Palmitoylation of γb protein directs a dynamic switch between Barley stripe mosaic virus replication and movement.

Viral replication and movement are intimately linked; however, the molecular mechanisms regulating the transition between replication and subsequent movement remain largely unknown. We previously demonstrated that the Barley stripe mosaic virus (BSMV) γb protein promotes viral replication and movement by interacting with the αa replicase and TGB1 movement proteins. Here, we found that γb is palmitoylated at Cys-10, Cys-19, and Cys-60 in Nicotiana benthamiana, which supports BSMV infection. Intriguingly, non-palmitoylated γb is anchored to chloroplast replication sites and enhances BSMV replication, whereas palmitoylated γb protein recruits TGB1 to the chloroplasts and forms viral replication-movement intermediate complexes. At the late stages of replication, γb interacts with NbPAT15 and NbPAT21 and is palmitoylated at the chloroplast periphery, thereby shifting viral replication to intracellular and intercellular movement. We also show that palmitoylated γb promotes virus cell-to-cell movement by interacting with NbREM1 to inhibit callose deposition at the plasmodesmata. Altogether, our experiments reveal a model whereby palmitoylation of γb directs a dynamic switch between BSMV replication and movement events during infection.

Comparative genomics and host range analysis of four Ralstonia pseudosolanacearum strains isolated from sunflower reveals genomic and phenotypic differences.

BACKGROUND: Bacterial wilt caused by Ralstonia solanacearum species complex (RSSC) is one of the devastating diseases in crop production, seriously reducing the yield of crops. R. pseudosolanacearum, is known for its broad infrasubspecific diversity and comprises 36 sequevars that are currently known. Previous studies found that R. pseudosolanacearum contained four sequevars (13, 14, 17 and 54) isolated from sunflowers sown in the same field. RESULTS: Here, we provided the complete genomes and the results of genome comparison of the four sequevars strains (RS639, RS642, RS647, and RS650). Four strains showed different pathogenicities to the same cultivars and different host ranges. Their genome sizes were about 5.84 ~ 5.94 Mb, encoding 5002 ~ 5079 genes and the average G + C content of 66.85% ~ 67%. Among the coding genes, 146 ~ 159 specific gene families (contained 150 ~ 160 genes) were found in the chromosomes and 34 ~ 77 specific gene families (contained 34 ~ 78 genes) in the megaplasmids from four strains. The average nucleotide identify (ANI) values between any two strains ranged from 99.05% ~ 99.71%, and the proportion of the total base length of collinear blocks accounts for the total gene length of corresponding genome was all more than 93.82%. Then, we performed a search for genomic islands, prophage sequences, the gene clusters macromolecular secretion systems, type III secreted effectors and other virulence factors in these strains, which provided detailed comparison results of their presence and distinctive features compared to the reference strain GMI1000. Among them, the number and types of T2SS gene clusters were different in the four strains, among which RS650 included all five types. T4SS gene cluster of RS639 and RS647 were missed. In the T6SS gene cluster, several genes were inserted in the RS639, RS647, and RS650, and gene deletion was also detected in the RS642. A total of 78 kinds of type III secreted effectors were found, which included 52 core and 9 specific effectors in four strains. CONCLUSION: This study not only provided the complete genomes of multiple R. pseudosolanacearum strains isolated from a new host, but also revealed the differences in their genomic levels through comparative genomics. Furthermore, these findings expand human knowledge about the range of hosts that Ralstonia can infect, and potentially contribute to exploring rules and factors of the genetic evolution and analyzing its pathogenic mechanism.

Barley stripe mosaic virus γb protein targets thioredoxin h-type 1 to dampen salicylic acid-mediated defenses.

Salicylic acid (SA) acts as a signaling molecule to perceive and defend against pathogen infections. Accordingly, pathogens evolve versatile strategies to disrupt the SA-mediated signal transduction, and how plant viruses manipulate the SA-dependent defense responses requires further characterization. Here, we show that barley stripe mosaic virus (BSMV) infection activates the SA-mediated defense signaling pathway and upregulates the expression of Nicotiana benthamiana thioredoxin h-type 1 (NbTRXh1). The γb protein interacts directly with NbTRXh1 in vivo and in vitro. The overexpression of NbTRXh1, but not a reductase-defective mutant, impedes BSMV infection, whereas low NbTRXh1 expression level results in increased viral accumulation. Similar with its orthologs in Arabidopsis (Arabidopsis thaliana), NbTRXh1 also plays an essential role in SA signaling transduction in N. benthamiana. To counteract NbTRXh1-mediated defenses, the BSMV γb protein targets NbTRXh1 to dampen its reductase activity, thereby impairing downstream SA defense gene expression to optimize viral cell-to-cell movement. We also found that NbTRXh1-mediated resistance defends against lychnis ringspot virus, beet black scorch virus, and beet necrotic yellow vein virus. Taken together, our results reveal a role for the multifunctional γb protein in counteracting plant defense responses and an expanded broad-spectrum antibiotic role of the SA signaling pathway.

