them are deleterious to their hosts. This, coupled with the fact that mycoviruses are very difficult to transmit between fungi (as they are only transmitted intracellularly), limits their use as control agents. Furthermore, this system is only useful for the control of plant diseases caused by fungi. Nevertheless, with the advent of new sequencing techniques, numerous novel mycoviruses are being identified in a multitude of fungi, offering new possibilities in this field .RNA interference (RNAi) is a gene silencing mechanism conserved among eukaryotes that regulates gene expression through the degradation of messenger RNA (mRNA) by the interaction of double-stranded RNA (dsRNA) molecules. Two strategies have been employed to exploit this mechanism for the control of plant pathogens: host-induced gene silencing (HIGS), which is based on the transgenic expression of specific interfering RNAs in the plant to confer resistance to a target pathogen, and spray-induced gene silencing (SIGS), where the topical application of specific dsRNA molecules degrades target genes in plant pathogens. The specificity of RNAi-based methods makes them a promising alternative to the use of conventional fungicides and pesticides. There is, however, greater interest in non-genetically modified (GM) exogenous approaches, such as SIGS, in light of some aspects of HIGS, especially the issues associated with public acceptance of genetically modified organisms (GMOs). In this work, we aim to review and discuss the use of RNA-based methods for plant disease management, focusing on mycoviruses and new RNAi-based approaches, especially SIGS, and the aspects to be considered to make them a viable option in the control and management of plant diseases. Sclerotinia sclerotiorum named Sclerotinia sclerotiorum hypovirulence-associated virus 1 (SsHADV-1) is capable of extracellular transmission [Mycoviruses are viruses that infect and replicate in fungi . First dsmission ,11, notasmission . Recentlsmission ,14, althSclerotinia sclerotiorum negative-strand RNA virus 1 (SsNSRV-1) was the first mycovirus characterized of this latter class [Botrytis cinerea [Fusarium graminearum [Viruses mostly store and replicate their genomes as RNA by encoding RNA-dependent RNA polymerases (RdRps). During viral infections, RdRps are involved in RNA template selection, RNA synthesis, and viral RNA preservation; hence, they are essential for virus survival . Accordier class , and sub cinerea ,19,20 anminearum . Since mCurvularia thermal tolerance virus that confers heat tolerance not only to the fungus it infects but also to the host plant of this latter [Most mycoviruses are considered cryptic: they remain latent and do not have any effect on their host ,22. Somes latter . Howevers latter . Such mys latter ,28,29,30s latter ,32,33,34s latter . B. cinerea [Entoleuca sp. [Fusarium sacchari and Fusarium andiyazi strains [Rhizoctonia solani isolates [Fusarium circinatum, characterizing some mitovirus [Heretobasidion [S. sclerotiorum that already harbored the SsHADV-1 virus [In recent years, the emergence of high-throughput sequencing technologies has made possible the identification of an unexpected number of new mycoviruses among fungi , a trend cinerea , 14 mycoeuca sp. , 10 myco strains , and a lisolates . Deep seitovirus . Similarbasidion , and two-1 virus . TherefoDespite these ongoing major advances in mycovirus detection and identification, phenotypic evaluation remains a slow and laborious process. In addition, unknown information about host range, replication, infection, and transmission is indispensable for the use of mycoviruses as effective biological control agents.Mycoviruses are transmitted vertically during sporogenesis and horizontally by cytoplasmic exchange during cell division, hyphal anastomosis, and mating . The absvic) [vc) types: whereas vegetative compatible isolates can successfully establish hyphal anastomosis, the interaction of vegetative incompatible isolates triggers localized programmed cell death, which hampers sharing of genetic material [vc type. However, phylogenetic evidence reveals occasional transmission across vegetatively incompatible strains [Cryphonectria parasitica, was identified in Cryphonectria sp. [Cryphonectria naterciae, a new virus able to cross the species barrier to infect Cryphonectria carpinicola and Cryphonectria radicalis was recently identified [Botrytis porri strains, has also been found in Botrytis squamosa and S. sclerotiorum, suggesting that this mycovirus may have different hosts [S. sclerotiorum that was identified and described as the first +ssRNA mycovirus capable of spreading across the vegetative compatibility barrier [vic strains was difficult in plates, it was much easier when the strains were inoculated in chestnuts [Vic is apparently the result of biological and ecological factors, and even a model has been proposed in which mycoviruses can spread to vegetatively incompatible strains and to other fungal species through plant fungus-mediated pathways facilitated by plant viruses [Hyphal anastomosis between two fungal isolates is genetically controlled by a self/non-self recognition system called “vegetative incompatibility” (vic) ,52. It rmaterial . As a re strains ,55. The tria sp. . In straentified . The Botnt hosts . In addint hosts  found a  barrier ,61. Brushestnuts . Vic is  viruses . Mycoviruses that negatively affect their hosts are rare and interesting from the plant protection viewpoint. Mycoviruses inducing hypovirulence in their host are rare but interesting from a plant protection point of view since hypovirulent pathogens are unable to cause severe disease . Since tCryphonectria parasitica, a destructive fungal pathogen causing chestnut blight. Van Alfen et al. discovered a hypovirulent strain of C. parasitica [C. parasitica was exploited to use CHV-1 as a biological control agent [C. parasitica, the most important for the biological control of chestnut blight being four species belonging to the family hypoviridae (+ssRNA): CHV-1 and CHV-2 in Europe; CHV-3 and CHV-4 in North America [C. parasitica virulence [C. parasitica have shown that the presence of CHV-1 affected a multitude of metabolic pathways: genes related to fungal metabolites, signaling pathways, virulence, and antiviral RNA silencing [C. parasitica are well-known [The first hypovirulence-inducing mycovirus was found in rasitica , in whicrasitica . This myrasitica . The posol agent ,68. Late America . CHV-1 h America . The CHV America . It has irulence ,73. RNA-ilencing . Howeverll-known , but thell-known . Botrytis cinerea, the causal agent of gray mold disease on more than 1400 plant species, including many economically important crops, the Botrytis cinerea partitivirus 2 (BcPV-2) was found in hypovirulent isolates in apple, cucumber, oilseed rape, strawberry, table grapes, tobacco, and tomato, suggesting that this mycovirus might be responsible of the hypovirulence [Colletotrichum liriopes, a fungus causing leaf anthracnose in many plants [C. parasitica, several mycoviruses triggering hypovirulence have been described in fungal pathogens. Hypovirulent strains of Ophiostoma novo-ulmi, the causing agent of the devastating Dutch Elm Disease in Ulmus spp., harbor a dsRNA virus called Ophiostoma novo-ulmi mitovirus (OnuMV) that reduces fungal growth in wounds caused by some beetles (vector) when feeding [Botryosphaeria dothidea, a pathogen of global importance for woody plant health, which infects a broad range of hosts. Three dsRNA mycoviruses producing an attenuated fungal growth and virulence have been discovered in hypovirulent isolates of B. dothidea: Botryosphaeria dothidea chrysovirus 1 (BdCV1) and Botryosphaeria dothidea partitivirus 1 (BdPV-1) [B. dothidea and mycoviruses involves the coupled action of the antiviral gene silencing pathway and micro-like RNAs-mediated regulation of target gene mRNA expression in B. dothidea [Following the discovery of CHV-1, mycovirus-mediated hypovirulence in fungal pathogens of plants ranging from grass to trees has been reported frequently . Recentlirulence . The Coly plants . In fore feeding . Another(BdPV-1)  and more(BdPV-1) . Recentldothidea . Howeverdothidea ,82. MycoTotiviridae, Chrysoviridae, Hypoviridae, Narnaviridae, and Reoviridae families [In a nutshell, all known mycoviruses causing hypovirulence in fungal hosts harbor dsRNA or ssRNA genomes, except for one that contains DNA ,33 and ifamilies ,83. A laHypovirulence-associated mycoviruses are biologically interesting for the control of diseases caused by their fungal hosts. Nevertheless, there are still many aspects that need attention and question their potential as biological control agents. Indeed, for efficient disease control, a mycovirus needs to: (a) cause hypovirulence in the host; (b) have a high vertical transmission rate through spores; (c) skip host mechanisms to control the spread and infection of viruses. In fungi, RNAi is known to play a critical role in antiviral defense. Indeed, studies with disruptive mutants of essential genes of the RNAi machinery show a severely weakened fungal phenotype . Howevervc types in the area of treatment. In the case of CHV-1 and C. parasitica, these requirements are fulfilled in Europe ; however, chestnut blight is so far the only disease that has been effectively controlled with an RNA mycovirus in the field [In addition, to enable effective horizontal transmission, the diversity of host vegetative compatibility types in the area of treatment must be low, and the hypovirulent strains must be compatible with the dominant he field ,102,103.https://serratus.io/ accessed on 16 August 2022) is a newly developed cloud computing infrastructure for ultra-high-throughput sequence alignment that facilitates the discovery of new viruses [One of the main challenges posed by virocontrol nowadays is the search for mycoviruses with a hypovirulent effect. Despite high-throughput sequencing technologies allowing rapid and massive identification of viruses and the recent and updated sequence banks, no known bioinformatics model predicts hypovirulence. For example, the Serratus platform  was first described by Fire et al. in the nematode  in 2006 . However elegans . Moreove elegans ,108 and a crassa , respecta crassa  and mamma crassa  as well. enzymes . Key pro enzymes . RNAi is initiated by small interfering RNA (siRNA) duplexes, which are 19–25 nucleotide-long double-stranded RNA molecules produced by DICER-mediated cleavage of