bZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection.

IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.

Soybean Mosaic Virus 6K1 Interactors Screening and GmPR4 and GmBI1 Function Characterization.

Host proteins are essential during virus infection, and viral factors must target numerous host factors to complete their infectious cycle. The mature 6K1 protein of potyviruses is required for viral replication in plants. However, the interaction between 6K1 and host factors is poorly understood. The present study aims to identify the host interacting proteins of 6K1. Here, the 6K1 of Soybean mosaic virus (SMV) was used as the bait to screen a soybean cDNA library to gain insights about the interaction between 6K1 and host proteins. One hundred and twenty-seven 6K1 interactors were preliminarily identified, and they were classified into six groups, including defense-related, transport-related, metabolism-related, DNA binding, unknown, and membrane-related proteins. Then, thirty-nine proteins were cloned and merged into a prey vector to verify the interaction with 6K1, and thirty-three of these proteins were confirmed to interact with 6K1 by yeast two-hybrid (Y2H) assay. Of the thirty-three proteins, soybean pathogenesis-related protein 4 (GmPR4) and Bax inhibitor 1 (GmBI1) were chosen for further study. Their interactions with 6K1 were also confirmed by bimolecular fluorescence complementation (BiFC) assay. Subcellular localization showed that GmPR4 was localized to the cytoplasm and endoplasmic reticulum (ER), and GmBI1 was located in the ER. Moreover, both GmPR4 and GmBI1 were induced by SMV infection, ethylene and ER stress. The transient overexpression of GmPR4 and GmBI1 reduced SMV accumulation in tobacco, suggesting their involvement in the resistance to SMV. These results would contribute to exploring the mode of action of 6K1 in viral replication and improve our knowledge of the role of PR4 and BI1 in SMV response.

Bax inhibitor 1 is a γ-secretase-independent presenilin-binding protein.

Presenilin is the catalytic subunit of γ-secretase, a four-component intramembrane protease responsible for the generation of ß-amyloid (Aß) peptides. Over 200 Alzheimer's disease-related mutations have been identified in presenilin 1 (PS1) and PS2. Here, we report that Bax-inhibitor 1 (BI1), an evolutionarily conserved transmembrane protein, stably associates with PS1. BI1 specifically interacts with PS1 in isolation, but not with PS1 in the context of an assembled γ-secretase. The PS1-BI1 complex exhibits no apparent proteolytic activity, as judged by the inability to produce Aß40 and Aß42 from the substrate APP-C99. At an equimolar concentration, BI1 has no impact on the proteolytic activity of γ-secretase; at a 200-fold molar excess, BI1 reduces γ-secretase activity nearly by half. Our biochemical study identified BI1 as a PS1-interacting protein, suggesting additional functions of PS1 beyond its involvement in γ-secretase.

The Bax inhibitor UvBI-1, a negative regulator of mycelial growth and conidiation, mediates stress response and is critical for pathogenicity of the rice false smut fungus Ustilaginoidea virens.

Bax inhibitor-1 (BI-1), an evolutionarily conserved protein, is a suppressor of cell death induced by the proapoptotic protein Bax and is involved in the response to biotic and abiotic stress in animals, plants and yeast. Rice false smut caused by Ustilaginoidea virens is one of the destructive rice diseases worldwide. Although BI-1 proteins are widely distributed across filamentous fungi, few of them are functionally characterized. In this study, we identified a BI-1 protein in U. virens, UvBI-1, which contains a predicted Bax inhibitor-1-like family domain and could suppress the cell death induced by Bax. By co-transformation of the CRISPR/Cas9 construct along with donor DNA fragment containing the hygromycin resistance gene, we successfully generated Uvbi-1 deletion mutants. The UvBI-1 deletion showed an increase in mycelia vegetative growth and conidiation, suggesting this gene acts as a negative regulator of the growth and conidiation. In addition, the Uvbi-1 mutants exhibited higher sensitivity to osmotic and salt stress, hydrogen peroxide stress, and cell wall or membrane stress than the wild-type strain. Furthermore, UvBI-1 deletion was found to cause increased production of secondary metabolites and loss of pathogenicity of U. virens. Taken together, our results demonstrate that UvBI-1 plays a negative role in mycelial growth and conidiation, and is critical for stress tolerance, cell wall integrity, secondary metabolites production and pathogenicity of U. virens. Therefore, this study provides new evidence on the conserved function of BI-1 among fungal organisms and other species.