Transcriptome and Metabolome Analyses Reveal That Jasmonic Acids May Facilitate the Infection of Cucumber Green Mottle Mosaic Virus in Bottle Gourd.

Cucumber green mottle mosaic virus (CGMMV) is a typical seed-borne tobamovirus that mainly infects cucurbit crops. Due to the rapid growth of international trade, CGMMV has spread worldwide and become a significant threat to cucurbit industry. Despite various studies focusing on the interaction between CGMMV and host plants, the molecular mechanism of CGMMV infection is still unclear. In this study, we utilized transcriptome and metabolome analyses to investigate the antiviral response of bottle gourd (Lagenaria siceraria) under CGMMV stress. The transcriptome analysis revealed that in comparison to mock-inoculated bottle gourd, 1929 differently expressed genes (DEGs) were identified in CGMMV-inoculated bottle gourd. Among them, 1397 genes were upregulated while 532 genes were downregulated. KEGG pathway enrichment indicated that the DEGs were mainly involved in pathways including the metabolic pathway, the biosynthesis of secondary metabolites, plant hormone signal transduction, plant-pathogen interaction, and starch and sucrose metabolism. The metabolome result showed that there were 76 differentially accumulated metabolites (DAMs), of which 69 metabolites were up-accumulated, and 7 metabolites were down-accumulated. These DAMs were clustered into several pathways, including biosynthesis of secondary metabolites, tyrosine metabolism, flavonoid biosynthesis, carbon metabolism, and plant hormone signal transduction. Combining the transcriptome and metabolome results, the genes and metabolites involved in the jasmonic acid and its derivatives (JAs) synthesis pathway were significantly induced upon CGMMV infection. The silencing of the allene oxide synthase (AOS) gene, which is the key gene involved in JAs synthesis, reduced CGMMV accumulation. These findings suggest that JAs may facilitate CGMMV infection in bottle gourd.

The Barley stripe mosaic virus γb protein promotes viral cell-to-cell movement by enhancing ATPase-mediated assembly of ribonucleoprotein movement complexes.

Nine genera of viruses in five different families use triple gene block (TGB) proteins for virus movement. The TGB modules fall into two classes: hordei-like and potex-like. Although TGB-mediated viral movement has been extensively studied, determination of the constituents of the viral ribonucleoprotein (vRNP) movement complexes and the mechanisms underlying their involvement in vRNP-mediated movement are far from complete. In the current study, immunoprecipitation of TGB1 protein complexes formed during Barley stripe mosaic virus (BSMV) infection revealed the presence of the γb protein in the products. Further experiments demonstrated that TGB1 interacts with γb in vitro and in vivo, and that γb-TGB1 localizes at the periphery of chloroplasts and plasmodesmata (PD). Subcellular localization analyses of the γb protein in Nicotiana benthamiana epidermal cells indicated that in addition to chloroplast localization, γb also targets the ER, actin filaments and PD at different stages of viral infection. By tracking γb localization during BSMV infection, we demonstrated that γb is required for efficient cell-to-cell movement. The N-terminus of γb interacts with the TGB1 ATPase/helicase domain and enhances ATPase activity of the domain. Inactivation of the TGB1 ATPase activity also significantly impaired PD targeting. In vitro translation together with co-immunoprecipitation (co-IP) analyses revealed that TGB1-TGB3-TGB2 complex formation is enhanced by ATP hydrolysis. The γb protein positively regulates complex formation in the presence of ATP, suggesting that γb has a novel role in BSMV cell-to-cell movement by directly promoting TGB1 ATPase-mediated vRNP movement complex assembly. We further demonstrated that elimination of ATPase activity abrogates PD and actin targeting of Potato virus X (PVX) and Beet necrotic yellow vein virus (BNYVV) TGB1 proteins. These results expand our understanding of the multifunctional roles of γb and provide new insight into the functions of TGB1 ATPase domains in the movement of TGB-encoding viruses.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China.

