Vacuolar Protein-Sorting Receptor MoVps13 Regulates Conidiation and Pathogenicity in Rice Blast Fungus Magnaporthe oryzae

Magnaporthe oryzae (synonym Pyricularia oryzae) is a filamentous fungal pathogen that causes major yield losses in cultivated rice worldwide. However, the mechanisms of infection of M. oryzae are not well characterized. The VPS13 proteins play vital roles in various biological processes in many eukaryotic organisms, including in the organization of actin cytoskeleton, vesicle trafficking, mitochondrial fusion, and phagocytosis. Nevertheless, the function of the Vps13 protein in plant pathogenic fungi has not been explored. Here, we analysed the biological functions of the Vps13 protein in the development and pathogenicity of M. oryzae. Deletion mutants of MoVps13 significantly reduced the conidiation and decreased the rate of fungal infection on hosts. Moreover, the loss of MoVps13 resulted in defective cell wall integrity (CWI) and plasma membrane (PM) homeostasis when treated with chemicals for inducing cell wall stress (200 mg/mL Congo Red or 0.005% SDS) and sphingolipid synthesis inhibitors (2 μM myriocin or 2 μM amphotericin B). This indicated that MoVps13 is also involved in cell wall synthesis and sphingolipid synthesis. Through immunoblotting, autophagic flux detection, co-localization, and chemical drug sensitivity assays, we confirmed the involvement of Movps13 in ER-phagy and the response to ER stress. Additionally, we generated the C-terminal structure of MoVps13 with high accuracy using the alphaflod2 database. Our experimental evidence indicates that MoVps13 is an important virulence factor that regulates the pathogenicity of M. oryzae by controlling CWI, lipid metabolism and the ER-phagy pathway. These results have expanded our knowledge about pathogenic fungi and will help exploration for novel therapeutic strategies against the rice blast fungus.

Coding Mutations in Vacuolar Protein-Sorting 4 AAA+ ATPase Endosomal Sorting Complexes Required for Transport Protein Homologs Underlie bc-2 and New bc-4 Gene Conferring Resistance to Bean Common Mosaic Virus in Common Bean

Bean common mosaic virus (BCMV) is a major disease in common bean (Phaseolus vulgaris L.). Host plant resistance is the most effective strategy to minimize crop damage against BCMV and the related Bean common mosaic necrosis virus (BCMNV). To facilitate breeding for resistance, we sought to identify candidate genes and develop markers for the bc-2 gene and the unknown gene with which it interacts. Genome-wide association study (GWAS) of the Durango Diversity Panel (DDP) identified a peak region for bc-2 on chromosome Pv11. Haplotype mapping narrowed the bc-2 genomic interval and identified Phvul.011G092700, a vacuolar protein-sorting 4 (Vps4) AAA+ ATPase endosomal sorting complexes required for transport (ESCRT) protein, as the bc-2 candidate gene. The race Durango Phvul.011G092700 gene model, bc-2[UI111], contains a 10-kb deletion, while the race Mesoamerican bc-2[Robust] consists of a single nucleotide polymorphism (SNP) deletion. Each mutation introduces a premature stop codon, and they exhibit the same interaction with the pathogroups (PGs) tested. Phvul.005G125100, another Vps4 AAA+ ATPase ESCRT protein, was identified as the candidate gene for the new recessive bc-4 gene, and the recessive allele is likely an amino acid substitution in the microtubule interacting and transport (MIT) domain. The two Vps4 AAA+ ATPase ESCRT proteins exhibit high similarity to the Zym Cucsa.385040 candidate gene associated with recessive resistance to Zucchini yellow mosaic virus in cucumber. bc-2 alone has no resistance effect but, when combined with bc-4, provides resistance to BCMV (except PG-V) but not BCMNV, and, when combined with bc-ud, provides resistance to BCMV (except BCMV PG-VII) and BCMNV. So instead of different resistance alleles (i.e., bc-2 and bc-22), there is only bc-2 with a differential reaction based on whether it is combined with bc-4 or bc-ud, which are tightly linked in repulsion. The new tools and enhanced understanding of this host-virus pathogen interaction will facilitate breeding common beans for resistance to BCMV and BCMNV.

Multiple genes in cis mediate the effects of a single chromatin accessibility variant on aberrant synaptic development and function in human neurons

Despite hundreds of risk loci from genome-wide association studies of neuropsychiatric disorders, causal variants/genes remain largely unknown. Here, in NEUROG2-induced human neurons, we identified 31 risk SNPs in 26 schizophrenia (SZ) risk loci that displayed allele-specific open chromatin (ASoC) and were likely to be functional. Editing the strongest ASoC SNP rs2027349 near vacuolar protein sorting 45 homolog (VPS45) altered the expression of VPS45, lncRNA AC244033.2, and a distal gene, C1orf54, in human neurons. Notably, the global gene expression changes in neurons were enriched for SZ risk and correlated with post-mortem brain gene expression signatures of neuropsychiatric disorders. Neurons carrying the risk allele exhibited increased dendritic complexity, synaptic puncta density, and hyperactivity, which were reversed by knocking-down distinct cis-regulated genes (VPS45, AC244033.2, or C1orf54), suggesting a phenotypic contribution from all three genes. Interestingly, transcriptomic analysis of knockdown cells suggested a non-additive effects of these genes. Our study reveals a compound effect of multiple genes at a single SZ locus on synaptic development and function, providing a mechanistic link between a non-coding SZ risk variant and disease-related cellular phenotypes.