Evaluation on the antiviral activity of genipin against white spot syndrome virus in crayfish.

White spot syndrome virus (WSSV) is a serious epidemic pathogen of crustaceans and cause severe economic losses to aquaculture. However, no commercial drugs presently available to control WSSV infection. Genipin (GN) is a bioactive compound extracted from the fruit of Gardenia jasminoides and exhibits potential antiviral activity. In the study, the antiviral activity of GN against WSSV was investigated in crayfish Procambarus clarkii and in shrimp Litopenaeus vannamei. In vitro antiviral test showed that GN could inhibit WSSV replication in crayfish and in shrimp, and the highest inhibition on WSSV was over 99% when treatment with 50 mg/kg of GN for 24 h. In vivo antiviral test proved that GN could be used to treat and prevent WSSV infection. GN could also effectively protect crayfish from WSSV infection by reducing the mortality rate of WSSV-infected crayfish. Moreover, GN attenuated the WSSV-induced oxidative stress and inflammatory by upregulation the expression of antioxidant-related genes and downregulation the expression of inflammatory-related genes, respectively. Mechanically, GN inhibited WSSV replication at least via decreasing STAT (signal transducer and activator of transcription) gene expression to block WSSV immediate-early gene ie1 transcription. Additionally, the inhibition of BI-1 (Bax inhibitor-1) gene expression also played an important role in the suppression of WSSV infection. In conclusion, GN represented a potential therapeutic and preventive agent to block WSSV infection.

Arabidopsis Bax inhibitor-1 interacts with enzymes related to very-long-chain fatty acid synthesis.

Bax inhibitor-1 (BI-1) is a widely conserved cell death regulator that confers resistance to environmental stress in plants. Previous studies suggest that Arabidopsis thaliana BI-1 (AtBI-1) modifies sphingolipids by interacting with cytochrome b5 (AtCb5), an electron-transfer protein. To reveal how AtBI-1 regulates sphingolipid synthesis, we screened yeast sphingolipid-deficient mutants and identified yeast ELO2 and ELO3 as novel enzymes that are essential for AtBI-1 function. ELO2 and ELO3 are condensing enzymes that synthesize very-long-chain fatty acids (VLCFAs), major fatty acids in plant sphingolipids. In Arabidopsis, we identified four ELO homologs (AtELO1-AtELO4), localized in the endoplasmic reticulum membrane. Of those AtELOs, AtELO1 and AtELO2 had a characteristic histidine motif and were bound to AtCb5-B. This result suggests that AtBI-1 interacts with AtELO1 and AtELO2 through AtCb5. AtELO2 and AtCb5-B also interact with KCR1, PAS2, and CER10, which are essential for the synthesis of VLCFAs. Therefore, AtELO2 may participate in VLCFA synthesis with AtCb5 in Arabidopsis. In addition, our co-immunoprecipitation/mass spectrometry analysis demonstrated that AtBI-1 forms a complex with AtELO2, KCR1, PAS2, CER10, and AtCb5-D. Furthermore, AtBI-1 contributes to the rapid synthesis of 2-hydroxylated VLCFAs in response to oxidative stress. These results indicate that AtBI-1 regulates VLCFA synthesis by interacting with VLCFA-synthesizing enzymes.

TMBIM-mediated Ca2+ homeostasis and cell death.

Ca2+ is a ubiquitous intracellular messenger that regulates numerous physiological activities in humans, animals, plants, and bacteria. Cytosolic Ca2+ is kept at a low level, but subcellular organelles such as the endoplasmic reticulum (ER) and Golgi apparatus maintain high-concentration Ca2+ stores. Under resting conditions, store Ca2+ homeostasis is dynamically regulated to equilibrate between active Ca2+ uptake and passive Ca2+ leak processes. The evolutionarily conserved Transmembrane BAX Inhibitor-1 Motif-containing (TMBIM) proteins mediate Ca2+ homeostasis and cell death. This review focuses on recent advances in functional and structural analysis of TMBIM proteins in regulation of the two related functions. The roles of TMBIM proteins in pathogen infection and cancer are also discussed with prospects for treatment. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.

