Nonpermissive Research Articles

Peripheral B Lymphocyte Serves as a Reservoir for the Persistently Covert Infection of Mandarin Fish Siniperca chuatsi Ranavirus

Mandarin fish ranavirus (MRV) is a distinctive member among the genus Ranavirus of the family Iridoviridae. The persistently covert infection of MRV was previously observed in a natural outbreak of MRV, but the underlying mechanism remains unclear. Here, we show that mandarin fish peripheral B lymphocytes are implemented as viral reservoirs to maintain the persistent infection. When mandarin fish were infected with a sublethal dosage of MRV under a nonpermissive temperature (19 °C) and a permissive temperature (26 °C), all of the fish in the 19 °C group survived and entered the persistent phase of infection, characterized by a very low viral load in white blood cells, whereas some of the fish died of MRV infection in the 26 °C group, and the survival fish then initiated a persistent infection status. Raising the temperature, vaccination and dexamethasone treatment can reactivate the quiescent MRV to replicate and result in partial mortality. The viral reservoir investigation showed that IgM+-labeled B lymphocytes, but not CD3Δ+-labeled T lymphocytes and MRC-1+-labeled macrophages, are target cells for the persistent infection of MRV. Moreover, the reactivation of the quiescent MRV was confirmed through a non-TLR5 signal pathway manner. Collectively, we demonstrate the presence of the B cell-dependent persistent infection of ranavirus, and provide a new clue for better understanding the complex infection mechanism of vertebrate iridovirus.

Genetic and molecular studies of fitC4 and its suppressors fitA76* and fit95 in Escherichia coli.

The fitA/pheS and fitB/pheT genes were previously proposed to function as transcription factors. The originally identified temperature sensitive (Ts) transcription-defective fitA76 mutant was shown to harbour a second mutation, fit95 (pheT) in addition to pheS5 (pheS; G293 → A293 transition). A new fit mutation namely, fitC4 (fitC locus) was identified in a Ts+ derivative of fitA76, namely JV4. Genetic mapping revealed that fitC4 mutation could be an extragenic suppressor, as it mapped at 39.01min while fitAB loci mapped at 38.7min on E. coli chromosome. Upon separation from JV4, fitC4 (Ts) failed to suppress the original fitA76 mutant (pheS5-fit95). Instead,JV4 harboured a modified form of fitA76 designated fitA76* (pheS4-fit95) with G293 → C293 transversion occurred at the same site of pheS5. The fitC4 and fitA76* mutations were genetically separated and reassembled to show that they both suppress each other as like in JV4. The separated fitC4 and fitA76* mutations behave like original fitA76 mutant in terms of transcription abnormality. This study focusses on further characterization of fitC4 and its accompanied mutations. The mutations fitC4, fitA76* and fitC4-fitA76* (reconstructed) are mobilized into new genetic backgrounds where the viability of these strains varied significantly. Growth and transcription abnormalities of fitC4 and fitA76* at 42°C are restored in the reconstructed strain (fitC4-fitA76*), but not the β-galactosidase induction. As direct evidence, fit95 is shown to suppress fitC4 in a rpoB201 mutation background where fit95 phenotype is completely stabilized. The implications of these results with reference to transcription control by Fit factors in vivo are discussed.

Dosage suppressors of gpn2ts mutants and functional insights into the role of Gpn2 in budding yeast.

Gpn2 is a highly conserved protein essential for the assembly of RNA polymerase II (RNAPII) in eukaryotic cells. Mutations in Gpn2, specifically Phe105Tyr and Leu164Pro, confer temperature sensitivity and significantly impair RNAPII assembly. Despite its crucial role, the complete range of Gpn2 functions remains to be elucidated. To further explore these functions, we conducted large-scale multicopy suppressor screening in budding yeast, aiming to identify genes whose overexpression could mitigate the growth defects of a temperature-sensitive gpn2 mutant (gpn2ts) at restrictive temperatures. We screened over 30,000 colonies harboring plasmids from a multicopy genetic library and identified 31 genes that rescued the growth defects of gpn2ts to various extents. Notably, we found that PAB1, CDC5, and RGS2 reduced the drug sensitivity of gpn2ts mutants. These findings lay a theoretical foundation for future studies on the function of Gpn2 in RNAPII assembly.

RNA Decay Assay: 5-Ethynyl-Uridine Labeling and Chasing.

