Development of an attenuated potato virus Y mutant carrying multiple mutations in helper-component protease for cross-protection.

Tobacco (Nicotiana tabacum) is one of the major cash crops in China. Potato virus Y (PVY), a representative member of the genus Potyvirus, greatly reduces the quality and yield of tobacco leaves by inducing veinal necrosis. Mild strain-mediated cross-protection is an attractive method of controlling diseases caused by PVY. Currently, there is a lack of effective and stable attenuated PVY mutants. Potyviral helper component-protease (HC-Pro) is a likely target for the development of mild strains. Our previous studies showed that the residues lysine at positions 124 and 182 (K124 and K182) in HC-Pro were involved in PVY virulence, and the conserved KITC motif in HC-Pro was involved in aphid transmission. In this study, to improve the stability of PVY mild strains, K at position 50 (K50) in KITC motif, K124, and K182 were separately substituted with glutamic acid (E), leucine (L), and arginine (R), resulting in a triple-mutant PVY-HCELR. The mutant PVY-HCELR had attenuated virulence and did not induce leaf veinal necrosis symptoms in tobacco plants and could not be transmitted by Myzus persicae. Furthermore, PVY-HCELR mutant was genetically stable after six serial passages, and only caused mild mosaic symptoms in tobacco plants even at 90 days post inoculation. The tobacco plants cross-protected by PVY-HCELR mutant showed high resistance to the wild-type PVY. This study showed that PVY-HCELR mutant was a promising mild mutant for cross-protection to control PVY.

Characterisation of the myosin light chain kinase (MLCK) gene of Locusta migratoria and the encoded MLCK.

Myosin light chain kinase (MLCK) is a dedicated kinase of myosin regulatory light chain (RLC), playing an essential role in the regulation of muscle contraction and cell motility. Much of the knowledge about MLCK comes from the study of vertebrate MLCK, and little is known about insect MLCK. Here, we identified the single MLCK gene in the locust Locusta migratoria, which spans over 1400 kb, includes 62 exons and accounts for at least five transcripts. We found that the five distinct transcripts of the locust MLCK gene are expressed in a tissue-specific manner, including three muscle-specific isoforms and two generic isoforms. To characterise the kinase activity of locust MLCK, we recombinantly expressed LmMLCK-G, the smallest locust MLCK isoform, in insect Sf9 cells. We demonstrated that LmMLCK-G is a Ca2+ /calmodulin-dependent kinase that specifically phosphorylates serine 50 of locust muscle myosin RLC (LmRLC). Additionally, we found that almost all LmRLC molecules in the flight muscle and the hindleg muscles of adult locusts are phosphorylated.

Spire2 and Rab11a synergistically activate myosin-5b motor function.

Cellular vesicle long-distance transport along the cytoplasmic actin network has recently been uncovered in several cell systems. In metaphase mouse oocytes, the motor protein myosin-5b (Myo5b) and the actin nucleation factor Spire are recruited to the Rab11a-positive vesicle membrane, forming a ternary complex of Myo5b/Spire/Rab11a that drives the vesicle long-distance transport to the oocyte cortex. However, the mechanism underlying the intermolecular regulation of the Myo5b/Spire/Rab11a complex remains unknown. In this study, we expressed and purified Myo5b, Spire2, and Rab11a proteins, and performed ATPase activity measurements, pulldown and single-molecule motility assays. Our results demonstrate that both Spire2 and Rab11a are required to activate Myo5b motor activity under physiological ionic conditions. The GTBM fragment of Spire2 stimulates the ATPase activity of Myo5b, while Rab11a enhances this activation. This activation occurs by disrupting the head-tail interaction of Myo5b. Furthermore, at the single-molecule level, we observed that the GTBM fragment of Spire2 and Rab11a coordinate to stimulate the Myo5b motility activity. Based on our results, we propose that upon association with the vesicle membrane, Myo5b, Spire2 and Rab11a form a ternary complex, and the inhibited Myo5b is synergistically activated by Spire2 and Rab11a, thereby triggering the long-distance transport of vesicles.