Wheat Bax Inhibitor-1 interacts with TaFKBP62 and mediates response to heat stress.

BACKGROUND: Heat stress is a severe environmental stress that affects plant growth and reduces yield. Bax inhibitor-1 (BI-1) is a cytoprotective protein that is involved in the response to biotic and abiotic stresses. The Arabidopsis (Arabidopsis thaliana) BI-1 mutants atbi1-1 and atbi1-2 are hypersensitive to heat stress, and AtBI-1 overexpression rescues thermotolerance deficiency in atbi1 plants. Nevertheless, the mechanism of BI-1 in plant thermotolerance is still unclear. RESULTS: We identified a wheat (Triticum aestivum L.) BI-1 gene, TaBI-1.1, which was highly upregulated in an RNA sequencing (RNA-seq) analysis of heat-treated wheat. The upregulation of TaBI-1.1 under heat stress was further demonstrated by real time quantitative PCR (qRT-PCR) and ß-glucuronidase (GUS) staining. Compared with the wild type Col-0, the atbi1-2 mutant is hypersensitive to heat stress, and constitutive expression of TaBI-1.1 in atbi1-2 (35S::TaBI-1.1/ atbi1-2) rescued the deficiency of atbi1-2 under heat stress. Furthermore, we identified TaFKBP62 as a TaBI-1.1-interacting protein that co-localized with TaBI-1.1 on the endoplasmic reticulum (ER) membrane and enhanced heat stress tolerance. Additionally, HSFA2, HSFB1, ROF1, HSP17.4B, HSP17.6A, HSP17.8, HSP70B, and HSP90.1 expression levels were suppressed in atbi1-2 plants under heat stress. In contrast, 35S::TaBI-1.1/atbi1-2 relieved the inhibitory effect of AtBI-1 loss of function. CONCLUSIONS: TaBI-1.1 interacted with TaFKBP62 and co-localized with TaFKBP62 on the ER membrane. Both TaBI-1.1 and AtBI-1 regulated the expression of heat-responsive genes and were conserved in plant thermotolerance.

New insights into the phylogeny of the TMBIM superfamily across the tree of life: Comparative genomics and synteny networks reveal independent evolution of the BI and LFG families in plants.

The Transmembrane BAX Inhibitor Motif containing (TMBIM) superfamily, divided into BAX Inhibitor (BI) and Lifeguard (LFG) families, comprises a group of cytoprotective cell death regulators conserved in prokaryotes and eukaryotes. However, no research has focused on the evolution of this superfamily in plants. We identified 685 TMBIM proteins in 171 organisms from Archaea, Bacteria, and Eukarya, and provided a phylogenetic overview of the whole TMBIM superfamily. Then, we used orthology and synteny network analyses to further investigate the evolution and expansion of the BI and LFG families in 48 plants from diverse taxa. Plant BI family forms a single monophyletic group; however, monocot BI sequences transposed to another genomic context during evolution. Plant LFG family, which expanded trough whole genome and tandem duplications, is subdivided in LFG I, LFG IIA, and LFG IIB major phylogenetic groups, and retains synteny in angiosperms. Moreover, two orthologous groups (OGs) are shared between bryophytes and seed plants. Other several lineage-specific OGs are present in plants. This work clarifies the phylogenetic classification of the TMBIM superfamily across the three domains of life. Furthermore, it sheds new light on the evolution of the BI and LFG families in plants providing a benchmark for future research.

Bax inhibitor-1 from orange spotted grouper, Epinephelus coioides involved in viral infection.

