Structure-based characterization and compound identification of the wild-type THF class-II riboswitch.

Riboswitches are conserved regulatory RNA elements participating in various metabolic pathways. Recently, a novel RNA motif known as the folE RNA motif was discovered upstream of folE genes. It specifically senses tetrahydrofolate (THF) and is therefore termed THF-II riboswitch. To unravel the ligand recognition mechanism of this newly discovered riboswitch and decipher the underlying principles governing its tertiary folding, we determined both the free-form and bound-form THF-II riboswitch in the wild-type sequences. Combining structural information and isothermal titration calorimetry (ITC) binding assays on structure-based mutants, we successfully elucidated the significant long-range interactions governing the function of THF-II riboswitch and identified additional compounds, including alternative natural metabolites and potential lead compounds for drug discovery, that interact with THF-II riboswitch. Our structural research on the ligand recognition mechanism of the THF-II riboswitch not only paves the way for identification of compounds targeting riboswitches, but also facilitates the exploration of THF analogs in diverse biological contexts or for therapeutic applications.

Bacillus cereus NJ01 induces SA- and ABA-mediated immunity against bacterial pathogens through the EDS1-WRKY18 module.

Emerging evidence suggests a beneficial role of rhizobacteria in ameliorating plant disease resistance in an environment-friendly way. In this study, we characterize a rhizobacterium, Bacillus cereus NJ01, that enhances bacterial pathogen resistance in rice and Arabidopsis. Transcriptome analyses show that root inoculation of NJ01 induces the expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes in Arabidopsis leaves. Genetic evidence showed that EDS1, PAD4, and WRKY18 are required for B. cereus NJ01-induced bacterial resistance. An EDS1-PAD4 complex interacts with WRKY18 and enhances its DNA binding activity. WRKY18 directly binds to the W box in the promoter region of the SA biosynthesis gene ICS1 and ABA biosynthesis genes NCED3 and NCED5 and contributes to the NJ01-induced bacterial resistance. Taken together, our findings indicate a role of the EDS1/PAD4-WRKY18 complex in rhizobacteria-induced disease resistance.

Phytochrome B inhibits the activity of phytochrome-interacting factor 7 involving phase separation.

Shade-intolerant plants sense changes in the light environment and trigger shade-avoidance syndrome in the presence of neighboring vegetation. Phytochrome-interacting factor 7 (PIF7) is an essential regulator that integrates shade signals into plant transcriptional networks. While the regulation of PIF7 under shade conditions has been well studied, the mechanism that represses PIF7 activity under white light remains ambiguous. Here, we report that PIF7 forms nuclear puncta containing phase-separated liquid-like condensates. Phytochrome B (phyB) then binds to dephosphorylated PIF7 and promotes its condensed phase of PIF7 under white light. The phyB-PIF7 condensate subsequently inhibits the DNA-binding activity of PIF7. However, shade inactivation of phyB causes the dissociation of phyB-PIF7 condensates and allows unbound PIF7 to promote the transcription of shade-induced genes. This reversible transcriptional condensation via phase separation provides sessile organisms with the flexibility of gene control to adapt to their surrounding environment.

Identification of Puccinia striiformis races from the spring wheat crop in Xinjiang, China.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a foliar disease that affects both winter and spring wheat crops in Xinjiang, China, which is linked to Central Asia. Race identification of Pst from spring wheat in Xinjiang was not done before. In this study, a total of 216 isolates were recovered from stripe rust samples of spring wheat in the region in 2021 and multiplied using the susceptible cultivar Mingxian 169. These isolates were tested on the Chinese set of 19 wheat differential lines for identifying Pst races. A total of 46 races were identified. Races Suwon-11-1, Suwon11-12, and CYR32 had high frequencies in the spring wheat region. The frequencies of virulence factors on differentials "Fulhard" and "Early Premium" were high (>95%), whereas the virulence factor to differential "Triticum spelta var. Album" (Yr5) was not detected, while virulence to other differentials showed variable frequency within different counties. The predominant races in winter wheat in the same season were also detected from spring wheat cultivars, indicating Pst spreading from winter wheat to spring wheat crops. Deploying resistance genes in spring and winter wheat cultivars is critical for control stripe rust.

Direct, ultrafast, and sensitive detection of environmental pathogenic microorganisms based on a graphene biosensor.

