A comparative analysis was carried out of published methods to assess seed viability using 2,3,5-triphenyltetrazolium chloride (TTC) based assays of seed batches. The tests were carried out on seeds of barley (Hordeum vulgare cv. Optic) as a model. We established that 10% [w/v] trichloroacetic acid (TCA)/methanol is superior to the acetone and methanol-only based methods: allowing the highest recovery of formazan and the lowest background optical density (OD) readings, across seed lots comprising different ratios of viable and dead seeds. The method allowed a linear-model to accurately capture the statistically significant relationship between the quantity of formazan that could be extracted using the method we developed and the seed temperature-response, and seed viability as a function of artificially aged seed lots. Other quality control steps are defined to help ensure the assay is robust and these are reported in a Standard Operating Procedure.
Somites arising from paraxial mesoderm are a hallmark of the segmented vertebrate body plan. They form sequentially during axis extension and generate musculoskeletal cell lineages. How paraxial mesoderm becomes regionalised along the axis and how this correlates with dynamic changes of chromatin accessibility and the transcriptome remains unknown. Here, we report a spatiotemporal series of ATAC-seq and RNA-seq along the chick embryonic axis. Footprint analysis shows differential coverage of binding sites for several key transcription factors, including CDX2, LEF1 and members of HOX clusters. Associating accessible chromatin with nearby expressed genes identifies cis-regulatory elements (CRE) for TCF15 and MEOX1. We determine their spatiotemporal activity and evolutionary conservation in Xenopus and human. Epigenome silencing of endogenous CREs disrupts TCF15 and MEOX1 gene expression and recapitulates phenotypic abnormalities of anterior–posterior axis extension. Our integrated approach allows dissection of paraxial mesoderm regulatory circuits in vivo and has implications for investigating gene regulatory networks.
Phytophthora spp. secrete vast arrays of effector molecules during infection to aid in host colonization. The CRN protein family forms an extensive repertoire of candidate effectors that accumulate in the host nucleus to perturb processes required for immunity. Here, we show that CRN12_997 from P. capsici binds a TCP transcription factor, SlTCP14-2, to inhibit its immunity-associated activity against Phytophthora. Co-immuno-precipitation and split-YFP studies confirm a specific CRN12_997-SlTCP14-2 interaction in vivo. Co-expression of CRN12_997 specifically counteracts the TCP14-enhanced immunity phenotype, suggesting CRN mediated perturbation of SlTCP14-2 function. We show that SlTCP14-2 associates with nuclear chromatin and that CRN12_997 diminishes SlTCP14-2 DNA-binding. Collectively, our data support a model in which SlTCP14-2 associates with chromatin to enhance immunity. The interaction between CRN12_997 and SlTCP14-2 reduces DNA-binding of the immune regulator. We propose that the modulation of SlTCP14-2 chromatin affinity, caused by CRN12-997, enhances susceptibility to Phytophthora capsici.
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