Bax inhibitor-1 (BI-1) is a conserved anti-apoptotic protein that suppresses endoplasmic reticulum (ER) stress-induced cell death. However, the function of fish BI-1 is not quite clear. In the present study, a bi-1 homolog (Ecbi-1) from orange spotted grouper, Epinephelus coioides was identified and its roles in viral infection were investigated. EcBI-1 encoded 237 amino acids protein, contained six transmembrane regions and a conservative C-terminus motif. Ecbi-1 predominantly expressed in kidney and spleen of healthy grouper. After SGIV stimulation, Ecbi-1 transcript was significantly increased in vitro. Subcellular localization analysis revealed that EcBI-1 was localized throughout the cytoplasm and co-localized with ER. Furthermore, overexpression of EcBI-1 suppressed SGIV infection induced cell death, caspase-3 activity and viral genes transcription. And C-terminus motif was critical for regulation roles of EcBI-1 during SGIV infection. In addition, EcBI-1 could interact with EcBNIP3 in vitro. Together, our data firstly demonstrated that fish BI-1 play important roles in response to viral infection.

PI3K signaling pathways modulated white spot syndrome virus (WSSV) replication in Procambarus clarkii.

The PI3K/AKT signaling pathway is commonly exploited to regulate viral replication and affect the fate of infected cells. In the present study, a PI3K-specific inhibitor (LY294002) was employed to pretreat crayfish to evaluate the effects of PI3K/AKT signaling pathway in WSSV replication. The results showed that the WSSV copy numbers in crayfish pretreated with LY294002 were significantly lower than those in Tris-HCl pretreatment crayfish on the sixth and tenth day after WSSV infection. In semigranular cells, the apoptosis rates were up-regulated on the third day post-WSSV infection, and a significantly lower proportion of apoptosis cells were observed in LY294002-pretreatment group. The expression level of Bax, Bax inhibitor-1 and lectin mRNA in haemocytes of crayfish were increased after WSSV infection. After the secondary stimulation with Tris-HCl, the Bax expression level in LY294002-pretreatment crayfish was significantly higher than that of crayfish pretreated with Tris-HCl on the third or sixth day, but the Toll and lectin mRNA expression decreased significantly on the third, sixth and tenth day. The Bax mRNA expression levels in LY294002-WSSV group were significantly higher than those in Tris-HCl-WSSV group on the third and tenth day. The Bax inhibitor-1 mRNA expression levels in LY294002-WSSV group were significantly lower than those in Tris-HCl-WSSV crayfish on the third day. These results together indicated that the hosts PI3K/AKT signaling pathway play positive roles in WSSV replication through the balance between host cell apoptois and innate immune responses. This information is helpful to further understand the role of PI3K/AKT signaling pathway on WSSV replication in Decapoda crustaceans.

A novel VIGS method by agroinoculation of cotton seeds and application for elucidating functions of GhBI-1 in salt-stress response.

KEY MESSAGE: A VIGS method by agroinoculation of cotton seeds was developed for gene silencing in young seedlings and roots, and applied in functional analysis of GhBI-1 in response to salt stress. Virus-induced gene silencing (VIGS) has been widely used to investigate the functions of genes expressed in mature leaves, but not yet in young seedlings or roots of cotton (Gossypium hirsutum L.). Here, we developed a simple and effective VIGS method for silencing genes in young cotton seedlings and roots by soaking naked seeds in Agrobacterium cultures carrying tobacco rattle virus (TRV)-VIGS vectors. When the naked seeds were soaked in Agrobacterium cultures with an OD600 of 1.5 for 90 min, it was optimal for silencing genes effectively in young seedlings as clear photo-bleaching phenotype in the newly emerging leaves of pTRV:GhCLA1 seedlings were observed at 12-14 days post inoculation. Silencing of GhPGF (cotton pigment gland formation) by this method resulted in a 90% decrease in transcript abundances of the gene in roots at the early development stage. We further used the tool to investigate function of GhBI-1 (cotton Bax inhibitor-1) gene in response to salt stress and demonstrated that GhBI-1 might play a protective role under salt stress by suppressing stress-induced cell death in cotton. Our results showed that the newly established VIGS method is a powerful tool for elucidating functions of genes in cotton, especially the genes expressed in young seedlings and roots.

Bax Inhibitor-1 Acts as an Anti-Influenza Factor by Inhibiting ROS Mediated Cell Death and Augmenting Heme-Oxygenase 1 Expression in Influenza Virus Infected Cells.