Eukaryotic RNA synthesis and degradation are intricately regulated, impacting on gene expression dynamics. RNA stability varies in individual transcripts and is modulated by trans-acting factors such as microRNAs, long noncoding RNAs, and RNA-binding proteins, which determine protein output and subsequent cellular processes. To measure RNA decay rate, accurate and reliable methodologies are essential in the field of RNA biology. Transcription inhibition and metabolic labeling enable comprehensive investigations on RNA decay, offering critical insights into dynamic regulation of RNA decay. Transcription shut-off has been employed widely by using various approaches, such as treatment with chemical inhibitors or generation of temperature-sensitive mutants of RNA polymerases. However, it has limitations, providing a static view and lacking real-time dynamics as well as precise measurement of decay rate. Metabolic labeling, a method of incorporating modified nucleotides into RNA transcripts, complements shut-off approaches, allowing selective monitoring of newly synthesized RNA and tracing decay intermediates. The purpose of the protocol described in this chapter is to assess the kinetics and statics of newly synthesized RNA and its decay by 5-ethynyl uridine labeling.

Potent Antimicrobial Azoles: Synthesis, In Vitro and In Silico Study.

Background/Objectives: The increase in fungal infections, both systemic and invasive, is a major source of morbidity and mortality, particularly among immunocompromised people such as cancer patients and organ transplant recipients. Because of their strong therapeutic activity and excellent safety profiles, azole antifungals are currently the most extensively used systemic antifungal drugs. Antibacterial properties of various topical antifungals, such as oxiconazole, which features oxime ether functionality, were discovered, indicating an exciting prospect in antimicrobial chemotherapy. Methods: In this study, eleven new oxime ether derivatives with the azole scaffold (5a-k) were synthesized and tested for their antimicrobial effects using the microdilution method to obtain broad-spectrum hits. Results: Although the title compounds showed limited efficacy against Candida species, they proved highly effective against dermatophytes. Compounds 5c and 5h were the most potent derivatives against Trichophyton mentagrophytes and Arthroderma quadrifidum, with minimum inhibitory concentration (MIC) values lower than those of the reference drug, griseofulvin. The MIC of 5c and 5h were 0.491 μg/mL and 0.619 μg/mL against T. mentagrophytes (MIC of griseofulvin: 2.52 μg/mL). The compounds were also tested against Gram-positive and Gram-negative bacteria. Briefly, 5c was the most active against Escherichia coli and Bacillus subtilis, with MIC values much better than that of ciprofloxacin (MIC of 5c = 1.56 μg/mL and 1.23 μg/mL, MIC of ciprofloxacin = 31.49 and 125.99 μg/mL, respectively). Molecular docking suggested a good fit in the active site of fungal lanosterol 14α-demethylase (CYP51) and bacterial FtsZ (Filamenting temperature-sensitive mutant Z) protein. Conclusions: As a result, the title compounds emerged as promising entities with broad antifungal and antibacterial effects, highlighting the utility of oxime ether function in the azole scaffold.

Characterization of the function of Adenovirus L4 gene products and their impact on AAV vector production

Efficient manufacturing of recombinant adenovirus-associated viral vectors is critical to the successful development of genomic medicines. We attempted to optimize AAV vector production in a producer cell line platform. In this system, helper functions required for AAV replication and production are provided via infection with a replication-competent wild-type Adenovirus. To evaluate strategies for reduction of replication and packaging of adenovirus as well as to understand the interplay of recombinant AAV and the helper virus during AAV vector production, wild-type adenovirus was compared to a mutant (Ad5ts149) containing a temperature-sensitive mutation in the DNA polymerase gene. Infection of a producer cell line with Ad5ts149 at the restrictive temperature reduced recombinant AAV titer and altered the pattern of AAV protein expression. Further investigation revealed that the adenoviral late L4-22K/33K gene products regulated both AAV rep/cap gene transcription and splicing of the rep/cap transcripts. Furthermore, the L4-33K gene products were found to impact AAV production in both the producer cell line and transient transfection platforms. Optimization of Adenovirus L4-22K/33K expression to facilitate efficient expression and splicing of AAV rep/cap transcripts therefore represents a unique opportunity to optimize AAV vector production.