First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 µg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3'/5'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3'), and TYMaV-F2/R2 (5'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3'/5'-ATGACCTAGCTTCAATAACAGTCGCG-3'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003). References: Kreuze, J. F., et al. 2009. Virology. 388: 1. Mi, S., et al. 2008. Cell. 133: 116. Xu, C., et al. 2017. J Virol. 91: 11. Zheng, Y., et al. 2017. Virology. 500: 130.

Anthracnose: A New Leaf Disease on Radermachera sinica (China Doll) in China.

Radermachera sinica (China doll) is a popular evergreen horticultural crop worldwide. However, little information has been provided to describe the anthracnose disease of R. sinica. In 2018, symptoms suspected of leaf anthracnose were observed on R. sinica in gardens and commercial greenhouses in Guangzhou, China. Lesions on diseased leaves showed thinned and grayish white centers, dark-brown to black borders, and raised black spots. Twenty-seven single-conidia isolates were obtained from symptomatic leaf lesions. Based on morphological characteristics and multilocus phylogenetic analysis, 19 isolates were identified as Colletotrichum siamense and six and two isolates were identified as C. fructicola and C. karstii, respectively. An in vivo pathogenicity test was conducted on leaves of R. sinica plants, and it was discovered that C. siamense was more aggressive under wounded conditions than under unwounded conditions, and caused symptomatic necrotic lesions on the leaf. Afterward, the same pathogen was reisolated from lesions of inoculated leaves to fulfill Koch's postulates. However, neither C. fructicola nor C. karstii caused visible lesions on leaves of R. sinica under wounded or unwounded conditions, indicating that they may be asymptomatic endophytes or opportunistic pathogens on R. sinica. To our knowledge, this study is the first report of Colletotrichum spp. associated with anthracnose disease on R. sinica in China.

Complete genome sequence of a previously undescribed monopartite begomovirus and betasatellite infecting Malvastrum coromandelianum in Cambodia.

A previously undescribed monopartite begomovirus was identified in Kampot province, Cambodia, in Malvastrum coromandelianum plants exhibiting yellow vein symptoms characteristic of begomovirus infections. The apparently full-length viral component was cloned and sequenced following enrichment of circular DNA by rolling-circle amplification and restriction enzyme digestion. The genome of the virus was 2737 nucleotides in length (KP188831) and exhibited an organization like that of other monopartite begomoviruses, sharing the highest nucleotide sequence similarity (87.7% identity) with ageratum yellow vein virus (AM940137). A satellite molecule was amplified from total DNA by PCR amplification, using the betasatellite-specific primer pair ß01/ß02. The satellite molecule (1346 nt, KP188832) had structural characteristics like those of other betasatellites associated with begomoviruses and shared the highest nucleotide sequence similarity (84.8% identity) with malvastrum yellow vein betasatellite (MN205547). According to the criteria established for species demarcation for classification of begomoviruses (family Geminiviridae) and betasatellites (family Tolecusatellitidae), respectively, the virus isolate from M. coromandelianum in Cambodia is a previously undescribed novel monopartite begomovirus, for which the name "malvastrum yellow vein Cambodia virus" (MaYVCV) is proposed, and the betasatellite is a previously undescribed novel betasatellite, for which the name "malvastrum yellow vein Cambodia betasatellite" (MaYVKHB) is proposed.

The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

Phosphorylation of TGB1 by protein kinase CK2 promotes barley stripe mosaic virus movement in monocots and dicots.

The barley stripe mosaic virus (BSMV) triple gene block 1 (TGB1) protein is required for virus cell-to-cell movement. However, little information is available about how these activities are regulated by post-translational modifications. In this study, we showed that the BSMV Xinjiang strain TGB1 (XJTGB1) is phosphorylated in vivo and in vitro by protein kinase CK2 from barley and Nicotiana benthamiana. Liquid chromatography tandem mass spectrometry analysis and in vitro phosphorylation assays demonstrated that Thr-401 is the major phosphorylation site of the XJTGB1 protein, and suggested that a Thr-395 kinase docking site supports Thr-401 phosphorylation. Substitution of Thr-395 with alanine (T395A) only moderately impaired virus cell-to-cell movement and systemic infection. In contrast, the Thr-401 alanine (T401A) virus mutant was unable to systemically infect N. benthamiana but had only minor effects in monocot hosts. Substitution of Thr-395 or Thr-401 with aspartic acid interfered with monocot and dicot cell-to-cell movement and the plants failed to develop systemic infections. However, virus derivatives with single glutamic acid substitutions at Thr-395 and Thr-401 developed nearly normal systemic infections in the monocot hosts but were unable to infect N. benthamiana systemically, and none of the double mutants was able to infect dicot and monocot hosts. The mutant XJTGB1T395A/T401A weakened in vitro interactions between XJTGB1 and XJTGB3 proteins but had little effect on XJTGB1 RNA-binding ability. Taken together, our results support a critical role of CK2 phosphorylation in the movement of BSMV in monocots and dicots, and provide new insights into the roles of phosphorylation in TGB protein functions.

