Size Restriction Is Required for Proper Functioning of a Bipartite Begomovirus AC4 Protein.

Many geminiviruses, including members of the genus Begomovirus, produce a protein known as C4 or AC4. Whereas C4/AC4 typically consists of more than 80 amino acid residues, a few are much shorter. The significance of these shorter C4/AC4 proteins in viral infection and why the virus maintains their abbreviated length is not yet understood. The AC4 of the begomovirus Tomato leaf curl Hsinchu virus contains only 65 amino acids, but it extends to 96 amino acids when the natural termination codon is replaced with a normal codon. We discovered that both interrupting and extending AC4 were harmful to tomato leaf curl Hsinchu virus (ToLCHsV). The extended AC4 (EAC4) also showed a reduced ability to promote the infection of the heterologous virus Potato virus X than the wild-type AC4. When the wild-type AC4 was fused with yellow fluorescent protein (AC4-YFP), it was predominantly found in chloroplasts, whereas EAC4-YFP was mainly localized to the cell periphery. These results suggest that ToLCHsV's AC4 protein is important for viral infection, and the virus may benefit from the abbreviated length, because it may lead to chloroplast localization. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Transcription start site mapping of geminiviruses using the in vitro cap-snatching of a tenuivirus.

Geminiviruses are a family of single-stranded DNA viruses that cause significant yield losses in crop production worldwide. Transcription start site (TSS) mapping is crucial in understanding the gene expression mechanisms of geminiviruses. However, this often requires costly and laborious experiments. Rice stripe virus (RSV) has a mechanism called cap-snatching, whereby it cleaves cellular mRNAs and uses the 5' cleavage product, a capped-RNA leader (CRL), as primers for transcription. Our previous work demonstrated that RSV snatches CRLs from geminiviral mRNAs in co-infected plants, providing a convenient and powerful approach to map the TSSs of geminiviruses. However, co-infections are not always feasible for all geminiviruses. In this study, we evaluated the use of in vitro cap-snatching of RSV for the same purpose, using tomato yellow leaf curl virus (TYLCV) as an example. We incubated RNA extracted from TYLCV-infected plants with purified RSV ribonucleoproteins in a reaction mixture that supports in vitro cap-snatching of RSV. The RSV mRNAs produced in the reaction were deep sequenced. The CRLs snatched by RSV allowed us to locate 28 TSSs in TYLCV. These results provide support for using RSV's in vitro cap-snatching to map geminiviral TSSs.

Distract Your Attention: Multi-Head Cross Attention Network for Facial Expression Recognition.

This paper presents a novel facial expression recognition network, called Distract your Attention Network (DAN). Our method is based on two key observations in biological visual perception. Firstly, multiple facial expression classes share inherently similar underlying facial appearance, and their differences could be subtle. Secondly, facial expressions simultaneously exhibit themselves through multiple facial regions, and for recognition, a holistic approach by encoding high-order interactions among local features is required. To address these issues, this work proposes DAN with three key components: Feature Clustering Network (FCN), Multi-head Attention Network (MAN), and Attention Fusion Network (AFN). Specifically, FCN extracts robust features by adopting a large-margin learning objective to maximize class separability. In addition, MAN instantiates a number of attention heads to simultaneously attend to multiple facial areas and build attention maps on these regions. Further, AFN distracts these attentions to multiple locations before fusing the feature maps to a comprehensive one. Extensive experiments on three public datasets (including AffectNet, RAF-DB, and SFEW 2.0) verified that the proposed method consistently achieves state-of-the-art facial expression recognition performance. The DAN code is publicly available.

PAs Regulate Early Somatic Embryo Development by Changing the Gene Expression Level and the Hormonal Balance in Dimocarpus longan Lour.