A strategy for identification and characterization of genic mutations using a temperature-sensitive chlorotic soybean mutant as an example.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "y24" phenotype results in smaller stature, weaker stems, and a smaller root system. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells, and RNA sequencing data indicate that it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice ortholog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean y24 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild-type Glyma.07G102300 in the knockout mutant of the Arabidopsis ortholog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the y24 phenotype. The variant analysis strategy used to identify the genes underlying this phenotype provides a template for the study of other soybean mutants.

Characterization of Ksg1 protein kinase-dependent phosphoproteome in the fission yeast S. pombe

Ksg1 is an essential protein kinase of the fission yeast S. pombe that belongs to the AGC kinase family and is homologous to the mammalian PDPK1 kinase. Previous studies have shown that Ksg1 functions in the nutrient-sensing TOR signaling pathway and is involved in the phosphorylation and activation of other AGC kinases, thereby affecting various downstream targets related to metabolism, cell division, stress response, and gene expression. To date, the molecular function of Ksg1 has been analyzed using its temperature sensitive mutants or mutants expressing its truncated isoforms, which are not always suitable for functional studies of Ksg1 and the identification of its targets. To overcome these limitations, we employed a chemical genetic strategy and used a conditional ksg1as mutant sensitive to an ATP analog. Combining this mutant with quantitative phosphoproteomics analysis, we identified 1986 phosphosites that were differentially phosphorylated when Ksg1as kinase was inhibited by an ATP analog. We found that proteins whose phosphorylation was dysregulated after inhibition of Ksg1as kinase were mainly represented by those involved in the regulation of cytokinesis, contractile ring contraction, cell division, septation initiation signaling cascade, intracellular protein kinase cascade, barrier septum formation, protein phosphorylation, intracellular signal transduction, cytoskeleton organization, cellular response to stimulus, or in RNA, ncRNA and rRNA processing. Importantly, proteins with significantly down-regulated phosphorylation were specifically enriched for R-X-X-S and R-X-R-X-X-S motifs, which are typical consensus substrate sequences for phosphorylation by the AGC family of kinases. The results of this study provide a basis for further analysis of the role of the Ksg1 kinase and its targets in S. pombe and may also be useful for studying Ksg1 orthologs in other organisms.

The Saccharomyces cerevisiae cell lysis mutant cly8 is a temperature-sensitive allele of ESP1.

The Saccharomyces cerevisiae mutants designated cly ( c ell ly sis) cause cell lysis at elevated temperatures. The cly8 mutation, previously localized to an 80 kilobase region between GCD2 and SPT6 on the right arm of chromosome VII, has been used for mutation mapping and in recombination assays, but its genetic identity has remained unknown. Whole genome sequencing of cly8 mutant and CLY8 wild-type strains revealed four missense mutations specific to the cly8 mutant strain in the GCD2 - SPT6 interval, three of these missense mutations affected essential genes. The esp1-G543E mutation, but not the two other missense mutations, recapitulated the temperature-sensitive growth, the cell lysis phenotype, and recessive nature of the cly8 mutation. The ESP1 gene encodes S. cerevisiae separase and is required for normal chromosome segregation during mitosis. Shifting cells containing the esp1-G543E mutation to the non-permissive temperature caused chromosome missegregation based on the analysis of DNA content by flow cytometry analysis. Taken together, these results indicate that the temperature-sensitive cly8 mutation is an allele of the essential ESP1 gene.

Identification of a novel alternate promoter element in the pheST operon of Escherichia coli.

Earlier work in this laboratory revealed that fitA was same as pheS as a recombinant clone, pSRJ5R1 harboring pheS+ gene complemented transcription-defective fitA76 Ts (temperature sensitive) mutant. However, this clone lacked the native promoter (NP)of pheST operon. A putative - 10 promoter like element was suggested to act as promoter in this clone. This work investigated the veracity of this putative promoter as well as its downstream regulatory region towards driving the pheS expression. Plasmid clones with promoter-mutations or -deletions were constructed by PCR-based cloning and their ability to complement fitA76 Ts mutant strains was checked. Chromosomal mutations were transferred into various genetic backgrounds via P1-transductions. Relative viability assays were performed to check the extent of complementation. Clones harboring point mutations (PM-pheS) or deletion (PD1-pheS) of - 10 region of the putative promoter did not abolish complementation of the fitA76 Ts phenotype. Subsequently, a novel alternate promoter (AP)was discovered by downstream deletion clone (PD2-pheS) which failed to complement. Keeping PD1-pheS intact but mutating initiation codon of pheS (ATG→TTG) failed to complement. Complementation ability of novel alternate promoter is poor in HfrC strain background unlike native promoter which complements well independent of strain background. A novel alternate-promoter of pheST operon was identified by mutational/deletional analyses and earlier reported putative - 10 promoter was shown to be dispensable. Alternate promoter is relA dependent.