longer double-stranded RNAs (dsRNA). Then, a single-stranded guide RNA is incorporated into the RNA-induced silencing complex (RISC), resulting in the endonucleolytic cleavage of the complementary mRNA, thereby regulating gene target expression ,114,115.RISC can potentially down-regulate any mRNA with perfect base complementarity to the seed region of the guide strand . The RNARNAi is believed to have evolved as a defense mechanism against viruses and transposable elements, which can affect the integrity of genomic DNA . ExposurRNAi machinery is not present in prokaryotes, but they have a functionally similar defense system to induce the inactivation of parasite genomes: they produce small non-coding RNAs that can up- or down-regulate mRNA stability and translation ,127. B. cinerea transfers RNAi effectors into host plant cells to induce silencing of host immune response genes and achieve infection [Arabidopsis plants were found to produce a pool of siRNAs contained in extracellular vesicles in response to Phytophthora capsici infection, leading to the impairment of pathogen development [P. capsici tried to suppress the RNAi response [RNA molecules can move across the cellular boundaries between hosts and their pathogens, pests, and parasites and induce gene silencing through RNAi . This confection , while snfection , exemplinfection ,125,134.nfection . Recentlnfection . Trans-kresponse .Host-induced gene silencing (HIGS), a term coined by Nowara et al. , exploitArabidopsis expressing dsRNAs targeting a nematode parasitism gene were resistant to root-knot nematode [in planta expression of gene-silencing dsRNAs [Blumeria graminis, in genetically modified barley [Phytophthora infestans [The first report on HIGS for plant protection showed that transgenic nematode . This wanematode ; signifig dsRNAs . In fungd barley . In oomynfestans . Since tnfestans ,147,148.® Pro (https://traits.bayer.com/corn/Pages/SmartStax-PRO.aspx accessed on 16 August 2022), which is a GM corn seed that deploys transgenic insecticidal proteins and RNAi to fight the corn rootworm [Over 170 studies on HIGS have been published so far . Howeverrootworm . This prrootworm . An increasing number of GM crops are being developed, risk assessed, and accepted by relevant international regulatory agencies . The EurC. elegans [RNAi response in pathogens induced by external RNAs uptaken from the environment, referred to as ‘environmental RNAi’, was first observed in  elegans ,160,161.B. cinerea to different post-harvest plant materials , they found that all plants treated with dsRNAs developed much weaker disease symptoms [F. graminearum conferred strong resistance against this pathogen [Fusarium culmorum, obtaining satisfactory results in reducing the growth of this fungus, which suggested that this technique had unprecedented potential for easy application against various fungal pathogens [F. graminearum testing HIGS and SIGS approaches [Brassica napus leaves infected by S. sclerotiorum, which they used to identify and design dsRNA molecules targeting genes associated with reactive oxygen species [SIGS has also been shown to be effective against fungi. When Wang et al. externally applied dsRNAs synthesized in vitro targeting Dicer-like proteins in the pathogen symptoms . Similarpathogen . Koch etathogens . Koch etproaches . They shproaches . McLough species . The top species ,167.SIGS technology can also be applied against oomycetes ,169. IntSIGS is likely to be technologically simple, safe for consumption, and socially accepted as it does not require the generation of GMOs. Moreover, it allows effective post-harvest control of fungi, which has not been well demonstrated with HIGS ,170. SIGSome challenges remain to be addressed to enable the large-scale production and commercial use of RNAi-based biopesticides and biofungicides: (1) the uptake efficiency, (2) the target choice, (3) the protection duration and stability of dsRNA, as well as (4) the safety, (5) the environmental implications, and (6) the economic competitiveness of SIGS.Sclerotinia sclerotiorum, Rhizoctonia solani, Aspergillus niger, and Verticillium dahliae). On the contrary, fungi that did not take up dsRNA, such as Colletotrichum gloeosporioides, did not experience a reduction in infection. The size of the dsRNA influences the uptake efficiency. Indeed, Höfle et al. found that molecules between 400 and 500 bp were most effective, while from 800 bp onwards, efficiency decreased, and molecules over 1500 bp had no silencing effect, suggesting they were not taken up by the fungus [The ability of the target pathogenic organisms to incorporate the dsRNA molecules The ability of the target pathogenic organisms to incorporate the dsRNA molecules is key to the success of this SIGS management strategy and determines its effectiveness. Recently, Qiao et al. assessed the uptake efficiency in different plant pathogenic fungi by applying fluorescein-labeled dsRNA and analyzing the fluorescent signal using a confocal microscope . They ese fungus . dsRNA me fungus . Interese fungus . Koch ete fungus . It is ae fungus ,178. Bete fungus . ConsequThe choice of effective target genes is essential for the success of the silencing and disease control strategy. First of