Characterization of individual HIV-1 budding event using ultra-fast atomic force microscopy reveals a multiplexed role for VPS4

The assembly and budding of newly formed human immunodeficiency virus-1 (HIV-1) particles occur at the plasma membrane of infected cells. Whereas the molecular basis for viral budding has been studied extensively, investigation of its spatiotemporal characteristics has been limited by the small dimensions (< 100 nm) of HIV particles and the fast kinetics of the process (a few minutes from bud formation to virion release). Here we applied ultra-fast atomic force microscopy to achieve real-time visualization of individual HIV-1 budding events from wildtype (WT) cell lines as well as from mutated lines lacking vacuolar protein sorting-4 (VPS4) or visceral adipose tissue-1 protein (VTA1). Using single-particle analysis, we show that HIV-1 bud formation follows two kinetic pathways (fast and slow) with each composed of three distinct phases (growth, stationary, decay). Notably, approximately 30% of events did not result in viral release and were characterized by the formation of short (rather than tall) particles that slowly decayed back into the cell membrane. These non-productive events became more abundant in VPS4 knockout cell lines. Strikingly, the absence of VPS4B, rather than VPS4A, increased the production of short viral particles, suggesting a role for VPS4B in earlier stages of HIV-1 budding than traditionally thought.

The Importance of Vacuolar Ion Homeostasis and Trafficking in Hyphal Development and Virulence in Candida albicans

The vacuole of Candida albicans plays a significant role in many processes including homeostasis control, cellular trafficking, dimorphic switching, and stress tolerance. Thus, understanding the factors affecting vacuole function is important for the identification of new drug targets needed in response to the world’s increasing levels of invasive infections and the growing issue of fungal drug resistance. Past studies have shown that vacuolar proton-translocating ATPases (V-ATPases) play a central role in pH homeostasis and filamentation. Vacuolar protein sorting components (VPS) regulate V-ATPases assembly and at the same time affect hyphal development. As well, vacuolar calcium exchange systems like Yvc1 and Pmc1 maintain cytosolic calcium levels while being affected by V-ATPases function. All these proteins play a role in the virulence and pathogenesis of C. albicans. This review highlights the relationships among V-ATPases, VPS, and vacuolar calcium exchange proteins while summarizing their importance in C. albicans infections.

A proteomics study of the subacute toxicity of rat brain after long-term exposure of Gelsemium elegans

Background: Gelsemium elegans (G. elegans) has been shown to have strong pharmacological and pharmacodynamic effects in relevant studies both in China and USA. G. elegans has been used as a traditional medicine to treat a variety of diseases and even has the potential to be an alternative to laboratory synthesized drugs. However, its toxicity severely limited its application and development. At present, there is little attention paid to protein changes in toxicity. Aim: This study investigated the toxicity effects after long-term exposure of G. elegans of the rat brain through proteomic. Method: 11 differential abundance proteins were detected, among which 8 proteins were higher in the G. elegans- exposure group than in the control group, including Ig-like domain-containing protein (N/A), receptor-type tyrosine-protein phosphatase C (Ptprc), disheveled segment polarity protein 3 (Dvl3), trafficking protein particle complex 12 (Trappc12), seizure-related 6 homolog-like (Sez6l), transmembrane 9 superfamily member 4 (Tm9sf4), DENN domain-containing protein 5A (Dennd5a) and Tle4, whereas the other 3 proteins do the opposite including Golgi to ER traffic protein 4 (Get4), vacuolar protein sorting 4 homolog B (Vps4b) and cadherin-related 23 (CDH23). Furthermore, we performed validation of WB analysis on the key protein CDH23. Result: Finally, only fewer proteins and related metabolic pathways were affected, indicating that there was no accumulative toxicity of G. elegans. G. elegans has the potential to develop and utilize of its pharmacological activity. CHD23, however, is a protein associated with hearing. Conclusion: Whether the hearing impairment is a sequela after G. elegans exposure remains to be further studied.