Phase-selective synthesis of Cu2ZnSnS4 nanocrystals using different sulfur precursors.

Cu2ZnSnS4 (CZTS) holds great promises as an absorber material for sustainable and low cost thin film solar cells. Kesterite and wurtzite are two common phases of CZTS. Until now, the synthesis and the growth of both phases are not clearly understood. In this work, kesterite CZTS nanoparticles, wurtzite CZTS nanoparticles as well as CZTS particles with a mixture of both structures were prepared by using elemental sulfur, 1-dodecanethiol, and thioacetamide, respectively. Time dependent studies were conducted and the reaction rate of sulfur source was found to be the key factor in determining the phase formation. Elemental sulfur reacts with oleylamine to produce highly reactive small molecule H2S, which leads to the formation of kesterite phase. The reaction pathways of the long alkane chain 1-dodecanethiol yield the formation of wurtzite phase via a binary phase. Thioacetamide yields a mixture of kesterite and wurtzite phase in the final product. The optical and electrical properties of kesterite and wurtzite CZTS were also evaluated.

Dissimilarity of megabenthic community structure between deep-water seamounts with cobalt-rich crusts: Case study in the northwestern Pacific Ocean.

As anthropogenic disturbance on deep-sea seamount ecosystems grows, there is an urgent need for a better understanding of the biodiversity and community structure in benthic ecosystems, which can vary at local and regional scales. A survey of the benthic megafauna on two adjacent deep-water seamounts in the northwestern Pacific Ocean was conducted, which are covered by cobalt-rich crusts, to assess the biodiversity patterns and dissimilarity of assemblage composition. Based on a multidisciplinary dataset generated from video recordings, multibeam bathymetry data, and near-bottom currents, environmental and spatial factors impacting the megabenthic communities were explored. Results showed that these two deep-water seamounts were dominated by hexactinellids, crinoids, and octocorals. The seamounts were able to support diverse and moderately abundant megafauna, with a total of 6436 individuals classified into 94 morphospecies. The survey covered a distance of 52.2 km across a depth range of 1421-3335 m, revealing multiple distinct megabenthic assemblages. The megabenthic communities of the two deep-water seamounts, with comparable environmental conditions, exhibited similarities in overall density, richness, and faunal lists, while dissimilarities in the relative abundance of taxa and assemblage composition. No gradual depth-related change in terms of abundance, richness, or species turnover was observed across the two seamounts, despite the statistical significance of depth in structuring the overall communities. The spatial distribution of megabenthic communities displayed a discontinuous and patchy pattern throughout the two deep-water seamounts. This patchiness was driven by the interactive effects of multiple environmental factors. Near-bottom currents and microhabitat features were the primary drivers influencing their dissimilarities in megabenthic community structure. This case study on the megabenthic community structure of two adjacent seamounts with cobalt-rich crusts can serve as an environmental baseline, providing a reference status for the conservation and management of seamount ecosystems, particularly valuable for areas being considered for deep-sea mining.

Pathogenicity analysis and seed transmission of watermelon virus A in bottle gourd.