Computational and in vitro analyses of the antibacterial effect of the ethanolic extract of Pluchea indica L. leaves.

The most common gram-negative, Escherichia coli, and gram-positive bacteria, Bacillus spp., have evolved different mechanisms that have caused the emergence of multi-drug resistance. As a result, drugs that block the bacterial growth cycle are needed. Here, in silico and in vitro studies were performed to assess compounds in the Pluchea indica leaf extract, a medicinal plant, that can inhibit bacterial proteins. Briefly, P. indica leaves were extracted using ethanol. The crude extract was then subjected to gas chromatography-mass spectrometry for metabolite screening. Molecular docking simulations with rhomboid protease (Rpro) (Protein data bank ID number: 3ZMI from E. coli and filamenting temperature-sensitive mutant Z (FtsZ) protein data bank ID number: 2VAM from Bacillus subtilis were performed. Moreover, the well diffusion method was used to confirm the antibacterial activity of P. indica leaf extract. A total of 10 compounds were identified in the P. indica extract and used for computational analysis. Based on drug-likeness prediction, P. indica compounds may be drug-like molecules. Binding affinity tests indicated that 10,10-Dimethyl-2,6-dimethylenebicyclo(7.2.0)undecan-5.β.-ol and 11,11-Dimethyl-4,8-dimethylenebicyclo(7.2.0)undecan-3-ol had the most negative values. Accordingly, these compounds may be potential ligands that bind to bacterial proteins. The root mean square fluctuation values was <2 Å, indicating stable fluctuation binding for the ligand-protein complex. According to in vitro antibacterial assays, a high concentration (50%) of the P. indica extract markedly inhibited E. coli and B. subtilis, with inhibitory zone diameters of 31.86±1.63 and 21.09±0.09 mm, respectively. Overall, the compounds in the P. indica leaf extract were identified as functional inhibitors of E. coli and B. subtilis proteins via in silico analysis. This may facilitate development of antibacterial agents.

Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.

Genetic and molecular characterization of fit95 mutation of Escherichia coli: evidence that fit95 is an allele of pheT.

The originally identified transcription-defective fitA76 temperature-sensitive (Ts) mutation defined an allele of pheS. Both fitA/pheS and fitB/pheT were previously proposed to function as transcription factors. Sequencing pheS region of the fitA76 mutant revealed the same G293→A293 transition found in the translation-defective pheS5 mutant. It was subsequently found that fitA76 harbored a second mutation (fit95) in addition to pheS5 mutation. The fit95 was found to be Ts on -salt media but was found unstable. In this investigation, genetic, physiological and molecular characterization of the fit95 mutation was carried out. The fit95 was genetically re-separated from the pheS5 mutation present in the fitA76 mutant and the same was subsequently mobilized into multiple genetic backgrounds to study its phenotypic modulations by altering the medium and supplements. Based on genetic studies, the unstable -salt Ts phenotype of the fit95 could be stabilized by the presence of rpoB201 mutation. Addition of glucose enhanced Ts phenotype in the presence of rpoB201 mutation, but citrate completely alleviated the Ts phenotype. Further, by series of complementation analyses and molecular cloning, the identity of fit95 was revealed as pheT gene which is part of pheST operon.

Whole-Genome and Poly(A)+Transcriptome Analysis of the Drosophila Mutant agnts3 with Cognitive Dysfunctions.

The temperature-sensitive Drosophila mutant agnts3 exhibits the restoration of learning defects both after heat shock (HS) and under hypomagnetic conditions (HMC). Previously, agnts3 was shown to have an increased level of LIM kinase 1 (LIMK1). However, its limk1 sequence did not significantly differ from that of the wild-type strain Canton-S (CS). Here, we performed whole-genome and poly(A)-enriched transcriptome sequencing of CS and agnts3 males normally, after HMC, and after HS. Several high-effect agnts3-specific mutations were identified, including MED23 (regulation of HS-dependent transcription) and Spn42De, the human orthologs of which are associated with intellectual disorders. Pronounced interstrain differences between the transcription profiles were revealed. Mainly, they included the genes of defense and stress response, long non-coding RNAs, and transposons. After HS, the differences between the transcriptomes became less pronounced. In agnts3, prosalpha1 was the only gene whose expression changed after both HS and HMC. The normal downregulation of prosalpha1 and Spn42De in agnts3 was confirmed by RT-PCR. Analysis of limk1 expression did not reveal any interstrain differences or changes after stress. Thus, behavioral differences between CS and agnts3 both under normal and stressed conditions are not due to differences in limk1 transcription. Instead, MED23, Spn42De, and prosalpha1 are more likely to contribute to the agnts3 phenotype.