Pathogenic microorganisms in the environment pose a serious threat to global human health. This study developed a reduced graphene oxide (rGO)-field effect transistor (FET) biosensor to realize the rapid and sensitive detection of pathogenic microorganisms. The rGO-FET sensors were prepared by in-situ thermal reduction method, and biorecognition elements were immobilized using a crosslinking agent to realize the surface functionalization of rGO. The rGO-FET biosensors can detect Escherichia coli O157:H7 as low as 1.4 CFU mL-1 within 46 s. The normalized current response was linearly correlated with E. coli concentration in the range of 1.4-1.4 × 107 CFU mL-1. The normalized current response of E. coli O157:H7 was about an order of magnitude higher than those of other microorganisms, indicating that the biosensor has good specificity. The current loss rates of the unmodified rGO-FET sensors and the biosensors modified with anti-E. coli O157:H7 after 30 days of storage at 4 °C were approximately 8% and 15%, respectively. Most importantly, the rGO-FET biosensors can directly detect real samples without pretreatment. Compared with other technologies, the rGO-FET biosensors can detect pathogenic microorganisms with a wider linear range in a shorter time, which is of great importance for the rapid warning and control of pathogenic microorganisms in the environment.

Halomonas flagellata sp. nov., a halophilic bacterium isolated from saline soil in Xinjiang.

A Gram-stain-negative, strictly aerobic, motile, slightly curved rod-shaped bacterium with multiple flagella, designated strain EGI 63088T, was isolated from a bulk soil of Kalidium foliatum, collected from Wujiaqu in Xinjiang Uighur Autonomous Region, PR China. The optimal growth temperature, salinity, and pH for strain EGI 63088T growth were 30 °C, 3% (w/v) NaCl and 8, respectively. Phylogenetic analysis using 16S rRNA gene sequences indicated that strain EGI 63088T showed the highest sequence similarities to Halomonas heilongjiangensis 9-2T (97.94%), H. lysinitropha 3(2)T (97.51%), and H. daqiaonensis CGMCC 1.9150T (97.08%). The average nucleotide identity and digital DNA-DNA hybridization values between the strain EGI 63088T and H. heilongjiangensis 9-2T were 89.03 and 41.10%, respectively. The DNA G + C content of the genome for strain EGI 63088T was 66.3 mol%. The most prevalent antibiotic resistance and virulence-related genes in Halomonas genomes were Streptomyces cinnamoneu EF-Tu mutant, pilT, and cheY, respectively. The predominant fatty acids of strain EGI 63088T were summed feature 8 (C18: 1 ω6c and/or C18: 1 ω7c), summed feature 3 (C16: 1 ω6c and/or C16: 1 ω7c), and C16: 0; its major respiratory quinone was ubiquinone-9 (Q-9), and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. According to the above results, strain EGI 63088T is considered a novel species of the genus Halomonas, for which the name Halomonas flagellata sp. nov. is proposed. The type strain is EGI 63088T (= KCTC 92047T = CGMCC 1.19133T).

The mechanism of UP1 binding and unfolding of human telomeric DNA G-quadruplex.

The human telomere contains multiple copies of the DNA sequence d(TTAGGG) which can fold into higher order intramolecular G-quadruplexes and regulate the maintenance of telomere length and chromosomal integrity. The nucleic acid binding protein heteronuclear ribonucleoprotein A1 (hnRNP A1) and its N-terminus proteolytic product UP1 have been shown to efficiently bind and unfold telomeric DNA G-quadruplex. However, the understanding of the molecular mechanism of the UP1 binding and unfolding telomeric G-quadruplexes is still limited. Here, we performed biochemical and biophysical characterizations of UP1 binding and unfolding of human telomeric DNA G-quadruplex d[AGGG(TTAGGG)3], and in combination of systematic site-direct mutagenesis of two tandem RNA recognition motifs (RRMs) in UP1, revealed that RRM1 is responsible for initial binding and unfolding, whereas RRM2 assists RRM1 to complete the unfolding of G-quadruplex. Isothermal titration calorimetry (ITC) and circular dichroism (CD) studies of the interactions between UP1 and DNA G-quadruplex variants indicate that the "TAG" binding motif in Loop2 of telomeric G-quadruplex is critical for UP1 recognition and G-quadruplex unfolding initiation. Together we depict a model for molecular mechanism of hnRNP A1 (UP1) binding and unfolding of the human telomeric DNA G-quadruplex.

