Structural basis of chorismate isomerization by Arabidopsis ISOCHORISMATE SYNTHASE1.

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semi-biotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes.

Three OsMYB36 members redundantly regulate Casparian strip formation at the root endodermis.

Plants have evolved a lignin-based Casparian strip (CS) in roots that restricts passive diffusion of mineral elements from the soil to the stele. However, the molecular mechanisms underlying CS formation in rice (Oryza sativa), which contains a CS at both the exodermis and endodermis, are poorly understood. Here, we demonstrate that CS formation at the rice endodermis is redundantly regulated by three MYELOBLASTOSIS (MYB) transcription factors, OsMYB36a, OsMYB36b, and OsMYB36c, that are highly expressed in root tips. Knockout of all three genes resulted in a complete absence of CS at the endodermis and retarded plant growth in hydroponic conditions and in soil. Compared with the wild-type, the triple mutants showed higher calcium (Ca) levels and lower Mn, Fe, Zn, Cu, and Cd levels in shoots. High Ca supply further inhibited mutant growth and increased Ca levels in shoots. Transcriptome analysis identified 1,093 downstream genes regulated by OsMYB36a/b/c, including the key CS formation gene OsCASP1 and other genes that function in CS formation at the endodermis. Three OsMYB36s regulate OsCASP1 and OsESB1 expression by directly binding to MYB-binding motifs in their promoters. Our findings thus provide important insights into the mechanism of CS formation at the endodermis and the selective uptake of mineral elements in roots.

Large-scale analysis of the N-terminal regulatory elements of the kinase domain in plant Receptor-like kinase family.

BACKGROUND: The N-terminal regulatory element (NRE) of Receptor-like kinases (RLKs), consisting of the juxtamembrane segment in receptor kinases (RKs) and the N-terminal extension segment in RLCKs, is a crucial component that regulates the activities of these proteins. However, the features and functions of the NRE have remained largely unexplored. Herein, we comprehensively analyze 510,233 NRE sequences in RLKs from 528 plant species, using information theory and data mining techniques to unravel their common characteristics and diversity. We also use recombinant RKs to investigate the function of the NRE in vitro. RESULTS: Our findings indicate that the majority of NRE segments are around 40-80 amino acids in length and feature a serine-rich region and a 14-amino-acid consensus sequence, 'FSYEELEKAT[D/N]NF[S/D]', which contains a characteristic α-helix and ST motif that connects to the core kinase domain. This conserved signature sequence is capable of suppressing FERONIA's kinase activity. A motif discovery algorithm identifies 29 motifs with highly conserved phosphorylation sites in RK and RLCK classes, especially the motif 'VGPWKpTGLpSGQLQKAFVTGVP' in LRR-VI-2 class. Phosphorylation of an NRE motif in an LRR-VI-2 member, MDIS1, modulates the auto-phosphorylation of its co-receptor, MIK1, indicating the potential role of NRE as a 'kinase switch' in RLK activation. Furthermore, the characterization of phosphorylatable NRE motifs improves the accuracy of predicting phosphorylatable sites. CONCLUSIONS: Our study provides a comprehensive dataset to investigate NRE segments from individual RLKs and enhances our understanding of the underlying mechanisms of RLK signal transduction and kinase activation processes in plant adaptation.

Uncovering interactions of mitochondrial proteins BNIP3 and BNIP3L with necroptosis-associated kinase RIPK3: Insights into kinase activation.

Mitochondria, an important organelle implicated in programmed cell death, assumes a crucial role in necroptosis. However, the regulatory mechanisms through which mitochondria participates in necroptosis are largely unknown. To address this knowledge gap, our study aimed to identify mitochondrial proteins that engage in interactions with receptor-interacting protein kinase 3 (RIPK3), a significant upstream kinase involved in necroptosis. Among the candidates, BNIP3 and BNIP3L exhibited significant higher binding scores to RIPK3 compared to others. Computational modeling revealed specific interactions, as RIPK3 specifically binds to a conserved α-helix region within BNIP3 and BNIP3L. Validation experiments confirmed the significance of these helical peptides for RIPK3 binding. Conserved peptides were also identified in BNIP3 and BNIP3L proteins from various animal species, including humans. The binding between human RIPK3 and BNIP3/BNIP3L peptides demonstrated perfect shape and charge complementation, with highly conserved interface residues. Moreover, peptide binding stabilized an active conformation of RIPK3, potentially enhancing its kinase activity. These findings uncover the interactions between RIPK3 and BNIP3/BNIP3L, providing insights into RIPK3 regulation and its role in necroptosis.