Polyamines (PAs) play an important regulatory role in many basic cellular processes and physiological and biochemical processes. However, there are few studies on the identification of PA biosynthesis and metabolism family members and the role of PAs in the transition of plant embryogenic calli (EC) into globular embryos (GE), especially in perennial woody plants. We identified 20 genes involved in PA biosynthesis and metabolism from the third-generation genome of longan (Dimocarpus longan Lour.). There were no significant differences between longan and other species regarding the number of members, and they had high similarity with Citrus sinensis. Light, plant hormones and a variety of stress cis-acting elements were found in these family members. The biosynthesis and metabolism of PAs in longan were mainly completed by DlADC2, DlSAMDC2, DlSAMDC3, DlSPDS1A, DlSPMS, DlCuAOB, DlCuAO3A, DlPAO2 and DlPAO4B. In addition, 0.01 mmol∙L-1 1-aminocyclopropane-1-carboxylic acid (ACC), putrescine (Put) and spermine (Spm), could promote the transformation of EC into GE, and Spm treatment had the best effect, while 0.01 mmol∙L-1 D-arginine (D-arg) treatment inhibited the process. The period between the 9th and 11th days was key for the transformation of EC into GE in longan. There were higher levels of gibberellin (GA), salicylic acid (SA) and abscisic acid (ABA) and lower levels of indole-3-acetic acid (IAA), ethylene and hydrogen peroxide (H2O2) in this key period. The expression levels in this period of DlADC2, DlODC, DlSPDS1A, DlCuAOB and DlPAO4B were upregulated, while those of DlSAMDC2 and DlSPMS were downregulated. These results showed that the exogenous ACC, D-arg and PAs could regulate the transformation of EC into GE in longan by changing the content of endogenous hormones and the expression levels of PA biosynthesis and metabolism genes. This study provided a foundation for further determining the physicochemical properties and molecular evolution characteristics of the PA biosynthesis and metabolism gene families, and explored the mechanism of PAs and ethylene for regulating the transformation of plant EC into GE.

The cap-snatching frequency of a plant bunyavirus from nonsense mRNAs is low but is increased by silencing of UPF1 or SMG7.

Bunyaviruses cleave host cellular mRNAs to acquire cap structures for their own mRNAs in a process called cap-snatching. How bunyaviruses interact with cellular mRNA surveillance pathways such as nonsense-mediated decay (NMD) during cap-snatching remains poorly understood, especially in plants. Rice stripe virus (RSV) is a plant bunyavirus threatening rice production in East Asia. Here, with a newly developed system allowing us to present defined mRNAs to RSV in Nicotiana benthamiana, we found that the frequency of RSV to target nonsense mRNAs (nsRNAs) during cap-snatching was much lower than its frequency to target normal mRNAs. The frequency of RSV to target nsRNAs was increased by virus-induced gene silencing of UPF1 or SMG7, each encoding a protein component involved in early steps of NMD (in an rdr6 RNAi background). Coincidently, RSV accumulation was increased in the UPF1- or SMG7-silenced plants. These data indicated that the frequency of RSV to target nsRNAs during cap-snatching is restricted by NMD. By restricting the frequency of RSV to target nsRNAs, NMD may impose a constraint to the overall cap-snatching efficiency of RSV. Besides a deeper understanding for the cap-snatching of RSV, these findings point to a novel role of NMD in plant-bunyavirus interactions.

Genome-wide identification and characterization of DEAD-box helicase family associated with early somatic embryogenesis in Dimocarpus longan Lour.

DEAD-box (DDX) proteins belong to the largest subfamily of RNA helicase SF2, which contributes to all biological processes of RNA metabolism in the plant kingdom. Till now, no significant data are available regarding studies on DDX in Somatic Embryogenesis (SE) of woody plants. It is important to investigate the biological function of the DlDDX family in longan SE. Thus, a comprehensive analysis of 58 longan DEAD-box (DlDDX) genes characterization was performed by genome-wide identification and transcript abundance validation analysis. Homologous evolution has revealed that some DlDDXs in longan had high sequence similarity with Mus musculus, Citrus and Saccharomyces cerevisiae, indicating that DlDDXs were highly conservative in the animal, plant, and microorganism. Remarkably, gene duplication, purifying selection, and alternative splicing events, and new auxiliary domains have likely contributed to the functional evolution of DlDDX, indicating that DlDDX appeared neofunctionalization in longan. Besides, DlDDX3, 15, 28, 36 might interact with protein complex (MAC3A, MAC3B, CDC5, CBP20) of miRNA biosynthesis. Notably, DlDDX28 contained a novel auxiliary domain (CAF-1 p150), which might contribute to DNA demethylation in longan early SE. 4 DlDDX genes significantly expressed not only in early SE and zygotic embryogenesis (ZE) but also up-regulated at high levels in 'Honghezi' and 'Quanlongbaihe' with abortive seeds, which are of great significance. Moreover, some DlDDXs presented abiotic stress-response dynamic expression patterns by ABA, SA, JA, and NaCl treatments during early SE. Hence, DEAD-box is essential to SE development and seed abortive in longan.