Structures and implications of the C962R protein of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

MYB44 regulates PTI by promoting the expression of EIN2 and MPK3/6 in Arabidopsis.

The plant signaling pathway that regulates pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) involves mitogen-activated protein kinase (MAPK) cascades that comprise sequential activation of several protein kinases and the ensuing phosphorylation of MAPKs, which activate transcription factors (TFs) to promote downstream defense responses. To identify plant TFs that regulate MAPKs, we investigated TF-defective mutants of Arabidopsis thaliana and identified MYB44 as an essential constituent of the PTI pathway. MYB44 confers resistance against the bacterial pathogen Pseudomonas syringae by cooperating with MPK3 and MPK6. Under PAMP treatment, MYB44 binds to the promoters of MPK3 and MPK6 to activate their expression, leading to phosphorylation of MPK3 and MPK6 proteins. In turn, phosphorylated MPK3 and MPK6 phosphorylate MYB44 in a functionally redundant manner, thus enabling MYB44 to activate MPK3 and MPK6 expression and further activate downstream defense responses. Activation of defense responses has also been attributed to activation of EIN2 transcription by MYB44, which has previously been shown to affect PAMP recognition and PTI development. AtMYB44 thus functions as an integral component of the PTI pathway by connecting transcriptional and posttranscriptional regulation of the MPK3/6 cascade.

Modular characterization of SARS-CoV-2 nucleocapsid protein domain functions in nucleocapsid-like assembly.

SARS-CoV-2 and its variants, with the Omicron subvariant XBB currently prevailing the global infections, continue to pose threats on public health worldwide. This non-segmented positive-stranded RNA virus encodes the multi-functional nucleocapsid protein (N) that plays key roles in viral infection, replication, genome packaging and budding. N protein consists of two structural domains, NTD and CTD, and three intrinsically disordered regions (IDRs) including the NIDR, the serine/arginine rich motif (SRIDR), and the CIDR. Previous studies revealed functions of N protein in RNA binding, oligomerization, and liquid-liquid phase separation (LLPS), however, characterizations of individual domains and their dissected contributions to N protein functions remain incomplete. In particular, little is known about N protein assembly that may play essential roles in viral replication and genome packing. Here, we present a modular approach to dissect functional roles of individual domains in SARS-CoV-2 N protein that reveals inhibitory or augmented modulations of protein assembly and LLPS in the presence of viral RNAs. Intriguingly, full-length N protein (NFL) assembles into ring-like architecture whereas the truncated SRIDR-CTD-CIDR (N182-419) promotes filamentous assembly. Moreover, LLPS droplets of NFL and N182-419 are significantly enlarged in the presence of viral RNAs, and we observed filamentous structures in the N182-419 droplets using correlative light and electron microscopy (CLEM), suggesting that the formation of LLPS droplets may promote higher-order assembly of N protein for transcription, replication and packaging. Together this study expands our understanding of the multiple functions of N protein in SARS-CoV-2.

Races CYR34 and Suwon11-1 of Puccinia striiformis f. sp. tritici Played an Important Role in Causing the Stripe Rust Epidemic in Winter Wheat in Yili, Xinjiang, China.

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici is a destructive disease. Its pathogen frequently adapts to newly invaded regions and overcomes resistance in wheat cultivars. This disease is especially important in China due to its favorable conditions for the stripe rust epidemic and the recombination population structure of pathogens. Xinjiang is a vast epidemic region in China, but very limited research on this disease has been performed in this region. In this study, we identified 25 races from 129 isolates collected from winter wheat fields from five different regions (Nileke, Xinyuan, Gongliu, Huocheng, and Qapqal) of Yili, Xinjiang, using the Chinese set of 19 differential wheat lines. All isolates were virulent on the differentials Fulhad and Early Premium, but no isolates were virulent on Yr5. Among the 25 races, Suwon11-1 was the most prevalent, followed by CYR34. Both races were found in four out of the five locations. It is important to continue monitoring stripe rust and its pathogen races in this region, as it forms a pathway between China and Central Asia. Collaborative research is essential for controlling stripe rust in this region, other regions in China, and neighboring countries.

Suppression of ETI by PTI priming to balance plant growth and defense through an MPK3/MPK6-WRKYs-PP2Cs module.

