Phosphate transporter PHT1;1 as a key determinant of phosphorus acquisition in Arabidopsis natural accessions

To understand the genetic basis in governing phosphorus (P) acquisition, we performed genome-wide association studies (GWAS) on a diversity panel of Arabidopsis thaliana by two primary determinants of P acquisition, phosphate (Pi)-uptake activity and PHOSPHATE TRANSPORTER 1 (PHT1) protein abundance. Association mapping revealed a shared significant peak on chromosome 5 (Chr5) where the PHT1;1/2/3 genes reside, suggesting a strong correlation between the regulation of Pi-uptake activity and PHT1 protein abundance. Genes encoding transcription factors, kinases, and a metalloprotease associated with both traits were also identified. Conditional GWAS followed by statistical analysis of genotype-dependent expression of PHT1;1 and transcription activity assays revealed an epistatic interaction between PHT1;1 and MYB DOMAIN PROTEIN 52 (MYB52) on Chr1. Analyses of F1 hybrids generated by crossing two subgroups of natural accessions carrying specific SNPs associated with PHT1;1 and MYB52 further revealed the strong effects of potential variants on PHT1;1 expression and Pi uptake activity. Notably, the soil P contents in A. thaliana habitats were found to coincide with PHT1;1 haplotype, underscoring how fine-tuning of the activity of P acquisition by natural variants allows plants to adapt to their environments. This study sheds light on the complex regulation of P acquisition and offers a framework to systematically assess the effectiveness of GWAS approaches in the study of quantitative traits.

EXTH-65. INSERTION OF MICRORNA TARGET SEQUENCES INTO RETROVIRAL REPLICATING VECTORS EFFECTIVELY RESTRICTS TRANSGENE EXPRESSION AND VIRAL REPLICATION IN HUMAN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Prodrug activator gene therapy with a retroviral replicating vector (RRV) has shown a highly favorable safety profile and long-term survival in early-phase trials for recurrent high-grade glioma. Overall endpoints were not met in a recent Phase 3 trial, but highly statistically significant survival was observed in prespecified patient subgroups as compared to randomized matched control patients receiving standard-of-care treatments, and further clinical evaluation is being focused on these subgroups. Additional strategies to enhance therapeutic potency may require two or more RRVs to deliver multiple transgenes simultaneously, but RRVs encoated with the same envelope protein will compete for the same cellular receptors, interfering with efficient co-infection. The current clinical vector (formerly Toca511, now DB107) is encoated by amphotropic murine leukemia virus (MLV) envelope, which binds to inorganic phosphate transporter PiT-2/SLC20A2. To switch RRV tropism, we developed RRVs pseudotyped with a heterologous envelope from Gibbon ape leukemia virus (GALV), which utilizes an alternative phosphate transporter, PiT-1/SLC20A1, for cell entry. Efficient co-infection of established and primary human glioblastoma cells with MLV- and GALV-pseudotyped RRV was achieved, without receptor competition. However, human hematopoietic stem/progenitor cells (HSPC) also express high levels of PiT-1, which may increase potential genotoxicity of GALV-pseudotyped RRV. Accordingly, as a novel strategy to restrict gene expression and replication in HSPC, we have developed and tested new RRV designs incorporating microRNA target sequences (miRT). Insertion of miRT recognized by hematopoietic lineage-specific microRNA-142-3p resulted in complete suppression of RRV replication in primary human CD34+ HSPC, with the percentage of infected cells below 1% after vector inoculation at multiplicities of infection (MOI) 0.05 and 0.1, and remaining below 3% even after inoculation at MOI 0.5 and 1.0. In vivo models have been developed for on-going studies to evaluate miRT-mediated restriction strategies for avoidance of potential genotoxicity by RRV in normal hematopoietic cells.