SOX9 in organogenesis: shared and unique transcriptional functions.

The transcription factor SOX9 is essential for the development of multiple organs including bone, testis, heart, lung, pancreas, intestine and nervous system. Mutations in the human SOX9 gene led to campomelic dysplasia, a haploinsufficiency disorder with several skeletal malformations frequently accompanied by 46, XY sex reversal. The mechanisms underlying the diverse SOX9 functions during organ development including its post-translational modifications, the availability of binding partners, and tissue-specific accessibility to target gene chromatin. Here we summarize the expression, activities, and downstream target genes of SOX9 in molecular genetic pathways essential for organ development, maintenance, and function. We also provide an insight into understanding the mechanisms that regulate the versatile roles of SOX9 in different organs.

Structural basis for zinc-induced activation of a zinc uptake transcriptional regulator.

The zinc uptake regulator (Zur) is a member of the Fur (ferric uptake regulator) family transcriptional regulators that plays important roles in zinc homeostasis and virulence of bacteria. Upon zinc perception, Zur binds to the promoters of zinc responsive genes and controls their transcription. However, the mechanism underlying zinc-mediated Zur activation remains unclear. Here we report a 2.2-Å crystal structure of apo Zur from the phytopathogen Xanthomonas campestris pv. campestris (XcZur), which reveals the molecular mechanism that XcZur exists in a closed inactive state before regulatory zinc binding. Subsequently, we present a 1.9-Å crystal structure of holo XcZur, which, by contrast, adopts an open state that has enough capacity to bind DNA. Structural comparison and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses uncover that binding of a zinc atom in the regulatory site, formed by the hinge region, the dimerization domain and the DNA binding domain, drives a closed-to-open conformational change that is essential for XcZur activation. Moreover, key residues responsible for DNA recognition are identified by site-directed mutagenesis. This work provides important insights into zinc-induced XcZur activation and valuable discussions on the mechanism of DNA recognition.

Ovotesticular disorders of sex development in FGF9 mouse models of human synostosis syndromes.

In mice, male sex determination depends on FGF9 signalling via FGFR2c in the bipotential gonads to maintain the expression of the key testis gene SOX9. In humans, however, while FGFR2 mutations have been linked to 46,XY disorders of sex development (DSD), the role of FGF9 is unresolved. The only reported pathogenic mutations in human FGF9, FGF9S99N and FGF9R62G, are dominant and result in craniosynostosis (fusion of cranial sutures) or multiple synostoses (fusion of limb joints). Whether these synostosis-causing FGF9 mutations impact upon gonadal development and DSD etiology has not been explored. We therefore examined embryonic gonads in the well-characterized Fgf9 missense mouse mutants, Fgf9S99N and Fgf9N143T, which phenocopy the skeletal defects of FGF9S99N and FGF9R62G variants, respectively. XY Fgf9S99N/S99N and XY Fgf9N143T/N143T fetal mouse gonads showed severely disorganized testis cords and partial XY sex reversal at 12.5 days post coitum (dpc), suggesting loss of FGF9 function. By 15.5 dpc, testis development in both mutants had partly recovered. Mitotic analysis in vivo and in vitro suggested that the testicular phenotypes in these mutants arise in part through reduced proliferation of the gonadal supporting cells. These data raise the possibility that human FGF9 mutations causative for dominant skeletal conditions can also lead to loss of FGF9 function in the developing testis, at least in mice. Our data suggest that, in humans, testis development is largely tolerant of deleterious FGF9 mutations which lead to skeletal defects, thus offering an explanation as to why XY DSDs are rare in patients with pathogenic FGF9 variants.

Crystal structure of transcription factor TGA7 from Arabidopsis.