A convenient in vivo cap donor delivery system to investigate the cap snatching of plant bunyaviruses.

Like their animal-infecting counterparts, plant bunyaviruses use capped RNA leaders cleaved from host cellular mRNAs to prime viral genome transcription in a process called cap-snatching, but in vivo systems to investigate the details of this process are lacking for them. Here, we report that Rice stripe tenuivirus (RSV) and Tomato spotted wilt tospovirus (TSWV) cleave capped RNA leaders from mRNAs transiently expressed by agroinfiltration, which makes it possible to artificially deliver defined cap donors to the two plant bunyaviruses with unprecedented convenience. With this system, some ideas regarding how plant bunyaviruses select and use capped RNA leaders can be tested easily. We were also able to obtain clear evidence that the capped RNA leaders selected by TSWV are generally longer than those by RSV. TSWV frequently uses the prime-and-realign mechanism in transcription primed by capped RNA leaders shorter than a certain length, like that has been demonstrated recently for RSV.

Molecular characterization and RSV Co-infection of Nicotiana benthamiana with three distinct begomoviruses.

Geminiviruses constitute a family of plant viruses with characteristic twinned quasi-icosahedral virions and a small circular DNA genome. Geminiviruses, especially begomoviruses, cause substantial economic losses in tropical and subtropical regions globally. Geminiviruses use the host's transcriptional mechanisms to synthesize their mRNAs. They are considered as an attractive model to understand the transcription mechanism of their host plants. Experiments were conducted to identify transcriptional start sites (TSSs) of the three begomoviruses, i.e., Cotton leaf curl Multan virus (CLCuMuV), Corchorus yellow vein virus (CoYVV), and Ramie mosaic virus (RamV). We first rub-inoculated Rice stripe tenuivirus (RSV), a segmented negative-sense RNA virus that uses cap-snatching to produce capped viral mRNAs, into N. benthamiana. After the inoculation, RSV-infected N. benthamiana were super-infected by CoYVV, CLCuMuV, or RamV, respectively. The capped-RNA leaders snatched by RSV were obtained by determining the 5'-ends of RSV mRNA with high throughput sequencing. Afterwards, snatched capped-RNA leaders of RSV were mapped onto the genome of each begomovirus and those matching the begomoviral genome were considered to come from the 5' ends of assumed begomoviral mRNAs. In this way, TSSs of begomoviruses were obtained. After mapping these TSSs onto the genome of the respective begomovirus, it was found very commonly that a begomovirus can use many different TSSs to transcribe the same gene, producing many different mRNA isoforms containing the corresponding open reading frames (ORFs).

A Periodic Event-Triggered Design of Robust Filtering for T-S Fuzzy Discrete-Time Systems.

Periodic event-triggered control (PETC) is a control strategy consisting of event-triggered control (ETC) and conventional periodic sampled-data control. By using event-triggering mechanisms (ETM) to periodically verify whether or not to transmit and compute the measured output, communication and computational datum are significantly reduced while still retaining a satisfactory performance. This paper investigates the PETC scheme of robust H ∞ filtering for a class of uncertain discrete-time Takagi-Sugeno (T-S) fuzzy systems, where the sample time is assumed to be a constant. To analyze the filtering problems of the PETC strategy, we present two frameworks based on perturbed linear and piecewise linear systems, to model filtering error systems. Sufficient conditions for the existence of a robust H ∞ filter are derived in the form of matrix inequalities (LMIs) under these two frameworks, respectively. Finally, a simulation example is used to testify to the effectiveness of the proposed approach.

Genetic diversity of the families Aeshnidae, Gomphidae and Libellulidae through COI gene from South China.