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) are required for host defense against pathogens. Although PTI and ETI are intimately connected, the underlying molecular mechanisms remain elusive. In this study, we demonstrate that flg22 priming attenuates Pseudomonas syringae pv. tomato DC3000 (Pst) AvrRpt2-induced hypersensitive cell death, resistance, and biomass reduction in Arabidopsis. Mitogen-activated protein kinases (MAPKs) are key signaling regulators of PTI and ETI. The absence of MPK3 and MPK6 significantly reduces pre-PTI-mediated ETI suppression (PES). We found that MPK3/MPK6 interact with and phosphorylate the downstream transcription factor WRKY18, which regulates the expression of AP2C1 and PP2C5, two genes encoding protein phosphatases. Furthermore, we observed that the PTI-suppressed ETI-triggered cell death, MAPK activation, and growth retardation are significantly attenuated in wrky18/40/60 and ap2c1 pp2c5 mutants. Taken together, our results suggest that the MPK3/MPK6-WRKYs-PP2Cs module underlies PES and is essential for the maintenance of plant fitness during ETI.

A novel high-affinity potassium transporter SeHKT1;2 from halophyte Salicornia europaea shows strong selectivity for Na+ rather than K.

High-affinity K+ transporters (HKTs) are known as transmembrane cation transporters and are involved in Na+ or Na+-K+ transport in plants. In this study, a novel HKT gene SeHKT1;2 was isolated and characterized from the halophyte, Salicornia europaea. It belongs to subfamily I of HKT and shows high homology with other halophyte HKT proteins. Functional characterization of SeHKT1;2 indicated that it contributes to facilitating Na+ uptake in Na+-sensitive yeast strains G19, however, cannot rescue the K+ uptake-defective phenotype of yeast strain CY162, demonstrating SeHKT1;2 selectively transports Na+ rather than K+. The addition of K+ along with NaCl relieved the Na+ sensitivity. Furthermore, heterologous expression of SeHKT1;2 in sos1 mutant of Arabidopsis thaliana increased salt sensitivity and could not rescued the transgenic plants. This study will provide valuable gene resources for improving the salt tolerance in other crops by genetic engineering.

Phase separation of SGS3 drives siRNA body formation and promotes endogenous gene silencing.

The generation of small interfering RNA (siRNA) involves many RNA processing components, including SUPPRESSOR OF GENE SILENCING 3 (SGS3), RNA-DEPENDENT RNA POLYMERASE 6 (RDR6), and DICER-LIKE proteins (DCLs). Nonetheless, how these components are coordinated to produce siRNAs is unclear. Here, we show that SGS3 forms condensates via phase separation in vivo and in vitro. SGS3 interacts with RDR6 and drives it to form siRNA bodies in cytoplasm, which is promoted by SGS3-targeted RNAs. Disrupting SGS3 phase separation abrogates siRNA body assembly and siRNA biogenesis, whereas coexpression of SGS3 and RDR6 induces siRNA body formation in tobacco and yeast cells. Dysfunction in translation and mRNA decay increases the number of siRNA bodies, whereas DCL2/4 mutations enhance their size. Purification of SGS3 condensates identifies numerous RNA-binding proteins and siRNA processing components. Together, our findings reveal that SGS3 phase separation-mediated formation of siRNA bodies is essential for siRNA production and gene silencing.

Identification of Aly1 and Aly2 as Modulators of Cytoplasmic pH in Saccharomyces cerevisiae.

The regulation of intracellular pH in yeast (Saccharomyces cerevisiae) cells is critical for cell function and viability. In yeast, protons (H+) can be excreted from the cell by plasma membrane ATPase PMA1 and pumped into vacuoles by vacuolar H+-ATPase. Because PMA1 is critical to the survival of yeast cells, it is unknown whether other compensatory components are involved in pH homeostasis in the absence of PMA1. To elucidate how intracellular pH is regulated independently of PMA1, we employed a screening approach by exposing the yeast haploid deletion mutant library (ver 4.0) to the selective plant plasma membrane H+-ATPase inhibitor PS-1, which we previously reported. After repeated screenings and verification, we identified two proteins, Aly1 and Aly2, that play a role in the regulation of intracellular pH when PMA1 is deficient. Our research uncovers a new perspective on the regulation of intracellular pH related to PMA1 and also preliminarily reveals a role for Aly1 and Aly2 in the regulation of intracellular pH.

Identifying the natural reserve area of Cistanche salsa under the effects of multiple host plants and climate change conditions using a maximum entropy model in Xinjiang, China.