TGA family of transcription factors play important roles in the systemic acquired resistance (SAR) in plants. In SAR, TGA7 binds to the activation sequence-1 (as-1) in the promoter region of SAR related genes and regulates their expressions in an NPR1 dependent manner. Despite its important roles in plant immunity, the molecular mechanism for DNA binding of TGA7 remains unclear. In the present work, we resolved the crystal structure of TGA7 dimers at a resolution of 2.06 Å, in which each monomer binds one molecule of palmitate. Further biochemical studies revealed that TGA7 specifically binds to the TGACG boxes of as-1 DNA in the form of homodimers, and it has specific requirements for the relative spacing and orientation of the two TGACG boxes. Moreover, we built a TGA7-DNA complex model and confirmed by site-directed mutagenesis that amino acid residue R109 in the DNA binding domain (DBD) of TGA7 is a key residue responsible for DNA recognition. Our work offers a good example for structural and functional studies of TGA proteins, and provides key clues to understand the DNA binding mechanism of TGA proteins in the SAR.

DWARF14 is a non-canonical hormone receptor for strigolactone.

Classical hormone receptors reversibly and non-covalently bind active hormone molecules, which are generated by biosynthetic enzymes, to trigger signal transduction. The α/ß hydrolase DWARF14 (D14), which hydrolyses the plant branching hormone strigolactone and interacts with the F-box protein D3/MAX2, is probably involved in strigolactone detection. However, the active form of strigolactone has yet to be identified and it is unclear which protein directly binds the active form of strigolactone, and in which manner, to act as the genuine strigolactone receptor. Here we report the crystal structure of the strigolactone-induced AtD14-D3-ASK1 complex, reveal that Arabidopsis thaliana (At)D14 undergoes an open-to-closed state transition to trigger strigolactone signalling, and demonstrate that strigolactone is hydrolysed into a covalently linked intermediate molecule (CLIM) to initiate a conformational change of AtD14 to facilitate interaction with D3. Notably, analyses of a highly branched Arabidopsis mutant d14-5 show that the AtD14(G158E) mutant maintains enzyme activity to hydrolyse strigolactone, but fails to efficiently interact with D3/MAX2 and loses the ability to act as a receptor that triggers strigolactone signalling in planta. These findings uncover a mechanism underlying the allosteric activation of AtD14 by strigolactone hydrolysis into CLIM, and define AtD14 as a non-canonical hormone receptor with dual functions to generate and sense the active form of strigolactone.

Crystal structure of the acyl acid amido synthetase GH3-8 from Oryza sativa.

The Gretchen Hagen 3 (GH3) family of acyl acid amido synthetases regulate the levels and activities of plant hormones containing carboxyl groups, thereby modulating diverse physiological responses. While structure-function relationships have been elucidated for dicotyledonous GH3s, the catalytic mechanism of monocotyledonous GH3 remains elusive. Rice (Oryza sativa) is a representative monocot, and its yield is controlled by the natural growth hormone IAA (indole-3-acetic acid). OsGH3-8 is a model GH3 enzyme that conjugates excess IAA to amino acids in an ATP-dependent manner, ensuring auxin homeostasis and regulating disease resistance, growth and development. Here, we report the crystal structure of OsGH3-8 protein in complex with AMP to uncover the molecular and structural basis for the activity of monocotyledonous GH3-8. Structural and sequence comparisons with other GH3 proteins reveal that the AMP/ATP binding sites are highly conserved. Molecular docking studies with IAA, the GH3-inhibitor Adenosine-5'-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP), and Aspartate provide important information for substrate binding and selectivity of OsGH3-8. Moreover, the observation that AIEP nearly occupies the entire binding site for AMP, IAA and amino acid, offers a ready explanation for the inhibitory effect of AIEP. Taken together, the present study provides vital insights into the molecular mechanisms of monocot GH3 function, and will help to shape the future designs of effective inhibitors.

Delicate structural coordination of the Severe Acute Respiratory Syndrome coronavirus Nsp13 upon ATP hydrolysis.

