YdiU is a novel UMPylation modification enzyme that plays an important role in regulating chaperones, flagellar synthesis, and iron absorption in Salmonella. Here, we report that YdiU plays an important role in regulating oxidative stress in Salmonella. The expression of ydiU was dramatically induced by hydrogen peroxide (H2O2) and the survivability of ∆ydiU was significantly stronger than that of WT under oxidative stress. Interestingly, in vitro and in vivo experiments showed that YdiU can interact with SodA and UMPylate it only in the presence of H2O2. Further study showed slight changes in the secondary structure of SodA under oxidative stress. Structural analysis of native protein and H2O2-treated protein showed that H2O2 treatment affected the conformation of Met24. The modification site Tyr12 is near Met24, and Tyr12 exposure may facilitate UMPylation by YdiU after H2O2 treatment. In addition, in vivo and in vitro experiments showed that YdiU negatively regulates SodA activity. Therefore, our results identify new biological functions of YdiU, highlight the importance of UMPylation in bacterial oxidation balance, and provide the theoretical basis to study Salmonella survival and pathogenesis.

Auxin response factors (ARFs) are key regulators of numerous aspects of plant growth and development through mediating auxin signaling. In this study, we conducted a comprehensive genome-wide analysis of ZmARFs to identify and validate all auxin response factor genes in maize. These ZmARF genes were categorized into four distinct groups (I-IV) based on phylogenetic analysis, revealing seven sister pairs. We presented detailed information on gene sequences, structures, chromosome locations, and conserved motifs of ZmARFs. Through transient expression assays, we identified transcriptional activators or repressors among ZmARFs. Notably, our study demonstrated, for the first time, that ZmARF3 acts as a positive regulator of adventitious roots development in maize. This study not only provides basic insights into the maize ARF gene family but also sheds light on the specific functions of ZmARF3, paving the way for a more precise understanding of ZmARFs' roles in plant growth and development in maize.

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

目的 鼠伤寒沙门菌效应蛋白SteC是沙门菌Ⅲ型分泌系统中唯一的一种激酶,然而其与以往发现的激酶同源性均较低,其发挥激酶功能的机制尚不清楚.本研究通过基因克隆、原核表达截短鼠伤寒沙门菌效应蛋白SteC-C端催化结构域,获得纯化蛋白并进行晶体培养,为揭示SteC作为激酶催化磷酸化的机制奠定基础.方法 以鼠伤寒沙门菌效应蛋白SteC生物信息学分析为基础,在SteC-C端催化结构域进行片段截取,并将此结构域中仅有的第276位半胱氨酸突变为丝氨酸,利用基因克隆技术分别构建SteC C1-pGl01和SteC C1C276S-pGl01两种重组质粒,在大肠埃希菌原核体系中进行表达.依次使用镍离子亲和层析柱、凝胶层析柱进行蛋白纯化.使用12种晶体试剂盒筛选适合SteC活性(Holo)与非活性(Apo)状态晶体生长的条件,从而获得SteC不同类型的蛋白质晶体,并使用additive试剂盒寻找更利于蛋白晶体生长的条件,从而获得单晶性良好的晶体.结果 鼠伤寒沙门菌效应蛋白SteC C1催化结构域相对分子质量大小为19.7×103,在原核表达体系中蛋白可溶性好、浓度高,但是蛋白性质不稳定存在二聚化现象,其突变体证明SteC Cys-276 是 SteC C1 导致二聚化的原因.两种蛋白均在 2.0 mol/LAmmonium sulfate,0.01 mol/L Magnesium sulfate heptahydrate,0.05 mol/LSodium cacodylate trihydrate(pH 6.5)条件结晶,且 SteCC1C276S 晶体结晶时间更快,添加0.1mol/L碘化钠试剂后更有助于蛋白晶体的生长.结论 成功构建SteC C1及SteC C1C276S原核表达系统并获得蛋白进行结晶培养.硫酸铵盐有助于SteC C1结构域及其突变体晶体的形成且突变体更容易结晶.SteC C1结构域蛋白晶体的培养为SteC蛋白质空间结构的解析及进一步揭示其催化磷酸化的分子机制奠定了基础.