Asymmetric and parallel subgenome selection co-shape common carp domestication.

BACKGROUND: The common carp (Cyprinus carpio) might best represent the domesticated allopolyploid animals. Although subgenome divergence which is well-known to be a key to allopolyploid domestication has been comprehensively characterized in common carps, the link between genetic architecture underlying agronomic traits and subgenome divergence is unknown in the selective breeding of common carps globally. RESULTS: We utilized a comprehensive SNP dataset in 13 representative common carp strains worldwide to detect genome-wide genetic variations associated with scale reduction, vibrant skin color, and high growth rate in common carp domestication. We identified numerous novel candidate genes underlie the three agronomically most desirable traits in domesticated common carps, providing potential molecular targets for future genetic improvement in the selective breeding of common carps. We found that independently selective breeding of the same agronomic trait (e.g., fast growing) in common carp domestication could result from completely different genetic variations, indicating the potential advantage of allopolyploid in domestication. We observed that candidate genes associated with scale reduction, vibrant skin color, and/or high growth rate are repeatedly enriched in the immune system, suggesting that domestication of common carps was often accompanied by the disease resistance improvement. CONCLUSIONS: In common carp domestication, asymmetric subgenome selection is prevalent, while parallel subgenome selection occurs in selective breeding of common carps. This observation is not due to asymmetric gene retention/loss between subgenomes but might be better explained by reduced pleiotropy through transposable element-mediated expression divergence between ohnologs. Our results demonstrate that domestication benefits from polyploidy not only in plants but also in animals.

A natural substitution of a conserved amino acid in eIF4E confers resistance against multiple potyviruses.

Eukaryotic translation initiation factor 4E (eIF4E), which plays a pivotal role in initiating translation in eukaryotic organisms, is often hijacked by the viral genome-linked protein to facilitate the infection of potyviruses. In this study, we found that the naturally occurring amino acid substitution D71G in eIF4E is widely present in potyvirus-resistant watermelon accessions and disrupts the interaction between watermelon eIF4E and viral genome-linked protein of papaya ringspot virus-watermelon strain, zucchini yellow mosaic virus or watermelon mosaic virus. Multiple sequence alignment and protein modelling showed that the amino acid residue D71 located in the cap-binding pocket of eIF4E is strictly conserved in many plant species. The mutation D71G in watermelon eIF4E conferred resistance against papaya ringspot virus-watermelon strain and zucchini yellow mosaic virus, and the equivalent mutation D55G in tobacco eIF4E conferred resistance to potato virus Y. Therefore, our finding provides a potential precise target for breeding plants resistant to multiple potyviruses.

Potato virus Y viral protein 6K1 inhibits the interaction between defense proteins during virus infection.

14-3-3 proteins play vital roles in plant defense against various pathogen invasions. To date, how 14-3-3 affects virus infections in plants remains largely unclear. In this study, we found that Nicotiana benthamiana 14-3-3h interacts with TRANSLATIONALLY CONTROLLED TUMOR PROTEIN (TCTP), a susceptibility factor of potato virus Y (PVY). Silencing of Nb14-3-3h facilitates PVY accumulation, whereas overexpression of Nb14-3-3h inhibits PVY replication. The antiviral activities of 3 Nb14-3-3h dimerization defective mutants are significantly decreased, indicating that dimerization of Nb14-3-3h is indispensable for restricting PVY infection. Our results also showed that the mutant Nb14-3-3hE16A, which is capable of dimerizing but not interacting with NbTCTP, has reduced anti-PVY activity; the mutant NbTCTPI65A, which is unable to interact with Nb14-3-3h, facilitates PVY replication compared with the wild-type NbTCTP, indicating that dimeric Nb14-3-3h restricts PVY infection by interacting with NbTCTP and preventing its proviral function. As a counter-defense, PVY 6K1 interferes with the interaction between Nb14-3-3h and NbTCTP by competitively binding to Nb14-3-3h and rescues NbTCTP to promote PVY infection. Our results provide insights into the arms race between plants and potyviruses.