To date, an effective therapeutic treatment that confers strong attenuation toward coronaviruses (CoVs) remains elusive. Of all the potential drug targets, the helicase of CoVs is considered to be one of the most important. Here, we first present the structure of the full-length Nsp13 helicase of SARS-CoV (SARS-Nsp13) and investigate the structural coordination of its five domains and how these contribute to its translocation and unwinding activity. A translocation model is proposed for the Upf1-like helicase members according to three different structural conditions in solution characterized through H/D exchange assay, including substrate state (SARS-Nsp13-dsDNA bound with AMPPNP), transition state (bound with ADP-AlF4-) and product state (bound with ADP). We observed that the ß19-ß20 loop on the 1A domain is involved in unwinding process directly. Furthermore, we have shown that the RNA dependent RNA polymerase (RdRp), SARS-Nsp12, can enhance the helicase activity of SARS-Nsp13 through interacting with it directly. The interacting regions were identified and can be considered common across CoVs, which provides new insights into the Replication and Transcription Complex (RTC) of CoVs.

Crystal Structure of the Epo1-Bem3 Complex for Bud Growth.

Tubules of the endoplasmic reticulum (ER) spread into the buds of yeast by an actin-based mechanism and, upon entry, become attached to the polarisome, a proteinaceous micro-compartment below the tip of the bud. The minimal tether between polarisome and cortical ER is formed by a protein complex consisting of Epo1, a member of the polarisome, Scs2, a membrane protein of the ER and Cdc42 guanosine triphosphatase-activating protein Bem3. Here, we report the crystal structure of a complex between Epo1 and Bem3. In addition, we characterize through the hydrogen/deuterium (H/D) exchange assay the interface between Scs2 and Epo1. Our findings provide a first structural insight into the molecular architecture of the link between cortical ER and the polarisome.

TMK4 receptor kinase negatively modulates ABA signaling by phosphorylating ABI2 and enhancing its activity.

In plants, clade A type 2C protein phosphatases (PP2CAs) have emerged as major players in abscisic acid (ABA)-regulated stress responses by inhibiting protein kinase activity. However, how different internal and external environmental signals modulate the activity of PP2CAs are not well known. The transmembrane kinase (TMK) protein 4 (TMK4), one member of a previously identified receptor kinase subfamily on the plasma membrane that plays vital roles in plant cell growth, directly interacts with PP2CAs member (ABA-Insensitive 2, ABI2). tmk4 mutant is hypersensitive to ABA in both ABA-inhibited seed germination and primary root growth, indicating that TMK4 is a negative regulator in ABA signaling pathway. Further analyses indicate that TMK4 phosphorylates ABI2 at three conserved Ser residues, thus enhancing the activity of ABI2. The phosphorylation-mimic ABI2S139DS140DS266D can complement but non-phosphorylated form ABI2S139AS140AS266A cannot complement ABA hypersensitive phenotype of the loss-of-function mutant abi1-2abi2-2. This study provides a previously unidentified mechanism for positively regulating ABI2 by a plasma membrane protein kinase.

Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

Brown adipose tissue (BAT) is an exclusive tissue of nonshivering thermogenesis. It is fueled by lipids and glucose and involved in energy and metabolic homeostasis. Intrauterine exposure to hyperglycemia during gestational diabetes mellitus may result in abnormal fetal development and metabolic phenotypes in adulthood. However, whether intrauterine hyperglycemia influences the development of BAT is unknown. In this study, mouse embryos were exposed to the intrauterine hyperglycemia environment by injecting streptozocin into pregnant mice at 1 d post coitum (dpc). The structure of BAT was examined by hematoxylin and eosin staining and immunohistochemical analysis. The glucose uptake in BAT was measured in vivo by [18F]-fluoro-2-deoxyglucose-micro-positron emission tomography. The gene expression in BAT was determined by real-time PCR, and the 5'-C-phosphate-G-3' site-specific methylation was quantitatively analyzed. Intrauterine hyperglycemia exposure resulted in the impaired structure of BAT and decreased glucose uptake function in BAT in adulthood. The expressions of the genes involved in thermogenesis and mitochondrial respiratory chain in BAT, such as Ucp1, Cox5b, and Elovl3, were down-regulated by intrauterine hyperglycemia exposure at 18.5 dpc and at 16 wk of age. Furthermore, higher methylation levels of Ucp1, Cox5b, and Elovl3 were found in offspring of mothers with streptozotocin-induced diabetes. Our results provide the evidence for enduring inhibitory effects of intrauterine hyperglycemia on BAT development in offspring. Intrauterine hyperglycemia is associated with increased DNA methylation of the BAT specific genes in offspring, which support an epigenetic involvement.-Yu, D.-Q., Lv, P.-P., Yan, Y.-S., Xu, G.-X., Sadhukhan, A., Dong, S., Shen, Y., Ren, J., Zhang, X.-Y., Feng, C., Huang, Y.-T., Tian, S., Zhou, Y., Cai, Y.-T., Ming, Z.-H., Ding, G.-L., Zhu, H., Sheng, J.-Z., Jin, M., Huang, H.-F. Intrauterine exposure to hyperglycemia retards the development of brown adipose tissue.