all, appropriate candidate genes of the target pathogen that are vital for its growth and pathogenicity must be identified . SecondlAs mentioned, several target pathogens, or several gene targets, can be silenced simultaneously. This is very useful as plant diseases are sometimes caused by a group of pathogens rather than by a single one. Silencing several genes or different regions of a gene at the same time can enhance the silencing effect and expand the target species .http://rna.tbi.univie.ac.at/cgi-bin/RNAxs/RNAxs.cgi accessed on 16 August 2022) [dsRNA molecules must be designed to optimize silencing efficiency: taking into account the secondary structure of the selected target sequence since complex RNA structures can prevent base-pairing between sRNA and the target, which inhibits cleavage of mRNA by the RISC; avoid secondary structure formation in the guide RNA, which can significantly reduce silencing effect ,185,186.st 2022) . Since it relies on the recognition of specific sequences, SIGS can be very specific for the target pathogen, which is important for disease management and represents a clear advantage over non-selective chemical products . HoweverTM when combined with dsRNA) [Several factors influence the duration of the silencing effect, limiting the use of SIGS as a control method. Several studies pointed out a short pathogen protection window post spray: 5–10 days, usually one week, with the efficacy of dsRNA decreasing over time ,176,195.h dsRNA) ,198, carh dsRNA) ,200, carh dsRNA) , a mix oh dsRNA) ; or goldh dsRNA) ,204, amoh dsRNA)  and someh dsRNA) . Anotherh dsRNA) ,207,208.h dsRNA) ,210. Theh dsRNA) ; therefoh dsRNA) ,212. All these formulations can protect sRNA and also be taken up by pathogens. Carriers’ features can be optimized to ensure efficient delivery: particle size, carrier shape, dsRNA dose, carrier-dsRNA complexing ratio, and treatment time. It was found that sheet-like clay dsRNA nanoparticles up to 50 nm in diameter were easily internalized . FurtherIn addition to inorganic and organic carriers, another delivery method being investigated is the use of vectors that have been genetically modified to produce dsRNAs. Niño-Sánchez et al. first demonstrated that silencing can be induced by the application of living or lysed bacteria engineered to produce biologically active dsRNAs targeting fungal genes . FurthersRNAs are normally present in all plant- and animal-derived food. In addition, sRNAs with sequences complementary to humans and animals have been found in crops widely consumed globally . Some apAppropriate construction of silencing molecules should prevent unintended consequences on the environment, such as the silencing of non-target organisms. SIGS-based products should not leave residues in soil, water, or plants due to the non-permanent nature of dsRNA molecules, and even in case it would, the natural occurrence of RNA in the environment hints that it would not be a problem ,220. In Escherichia coli HT115(DE3) strain deficient in the enzyme that degrades dsRNAs in prokaryotes [RNAi gene silencing in vivo requires a large amount of dsRNA to be effective: 2–10 g of dsRNA per hectare, as predicted by Zotti et al. . Fortunakaryotes ,224, coskaryotes . Thus, lkaryotes , insectskaryotes , crustackaryotes , mammalikaryotes , and filkaryotes . Howeverkaryotes ,231,232.Varroa destructor mites in honeybees. It has been submitted by Bayer for registration in 2019, being the first submission to the U.S. Environmental Protection Agency for an exogenously applied dsRNA biopesticidal active ingredient in the industry [The multinational companies Monsanto, which has developed a line of RNA-based compounds known as “BioDirect”; Syngenta (Chem-China) and Bayer Crop-Science, which has now acquired Monsanto; are working on the development of dsRNA compounds. BioDirect (dsRNA-based) has been developed to control industry .https://www.greenlightbiosciences.com/ accessed on 16 August 2022), RNAissance (https://www.rnaissanceag.net/ accessed on 16 August 2022), and Genolution (http://genolution.co.kr/ accessed on 16 August 2022) are working on low-cost dsRNA synthesis. RNAissance, AgroSpheres (https://www.agrospheres.com/ accessed on 16 August 2022), Nanosur (http://www.nanosur.com/ accessed on 16 August 2022), and Agrisome (TrilliumAg) (http://www.trilliumag.com/ accessed on 16 August 2022) work on the protection of RNA molecules from degradation [Start-up companies are also involved in the task of exploiting biotechnology to offer cost-effective solutions to the challenges posed by SIGS. For example, GreenLight Biosciences   and SsHAOn the other hand, the discovery and subsequent findings on RNAi have expanded our understanding of gene regulation, and it has been widely used as a research tool to elucidate the function of genes . More thIn crops and forests, biological control by RNA is a beneficial alternative to using fungicides or pesticides, but it must also be cost-effective and easy to apply, especially in forest disease management, where the cost-benefit balance is crucial owing to the narrow profit margin.Insights reviewed here and future discoveries will provide opportunities to learn and improve these innovative approaches to plant protection.