Fungal oxysterol-binding protein-related proteins promote pathogen virulence and activate plant immunity

Oxysterol-binding protein-related proteins (ORPs) are a conserved class of lipid transfer proteins that are closely involved in multiple cellular processes in eukaryotes but their roles in plant-pathogen interactions are mostly unknown. We showed that transient expression of ORPs of Magnaporthe oryzae (MoORPs) in Nicotiana benthamina plants triggered oxidative burst and cell death; treatment of tobacco Bright Yellow-2 suspension cells with recombinant MoORPs elicited the production of reactive oxygen species. Despite that ORPs are normally described as intracellular proteins, we detected MoORPs in fungal cultural filtrates and intercellular fluids from barley plants infected with the fungus. More importantly, infiltration of Arabidopsis plants with recombinant Arabidopsis or fungal ORPs activated oxidative burst, callose deposition, PR1 gene expression, and enhanced plant disease resistance, implying that ORPs may function as endogenous and exogenous danger signals triggering plant innate immunity. Extracellular application of fungal ORPs exerted an opposite impact on salicylic acid and jasmonic acid/ethylene signaling pathways. The Brassinosteroid Insensitive 1-associated Kinase 1 was dispensable for the ORP-activated defense. Besides, simultaneous knockout of MoORP1 and MoORP3 abolished fungal colony radial growth and conidiation, whereas double knockout of MoORP1 and MoORP2 compromised fungal virulence on barley and rice plants. These observations collectively highlight the multifaceted role of MoORPs in the modulation of plant innate immunity and promotion of fungal development and virulence in M. oryzae.

PI4P/PS countertransport by ORP10 at ER–endosome membrane contact sites regulates endosome fission

Membrane contact sites (MCSs) serve as a zone for nonvesicular lipid transport by oxysterol-binding protein (OSBP)-related proteins (ORPs). ORPs mediate lipid countertransport, in which two distinct lipids are transported counterdirectionally. How such lipid countertransport controls specific biological functions, however, remains elusive. We report that lipid countertransport by ORP10 at ER–endosome MCSs regulates retrograde membrane trafficking. ORP10, together with ORP9 and VAP, formed ER–endosome MCSs in a phosphatidylinositol 4-phosphate (PI4P)-dependent manner. ORP10 exhibited a lipid exchange activity toward its ligands, PI4P and phosphatidylserine (PS), between liposomes in vitro, and between the ER and endosomes in situ. Cell biological analysis demonstrated that ORP10 supplies a pool of PS from the ER, in exchange for PI4P, to endosomes where the PS-binding protein EHD1 is recruited to facilitate endosome fission. Our study highlights a novel lipid exchange at ER–endosome MCSs as a nonenzymatic PI4P-to-PS conversion mechanism that organizes membrane remodeling during retrograde membrane trafficking.

Potential Prognostic Biomarkers of OSBPL Family Genes in Patients with Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly fatal malignancy with poor survival outcomes. In addition, oxysterol-binding protein-like (OSBPL) family members are reported to be involved in lipid binding and transport and play critical roles in tumorigenesis. However, relationships between PDAC and OSBPL family members have not comprehensively been elucidated. In this study, we used the Oncomine and GEPIA 2 databases to analyze OSBPL transcription expressions in PDAC. The Kaplan–Meier plotter and TIMER 2.0 were used to assess the relationships between overall survival (OS) and immune-infiltration with OSBPL family members. Co-expression data from cBioPortal were downloaded to assess the correlated pathways with OSBPL gene family members using DAVID. The expressions of OSBPL3, OSBPL8, OSBPL10, and OSBPL11 were found to be highly upregulated in PDAC. Low expressions of OSBPL3, OSBPL8, and OSBPL10 indicated longer OS. The functions of OSBPL family members were mainly associated with several potential signaling pathways in cancer cells, including ATP binding, integrin binding, receptor binding, and the renin-angiotensin system (RAS) signaling pathway. The transcription levels of OSBPL gene family members were connected with several immune infiltrates. Collectively, OSBPL family members are influential biomarkers for the early diagnosis of PDAC and have prognostic value, with the promise of precise treatment of PDAC in the future.