Seed transmission is among the primary strategies utilized by plant viruses for long-distance dissemination, leading to the widespread occurrence of viral diseases globally. Watermelon virus A (WVA) is a novel wamavirus first found in watermelon. However, the pathogenicity and transmission mode of WVA are still unclear. Our previous work found that the incidence of WVA in bottle gourd is very high. Based on that, the pathogenicity and seed transmission mode of WVA in bottle gourd were studied. Compared with healthy plant, bottle gourd infected by WVA showed no visible disease symptom. Moreover, in the seeds of 20 bottle gourd cultivars, the occurrence of WVA varies from 0 to 90%, and one cultivar even reaches 100%. We also found that the transmission rate from seeds to the resulting seedlings was 100%. Furthermore, WVA was present in both the seed coat and embryo, and seed disinfection cannot eliminate WVA. Besides the seed and leaf, WVA can also be detected in stem, flower, and fruit, but not in the root. To our surprise, the level of transmission from WVA-infected plants to seeds was more than 85%. In addition, the viral accumulations of both WVA and CGMMV were increased in plants with co-infection of WVA and CGMMV. Taken together, these findings reveal that WVA is a seed-transmitted virus which causes no disease symptom in bottle gourd, and there may be synergism between WVA and CGMMV.

Improvement of plant resistance to geminiviruses via protein de-S-acylation.

Geminiviruses are an important group of viruses that infect a variety of plants and result in heavy agricultural losses worldwide. The homologs of C4 (or L4) in monopartite geminiviruses and AC4 (or AL4) in bipartite geminiviruses are critical viral proteins. The C4 proteins from several geminiviruses are the substrates of S-acylation, a dynamic post-translational modification, for the maintenance of their membrane localization and function in virus infection. Here we initiated a screening and identified a plant protein ABAPT3 (Alpha/Beta Hydrolase Domain-containing Protein 17-like Acyl Protein Thioesterase 3) as the de-S-acylation enzyme of C4 encoded by BSCTV (Beet severe curly top virus). Overexpression of ABAPT3 reduced the S-acylation of BSCTV C4, disrupted its plasma membrane localization, inhibited its function in pathogenesis, and suppressed BSCTV infection. Because the S-acylation motifs are conserved among C4 from different geminiviruses, we tested the effect of ABAPT3 on the C4 protein of ToLCGdV (Tomato leaf curl Guangdong virus) from another geminivirus genus. Consistently, ABAPT3 overexpression also disrupted the S-acylation, subcellular localization, and function of ToLCGdV C4, and inhibited ToLCGdV infection. In summary, we provided a new approach to globally improve the resistance to different types of geminiviruses in plants via de-S-acylation of the viral C4 proteins and it can be extendedly used for suppression of geminivirus infection in crops.

Study on the Influence of Accelerated Aging on the Properties of an RTV Anti-Pollution Flashover Coating.

The purpose of this work is to study the accelerated aging behavior of a room-temperature vulcanized (RTV) silicone rubber anti-pollution flashover coating. Red, blue and gray RTV rubber samples were selected to prepare coatings on the surface of stainless-steel sheets. The accelerated aging test was carried out in an aging test chamber according to a four-step program cycle. After the completion of different aging tests, the color difference, glossiness, surface micromorphology, wettability, insulation performance and other parameters of the samples were measured using colorimetry, infrared spectrometry, scanning electron microscopy, and a high-voltage breakdown tester. The results showed that with the increase in aging time, the color difference ∆Eab of the coatings increased. The G60 gloss value decreased gradually and tended to be saturated after 60 cycles. After the aging tests, the RTV coating surface had holes, cracks, peeling and other damage to varying degrees. The C:Si atomic ratio was less than 2, and the hydrophobicity was obviously deteriorated. After aging, the electrical strength of the three RTV coatings decreased significantly. It can be concluded that during the accelerated aging test, the RTV coating had cross-linking and oxidation reactions, and the internal deterioration and surface damage of the coating led to changes in its color, luster, morphology, insulation strength, etc.

Conservation and evolution in and among SRF- and MEF2-type MADS domains and their binding sites.

Serum response factor (SRF) and myocyte enhancer factor 2 (MEF2) represent two types of members of the MCM1, AGAMOUS, DEFICIENS, and SRF (MADS)-box transcription factor family present in animals and fungi. Each type has distinct biological functions, which are reflected by the distinct specificities of the proteins bound to their cognate DNA-binding sites and activated by their respective cofactors. However, little is known about the evolution of MADS domains and their DNA-binding sites. Here, we report on the conservation and evolution of the two types of MADS domains with their cognate DNA-binding sites by using phylogenetic analyses. First, there are great similarities between the two types of proteins with amino acid positions highly conserved, which are critical for binding to the DNA sequence and for the maintenance of the 3D structure. Second, in contrast to MEF2-type MADS domains, distinct conserved residues are present at some positions in SRF-type MADS domains, determining specificity and the configuration of the MADS domain bound to DNA sequences. Furthermore, the ancestor sequence of SRF- and MEF2-type MADS domains is more similar to MEF2-type MADS domains than to SRF-type MADS domains. In the case of DNA-binding sites, the MEF2 site has a T-rich core in one DNA sequence and an A-rich core in the reverse sequence as compared with the SRF site, no matter whether where either A or T is present in the two complementary sequences. In addition, comparing SRF sites in the human and the mouse genomes reveals that the evolution rate of CArG-boxes is faster in mouse than in human. Moreover, interestingly, a CArG-like sequence, which is probably functionless, could potentially mutate to a functional CArG-box that can be bound by SRF and vice versa. Together, these results significantly improve our knowledge on the conservation and evolution of the MADS domains and their binding sites to date and provide new insights to investigate the MADS family, which is not only on evolution of MADS factors but also on evolution of their binding sites and even on coevolution of MADS factors with their binding sites.