The crystal structure of the phytopathogenic bacterial sensor PcrK reveals different cytokinin recognition mechanism from the plant sensor AHK4.

Plant cytokinins (CKs) are essential for many central cellular processes and play important roles in the interaction between bacteria and plants. Perception of CK is executed by the CHASE domain in the histidine kinase sensors of a class of two-component regulatory systems. Despite advances in understanding the structural basis for CK perception by the sensor AHK4 in Arabidopsis, the molecular mechanism of CK binding by other sensors is unclear. Here, we report the crystal structure of the CHASE domain in the histidine kinase PcrK of the bacterial plant pathogen Xanthomonas campestris pathovar campestris, which senses plant CK, determined at 2.55 Šresolution. The structure reveals that the PcrK has an AHK4-like overall topology and assembles into a homodimer. Strikingly, detailed structural analysis unveils two unique features of the PcrK ligand binding pocket: the size of the pocket is restricted for CK binding, and the PcrK applies a positively charged arginine but not a negatively charged aspartate to recognize the ligand. We propose a model to explain how the PcrK accommodates CK-sized compounds through conformational changes, providing a potential mechanistic framework for understanding ligand recognition by the PcrK.

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus.

Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 in vitro Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins.IMPORTANCE The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.

The Gram-negative phytopathogen Xanthomonas campestris pv. campestris employs a 5'UTR as a feedback controller to regulate methionine biosynthesis.

The synthesis of methionine is critical for most bacteria. It is known that cellular methionine has a feedback effect on the expression of met genes involved in de novo methionine biosynthesis. Previous studies revealed that Gram-negative bacteria control met gene expression at the transcriptional level by regulator proteins, while most Gram-positive bacteria regulate met genes at post-transcriptional level by RNA regulators (riboregulators) located in the 5'UTR of met genes. However, despite its importance, the methionine biosynthesis pathway in the Gram-negative Xanthomonas genus that includes many important plant pathogens is completely uncharacterized. Here, we address this issue using the crucifer black rot pathogen Xanthomonas campestris pv. campestris (Xcc), a model bacterium in microbe-plant interaction studies. The work identified an operon (met) involved in de novo methionine biosynthesis in Xcc. Disruption of the operon resulted in defective growth in methionine-limited media and in planta. Western blot analysis revealed that the expression of the operon is dependent on methionine levels. Further molecular analyses demonstrated that the 5'UTR, but not the promoter of the operon, is involved in feedback regulation on operon expression in response to methionine availability, providing an example of a Gram-negative bacterium utilizing a 5'UTR region to control the expression of the genes involved in methionine biosynthesis.

Rating the quality of evidence: the GRADE system in systematic reviews/meta-analyses of AKI.