Osh6 Revisited: Control of PS Transport by the Concerted Actions of PI4P and Sac1 Phosphatase

Osh6, a member of the oxysterol-binding protein–related protein (ORP) family, is a lipid transport protein that is involved in the transport of phosphatidylserine (PS) between the endoplasmic reticulum (ER) and the plasma membrane (PM). We used a biophysical approach to characterize its transport mechanism in detail. We examined the transport of all potential ligands of Osh6. PI4P and PS are the best described lipid cargo molecules; in addition, we showed that PIP2 can be transported by Osh6 as well. So far, it was the exchange between the two cargo molecules, PS and PI4P, in the lipid-binding pocket of Osh6 that was considered an essential driving force for the PS transport. However, we showed that Osh6 can efficiently transport PS along the gradient without the help of PI4P and that PI4P inhibits the PS transport along its gradient. This observation highlights that the exchange between PS and PI4P is indeed crucial, but PI4P bound to the protein rather than intensifying the PS transport suppresses it. We considered this to be important for the transport directionality as it prevents PS from returning back from the PM where its concentration is high to the ER where it is synthesized. Our results also highlighted the importance of the ER resident Sac1 phosphatase that enables the PS transport and ensures its directionality by PI4P consumption. Furthermore, we showed that the Sac1 activity is regulated by the negative charge of the membrane that can be provided by PS or PI anions in the case of the ER membrane.

OSBPL5 expression associates with survival in triple negative breast cancer.

We mined published microarray data (1) to understand the most significant gene expression differences in the tumors of triple negative breast cancer patients based on survival following treatment: dead or alive. We observed significant transcriptome-wide differential expression of oxysterol binding protein-like 5, encoded by OSBPL5 when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, OSBPL5 expression was correlated with overall survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. OSBPL5 may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.

Loss of the GARP but not EARP protein complex drives Golgi sterol overload during dendrite remodeling

Membrane trafficking is essential for sculpting neuronal morphology. The GARP and EARP complexes are conserved tethers that regulate vesicle trafficking in the secretory and endolysosomal pathways, respectively. Both complexes contain the Vps51, Vps52, and Vps53 proteins, and a complex-specific protein: Vps54 in GARP and Vps50 in EARP. In Drosophila, we find that both complexes are required for dendrite morphogenesis during developmental remodeling of multidendritic class IV da (c4da) neurons. Having found that sterol accumulates at the trans-Golgi network (TGN) in Vps54KO/KO neurons, we investigated genes that regulate sterols and related lipids at the TGN. Overexpression of oxysterol binding protein (Osbp) or knockdown of the PI4K four wheel drive (fwd) exacerbates the Vps54KO/KO phenotype, whereas eliminating one allele of Osbp rescues it, suggesting that excess sterol accumulation at the TGN is, in part, responsible for inhibiting dendrite regrowth. These findings distinguish the GARP and EARP complexes in neurodevelopment and implicate vesicle trafficking and lipid transfer pathways in dendrite morphogenesis.

Oxysterol-Binding Protein 2 Promotes Pancreatic Ductal Adenocarcinoma Progression Through Epithelial-Mesenchymal Transition Process

Oxysterol-binding protein 2 (OSBP2) is crucial for the promotion of growth and development of cancers, however, its effects in pancreatic ductal adenocarcinoma (PDAC) were still unclear. Here we report the evidence that OSBP2 works as an efficient tumor-associated protein to lead to PDAC extremely malignant characters. We discovered that raised OSBP2 expression in primary tumors was associated with shorter survival in PDAC patients. Therefore, we used immunohistochemistry to analysis the levels of OSBP2 expression in PDAC tissues and adjacent para-cancer tissues. We used wound-healing assay and transwell assay to evaluate the effects of OSBP2 on PDAC cells’ (ASPC-1 and BXPC-3) migration and invasion, respectively. Using CCK-8 and Annexin V / PI double staining, we evaluated the effects of OSBP2 on PDAC cells’ proliferation and apoptosis, respectively. We also explored the effect of OSBP2 on chemosensitivity (gemcitabine and 5-Fluorouracil). And we further validated these findings in vivo of mice. At last, we used western blot to analysis the effect of OSBP2 on PDAC cells’ phenotype. We proved that OSBP2 overexpression promoted PDAC cells’ migration, invasion, proliferation and chemotherapy resistance, decreased apoptosis. OSBP2 overexpression downregulated E-cadherin, and upregulated the levels of N-cadherin, vimentin, Snail, Slug, ZEB1 and β-Catenin. Taken collectively, our findings indicated that OSBP2 exhibited overexpression in PDAC, and upregulation of OSBP2 may promote the progression of PDAC. OSBP2 may have potential diagnostic and therapeutic values in PDAC.