Influenza virus remains a major health concern worldwide, and there have been continuous efforts to develop effective antivirals despite the use of annual vaccination programs. The purpose of this study was to determine the anti-influenza activity of Bax inhibitor-1 (BI-1). Madin-Darby Canine Kidney (MDCK) cells expressing wild type BI-1 and a non-functional BI-1 mutant, BI-1 ∆C (with the C-terminal 14 amino acids deleted) were prepared and infected with A/PR/8/34 influenza virus. BI-1 overexpression led to the suppression of virus-induced cell death and virus production compared to control Mock or BI-1 ∆C overexpression. In contrast to BI-1 ∆C-overexpressing cells, BI-1-overexpressing cells exhibited markedly reduced virus-induced expression of several viral genes, accompanied by a substantial decrease in ROS production. We found that treatment with a ROS scavenging agent, N-acetyl cysteine (NAC), led to a dramatic decrease in virus production and viral gene expression in control MDCK and BI-1 ∆C-overexpressing cells. In contrast, NAC treatment resulted in the slight additional suppression of virus production and viral gene expression in BI-1-overexpressing cells but was statistically significant. Moreover, the expression of heme oxygenase-1 (HO-1) was also significantly increased following virus infection in BI-1-overexpressing cells compared to control cells. Taken together, our data suggest that BI-1 may act as an anti-influenza protein through the suppression of ROS mediated cell death and upregulation of HO-1 expression in influenza virus infected MDCK cells.

Melatonin restricts Pb-induced PCD by enhancing BI-1 expression in tobacco suspension cells.

Melatonin is a conserved substance, which was discovered in the evolutionary distant organisms like bacteria, plants, invertebrates and vertebrates. Recent studies have shown that melatonin despite its possible role in photoperiod processes, has been found to be a direct free radical scavenger and an indirect antioxidant. In this report the impact of exogenous melatonin on the Bax inhibitor-1 (BI-1) expression level in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) suspension cells exposed to lead was examined. BI-1 is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. Our results showed that pretreatment with 200 nm melatonin, expressing BI-1 and fortified tobacco suspension cells against damages induced by lead. The obtained results revealed, that melatonin significantly increases BY-2 cells proliferation and protects BY-2 cells against death. Moreover, the conducted analyses showed for the first time that the protective effect of melatonin may be connected not only with its antioxidant properties but also with its direct impact on elevating BI-1 expression and lead-induced programmed cell death (PCD) restriction.

Salicylic acid induced cysteine protease activity during programmed cell death in tomato plants.

The hypersensitive response (HR), a type of programmed cell death (PCD) during biotic stress is mediated by salicylic acid (SA). The aim of this work was to reveal the role of proteolysis and cysteine proteases in the execution of PCD in response of SA. Tomato plants were treated with sublethal (0.1 mM) and lethal (1 mM) SA concentrations through the root system. Treatment with 1 mM SA increased the electrolyte leakage and proteolytic activity and reduced the total protein content of roots after 6 h, while the proteolytic activity did not change in the leaves and in plants exposed to 0.1 mM SA. The expression of the papain-type cysteine protease SlCYP1, the vacuolar processing enzyme SlVPE1 and the tomato metacaspase SlMCA1 was induced within the first three hours in the leaves and after 0.5 h in the roots in the presence of 1 mM SA but the transcript levels did not increase significantly at sublethal SA. The Bax inhibitor-1 (SlBI-1), an antiapoptotic gene was over-expressed in the roots after SA treatments and it proved to be transient in the presence of sublethal SA. Protease inhibitors, SlPI2 and SlLTC were upregulated in the roots by sublethal SA but their expression remained low at 1 mM SA concentration. It is concluded that in contrast to leaves the SA-induced PCD is associated with increased proteolytic activity in the root tissues resulting from a fast up-regulation of specific cysteine proteases and down-regulation of protease inhibitors.

Human and viral Golgi anti-apoptotic proteins (GAAPs) oligomerize via different mechanisms and monomeric GAAP inhibits apoptosis and modulates calcium.