BACKGROUND: The method of systematic reviews/meta-analyses (SRs/MAs) has been widely used in acute kidney injury (AKI) studies. However, it is not quite clear about the quality of the evidence and existing problems. OBJECTIVES: To grade the evidence quality of published SRs/MAs of AKI by using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system, understand the current situation of evidence rating and analyze the possible problems. METHODS: Researchers systematically searched for articles about SRs/MAs of AKI published in the following four Chinese databases and four English databases, including Chinese Biomedicine Literature Database, Wanfang Database, China National Knowledge Internet Database, VIP Database, Pubmed, EMBASE, the Cochrane Library and Web of Science. RESULTS: Totally, 81 SRs/MAs were included in this study and the overall quality of evidence was not satisfactory. The number of literatures of low and very low evidence quality was 33 (40.7%) and 41 (50.6%), respectively. Limitation was the main factor which caused the quality of research evidence degrading (92.6%), and other degradation factors were inconsistency (56.8%), publication bias (44.4%), indirectness (35.8%) and imprecision (32.1%). The quality of evidence for AKI has been significantly improved after the publication of the GRADE system in 2004. CONCLUSIONS: Since 2004 when the GRADE system was published, the quality of evidence of AKI has been increased clearly. But quality of AKI evidence of SRs/MAs for intervention is still not satisfactory. Limitation and inconsistency were two major factors leading to degradation.

A role for TRPC3 in mammalian testis development.

SOX9 is a key transcription factor for testis determination and development. Mutations in and around the SOX9 gene contribute to Differences/Disorders of Sex Development (DSD). However, a substantial proportion of DSD patients lack a definitive genetic diagnosis. SOX9 target genes are potentially DSD-causative genes, yet only a limited subset of these genes has been investigated during testis development. We hypothesize that SOX9 target genes play an integral role in testis development and could potentially be causative genes in DSD. In this study, we describe a novel testicular target gene of SOX9, Trpc3. Trpc3 exhibits high expression levels in the SOX9-expressing male Sertoli cells compared to female granulosa cells in mouse fetal gonads between embryonic day 11.5 (E11.5) and E13.5. In XY Sox9 knockout gonads, Trpc3 expression is markedly downregulated. Moreover, culture of E11.5 XY mouse gonads with TRPC3 inhibitor Pyr3 resulted in decreased germ cell numbers caused by reduced germ cell proliferation. Trpc3 is also expressed in endothelial cells and Pyr3-treated E11.5 XY mouse gonads showed a loss of the coelomic blood vessel due to increased apoptosis of endothelial cells. In the human testicular cell line NT2/D1, TRPC3 promotes cell proliferation and controls cell morphology, as observed by xCELLigence and HoloMonitor real-time analysis. In summary, our study suggests that SOX9 positively regulates Trpc3 in mouse testes and TRPC3 may mediate SOX9 function during Sertoli, germ and endothelial cell development.

Structural and biochemical basis of FLS2-mediated signal activation and transduction in rice.

The receptor-like kinase FLAGELLIN-SENSITIVE 2 (FLS2) functions as a bacterial flagellin receptor localized on the cell membrane of plants. In Arabidopsis, the co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) cooperates with FLS2 to detect the flagellin epitope flg22, resulting in formation of a signaling complex that triggers plant defense responses. However, the co-receptor responsible for recognizing and signaling the flg22 epitope in rice remains to be determined, and the precise structural mechanism underlying FLS2-mediated signal activation and transduction has not been clarified. This study presents the structural characterization of a kinase-dead mutant of the intracellular kinase domain of OsFLS2 (OsFLS2-KDD1013A) in complex with ATP or ADP, resolved at resolutions of 1.98 Å and 2.09 Å, respectively. Structural analysis revealed that OsFLS2 can adopt an active conformation in the absence of phosphorylation, although it exhibits only weak basal catalytic activity for autophosphorylation. Subsequent investigations demonstrated that OsSERK2 effectively phosphorylates OsFLS2, which reciprocally phosphorylates OsSERK2, leading to complete activation of OsSERK2 and rapid phosphorylation of the downstream substrate receptor-like cytoplasmic kinases OsRLCK176 and OsRLCK185. Through mass spectrometry experiments, we successfully identified critical autophosphorylation sites on OsSERK2, as well as sites transphosphorylated by OsFLS2. Furthermore, we demonstrated the interaction between OsSERK2 and OsFLS2, which is enhanced in the presence of flg22. Genetic evidence suggests that OsRLCK176 and OsRLCK185 may function downstream of the OsFLS2-mediated signaling pathway. Our study reveals the molecular mechanism by which OsFLS2 mediates signal transduction pathways in rice and provides a valuable example for understanding RLK-mediated signaling pathways in plants.