Cortactin knockdown results in disruption of basal TBCs and alters turnover of Sertoli cell ESs in R. norvegicus

Here we explore the prediction that long-term knockdown of cortactin (CTTN), a component of tubulobulbar complexes (TBCs), disrupts TBCs in Sertoli cells and alters the turnover of basal ectoplasmic specializations (ESs). In rats, intratesticular injections of siRNA targeting CTTN (siCTTN) in one testis and non-targeting siRNA (siControl) in the contralateral testis were done on days 0, 2, 4, 6, and 8. The experiment was terminated on day 9 and testes were analyzed by either Western Blotting, or by stimulated emission depletion (STED), electron and/or conventional fluorescence microscopy. Levels of CTTN were successfully knocked down in experimental testes compared to controls. When cryo-sections were labeled for actin filaments, or CTTN, and oxysterol binding protein–related protein 9 (ORP9) and analyzed by STED microscopy, TBCs were “less distinct” than in tubules of the same stages from control testes. When analyzed by electron microscopy, redundant clumps of basal actin filament containing ESs were observed in experimental sections. Using labeling of actin filaments in ESs, thresholding techniques were used to calculate the number of pixels above threshold per unit length of tubule wall in seminiferous tubules at Stage VII. Median values were higher in experimental testes relative to controls in the four animals analyzed. Although we detected subtle differences in ES turnover, we were unable to demonstrate changes in spermatocyte translocation or in the levels of junction proteins at the sites. Our results are the first to demonstrate that perturbation of basal TBCs alters the turnover of actin related junctions (ESs).

Antimicrobial Resistance and Sensitivity of Phytophthora agathidicida

Members of the oomycete genus Phytophthora are highly infectious plant pathogens. P. agathidicida affects the New Zealand native keystone species Agathis australis(kauri) and is the cause of kauri dieback. The complex oomycete lifecycle makes Phytophthora infections hard to manage. The current management of kauri dieback has been limited and antimicrobial resistance is a concern. Phosphite agrichemical preparations are commonly used in the control of Phytophthora diseases, including kauri dieback. However, phosphite is not the only option; the agrichemicals oxathiapiprolin, and the plant-derived natural products polygodial and falcarindiol, have also been shown to have activity against P. agathidicida. The overall goal of this thesis was to further explore aspects of sensitivity and resistance of P. agathidicidatowards these four compounds.In New Zealand, there are three commercially available phosphite preparations, Agri-Fos 600, Phosgard, and Foschek. All previous studies have used Agri-Fos 600, so the first aim was to determine whether the particular formulation altered anti-oomycete activity. No significant difference was found between the 50% inhibitory concentrations (EC50 values) for the three formulations. Interestingly, however, formulating polygodial and falcarindiol with the surfactants and other non-phosphite ingredients of Foschek led to a significant increase in their inhibitory effects. The second aim of this thesis was to implement a serial passaging protocol for P. agathidicida and attempt to isolate mutants with increased resistance to phosphite, polygodial or falcarindiol. Serial passaging was carried out on amended agar plated with increasing concentrations of each chemical. However, even after 7 passages, over 16-18 weeks of growth, no mutants with increased resistance were isolated. This could be due to the complicated modes of action of the polygodial, falcarindioland phosphite, which makes it likely that several specific mutations are required to effect resistance. <br>IIOxathiapiprolin is a highly potent, new anti-oomycete agrichemical. It targets the Phytophthora oxysterol binding protein (OSBP) related protein (ORP1). Mutations in this protein are known to give oxathiapiprolin resistance in other species of Phytophthora; however, the P. agathidicida protein (PaORP1) has never been studied. In this work, the gene for PaORP1 was partially sequenced from five P. agathidicida isolates. None contained any of the known resistance mutations. A new protocol for expressing PaORP1 in E. coli and purifying it using immobilised metal affinity chromatography was also developed. After optimisation, this protocol yielded up to 30 mg of purified protein per litre of E. coli culture and is the first successful example of heterologously expressing and purifying any P. agathidicida protein. In future, this will allow the biomolecular interaction between PaORP1 and oxathiapiprolin to be studied in more detail. Overall, the work presented in this thesis assessed commercial formulations of phosphite, established a directed evolution protocol for studying resistance in P. agathidicida, and reported the first in vitro characterisation of a P. agathidicidaprotein. This research suggests that commercial formulation of plant-derived natural products may be a powerful new approach for combatting kauri dieback and, promisingly, also suggests that the risk of developing resistance to these compounds might be low.