Alternative relay regulates the adenosine triphosphatase activity of Locusta migratoria striated muscle myosin.

Locust (Locusta migratoria) has a single striated muscle myosin heavy chain (Mhc) gene, which contains 5 clusters of alternative exclusive exons and 1 differently included penultimate exon. The alternative exons of Mhc gene encode 4 distinct regions in the myosin motor domain, that is, the N-terminal SH3-like domain, one lip of the nucleotide-binding pocket, the relay, and the converter. Here, we investigated the role of the alternative regions on the motor function of locust muscle myosin. Using Sf9-baculovirus protein expression system, we expressed and purified 5 isoforms of the locust muscle myosin heavy meromyosin (HMM), including the major isoform in the thorax dorsal longitudinal flight muscle (FL1) and 4 isoforms expressed in the abdominal intersegmental muscle (AB1 to AB4). Among these 5 HMMs, FL1-HMM displayed the highest level of actin-activated adenosine triphosphatase (ATPase) activity (hereafter referred as ATPase activity). To identify the alternative region(s) responsible for the elevated ATPase activity of FL1-HMM, we produced a number of chimeras of FL1-HMM and AB4-HMM. Substitution with the relay of AB4-HMM (encoded by exon-14c) substantially decreased the ATPase activity of FL1-HMM, and conversely, the relay of FL1-HMM (encoded by exon-14a) enhanced the ATPase activity of AB4-HMM. Mutagenesis showed that the exon-14a-encoded residues Gly474 and Asn509 are responsible for the elevated ATPase activity of FL1-HMM. Those results indicate that the alternative relay encoded by exon-14a/c play a key role in regulating the ATPase activity of FL1-HMM and AB4-HMM.

m5C-dependent cross-regulation between nuclear reader ALYREF and writer NSUN2 promotes urothelial bladder cancer malignancy through facilitating RABL6/TK1 mRNAs splicing and stabilization.

The significance of 5-methylcytosine (m5C) methylation in human malignancies has become an increasing focus of investigation. Here, we show that m5C regulators including writers, readers and erasers, are predominantly upregulated in urothelial carcinoma of the bladder (UCB) derived from Sun Yat-sen University Cancer Center and The Cancer Genome Atlas cohort. In addition, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) as a methyltransferase and Aly/REF export factor (ALYREF) as a nuclear m5C reader, are frequently coexpressed in UCB. By applying patient-derived organoids model and orthotopic xenograft mice model, we demonstrate that ALYREF enhances proliferation and invasion of UCB cells in an m5C-dependent manner. Integration of tanscriptome-wide RNA bisulphite sequencing (BisSeq), RNA-sequencing (RNA-seq) and RNA Immunoprecipitation (RIP)-seq analysis revealed that ALYREF specifically binds to hypermethylated m5C site in RAB, member RAS oncogene family like 6 (RABL6) and thymidine kinase 1 (TK1) mRNA via its K171 domain. ALYREF controls UCB malignancies through promoting hypermethylated RABL6 and TK1 mRNA for splicing and stabilization. Moreover, ALYREF recognizes hypermethylated m5C site of NSUN2, resulting in NSUN2 upregulation in UCB. Clinically, the patients with high coexpression of ALYREF/RABL6/TK1 axis had the poorest overall survival. Our study unveils an m5C dependent cross-regulation between nuclear reader ALYREF and m5C writer NSUN2 in activation of hypermethylated m5C oncogenic RNA through promoting splicing and maintaining stabilization, consequently leading to tumor progression, which provides profound insights into therapeutic strategy for UCB.