Golgi anti-apoptotic proteins (GAAPs) are hydrophobic proteins resident in membranes of the Golgi complex. They protect cells from a range of apoptotic stimuli, reduce the Ca(2+) content of intracellular stores, and regulate Ca(2+) fluxes. GAAP was discovered in camelpox virus, but it is highly conserved throughout evolution and encoded by all eukaryote genomes examined. GAAPs are part of the transmembrane Bax inhibitor-containing motif (TMBIM) family that also includes other anti-apoptotic and Ca(2+)-modulating membrane proteins. Most TMBIM members show multiple bands when analyzed by SDS-PAGE, suggesting that they may be oligomeric. However, the molecular mechanisms of oligomerization, the native state of GAAPs in living cells and the functional significance of oligomerization have not been addressed. TMBIM members are thought to have evolved from an ancestral GAAP. Two different GAAPs, human (h) and viral (v)GAAP were therefore selected as models to examine oligomerization of TMBIM family members. We show that both hGAAP and vGAAP in their native states form oligomers and that oligomerization is pH-dependent. Surprisingly, hGAAP and vGAAP do not share the same oligomerization mechanism. Oligomerization of hGAAP is independent of cysteines, but oligomerization of vGAAP depends on cysteines 9 and 60. A mutant vGAAP that is unable to oligomerize revealed that monomeric vGAAP retains both its anti-apoptotic function and its effect on intracellular Ca(2+) stores. In conclusion, GAAP can oligomerize in a pH-regulated manner, and monomeric GAAP is functional.

ER stress-induced cell death mechanisms.

The endoplasmic-reticulum (ER) stress response constitutes a cellular process that is triggered by a variety of conditions that disturb folding of proteins in the ER. Eukaryotic cells have developed an evolutionarily conserved adaptive mechanism, the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. In cases where ER stress cannot be reversed, cellular functions deteriorate, often leading to cell death. Accumulating evidence implicates ER stress-induced cellular dysfunction and cell death as major contributors to many diseases, making modulators of ER stress pathways potentially attractive targets for therapeutics discovery. Here, we summarize recent advances in understanding the diversity of molecular mechanisms that govern ER stress signaling in health and disease. This article is part of a Special Section entitled: Cell Death Pathways.

CYP83A1 is required for metabolic compatibility of Arabidopsis with the adapted powdery mildew fungus Erysiphe cruciferarum.

Aliphatic glucosinolates function in the chemical defense of Capparales. The cytochrome P450 83A1 monooxygenase (CYP83A1) catalyzes the initial conversion of methionine-derived aldoximes to thiohydroximates in the biosynthesis of glucosinolates, and thus cyp83a1 mutants have reduced levels of aliphatic glucosinolates. Loss of CYP83A1 function leads to dramatically reduced parasitic growth of the biotrophic powdery mildew fungus Erysiphe cruciferarum on Arabidopsis thaliana. The cyp83a1 mutants support less well the germination and appressorium formation of E. cruciferarum on the leaf surface and post-penetration conidiophore formation by the fungus. By contrast, a myb28-1 myb29-1 double mutant, which totally lacks aliphatic glucosinolates, shows a wild-type level of susceptibility to E. cruciferarum. The cyp83a1 mutants also lack very-long-chain aldehydes on their leaf surface. Such aldehydes support appressorium formation by E. cruciferarum in vitro. In addition, when chemically complemented with the C26 aldehyde n-hexacosanal, cyp83a1 mutants can again support appressorium formation. The mutants further accumulate 5-methylthiopentanaldoxime, the potentially toxic substrate of CYP83A1. Loss of powdery mildew susceptibility by cyp83a1 may be explained by a reduced supply of the fungus with inductive signals from the host and an accumulation of potentially fungitoxic metabolites.

UvATG6 Interacts with BAX Inhibitor 1 Proteins and Plays Critical Roles in Growth, Conidiation, and Virulence in Ustilaginoidea virens.