Mitochondrial phosphate transporter and methyltransferase genes contribute to Fusarium head blight Type II disease resistance and grain development in wheat

Fusarium head blight (FHB) is an economically important disease of wheat that results in yield loss and grain contaminated with fungal mycotoxins that are harmful to human and animal health. Herein we characterised two wheat genes involved in the FHB response in wheat: a wheat mitochondrial phosphate transporter (TaMPT) and a methyltransferase (TaSAM). Wheat has three sub-genomes (A, B, and D) and gene expression studies demonstrated that TaMPT and TaSAM homoeologs were differentially expressed in response to FHB infection and the mycotoxigenic Fusarium virulence factor deoxynivalenol (DON) in FHB resistant wheat cv. CM82036 and susceptible cv. Remus. Virus-induced gene silencing (VIGS) of either TaMPT or TaSAM enhanced the susceptibility of cv. CM82036 to FHB disease, reducing disease spread (Type II disease resistance). VIGS of TaMPT and TaSAM significantly reduced grain number and grain weight. This indicates TaSAM and TaMPT genes also contribute to grain development in wheat and adds to the increasing body of evidence linking FHB resistance genes to grain development. Hence, Fusarium responsive genes TaSAM and TaMPT warrant further study to determine their potential to enhance both disease resistance and grain development in wheat.

Mast cell regranulation involves a metabolic switch promoted by the interaction between mTORC1 and a glucose-6-phosphate transporter

Mast cells (MCs) are highly granulated tissue resident hematopoietic cells and because of their capacity to degranulate and release many proinflammatory mediators, they are major effectors of chronic inflammatory disorders including asthma and urticaria. As MCs have the unique capacity to reform their granules following degranulation in vitro, their potential to undergo multiple cycles of degranulation and regranulation in vivo has been linked to their pathogenesis. However, it is not known what factors regulate MC regranulation let alone if MC regranulation occurs in vivo. Here, we report that IgE-sensitized mice can undergo multiple bouts of regranulation, following repeated anaphylactic reactions. mTORC1, a critical nutrient sensor that activates protein and lipid synthesis, was found necessary for MC regranulation. mTORC1 activity in MCs was regulated by a glucose-6-phosphate transporter, Slc37a2, which was found to be necessary for increased glucose-6-phosphate and ATP levels during regranulation, two upstream signals of mTOR. Slc37a2 is highly expressed at the cell periphery early during regranulation where it appears to colocalize with mTORC1. Additionally, this transporter was found to concentrate extracellular metabolites within endosomes which are trafficked directly into nascent granules. Thus, the metabolic switch associated with MC regranulation is mediated by the interactions of a cellular metabolic sensor and a transporter of extracellular metabolites into MC granules.

Single Cell App: An App for Single Cell RNA-sequencing Data Visualization, Comparison and Discovery

SummaryThe Single Cell App is a cloud-based application that allows visualisation and comparison of scRNA-seq data and is scalable according to use. Users upload their own or publicly available scRNA-seq datasets after pre-processing to be visualised using a web browser. The data can be viewed in two colour modes, Cluster - representing cell identity, and Values – level of expression, and data queried using keyword or gene identification number(s). Using the app to compare four different studies we determined that some genes frequently used as cell-type markers are in fact study specific. Phosphate transporter and hormone response genes were exemplary investigated with the app. This showed that the apparent cell specific expression of PHO1;H3 differed between GFP-tagging and scRNA-seq studies. Some phosphate transporter genes were induced by protoplasting, they retained cell specificity, indicating that cell specific stress responses (i.e. protoplasting). Examination of the cell specificity of hormone response genes revealed that 132 hormone responsive genes display restricted expression and that the jasmonate response gene TIFY8 is expressed in endodermal cells which differs from previous reports. It also appears that JAZ repressors have cell-type specific functions. These differences, identified using the Single Cell App, highlight the need for resources to enable researchers to find common and different patterns of cell specific gene expression. Thus, the Single Cell App enables researchers to form new hypothesis, perform comparative studies, allows for easy re-use of data for this emerging technology to provide novel avenues to crop improvement.