Cistanche salsa (C. A. Mey.) G. Beck, a holoparasitic desert medicine plant with multiple hosts, is regarded as a potential future desert economic plant. However, as a result of excessive exploitation and poaching, its wild resources have become scarce. Thus, before developing its desert economic value, this plant has to be protected, and the identification of its natural reserve is currently the top priority. However, in previous nature reserve prediction studies, the influence of host plants has been overlooked, particularly in holoparasitic plants with multiple hosts. In this study, we sought to identify the conservation areas of wild C. salsa by considering multiple host-plant interactions and climate change conditions using the MaxEnt model. Additionally, a Principal Component Analysis (PCA) was used to reduce the autocorrelation between environmental variables. The effects of the natural distribution of the host plants in terms of natural distribution from the perspective of niche similarities and extrapolation detection were considered by filtering the most influential hosts: Krascheninnikovia ceratoides (Linnaeus), Gueldenstaedt, and Nitraria sibirica Pall. Additionally, the change trends in these hosts based on climate change conditions combined with the change trends in C. salsa were used to identify a core protection area of 126483.5 km2. In this article, we corrected and tried to avoid some of the common mistakes found in species distribution models based on the findings of previous research and fully considered the effects of host plants for multiple-host holoparasitic plants to provide a new perspective on the prediction of holoparasitic plants and to provide scientific zoning for biodiversity conservation in desert ecosystems. This research will hopefully serve as a significant reference for decision-makers.

The Metabolic Potential of Endophytic Actinobacteria Associated with Medicinal Plant Thymus roseus as a Plant-Growth Stimulator.

Bio-fertilizer practice considers not only economical but also environmentally friendly, sustainable agriculture. Endophytes can play important beneficiary roles in plant development, directly, indirectly, or synergistically. In this study, the majority of our endophytic actinobacteria were able to possess direct plant growth-promoting (PGP) traits, including auxin (88%), ammonia (96%), siderophore production (94%), and phosphate solubilization (24%), along with cell-wall degrading enzymes such as protease (75%), cellulase (81%), lipase (81%), and chitinase (18%). About 45% of tested strains have an inhibitory effect on the phytopathogen Fusarium oxysporum, followed by 26% for Verticillium dahlia. Overall, our results showed that strains XIEG63 and XIEG55 were the potent strains with various PGP traits that caused a higher significant increase (p ≤ 0.05) in length and biomass in the aerial part and roots of tomato and cotton, compared to the uninoculated plants. Our data showed that the greatest inhibition percentages of two phytopathogens were achieved due to treatment with strains XIEG05, XIEG07, XIEG45, and XIEG51. The GC-MS analysis showed that most of the compounds were mainly alkanes, fatty acid esters, phenols, alkenes, and aromatic chemicals and have been reported to have antifungal activity. Our investigation emphasizes that endophytic actinobacteria associated with medicinal plants might help reduce the use of chemical fertilization and potentially lead to increased agricultural productivity and sustainability.

Cryo-EM structures of human m6A writer complexes.

N6-methyladenosine (m6A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m6A "writer" consists of the catalytic subunit m6A-METTL complex (MAC) and the regulatory subunit m6A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA. Furthermore, the 4.4-Å resolution cryo-EM map of the MACOM-MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m6A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m6A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m6A writer complex.

Structural insights into molecular mechanism for N6-adenosine methylation by MT-A70 family methyltransferase METTL4.

METTL4 belongs to a subclade of MT-A70 family members of methyltransferase (MTase) proteins shown to mediate N6-adenosine methylation for both RNA and DNA in diverse eukaryotes. Here, we report that Arabidopsis METTL4 functions as U2 snRNA MTase for N6-2'-O-dimethyladenosine (m6Am) in vivo that regulates flowering time, and specifically catalyzes N6-methylation of 2'-O-methyladenosine (Am) within a single-stranded RNA in vitro. The apo structures of full-length Arabidopsis METTL4 bound to S-adenosyl-L-methionine (SAM) and the complex structure with an Am-containing RNA substrate, combined with mutagenesis and in vitro enzymatic assays, uncover a preformed L-shaped, positively-charged cavity surrounded by four loops for substrate binding and a catalytic center composed of conserved residues for specific Am nucleotide recognition and N6-methylation activity. Structural comparison of METTL4 with the mRNA m6A enzyme METTL3/METTL14 heterodimer and modeling analysis suggest a catalytic mechanism for N6-adenosine methylation by METTL4, which may be shared among MT-A70 family members.