Antimicrobial Resistance and Sensitivity of Phytophthora agathidicida

Members of the oomycete genus Phytophthora are highly infectious plant pathogens. P. agathidicida affects the New Zealand native keystone species Agathis australis(kauri) and is the cause of kauri dieback. The complex oomycete lifecycle makes Phytophthora infections hard to manage. The current management of kauri dieback has been limited and antimicrobial resistance is a concern. Phosphite agrichemical preparations are commonly used in the control of Phytophthora diseases, including kauri dieback. However, phosphite is not the only option; the agrichemicals oxathiapiprolin, and the plant-derived natural products polygodial and falcarindiol, have also been shown to have activity against P. agathidicida. The overall goal of this thesis was to further explore aspects of sensitivity and resistance of P. agathidicidatowards these four compounds.In New Zealand, there are three commercially available phosphite preparations, Agri-Fos 600, Phosgard, and Foschek. All previous studies have used Agri-Fos 600, so the first aim was to determine whether the particular formulation altered anti-oomycete activity. No significant difference was found between the 50% inhibitory concentrations (EC50 values) for the three formulations. Interestingly, however, formulating polygodial and falcarindiol with the surfactants and other non-phosphite ingredients of Foschek led to a significant increase in their inhibitory effects. The second aim of this thesis was to implement a serial passaging protocol for P. agathidicida and attempt to isolate mutants with increased resistance to phosphite, polygodial or falcarindiol. Serial passaging was carried out on amended agar plated with increasing concentrations of each chemical. However, even after 7 passages, over 16-18 weeks of growth, no mutants with increased resistance were isolated. This could be due to the complicated modes of action of the polygodial, falcarindioland phosphite, which makes it likely that several specific mutations are required to effect resistance. <br>IIOxathiapiprolin is a highly potent, new anti-oomycete agrichemical. It targets the Phytophthora oxysterol binding protein (OSBP) related protein (ORP1). Mutations in this protein are known to give oxathiapiprolin resistance in other species of Phytophthora; however, the P. agathidicida protein (PaORP1) has never been studied. In this work, the gene for PaORP1 was partially sequenced from five P. agathidicida isolates. None contained any of the known resistance mutations. A new protocol for expressing PaORP1 in E. coli and purifying it using immobilised metal affinity chromatography was also developed. After optimisation, this protocol yielded up to 30 mg of purified protein per litre of E. coli culture and is the first successful example of heterologously expressing and purifying any P. agathidicida protein. In future, this will allow the biomolecular interaction between PaORP1 and oxathiapiprolin to be studied in more detail. Overall, the work presented in this thesis assessed commercial formulations of phosphite, established a directed evolution protocol for studying resistance in P. agathidicida, and reported the first in vitro characterisation of a P. agathidicidaprotein. This research suggests that commercial formulation of plant-derived natural products may be a powerful new approach for combatting kauri dieback and, promisingly, also suggests that the risk of developing resistance to these compounds might be low.