Salmonella infection kills more than 300,000 people every year. After infection, Salmonella mainly parasitizes host cells, as it prevents host cell pyroptosis by turning off the synthesis of flagellar antigen.

Precise spatiotemporal control of the timing and extent of asymmetric cell divisions (ACDs) is essential for plant development. In the Arabidopsis root, ground tissue maturation involves an additional ACD of the endo-dermis that maintains the inner cell layer as the endodermis and generates the middle cortex to the outside. Through regulation of the cell cycle regulator CYCLIND6;1 (CYCD6;1), the transcription factors SCARECROW (SCR) and SHORT-ROOT (SHR) play critical roles in this process. In the present study, we found that loss of function of NAC1, a NAC transcription factor family gene, causes markedly increased periclinal cell divisions in the root endodermis. Importantly, NAC1 directly represses the transcription of CYCD6;1 by recruiting the co-repressor TOPLESS (TPL), creating a fine-tuned mechanism to maintain proper root ground tissue patterning by limiting production of middle cortex cells. Biochemical and genetic analyses further showed that NAC1 physically interacts with SCR and SHR to restrict excessive periclinal cell divisions in the endo-dermis during root middle cortex formation. Although NAC1-TPL is recruited to the CYCD6;1 promoter and represses its transcription in an SCR-dependent manner, NAC1 and SHR antagonize each other to regu-late the expression of CYCD6;1. Collectively, our study provides mechanistic insights into how the NAC1-TPL module integrates with the master transcriptional regulators SCR and SHR to control root ground tissue patterning by fine-tuning spatiotemporal expression of CYCD6;1 in Arabidopsis.

Upon entering host cells, Salmonella quickly turns off flagella biogenesis to avoid recognition by the host immune system. However, it is not clear which host signal(s) Salmonella senses to initiate flagellum control. Here, we demonstrate that the acid signal can suppress flagella synthesis and motility of Salmonella, and this occurs after the transcription of master flagellar gene flhDC and depends on the anti-FlhDC factor YdiV. YdiV expression is activated after acid treatment. A global screen with ydiV promoter DNA and total protein from acid-treated Salmonella revealed a novel regulator of YdiV, the acid-related transcription factor CadC. Further studies showed that CadCC, the DNA binding domain of CadC, directly binds to a 33 nt region of the ydiV promoter with a 0.2 μM KD affinity. Furthermore, CadC could separate H-NS-ydiV promoter DNA complex to form CadC-DNA complex at a low concentration. Structural simulation and mutagenesis assays revealed that H43 and W106 of CadC are essential for ydiV promoter binding. No acid-induced flagellum control phenotype was observed in cadC mutant or ydiV mutant strains, suggesting that flagellum control during acid adaption is dependent on CadC and YdiV. The intracellular survival ability of cadC mutant strain decreased significantly compared with WT strain while the flagellin expression could not be effectively controlled in the cadC mutant strain when surviving within host cells. Together, our results demonstrated that acid stress acts as an important host signal to trigger Salmonella flagellum control through the CadC-YdiV-FlhDC axis, allowing Salmonella to sense a hostile environment and regulate flagellar synthesis during infection.