[Effects of Different Topdressing Nitrogen Rates on Soil Fungal Community Structure and Ecological Network in Wheat Field Under Crop Residue Retention].

Crop residue retention and fertilizer application are the main sources of soil nutrient input in fields. Crop residue retention combined with appropriate fertilizer application rates could provide necessary nutrients for crop production under the premise of environmentally friendly conditions. The aim of this study was to clarify the influence of different topdressing nitrogen rates on the soil fungal community in a wheat field under crop residue retention and to evaluate the rationality of nitrogen fertilizer management in winter wheat from the perspective of soil ecological function. On the basis of full straw retention and 150 kg·hm-2 basal nitrogen, treatments with five topdressing nitrogen rates (0, 37.5, 75, 112.5, and 150 kg·hm-2) were set up. The abundance, diversity, structure, and ecological network of soil fungal communities were analyzed using real-time fluorescence quantitative PCR and high-throughput sequencing, and the main soil physical and chemical factors driving the change in soil fungal communities were explored. The results showed that, compared with the no topdressing nitrogen and low topdressing nitrogen rate treatments, high topdressing nitrogen rate treatments increased soil total nitrogen and mineral nitrogen and decreased soil pH, total phosphorus, available phosphorus, and available potassium. Compared with the no topdressing nitrogen treatments, the 37.5-150 kg·hm-2 topdressing nitrogen treatments significantly increased soil fungal community abundance (P<0.05), whereas there was no significant difference among different topdressing nitrogen treatments (P>0.05). The Heip index and Shannon index of soil fungal communities decreased gradually with the increase in topdressing nitrogen rate, and the Sobs index, Heip index, and Shannon index of soil fungal communities in the treatment with 150 kg·hm-2 topdressing nitrogen were significantly lower than those of 0-75 kg·hm-2 topdressing nitrogen treatments (P<0.05). Principal component analysis and similarity analysis showed that there were significant differences in soil fungal community structure under different topdressing nitrogen rate treatments (P<0.05). With the increase in topdressing nitrogen rate, the number of network edges and average number of neighbors of soil fungal ecological network increased first and then decreased, and the network complexity of 37.5 kg·hm-2 topdressing nitrogen treatments was the highest. Compared with 0-75 kg·hm-2 topdressing nitrogen treatments, 112.5 kg·hm-2 and 150 kg·hm-2 topdressing nitrogen treatments increased the characteristic path length of the soil fungal ecological network, whereas it decreased the network density. With the increase in topdressing nitrogen rate, the relative abundance of soil saprotrophs gradually increased, and the pathotroph-saprotroph-symbiotroph relative abundance gradually decreased. Redundancy analysis showed that soil pH, total phosphorus, mineral nitrogen, available phosphorus, and available potassium were the main soil physicochemical factors affecting the soil fungal community structure in the wheat field under different topdressing nitrogen rate treatments. In conclusion, on the basis of straw retention and basal nitrogen, topdressing nitrogen at the wheat jointing stage could change the diversity, structure, and species composition of the soil fungal community, in turn affecting the soil fungal ecological network and function, and high topdressing nitrogen rates could reduce soil fungal community diversity, ecological network complexity, and network density.

Impact of the eHealth literacy, knowledge and attitudes on COVID-19 prevention behavior among residents in the second year of the COVID-19 pandemic: A cross-sectional study in Anhui Province, China.