Adult dragonflies (Anisoptera) were collected from different localities of South China covering eight provinces. Representative sequences were sixty-one, including 16 species, 11 genera and three families (Aeshnidae, Gomphidae and Libellulidae), under cytochrome oxidase subunit I (COI) gene. After alignment of sequences by BioEdit v6, genetic interaction and divergence were computed by MEGA 7 whereas all the indices of genetic diversity were calculated by DnaSP v5 software. Phylogenetic trees were constructed through Neighbor-Joining method under Jukes-Cantor model, and all species of respective families were assembled with each other into individual groups. Maximum divergence was observed by Trithemis genus (18.69%), followed by Orthetrum genus (18.16%), whereas a minimum value of divergence was noted for Pantala genus (0.31%). On the other hand, maximum genetic diversity was recorded for Orthetrum genus up to 142 mutations, followed by Trithemis genus (126 mutations), while the minimum value (two mutations) was observed for Pantala genus. Genetic diversity for overall and Libellulidae family sequences was much higher, up to 404 mutations and 344 mutations, respectively. Current results suggest a high diversity of odonates in the South China region and results are valuable in gaining a total obligation of the diversity of Asian odonates and conservation measures of this insect group.

Complete genome sequences of three novel cycloviruses identified in a dragonfly (Odonata: Anisoptera) from China.

Three cycloviruses (genus Cyclovirus, family Circoviridae) were recovered from a dragonfly (Odonata: Anisoptera) captured in Fuzhou, China. The three cycloviruses, named dragonfly associated cyclovirus 9, 10 and 11 (DfCyV-9, -10, -11), respectively, show 56.1-79.6% genome-wide identity to known cycloviruses and 61.6-65.1% among themselves. Thus, according to the current species demarcation criteria, they represent three novel cycloviruses. Notably, DfCyV-10 has a predicted replication-associated protein (Rep) that is most similar to that of bat associated cyclovirus 2 (BatACyV-2), a cyclovirus discovered in China, with 79.4% amino acid sequence identity, but a putative capsid protein (Cp) most similar to that of BatACyV-10, a cyclovirus discovered in Brazil, with 71.7% amino acid sequence identity. These data are useful for understanding the diversity and evolution of cycloviruses, especially those found in insects.

A nation-wide genetic survey revealed a complex population structure of Bemisia tabaci in Pakistan.

The whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex distributed worldwide. In Pakistan, B. tabaci poses a serious threat to agriculture production. To understand its diversity in Pakistan, a large-scale sampling was conducted from various locations of all four provinces of the country and Mitochondrial cytochrome oxidase I (mtCOI) gene sequencing was used to determine the whiteflies genetically. The study revealed the presence of five different cryptic species in Pakistan namely Asia II-1, Asia II-5, Asia II-7, Asia II-8 and MEAM-1, respectively. Among them, Asia II-1, which was previously reported from a few areas in the country, had been found now to be prevalent all over the country covering 88.7% of all the sequenced samples. Based on the mtCOI sequences and genetic distance analyses, the diversity of Asia II-1 was much greater than all other cryptic species, which exist only in small patches.

Genetic diversity of begomoviruses in Pakistan captured through a vector based survey.

Begomoviruses (Geminiviridea), transmitted by whiteflies, constitute one of the most dangerous groups of plant viruses posing a severe threat to economically important crops in tropical and sub-tropical areas. In this study, whiteflies were collected from various locations all over Pakistan. The begomoviruses carried by these whiteflies were detected by PCR with the degenerative primers pair AV94/Dep3. Analysis of the 177 sequences obtained in our study, revealed 14 distinct begomovirus species, including five which were not previously reported in this country. Putative novel strains of Corchorus yellow vein virus (CoYVV) and Chilli leaf curl virus (ChiLCV) showing less than 90% identity with the previously available taxa were also identified. The greatest number of begomoviruses per single site was detected in Sindh province, where up to five different begomovirus species were identified from the same cropping field. Moreover, Cotton leaf curl Multan virus - Rajasthan (CLCuMuV-Ra) was found prevalent in all the cotton growing areas. The data reported here may be useful in the development of control measures against begomoviruses.

Genetic interaction and diversity of the families Libellulidae and Gomphidae through COI gene from China and Pakistan.