Cotton leaf curl Multan virus differentially regulates innate antiviral immunity of whitefly (Bemisia tabaci) vector to promote cryptic species-dependent virus acquisition.

Begomoviruses represent the largest group of economically important, highly pathogenic, DNA plant viruses that contribute a substantial amount of global crop disease burden. The exclusive transmission of begomoviruses by whiteflies (Bemisia tabaci) requires them to interact and efficiently manipulate host responses at physiological, biological and molecular scales. However, the molecular mechanisms underlying complex begomovirus-whitefly interactions that consequently substantiate efficient virus transmission largely remain unknown. Previously, we found that whitefly Asia II 7 cryptic species can efficiently transmit cotton leaf curl Multan virus (CLCuMuV) while MEAM1 cryptic species is a poor carrier and incompetent vector of CLCuMuV. To investigate the potential mechanism/s that facilitate the higher acquisition of CLCuMuV by its whitefly vector (Asia II 7) and to identify novel whitefly proteins that putatively interact with CLCuMuV-AV1 (coat protein), we employed yeast two-hybrid system, bioinformatics, bimolecular fluorescence complementation, RNA interference, RT-qPCR and bioassays. We identified a total of 21 Asia II 7 proteins putatively interacting with CLCuMuV-AV1. Further analyses by molecular docking, Y2H and BiFC experiments validated the interaction between a whitefly innate immunity-related protein (BTB/POZ) and viral AV1 (coat protein). Gene transcription analysis showed that the viral infection significantly suppressed the transcription of BTB/POZ and enhanced the accumulation of CLCuMuV in Asia II 7, but not in MEAM1 cryptic species. In contrast to MEAM1, the targeted knock-down of BTB/POZ substantially reduced the ability of Asia II 7 to acquire and accumulate CLCuMuV. Additionally, antiviral immune signaling pathways (Toll, Imd, Jnk and Jak/STAT) were significantly suppressed following viral infection of Asia II 7 whiteflies. Taken together, the begomovirus CLCuMuV potentiates efficient virus accumulation in its vector B. tabaci Asia II 7 by targeting and suppressing the transcription of an innate immunity-related BTB/POZ gene and other antiviral immune responses in a cryptic species-specific manner.

Predicting willingness to donate blood based on machine learning: two blood donor recruitments during COVID-19 outbreaks.

Machine learning methods are a novel way to predict and rank donors' willingness to donate blood and to achieve precision recruitment, which can improve the recruitment efficiency and meet the challenge of blood shortage. We collected information about experienced blood donors via short message service (SMS) recruitment and developed 7 machine learning-based recruitment models using PyCharm-Python Environment and 13 features which were described as a method for ranking and predicting donors' intentions to donate blood with a floating number between 0 and 1. Performance of the prediction models was assessed by the Area under the receiver operating characteristic curve (AUC), accuracy, precision, recall, and F1 score in the full dataset, and by the accuracy in the four sub-datasets. The developed models were applied to prospective validations of recruiting experienced blood donors during two COVID-19 pandemics, while the routine method was used as a control. Overall, a total of 95,476 recruitments via SMS and their donation results were enrolled in our modelling study. The strongest predictor features for the donation of experienced donors were blood donation interval, age, and donation frequency. Among the seven baseline models, the eXtreme Gradient Boosting (XGBoost) and Support vector machine models (SVM) achieved the best performance: mean (95%CI) with the highest AUC: 0.809 (0.806-0.811), accuracy: 0.815 (0.812-0.818), precision: 0.840 (0.835-0.845), and F1 score of XGBoost: 0.843 (0.840-0.845) and recall of SVM: 0.991 (0.988-0.994). The hit rate of the XGBoost model alone and the combined XGBoost and SVM models were 1.25 and 1.80 times higher than that of the conventional method as a control in 2 recruitments respectively, and the hit rate of the high willingness to donate group was 1.96 times higher than that of the low willingness to donate group. Our results suggested that the machine learning models could predict and determine the experienced donors with a strong willingness to donate blood by a ranking score based on personalized donation data and demographical details, significantly improve the recruitment rate of blood donors and help blood agencies to maintain the blood supply in emergencies.