Objective: The pandemic of COVID-19 continues to challenge people's health worldwide. In the second year of the pandemic, adherence to COVID-19 preventive behavior is key to continuing efforts to overcome the epidemic. This study aims to assess the COVID-19-related knowledge, attitude, and prevention behavior (KAP) and electronic health literacy (eHealth literacy) among Anhui residents in China. Methods: From January 30 to March 27, 2021, the cross-sectional study was performed among Anhui residents in China, including 16 cities. An online survey was adopted to assess KAP regarding COVID-19, and eHealth, involving a total of 2,122 citizens. Following informed consent, residents were recruited by convenience sampling. Frequencies and proportions were calculated. Additionally, Mann-Whitney U tests were used to analyze the variables. Independent predictors of preventive behavior of COVID-19 were ascertained using a multivariable logistic regression model. Result: Residents demonstrated good knowledge, positive attitudes, acceptable practices, and good eHealth literacy. Online news and WeChat are the main health information resources. Citizens who had good knowledge, a positive attitude, good eHealth, and did not participate in the online lectures or training COVID-19 were more likely to take preventive measures. Those with poor health, who were male, did not have family members working in health care facilities, and did not work in a face-to-face environment were less likely to take precautions. Compared with a master's degree and above, participants with middle school education level and below took preventive behavior sometimes. Residents who browse the COVID-19 webpage <15 min weekly seldom took preventive actions. Conclusion: The study showed that in the second year of the COVID-19 pandemic, Chinese residents had adequate knowledge of COVID-19, positive attitudes, appropriate preventive practices, and basic eHealth literacy. To prevent the rebound of the COVID-19 epidemic, the government and health agencies should inform citizens concerning which information channels or websites to use and assist the underprivileged population who lacks basic infrastructure. In addition, increasing the level of knowledge and attitude, enhancing eHealth literacy and the Health Belief Model (HBM), and implementing the Health Code were seen as ways to reinforce adherence to preventive behavior. Targeting men, implementing public awareness campaigns, community engagement strategies, and health education programs are recommended.

[Soil bacterial communities of different crop rotations and yield of succeeding wheat.] / 不同轮作茬口土壤细菌群落及后茬小麦产量.

Evaluating ecological sustainability and crop productivity of different crop rotation patterns could provide theoretical support for adjusting and optimizing crop planting structure. We set seven treatments with different rotation crops and periods. We used real-time quantitative PCR to determine the abundance of soil bacterial community and 16S rRNA gene amplicon high-throughput sequencing technology to analyze diversity and taxa composition of soil bacterial community. Both soil available nutrients and succeeding wheat yield were measured. The results showed that, compared with the rotation with summer maize, the rotations with summer peanut or soybean in diffe-rent periods reduced soil organic carbon, mineral nitrogen, and available potassium, but significantly increased soil available phosphorus. The 16S rRNA gene copy numbers of soil bacteria in the treatments of rotations with summer peanut or soybean in different periods were significantly decreased, while community richness and diversity were increased. Different rotation crops significantly changed the structure and taxonomic composition of soil bacterial community. Compared with the rotation with summer maize, the rotations with summer soybean in different periods significantly increased the 1000-grain weight and grain yield of succeeding winter wheat. In conclusion, rotations with summer peanut or soybean in different periods could increase soil available phosphorus content and bacterial diversity, and significantly change soil bacterial community structure. In particular, rotation with summer soybean performed best in promoting yield formation of succeeding winter wheat.

Global airborne bacterial community-interactions with Earth's microbiomes and anthropogenic activities.

Airborne bacteria are an influential component of the Earth's microbiomes, but their community structure and biogeographic distribution patterns have yet to be understood. We analyzed the bacterial communities of 370 air particulate samples collected from 63 sites around the world and constructed an airborne bacterial reference catalog with more than 27 million nonredundant 16S ribosomal RNA (rRNA) gene sequences. We present their biogeographic pattern and decipher the interlacing of the microbiome co-occurrence network with surface environments of the Earth. While the total abundance of global airborne bacteria in the troposphere (1.72 × 1024 cells) is 1 to 3 orders of magnitude lower than that of other habitats, the number of bacterial taxa (i.e., richness) in the atmosphere (4.71 × 108 to 3.08 × 109) is comparable to that in the hydrosphere, and its maximum occurs in midlatitude regions, as is also observed in other ecosystems. The airborne bacterial community harbors a unique set of dominant taxa (24 species); however, its structure appears to be more easily perturbed, due to the more prominent role of stochastic processes in shaping community assembly. This is corroborated by the major contribution of surface microbiomes to airborne bacteria (averaging 46.3%), while atmospheric conditions such as meteorological factors and air quality also play a role. Particularly in urban areas, human impacts weaken the relative importance of plant sources of airborne bacteria and elevate the occurrence of potential pathogens from anthropogenic sources. These findings serve as a key reference for predicting planetary microbiome responses and the health impacts of inhalable microbiomes with future changes in the environment.

