Journal: bioRxiv

Article Title: Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1)

doi: 10.1101/2021.02.08.430285

Figure Lengend Snippet: Yeast complementation assay and root 55 Fe 2+ uptake rates. (A) Growth of WT and the fet3fet4 Fe transport-defective mutant transformed with a modified pFL61 plasmid as empty vector (eV), or containing the IRT1 , IRT1 S206A or IRT1 H232A cDNA, respectively. Ten-fold serial dilutions of liquid cultures were spotted on SD (-Ura) medium (pH 5.7) supplemented with 0.5 mM FeSO 4 or left unamended. (B) Uptake rates determined in hydroponically cultivated 7.5 to 8.5-w-old plants over a 13.5-min period. Before the initiation of uptake assays, plants were exposed to Fe deficiency (0 μM FeHBED) for 6 d, subsequent to an initial cultivation period in 1x modified Hoagland‘s solution containing 10 μM FeHBED (WT and irt1 IRT1) or 50 μM FeHBED (all other genotypes) for 6 to 7 weeks. Bars represent arithmetic mean ± SD ( n = 4 to 5 plants). Data show one experiment representative of three independent experiments (see Supplemental Table S2).

Article Snippet: Yeast Constructs, Strains, and Growth The IRT1 coding cDNA sequence, including translational start and stop codon, was amplified from the corresponding transgenic plant line by RT-PCR with IRT1-ATG_F 5’-CACCATGGCTTCAAATTCAGCAC-3’ and IRT1-cDNA_TAA_R 5’- TTAAGCCCATTTGGCGATAATCG-3’, introduced into pENTR-D/TOPO (Invitrogen) and cloned into the yeast expression vector pFL61-Gateway ( ) using LR clonase (Invitrogen) according to the manufacturer’s protocol.

Techniques: Mutagenesis, Transformation Assay, Modification, Plasmid Preparation

Journal: Autophagy

Article Title: Features of autophagic cell death in Plasmodium liver-stage parasites

doi: 10.4161/auto.23689

Figure Lengend Snippet: Figure 7. PbAtg8 does not localize to autophagosomes in dying parasites ( A ) and cannot complement yeast Atg8 ( B ). ( A ) HepG2 cells were infected with P. berghei parasites constitutively expressing mCherry (mCherry). 60 hpi the cells were fixed and stained with an anti-PbACP antiserum (ACP) to label the apicoplast and an anti-PbAtg8 antiserum (PbAtg8) to monitor the localization of this protein during parasite cell death. DNA was labeled with DAPI. A representative parasite showing strong vacuolization is depicted. A higher magnification of some important details is presented in the merged image. Scale bar: 10 µm: CPS. ( B ) Scatg8Δ and wt strains were transformed with empty pFL61 plasmid or the same plasmid containing PbAtg8 or ScAtg8 as a positive control. Western blot analysis was performed with the transformed strains using anti-aminopeptidase I antibodies. Transport of prApe1 to the vacuole where it matures (Ape1) only takes place in the presence of a functional autophagy pathway. The prApe1 and the Ape1 bands are marked with arrows.

Article Snippet: The fragments were cloned into the yeast expression vector pFL61 via NotI (New England Biolabs, R0189) and the resulting plasmids used to transform Scatg8Δ WCG strains of S. cerevisiae by the acetate method.

Techniques: Infection, Expressing, Staining, Labeling, Transformation Assay, Plasmid Preparation, Positive Control, Western Blot, Functional Assay