Unraveling the Spatiotemporal Distribution of VPS13A in the Mouse Brain

Loss-of-function mutations in the human vacuolar protein sorting the 13 homolog A (VPS13A) gene cause Chorea-acanthocytosis (ChAc), with selective degeneration of the striatum as the main neuropathologic feature. Very little is known about the VPS13A expression in the brain. The main objective of this work was to assess, for the first time, the spatiotemporal distribution of VPS13A in the mouse brain. We found VPS13A expression present in neurons already in the embryonic stage, with stable levels until adulthood. VPS13A mRNA and protein distributions were similar in the adult mouse brain. We found a widespread VPS13A distribution, with the strongest expression profiles in the pons, hippocampus, and cerebellum. Interestingly, expression was weak in the basal ganglia. VPS13A staining was positive in glutamatergic, GABAergic, and cholinergic neurons, but rarely in glial cells. At the cellular level, VPS13A was mainly located in the soma and neurites, co-localizing with both the endoplasmic reticulum and mitochondria. However, it was not enriched in dendritic spines or the synaptosomal fraction of cortical neurons. In vivo pharmacological modulation of the glutamatergic, dopaminergic or cholinergic systems did not modulate VPS13A concentration in the hippocampus, cerebral cortex, or striatum. These results indicate that VPS13A has remarkable stability in neuronal cells. Understanding the distinct expression pattern of VPS13A can provide relevant information to unravel pathophysiological hallmarks of ChAc.

VPS52 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 vacuolar protein sorting 52, encoded by VPS52 when comparing the primary tumors of triple negative breast cancer patients dead or alive. Importantly, VPS52 expression was correlated with recurrence-free survival in basal subtype breast cancer, a molecular subtype sharing significant overlap with triple negative breast cancer. VPS52 may be of relevance as a biomarker or as a molecule of interest in understanding the etiology or progression of triple negative breast cancer.

A Novel DCL2-Dependent Micro-Like RNA Vm-PC-3p-92107_6 Affects Pathogenicity by Regulating the Expression of Vm-VPS10 in Valsa mali

Dicer proteins are mainly responsible for generating small RNAs (sRNAs), which are involved in gene silencing in most eukaryotes. In previous research, two DCL proteins in Valsa mali, the pathogenic fungus causing apple tree Valsa canker, were found associated with both the pathogenicity and generation of sRNAs. In this study, the differential expression of small interfering RNAs (siRNAs) and miRNA-like RNAs (milRNAs) was analyzed based on the deep sequencing of the wild type and Vm-DCL2 mutant, respectively. Overall, the generation of 40 siRNAs and 18 milRNAs was evidently associated with Vm-DCL2. The target genes of milRNAs were then identified using degradome sequencing; according to the prediction results, most candidate targets are related to pathogenicity. Further, expression of Vm-PC-3p-92107_6 was confirmed in the wild type but not in the Vm-DCL2 mutant. Moreover, the pathogenicity of Vm-PC-3p-92107_6 deletion mutants (ΔVm-PC-3p-92107_6) and the over-expression transformants (Vm-PC-3p-92107_6-OE) was significantly increased and decreased, respectively. Based on those degradome results, vacuolar protein sorting 10 (Vm-VPS10) was identified as the target of Vm-PC-3p-92107_6. Co-expression analysis in tobacco leaves further confirmed that Vm-PC-3p-92107_6 could suppress the expression of Vm-VPS10. Meanwhile, the expression levels of Vm-PC-3p-92107_6 and Vm-VPS10 displayed divergent trends in ΔVm-PC-3p-92107_6 and Vm-PC-3p-92107_6-OE, respectively. Perhaps most importantly, ΔVm-VPS10 featured a significant reduction in pathogenicity. Taken together, our results indicate that a DCL2-dependent milRNA Vm-PC-3p-92107_6 plays roles in pathogenicity by regulating the expression of Vm-VPS10. This study lays a foundation for the comprehensive analysis of pathogenic mechanisms of V. mali and deepens our understanding of the generation and function of fungal sRNA.

VPS35 Downregulation Alters Degradation Pathways in Neuronal Cells

Background: The vacuolar protein sorting 35 (VPS35) is the main component of the retromer recognition core complex system which regulates intracellular cargo protein sorting and trafficking. Downregulation of VPS35 has been linked to the pathogenesis of neurodegenerative disorders such Alzheimer’s and Parkinson’s diseases via endosome dysregulation. Objective: Here we show that the genetic manipulation of VPS35 affects intracellular degradation pathways. Methods: A neuronal cell line expressing human APP Swedish mutant was used. VPS35 silencing was performed treating cells with VPS35 siRNA or Ctr siRNA for 72 h. Results: Downregulation of VPS35 was associated with alteration of autophagy flux and intracellular accumulation of acidic and ubiquitinated aggregates suggesting that dysfunction of the retromer recognition core leads to a significant alteration in both pathways. Conclusion: Taken together, our data demonstrate that besides cargo sorting and trafficking, VPS35 by supporting the integral function of the retromer complex system plays an important role also as a critical regulator of intracellular degradation pathways.