Insect Sf9 cells are suitable for functional expression of insect, but not vertebrate, striated muscle myosin.

Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.

[Effects of different peanut-cotton intercropping modes on physiological characteristics and yield of peanut in late growth stage]. / 不同花生棉花间作模式对花生生育后期生理特性及产量的影响.

Using peanut cultivar Huayu 25 and cotton cultivar Liaomian 19 as experimental material, we examined the effects of different intercropping patterns on physiological characteristics of peanut in later growth stage, yield and economic benefit, based on an experiment with five treatments, including intercropping modes of 4 rows peanut and 4 rows cotton (H4M4), 6 rows peanut and 4 rows cotton (H6M4), 4 rows peanut and 4 rows cotton (H4M2), sole peanut (DH) and sole cotton (DM). The results showed that intercropping mode increased the length of main stem and branches of peanut, but decreased green leaves number of main steam, leaf area index, and total dry matter accumulation. Among the intercropping modes, chlorophyll content, chlorophyll fluorescence parameters, root vigor, nitrate reductase activity under H6M4 and H4M2 were significantly higher than that under H4M4, as well as higher superoxide dismutase, peroxidase, catalase activity and decreased malondialdehyde content. Intercropping significantly reduced peanut and cotton yields, but enhanced the gross economic output value. The yield reduction of H6M4 was the lowest and the economic output was the highest among all the intercropping modes. In addition, the land equivalent ratio of H6M4 was greater than 1, indicating the obvious advantage of intercropping. Our results indicated that appropriate reduction of the ratio of cotton under the peanut-cotton intercropping systems could strengthen root vigor and increased nitrate reductase activity, promote nutrient absorption capacity, reduce senescence, and increase the economic output.

Corrigendum: Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

[This corrects the article DOI: 10.3389/fpls.2021.788963.].

[Effects of temperature on growth, survival, and reproduction of Conogethes punctiferalis feeding on corn kernels]. / 温度对取食玉米籽粒桃蛀螟生长发育、存活和生殖的影响.

In order to examine the effects of temperature on the growth, survival, and reproduction of Conogethes punctiferalis (Lepidoptera: Crambidae) feeding on corn kernels, the life table parameters of C. punctiferalis at 21, 24, 27, 30 ℃ were calculated based on the age-stage two-sex life table theory, and then population dynamics of C. punctiferalis was projected based on these parameters. The results showed that the life cycles of the pest could be finished at all the four temperature treatments. The developmental duration of each stage shortened with increasing temperature, and there were significant differences among treatments. The highest fecundity (116.7 eggs per female), preadult survival rate (84.7%), and proportion of female (0.46) were observed at 24 ℃. The intrinsic rates of increase at 24, 27, 30 ℃ were 0.1059, 0.1101, and 0.1045 d-1, respectively. The finite rates of increase were 1.1117, 1.1164, and 1.1102 d-1, respectively. There was no significant difference among the three treatments, but the r and λ values were significantly higher than those at 21 ℃. The net reproductive rates (R0) at 21, 24, 27, 30 ℃ were 17.3, 53.7, 36.9, and 19.8, respectively. R0 at 24 ℃ was the highest. It suggested that survival rates, fecundities and female proportions of C. punctiferalis population were high at 24-27 ℃, which was the suitable temperature range for its growth, survival, and reproduction.