Single-strand DNA-binding protein suppresses illegitimate recombination in Escherichia coli, acting in synergy with RecQ helicase

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression.

Elevated temperature affects the expression of signaling molecules in quail testes meiosis I prophase, but spermatogenesis remains normal

Spermatogenesis in eukaryotes is a process that occurs within a very narrow temperature threshold, typically not exceeding 36 °C. SPO11 was isolated from the temperature-sensitive mutant receptor of Saccharomyces cerevisiae and is thought to be the only protein that functions during meiosis. This suggested that SPO11 may be the key protein that influenced the temperature of spermatogenesis not exceeding 36 °C. Elevated temperatures typically damage the spermatogenic cells. Birds have a core body temperature of 41–42 °C, and their testis are located inside their bodies, providing an alternative perspective to investigate the potential impact of temperature threshold on spermatogenesis. The objective of this study was to ascertain whether elevated ambient temperatures affect spermatogenesis in birds and whether SPO11 is the key gene affecting the temperature threshold for spermatogenesis. STRA8, SCP3, SPO11, γ-H2AX, and RAD51 were all crucial components in the process of meiotic initiation, synapsis, DNA double-strand break (DSB) induction, homologous chromosome crossover recombination, and repair of DSB. In this study, 39-day-old Japanese quail were subjected to heat stress (HS) at 38 °C for 8 h per day for 3 (3d HS) and 13 (13d HS) consecutive days and analyzed the expression of meiotic signaling molecules (STRA8, SCP3, SPO11, γ-H2AX, and RAD51) using molecular biology techniques, including Immunohistochemistry (IHC), Western Blot (WB), and Real-time Quantitative Polymerase Chain Reaction (qRT-PCR). We found that spermatogenesis was normal in both groups exposed to HS. Meiotic signaling molecules were expressed normally in the 3d HS group. All detected signaling molecules were normally expressed in the 13d HS group, except for SPO11, which showed a significant increase in expression, indicating that SPO11 was temperature-sensitive. We examined the localized expression of each meiotic signaling molecule in quail testis, explored the temperature sensitivity of SPO11, and determined that quail testis can undergo normal spermatogenesis at ambient temperatures exceeding 36 °C. This study concluded that SPO11 is not the key protein influencing spermatogenesis in birds. These findings enhance our understanding of avian spermatogenesis.

Synergistic rescue of temperature-sensitive p53 mutants by hypothermia and arsenic trioxide.

The p53 tumor suppressor is inactivated by mutations in about 50% of tumors. Rescuing the transcriptional function of mutant p53 has potential therapeutic benefits. Approximately 15% of p53 mutants are temperature sensitive (TS) and regain maximal activity at 32°C. Proof of concept study showed that induction of 32°C hypothermia in mice restored TS mutant p53 activity and inhibited tumor growth. However, 32°C is the lower limit of therapeutic hypothermia procedures for humans. Higher temperatures are preferable but result in suboptimal TS p53 activation. Recently, arsenic trioxide (ATO) was shown to rescue the conformation of p53 structural mutants by stabilizing the DNA binding domain. We examined the responses of 17 frequently observed p53 TS mutants to functional rescue by temperature shift and ATO. The results showed that ATO only rescued mild p53 TS mutants with high basal activity at 37°C. Mild TS mutants showed a common feature of regaining significant activity at the semi-permissive temperature of 35°C and could be further stimulated by ATO at 35°C. TS p53 rescue by ATO was antagonized by the cellular redox mechanism and was rapidly reversible. Inhibition of glutathione (GSH) biosynthesis enhanced ATO rescue efficiency and sustained p53 activity after ATO washout. The results suggest that mild TS p53 mutants are uniquely responsive to functional rescue by ATO due to small thermostability deficits and inherent potential to regain active conformation. Combining mild hypothermia and ATO may provide an effective and safe procedure for targeting tumors with p53 TS mutations.