Autophagy and apoptosis are evolutionarily conserved catabolic processes involved in regulating development and cellular homeostasis. Bax inhibitor 1 (BI-1) and autophagy protein 6 (ATG6) perform essential functions in these roles, such as cellular differentiation and virulence in various filamentous fungi. However, the functions of ATG6 and BI-1 proteins in development and virulence in the rice false smut fungus Ustilaginoidea virens are still poorly understood. In this study, UvATG6 was characterized in U. virens. The deletion of UvATG6 almost abolished autophagy in U. virens and reduced growth, conidial production and germination, and virulence. Stress tolerance assays showed that UvATG6 mutants were sensitive to hyperosmotic, salt, and cell wall integrity stresses but were insensitive to oxidative stress. Furthermore, we found that UvATG6 interacted with UvBI-1 or UvBI-1b and suppressed Bax-induced cell death. We previously found that UvBI-1 could suppress Bax-induced cell death and was a negative regulator of mycelial growth and conidiation. Unlike UvBI-1, UvBI-1b could not suppress cell death. UvBI-1b-deleted mutants exhibited decreased growth and conidiation, while the UvBI-1 and UvBI-1b double deletion reduced the phenotype, indicating that UvBI-1 and UvBI-1b antagonistically regulate mycelial growth and conidiation. In addition, the UvBI-1b and double mutants exhibited decreased virulence. Our results provide evidence of the cross talk of autophagy and apoptosis in U. virens and give clues for studying other phytopathogenic fungi. IMPORTANCE Ustilaginoidea virens causes destructive panicle disease in rice, significantly threatening agricultural production. UvATG6 is required for autophagy and contributes to growth, conidiation, and virulence in U. virens. Additionally, it interacts with the Bax inhibitor 1 proteins UvBI-1 and UvBI-1b. UvBI-1 suppresses cell death induced by Bax, unlike UvBI-1b. UvBI-1 negatively regulates growth and conidiation, while UvBI-1b is required for these phenotypes. These results indicate that UvBI-1 and UvBI-1b may antagonistically regulate growth and conidiation. In addition, both of them contribute to virulence. Additionally, our results suggest cross talk between autophagy and apoptosis, contributing to the development, adaptability, and virulence of U. virens.

[Long noncoding RNA H19 promotes vascular calcification by repressing the Bax inhibitor 1/optic atrophy 1 pathway].

OBJECTIVE: To investigate whether long noncoding RNA H19 (lncRNA H19) induces vascular calcification by promoting calcium deposition, osteogenic differentiation and apoptosis via inhibiting the Bax inhibitor 1/optic atrophy 1 (BI-1/ OPA1) pathway. METHODS: ß-glycerophosphate and calcium chloride were used to induce calcification in rat vascular smooth muscle cells (VSMCs), and the effects of siH19, alone or in combination with BI-1 or OPA1 knockdown, on calcification of the cells were investigated. Osteogenic differentiation was assessed by measuring Runt-related transcription factor 2 (Runx-2) and bone morphogenetic protein 2 (BMP-2) expression with Western blotting, and cell apoptosis was evaluated by TUNEL staining and Western blotting. An ApoE-/- diabetic mouse model with high-fat feeding for 32 weeks were given an intraperitoneal injection of siH19, and the changes in calcium deposition in the aortic arch were examined using Alizarin red S staining and von Kossa staining. RESULTS: In rat VSMCs with calcification, the expression of lncRNA H19 was significantly increased, and the expressions of BI- 1 and OPA1 were significantly decreased. Downregulation of lncRNA H19 significantly increased the expressions of BI-1 and OPA1 proteins in the cells, and BI-1 knockdown further reduced OPA1 expression (P<0.001). The cells treated with siH19 showed total disappearance of the calcified nodules with significantly reduced expressions of Runx-2, BMP-2 and cleaved caspase-3 and a lowered cell apoptosis rate (P<0.001). Calcified nodules were again observed in the cells with lncRNA H19 knockdown combined with BI-1 or OPA1 knockdown, and the expressions of Runx-2, BMP-2, cleaved-caspase-3 and cell apoptosis rate all significantly increased (P<0.001). In the diabetic mouse model with high-fat feeding, siH19 treatment significantly reduced the calcification area and increased mRNA expressions of BI-I and OPA1 in the aortic arch. CONCLUSION: LncRNA H19 promotes vascular calcification possibly by promoting calcium deposition, osteogenic differentiation and cell apoptosis via inhibiting the BI-1/OPA1 pathway.