Molecular basis of Wnt biogenesis, secretion, and Wnt7-specific signaling.

Wnt proteins are enzymatically lipidated by Porcupine (PORCN) in the ER and bind to Wntless (WLS) for intracellular transport and secretion. Mechanisms governing the transfer of these low-solubility Wnts from the ER to the extracellular space remain unclear. Through structural and functional analyses of Wnt7a, a crucial Wnt involved in central nervous system angiogenesis and blood-brain barrier maintenance, we have elucidated the principles of Wnt biogenesis and Wnt7-specific signaling. The Wnt7a-WLS complex binds to calreticulin (CALR), revealing that CALR functions as a chaperone to facilitate Wnt transfer from PORCN to WLS during Wnt biogenesis. Our structures, functional analyses, and molecular dynamics simulations demonstrate that a phospholipid in the core of Wnt-bound WLS regulates the association and dissociation between Wnt and WLS, suggesting a lipid-mediated Wnt secretion mechanism. Finally, the structure of Wnt7a bound to RECK, a cell-surface Wnt7 co-receptor, reveals how RECKCC4 engages the N-terminal domain of Wnt7a to activate Wnt7-specific signaling.

Phytophthora sojae effector Avr1d functions as an E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection.

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

Mechanism of phosphate sensing and signaling revealed by rice SPX1-PHR2 complex structure.

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP6, SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2.

Phytophthora sojae Effector PsAvh240 Inhibits Host Aspartic Protease Secretion to Promote Infection.

Plants secrete defense molecules into the extracellular space (the apoplast) to combat attacking microbes. However, the mechanisms by which successful pathogens subvert plant apoplastic immunity remain poorly understood. In this study, we show that PsAvh240, a membrane-localized effector of the soybean pathogen Phytophthora sojae, promotes P. sojae infection in soybean hairy roots. We found that PsAvh240 interacts with the soybean-resistant aspartic protease GmAP1 in planta and suppresses the secretion of GmAP1 into the apoplast. By solving its crystal structure we revealed that PsAvh240 contain six α helices and two WY motifs. The first two α helices of PsAvh240 are responsible for its plasma membrane-localization and are required for PsAvh240's interaction with GmAP1. The second WY motifs of two PsAvh240 molecules form a handshake arrangement resulting in a handshake-like dimer. This dimerization is required for the effector's repression of GmAP1 secretion. Taken together, these data reveal that PsAvh240 localizes at the plasma membrane to interfere with GmAP1 secretion, which represents an effective mechanism by which effector proteins suppress plant apoplastic immunity.

Interactions between rGO/TNT nanocomposites and cells: Regulation of cell morphology, uptake, cytotoxicity, adhesion and migration.

Reduced graphene oxide/titanium dioxide nanotube (rGO/TNT) composites have superior properties, such as a large surface area, extraordinary mechanical strength, high carrier mobility, etc. However, the biosafety and biocompatibility of these composites, such as their influences on cell viability and cell functions, which are of paramount importance, are still not fully addressed. In this study, rGO/TNT nanocomposites were successfully synthesized through a modified hydrothermal treatment method. Then, the interactions between the rGO/TNT nanocomposites and Raw264.7 mouse monocyte-macrophage cells were further investigated. The results show that the rGO/TNT nanocomposites could be internalized by Raw264.7 cells and mainly gathered inside the cytoplasm. No rGO/TNT nanocomposites were observed in the nucleus. Moreover, the rGO/TNT nanocomposites exhibited low cytotoxicity toward Raw264.7 cells at a lower dose, though they may exhibit cytotoxicity to some extent at very high concentrations. In addition, the uptake of the nanocomposites influenced the cell cytoskeleton organization, while the cell adhesion and migration abilities were also impaired.

Synthesis of polymer-functionalized nanoscale graphene oxide with different surface charge and its cellular uptake, biosafety and immune responses in Raw264.7 macrophages.

Polymer-functionalized graphene oxide (GO) has superior properties such as large surface area, extraordinary mechanical strength, high carrier mobility, good stability in physiological media and low cytotoxicity, making it an attractive material for drug and gene delivery. Herein, we successfully synthesized GO with an average size of 168.3 nm by a modified Hummers' method. Branched polyethylenimine (PEI) and 6-armed polyethylene glycol (PEG) functionalized GO complexes (GO-PEI and GO-PEG) with different zeta potentials of 47.2 mV and -43.0 mV, respectively, were successfully synthesized through amide linkages between the COOH groups of GO and the NH2 groups of PEI and PEG. Then, the interactions between GO-PEI and GO-PEG complexes and Raw264.7 mouse monocyte-macrophage cells were investigated. The GO-PEI and GO-PEG complexes could both be internalized by Raw264.7 cells. However, compared with the GO-PEG complex, the GO-PEI complex showed higher intracellular delivery efficiency in Raw264.7 cells. Moreover, it was found that the GO-PEI complex not only gathered in endosomes but also in the cytoplasm, whereas GO-PEG gathered in endosomes only. The MTT tests showed that both GO-PEI and GO-PEG complexes exhibited very low cytotoxicity towards Raw264.7 cells when at a low concentration. The cellular immune response test demonstrated the GO-PEG complex enhanced the secretion of IL-6, illustrating it was more stimulus towards macrophage cells. The above results indicated that the GO-PEI complex, with a positive surface charge, demonstrated better potential to be used in effective drug and gene delivery.