Impact of enhanced recovery nursing combined with limb training on knee joint function and neurological function after total knee arthroplasty in patients with knee osteoarthritis.

OBJECTIVE: To investigate the impact of enhanced recovery after surgery (ERAS) nursing combined with limb training on knee joint function and neurological function after total knee arthroplasty in patients with knee osteoarthritis (KOA). METHODS: Eighty-six patients with KOA after TKA were randomly divided into two groups, group A and group B, with 43 patients in each group. Group A was given ERAS nursing, and group B was given limb rehabilitation training combined with ERAS nursing. The changes in knee joint function and neurological function were observed. RESULTS: There was no significant difference in the time to get out of bed for the first time, first bowel movement time after the surgery, hospital stay and hospital costs between the two groups (P>0.05). There was no significant difference in VAS scores between the two groups before the operation and 1 d after the operation (P>0.05). Three days and seven days after the operation, the VAS scores in the two groups both decreased, and the VAS scores of group B were higher than those of group A (P<0.05). There was no significant difference in the excellent rate of Judet scores and Lysholm scores between the two groups (P>0.05), but the two indicators in the two groups all increased at three and six months after the operation, and the two indicators in group B were higher than those of group A (P<0.05). There was no significant difference in NIHSS scores between the two groups before the operation (P>0.05). Fifteen and thirty days after the operation, the NIHSS scores of the two groups both decreased, and the NIHSS scores of group B were lower than those of group A (P<0.05). After the nursing care, the scores of health knowledge level, self-care concept, self-care responsibility and self-care skills in group B were higher than those in group A (P<0.05). The incidence of complications in group B during nursing was lower than group A (P<0.05). CONCLUSION: The enhanced recovery after surgery nursing combined with limb training has a better effect on KOA patients after TKA. It can significantly improve knee joint function, limb motor ability and neurological function, increase patients' cognition of disease and reduce the incidence of complications, compared with simple enhanced recovery after surgery nursing.

C4, the Pathogenic Determinant of Tomato Leaf Curl Guangdong Virus, May Suppress Post-transcriptional Gene Silencing by Interacting With BAM1 Protein.

Tomato leaf curl Guangdong virus (ToLCGdV) is a begomovirus associated with a Tomato yellow leaf curl disease (TYLCD) epidemic in Guangdong province, China. Being the least conserved protein among geminivirus proteins, the function of C4 during ToLCGdV infection has not been elucidated. In this study, the infectious clones of ToLCGdV and a ToLCGdV mutant (ToLCGdVmC4) with disrupted C4 ORF were constructed. Although ToLCGdV and ToLCGdVmC4 could infect Nicotiana benthamiana and tomato plants, ToLCGdVmC4 elicited much milder symptoms compared with ToLCGdV. To further verify the role of C4 in viral pathogenesis, C4 was expressed in N. benthamiana from Potato virus X (PVX) vector. The results showed that ToLCGdV C4 enhanced the pathogenicity of PVX and induced more severe developmental abnormalities in plants compared with PVX alone or PVX-mC4. In addition, ToLCGdV C4 suppresses systemic gene silencing in the transgenic N. benthamiana line 16c, but not local gene silencing induced by sense GFP in wild-type N. benthamiana plants. Moreover, C4 suppresses transcriptional gene silencing (TGS) by reducing the DNA methylation level of 35S promoter in 16c-TGS N. benthamiana plants. Furthermore, C4 could also interact with the receptor-like kinase (RLK) BARELY ANY MERISTEM 1 (BAM1), suggesting that C4 may suppress gene silencing by interfering with the function of BAM1 in the cell-to-cell spread of RNAi. All these results suggest that C4 is a pathogenic determinant of ToLCGdV, and C4 may suppress post-transcriptional gene silencing (PTGS) by interacting with BAM1.