A total of 300 dragonflies (Odonata) were collected from six different localities of China and Pakistan. Sixty seven representative samples were selected to sequence their mitochondrial cytochrome oxidase subunit I (COI). An examination of the resultant sequences identified 21 different dragonfly species, belonging to 15 distinct genera, two families, Libellulidae and Gomphidae. Sequence alignment was executed using Clustal-W in BioEdit v6. The phylogenetic tree was constructed through Neighbor-joining method by using Jukes-Cantor model, and genetic divergence was calculated via Kimura 2-parameter using MEGA7, while Genetic diversity was calculated by DnaSP v5. The maximum genetic divergence was observed for Crocothemis servilia, at 20.49%, followed by Libellulidae sp. with 22.30% while minimum divergence (0.82%) was observed for Melligomphus ardens. Likewise, a significant genetic diversity was observed for all species. However, Crocothemis servilia species presented maximum value (176 mutations) followed by Libellulidae spp. (150 mutations), whereas minimum value (3 mutations) was observed by Orthetrum testaceum. Interestingly, the diversity of C. servilia, all of which are collected from a single location of China, is much higher than those from Pakistan, which were collected from 5 different places with a spatial distance exceeding 500 Kms. Our results are useful in gaining a full appreciation of the global diversity of dragonflies and the development of conservation measures of this insect.

Rice Stripe Tenuivirus Has a Greater Tendency To Use the Prime-and-Realign Mechanism in Transcription of Genomic than in Transcription of Antigenomic Template RNAs.

Most segmented negative-sense RNA viruses employ a process termed cap snatching, during which they snatch capped RNA leaders from host cellular mRNAs and use the snatched leaders as primers for transcription, leading to the synthesis of viral mRNAs with 5' heterogeneous sequences (HSs). With traditional methods, only a few HSs can be determined, and identification of their donors is difficult. Here, the mRNA 5' ends of Rice stripe tenuivirus (RSV) and Rice grassy stunt tenuivirus (RGSV) and those of their host rice were determined by high-throughput sequencing. Millions of tenuiviral HSs were obtained, and a large number of them mapped to the 5' ends of corresponding host cellular mRNAs. Repeats of the dinucleotide AC, which are complementary to the U1G2 of the tenuiviral template 3'-U1G2U3G4UUUCG, were found to be prevalent at the 3' termini of tenuiviral HSs. Most of these ACs did not match host cellular mRNAs, supporting the idea that tenuiviruses use the prime-and-realign mechanism during cap snatching. We previously reported a greater tendency of RSV than RGSV to use the prime-and-realign mechanism in transcription with leaders cap snatched from a coinfecting reovirus. Besides confirming this observation in natural tenuiviral infections, the data here additionally reveal that RSV has a greater tendency to use this mechanism in transcribing genomic than in transcribing antigenomic templates. The data also suggest that tenuiviruses cap snatch host cellular mRNAs from translation- and photosynthesis-related genes, and capped RNA leaders snatched by tenuiviruses base pair with U1/U3 or G2/G4 of viral templates. These results provide unprecedented insights into the cap-snatching process of tenuiviruses.IMPORTANCE Many segmented negative-sense RNA viruses (segmented NSVs) are medically or agriculturally important pathogens. The cap-snatching process is a promising target for the development of antiviral strategies against this group of viruses. However, many details of this process remain poorly characterized. Tenuiviruses constitute a genus of agriculturally important segmented NSVs, several members of which are major viral pathogens of rice. Here, we for the first time adopted a high-throughput sequencing strategy to determine the 5' heterogeneous sequences (HSs) of tenuiviruses and mapped them to host cellular mRNAs. Besides providing deep insights into the cap snatching of tenuiviruses, the data obtained provide clear evidence to support several previously proposed models regarding cap snatching. Curiously and importantly, the data here reveal that not only different tenuiviruses but also the same tenuivirus synthesizing different mRNAs use the prime-and-realign mechanism with different tendencies during their cap snatching.

The Cap Snatching of Segmented Negative Sense RNA Viruses as a Tool to Map the Transcription Start Sites of Heterologous Co-infecting Viruses.