Bacterial flagellin activates the host immune system and triggers pyroptosis. Salmonella reduces flagellin expression when it survives within host cells. Here, we found that the UMPylator YdiU significantly altered the Salmonella flagellar biogenesis process upon host cell entry. The expression levels of class II and class III flagellar genes, but not the class I flagellar genes flhDC, were dramatically increased in a ΔydiU strain compared to wild-type (WT) Salmonella in a host-simulating environment. A direct interaction between YdiU and FlhDC was detected by bacterial two-hybrid assay. Furthermore, YdiU efficiently catalyzed the UMPylation of FlhC but not FlhD, FliA, or FliC. UMPylation of FlhC completely eliminated its DNA-binding activity. In vivo experiments showed that YdiU was required and sufficient for Salmonella flagellar control within host cells. Mice infected with the ΔydiU strain died much earlier than WT strain-infected mice and developed much more severe inflammation and injury in organs and much higher levels of cytokines in blood, demonstrating that early host death induced by the ΔydiU strain is probably due to excessive inflammation. Our results indicate that YdiU acts as an essential factor of Salmonella to mediate host immune escape. IMPORTANCE Salmonella is an important facultative pathogen of foodborne illness and typhoid fever in humans. Flagella allow bacterial motility and are required for Salmonella to successfully invade the host cells. In parallel, flagellin triggers the host immune system. Salmonella reduces flagellar biogenesis to avoid detection within host cells by a largely unknown mechanism. Here, we report that the UMPylator YdiU inhibits flagellin expression in response to host signals in an UMPylation-dependent manner. The target of YdiU is the major flagellar transcription factor FlhDC. YdiU UMPylates the FlhC subunit on its Ser31 residue and prevents FlhDC from binding to flagellar genes, thus switching off flagellar biogenesis. Our results reveal a novel mechanism by which Salmonella adopts posttranslational modification to shut down flagellar synthesis as a strategy to achieve immune escape.

Gravitropism is an essential adaptive response of land plants. Asymmetric auxin gradients across plant organs, interpreted by multiple auxin signaling components including AUXIN RESPONSE FACTOR7 (ARF7), trigger differential growth and bending response. However, how this fundamental process is strictly maintained in nature remains unclear. Here, we report that gravity stimulates the transcription of METHYL ESTERASE17 (MES17) along the lower side of the hypocotyl via ARF7-dependent auxin signaling. The asymmetric distribution of MES17, a methyltransferase that converts auxin from its inactive form methyl indole-3-acetic acid ester (MeIAA) to its biologically active form free-IAA, enhanced the gradient of active auxin across the hypocotyl, which in turn reversely amplified the asymmetric auxin responses and differential growth that shape gravitropic bending. Taken together, our findings reveal the novel role of MES17-mediated auxin homeostasis in gravitropic responses and identify an ARF7-triggered feedback mechanism that reinforces the asymmetric distribution of active auxin and strictly controls gravitropism in plants.

Salmonella is the major pathogen causing bacterial enteric illness in both humans and animals. Iron availability is strictly controlled upon Salmonella entry into host cells.

Maize height is determined by the number of nodes and the length of internodes. Node number is driven by intercalary meristem formation and internode length by intercalary cell elongation, respectively. However, mechanisms regulating establishment of nodes and internode growth are unclear. We screened EMS-induced maize mutants and identified a dwarf mutant zm66, linked to a single base change in TERMINAL EAR 1 (ZmTE1). Detailed phenotypic analysis revealed that zm66 (zmte1-2) has shorter internodes and increased node numbers, caused by decreased cell elongation and disordered intercalary meristem formation, respectively. Transcriptome analysis showed that auxin signalling genes are also dysregulated in zmte1-2, as are cell elongation and cell cycle-related genes. This argues that ZmTE1 regulates auxin signalling, cell division, and cell elongation. We found that the ZmWEE1 kinase phosphorylates ZmTE1, thus confining it to the nucleus and probably reducing cell division. In contrast, the ZmPP2Ac-2 phosphatase promotes dephosphorylation and cytoplasmic localization of ZmTE1, as well as cell division. Taken together, ZmTE1, a key regulator of plant height, is responsible for maintaining organized formation of internode meristems and rapid cell elongation. ZmWEE1 and ZmPP2Ac-2 might balance ZmTE1 activity, controlling cell division and elongation to maintain normal maize growth.