Conditional nmy-1 and nmy-2 alleles establish that nonmuscle myosins are required for late Caenorhabditis elegans embryonic elongation.

The elongation of Caenorhabditis elegans embryos allows examination of mechanical interactions between adjacent tissues. Muscle contractions during late elongation induce the remodeling of epidermal circumferential actin filaments through mechanotransduction. Force inputs from the muscles deform circumferential epidermal actin filament, which causes them to be severed, eventually reformed, and shortened. This squeezing force drives embryonic elongation. We investigated the possible role of the nonmuscle myosins NMY-1 and NMY-2 in this process using nmy-1 and nmy-2 thermosensitive alleles. Our findings show these myosins act redundantly in late elongation, since double nmy-2(ts); nmy-1(ts) mutants immediately stop elongation when raised to 25°C. Their inactivation does not reduce muscle activity, as measured from epidermis deformation, suggesting that they are directly involved in the multistep process of epidermal remodeling. Furthermore, NMY-1 and NMY-2 inactivation is reversible when embryos are kept at the nonpermissive temperature for a few hours. However, after longer exposure to 25°C double mutant embryos fail to resume elongation, presumably because NMY-1 was seen to form protein aggregates. We propose that the two C. elegans nonmuscle myosin II act during actin remodeling either to bring severed ends or hold them.

GLUTAMYL-tRNA SYNTHETASE 1 deficiency confers thermosensitive male sterility in rice by affecting reactive oxygen species homeostasis.

Temperature is one of the key environmental factors influencing crop fertility and yield. Understanding how plants sense and respond to temperature changes is, therefore, crucial for improving agricultural production. In this study, we characterized a temperature-sensitive male sterile mutant in rice (Oryza sativa), glutamyl-tRNA synthetase 1-2 (ers1-2), that shows reduced fertility at high temperatures and restored fertility at low temperatures. Mutation of ERS1 resulted in severely delayed pollen development and meiotic progression at high temperatures, eventually leading to male sterility. Moreover, meiosis-specific events, including synapsis and crossover formation, were also delayed in ers1-2 compared with the wild type. However, these defects were all mitigated by growing ers1-2 at low temperatures. Transcriptome analysis and measurement of ascorbate, glutathione, and hydrogen peroxide (H2O2) contents revealed that the delayed meiotic progression and male sterility in ers1-2 were strongly associated with changes in reactive oxygen species (ROS) homeostasis. At high temperatures, ers1-2 exhibited decreased accumulation of ROS scavengers and overaccumulation of ROS. In contrast, at low temperatures, the antioxidant system of ROS was more active, and ROS contents were lower. These data suggest that ROS homeostasis in ers1-2 is disrupted at high temperatures but restored at low temperatures. We speculate that ERS1 dysfunction leads to changes in ROS homeostasis under different conditions, resulting in delayed or rescued meiotic progression and thermosensitive male fertility. ers1-2 may hold great potential as a thermosensitive material for crop heterosis breeding.

The MKK3 MAPK cascade integrates temperature and after-ripening signals to modulate seed germination

The timing of seed germination is controlled by the combination of internal dormancy and external factors. Temperature is a major environmental factor for seed germination. The permissive temperature range for germination is narrow in dormant seeds and expands during after-ripening (AR) (dormancy release). Quantitative trait loci analyses of preharvest sprouting in cereals have revealed that MKK3, a mitogen-activated protein kinase (MAPK) cascade protein, is a negative regulator of grain dormancy. Here, we show that the MAPKKK19/20-MKK3-MPK1/2/7/14 cascade modulates the germination temperature range in Arabidopsis seeds by elevating the germinability of the seeds at sub- and supraoptimal temperatures. The expression of MAPKKK19 and MAPKKK20 is induced around optimal temperature for germination in after-ripened seeds but repressed in dormant seeds. MPK7 activation depends on the expression levels of MAPKKK19/20, with expression occurring under conditions permissive for germination. Abscisic acid (ABA) and gibberellin (GA) are two major phytohormones which are involved in germination control. Activation of the MKK3 cascade represses ABA biosynthesis enzyme gene expression and induces expression of ABA catabolic enzyme and GA biosynthesis enzyme genes, resulting in expansion of the germinable temperature range. Our data demonstrate that the MKK3 cascade integrates temperature and AR signals to phytohormone metabolism and seed germination.