Identification of the transcription start sites (TSSs) of a virus is of great importance to understand and dissect the mechanism of viral genome transcription but this often requires costly and laborious experiments. Many segmented negative-sense RNA viruses (sNSVs) cleave capped leader sequences from a large variety of mRNAs and use these cleaved leaders as primers for transcription in a conserved process called cap snatching. The recent developments in high-throughput sequencing have made it possible to determine most, if not all, of the capped RNAs snatched by a sNSV. Here, we show that rice stripe tenuivirus (RSV), a plant-infecting sNSV, co-infects Nicotiana benthamiana with two different begomoviruses and snatches capped leader sequences from their mRNAs. By determining the 5' termini of a single RSV mRNA with high-throughput sequencing, the 5' ends of almost all the mRNAs of the co-infecting begomoviruses could be identified and mapped on their genomes. The findings in this study provide support for the using of the cap snatching of sNSVs as a tool to map viral TSSs.

Complete sequence of a double-stranded RNA from the phytopathogenic fungus Erysiphe cichoracearum that might represent a novel endornavirus.

A double-stranded RNA (dsRNA) HBJZ1506 recovered from the phytopathogenic fungus Erysiphe cichoracearum infecting Calendula officinalis in Jingzhou, Hubei Province, China, was sequenced. HBJZ1506 comprises 11,908 nucleotides (nt) and contains a 11,859-nt-long open reading frame (ORF) coding for a polypeptide that is 61 % identical to that of a putative endornavirus named grapevine endophyte endornavirus (GeEV). The putative polyprotein has an RNA-dependent RNA polymerase (RdRp) domain and an RNA helicase domain, which show homology to and have an arrangement that is similar to that of their counterparts in approved or putative endornaviruses. In a phylogenetic tree constructed using amino acid sequences of the RdRp region of HBJZ1506 and selected endornaviruses, HBJZ1506 clustered with endornaviruses and formed a well-supported monophyletic branch with GeEV. These results suggest that HBJZ1506 might represent a novel endornavirus, for which the name Erysiphe cichoracearum endornavirus (EcEV) is proposed.

Effects of timolol-related ophthalmic solutions on cultured human conjunctival cells.

PURPOSE: To investigate the inhibitory effects of drugs containing timolol on the proliferation of human conjunctival cells in vitro. METHODS: Timoptol, Timoptol XE, Rysmon TG, and Timabak solutions were used. These commercially available drugs were diluted to 1/30, 1/100, and 1/300, and their effects on cell morphology, cell count, and cell activity were investigated. The effects of drugs containing benzalkonium chloride (BAK) as well as those of the Rysmon TG vehicle alone were also assessed. RESULTS: At 1/30 dilution, cells treated with Timoptol and Timoptol XE showed cell deformation. Timoptol and Timoptol XE also caused a significant decrease in the number of cells at 1/100 and 1/30 dilutions. Cell activity decreased in a concentration-dependent manner after the addition of either Timoptol or Timoptol XE. Rysmon TG and Timabak showed significantly higher cell activity than Timoptol or Timoptol XE at both 1/100 and 1/30 dilutions. The cell count increased in a concentration-dependent manner in the BAK-free group, while cell activity decreased in a concentration-dependent manner in the cultures in the BAK-containing group. CONCLUSIONS: Compared with Timoptol and Timoptol XE, Rysmon TG and Timabak showed milder toxicity on human conjunctival cells in vitro.

Relationship between central corneal thickness and visual field defect in open-angle glaucoma.

PURPOSE: To explore the relationship between central corneal thickness (CCT) and visual field defect in open-angle glaucoma (OAG). METHODS: In this cross-sectional study, we tested 344 eyes in 344 eligible patients, including 233 with normal-tension glaucoma (NTG) and 111 with primary open-angle glaucoma (POAG). The association among variables, especially that between visual field defect and CCT, was probed by multivariate regression in eyes with NTG or POAG, and in all eyes. All eyes were divided into early, moderate, or severe visual field defect groups according to Anderson's classification. Statistical analysis was performed for all cases, and for the three CCT groups. RESULTS: Multivariate regression analysis revealed an association between CCT and visual field defect in eyes with NTG but not in eyes with POAG or in all eyes. The eyes with early visual field defect had greater CCT than did those with severe visual field defect (533.2 versus 519.0 microm). The eyes with greater CCT had better visual field indices than did those with thinner CCT (-6.91 versus -9.17 dB). CONCLUSIONS: Central corneal thickness is a factor associated with the status of the visual field defect: a greater CCT is associated with a better visual field index. Other factors such as the glaucoma subtype play a role in the effect of CCT on visual field defect.