Cytokinins are phytohormones that regulate plant development, growth, and responses to stress. In particular, cytokinin has been reported to negatively regulate plant adaptation to high salinity; however, the molecular mechanisms that counteract cytokinin signaling and enable salt tolerance are not fully understood. Here, we provide evidence that salt stress induces the degradation of the cytokinin signaling components Arabidopsis (Arabidopisis thaliana) response regulator 1 (ARR1), ARR10 and ARR12. Furthermore, the stress-activated mitogen-activated protein kinase 3 (MPK3) and MPK6 interact with and phosphorylate ARR1/10/12 to promote their degradation in response to salt stress. As expected, salt tolerance is decreased in the mpk3/6 double mutant, but enhanced upon ectopic MPK3/MPK6 activation in an MKK5(DD) line. Importantly, salt hypersensitivity phenotypes of the mpk3/6 line were significantly alleviated by mutation of ARR1/12. The above results indicate that MPK3/6 enhance salt tolerance in part via their negative regulation of ARR1/10/12 protein stability. Thus, our work reveals a new molecular mechanism underlying salt-induced stress adaptation and the inhibition of plant growth, via enhanced degradation of cytokinin signaling components.

Aluminium (Al) stress is a major limiting factor for worldwide crop production in acid soils. In Arabidopsis thaliana, the TAA1-dependent local auxin biosynthesis in the root-apex transition zone (TZ), the major perception site for Al toxicity, is crucial for the Al-induced root-growth inhibition, while the mechanism underlying Al-regulated auxin accumulation in the TZ is not fully understood. In the present study, the role of auxin transport in Al-induced local auxin accumulation in the TZ and root-growth inhibition was investigated. Our results showed that PIN-FORMED (PIN) proteins such as PIN1, PIN3, PIN4 and PIN7 and AUX1/LAX proteins such as AUX1, LAX1 and LAX2 were all ectopically up-regulated in the root-apex TZ in response to Al stress and coordinately regulated local auxin accumulation in the TZ and root-growth inhibition. The ectopic up-regulation of PIN1 in the TZ under Al stress was regulated by both ethylene and auxin, with auxin signalling acting downstream of ethylene. Al-induced PIN1 up-regulation and auxin accumulation in the root-apex TZ was also regulated by the calossin-like protein BIG. Together, our results provide insight into how Al stress induces local auxin accumulation in the TZ and root-growth inhibition through the local regulation of auxin transport.

Ribosome assembly factors guide the complex process by which ribosomal proteins and the ribosomal RNAs form a functional ribosome. However, the assembly of plant plastid ribosomes is poorly understood. In the present study, we discovered a maize (Zea mays) plastid ribosome assembly factor based on our characterization of the embryo defective 15 (emb15) mutant. Loss of function of Emb15 retards embryo development at an early stage, but does not substantially affect the endosperm, and causes an albino phenotype in other genetic backgrounds. EMB15 localizes to plastids and possesses a ribosome maturation factor M (RimM) domain in the N-terminus and a predicted UDP-GlcNAc pyrophosphorylase domain in the C-terminus. The EMB15 RimM domain originated in bacteria and the UDP-GlcNAc pyrophosphorylase domain originated in fungi; these two domains came together in the ancestor of land plants during evolution. The N-terminus of EMB15 complemented the growth defect of an Escherichia coli strain with a RimM deletion and rescued the albino phenotype of emb15 homozygous mutants. The RimM domain mediates the interaction between EMB15 and the plastid ribosomal protein PRPS19. Plastid 16S rRNA maturation is also significantly impaired in emb15. These observations suggest that EMB15 functions in maize seed development as a plastid ribosome assembly factor, and the C-terminal domain is not important under normal conditions.

In order to study the influence of quantitative magnetic susceptibility mapping (QSM) on them. A 2.5D Attention U-Net Network based on multiple input and multiple output, a method for segmenting RN, SN, and STN regions in high-resolution QSM images is proposed, and deep learning realizes accurate segmentation of deep nuclei in brain QSM images. Experimental results show data first cuts each layer of 0 100 case data, based on the image center, from 384 × 288 to the size of 128 × 128. Image combination: each layer of the image in the layer direction combines with two adjacent images into a 2.5D image, i.e., (It − m It; It + i), where It represents the layer i image. At this time, the size of the image changes from 128 × 128 to 128 × 128 × 3, in which 3 represents three consecutive layers of images. The SNR of SWP I to STN is twice that of SWI. The small deep gray matter nuclei (RN, SN, and STN) in QSM images of the brain and the pancreas with irregular shape and large individual differences in abdominal CT images can be automatically segmented.

SUMMARYSensing stressful conditions and adjusting cellular metabolism to adapt to the environment is essential for bacteria to survive in variable situations. Here, we describe a new stress-related protein YdiU, and characterize YdiU as an enzyme that catalyzes the covalent attachment of uridine 5’-monophosphate to a protein tyrosine/histidine residue—a novel modification defined as UMPylation. Mn2+serves as an essential co-factor for YdiU-mediated UMPylation. UTP and Mn2+-binding converts YdiU to an aggregate-prone state facilitating the recruitment of chaperones. The UMPylation of chaperones prevents them from binding co-factors or clients, thereby impairing their function. Consistent with the recent finding that YdiU acts as an AMPylator, we further demonstrate that the self-AMPylation of YdiU padlocks its chaperone-UMPylation activity. The detailed mechanism is proposed based on Apo-YdiU, YdiU-ATP, YdiU-AMP crystal structures and molecular dynamics simulation models of YdiU-UTP and YdiU-UTP-peptide.In vivodata demonstrate that YdiU effectively protectsSalmonellafrom stress-induced ATP depletion through UMPylation.HighlightsYdiU involves in stress-resistance ofSalmonella.YdiU mediates protein UMPylation in a Mn2+-dependent manner.Structural insights into YdiU-mediated UMPylation.UMPylation of chaperones by YdiU modulates their function.

Shade avoidance syndrome (SAS) arises in densely growing plants that compete for light. In Arabidopsis thaliana, phytochrome interacting factor (PIF) proteins link the perception of shade to stem elongation via auxin production. Here, we report that PIFs inhibit the shade-induced expression of AUXIN RESPONSE FACTOR 18 (ARF18), and ARF18 represses auxin signaling. Therefore, PIF-mediated inhibition of ARF18 enhances auxin-dependent hypocotyl elongation in simulated shade. Furthermore, we show that both PIFs and ARF18 directly repress qua-quine starch (QQS), which controls the allocation of carbon and nitrogen. Shade-repressed QQS attenuates the conversion of starch to protein and thus reduced leaf area. Our results suggest that PIF-dependent gene regulation coordinates multiple SAS responses, including altered stem growth via ARF18, as well as altered leaf growth and metabolism via QQS.

[This corrects the article DOI: 10.1371/journal.pgen.1008044.].

目的 优化鼠疫耶尔森菌YopJ蛋白基因密码子并在大肠埃希菌中进行异源表达纯化. 方法 依据大肠埃希菌密码子偏好性对鼠疫耶尔森菌YopJ基因进行全基因序列优化,并设计引物.分别以密码子优化前后的鼠疫耶尔森菌YopJ蛋白的基因为模板,将包含YopJ(22aa-288aa)和YopJ(22aa-250aa)的基因片段连接到载体pGI01中.重组质粒转化到大肠埃希菌BL21菌株中,扩大培养后,IPTG诱导目的蛋白表达.提取大肠埃希菌表达蛋白并使用镍离子柱进行纯化. 结果 鼠疫耶尔森菌YopJ密码子优化前YopJ(22aa-288aa)及YopJ(22aa-250aa)在大肠埃希菌内表达水平较低,密码子优化后YopJ(22aa-288aa)及YopJ(22aa-250aa)在大肠埃希菌内大量表达可溶性YopJ蛋白. 结论 对鼠疫耶尔森菌YopJ蛋白进行基因密码子优化能够提高其在大肠埃希菌内的表达量.获得的鼠疫耶尔森菌YopJ蛋白具有可溶性,为进一步研究其结构和功能奠定了基础.