Journal: Nature Communications

Article Title: The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins

doi: 10.1038/s41467-018-04149-0

Figure Lengend Snippet: Rsp3 interacts with maize secreted AFP1 protein. a Amino acid sequence alignment of maize AFP1, APF2, and G. biloba Gnk2. The signal peptide is underlined with a blue line. Conserved amino acids are highlighted in black. The DUF26 domains (C-X 8 -C-X 2 -C) are indicated by red dashed boxes. The red and green arrows indicate residues putatively involved in mannose binding in the N- or C-terminal domains of AFP1 and APF2, respectively. b Secreted Rsp3 interacts with purified AFP1. Leaf tissues of N. benthamiana infiltrated with A. tumefaciens carrying either a AFP1-His expressing plasmid or an empty vector (EV) as negative control were subjected to NTA-affinity purification. Prior to protein elution the NTA-agarose beads were mixed with culture supernatants of SG200Δrsp3 expressing the indicated Rsp3-HA variants under control of the otef promoter. The input and bound proteins were detected by western blot using anti-His-HRP and anti-HA antibodies. The asterisk (*) indicates a truncated form of the Rsp3 Δ412-869 -HA. c Secreted Rsp3 Um-Sr hybrid protein interacts with purified AFP1. Culture supernatants of SG200Δrsp3 expressing the indicated Rsp3-HA variants were incubated with AFP1-His as described in b and interaction was shown by western blot as in b . The experiments in b and c were repeated three times and one representative experiment is shown. Full blots are shown in Supplementary Fig.

Article Snippet: Beads with bound proteins were boiled and subjected to western blotting analysis using anti-His antibodies conjugated to horseradish peroxidase (HRP) (1:8000 dilution, Qiagen Cat#34460).

Techniques: Sequencing, Binding Assay, Purification, Expressing, Plasmid Preparation, Negative Control, Affinity Purification, Control, Western Blot, Incubation

Journal: Nature Communications

Article Title: The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins

doi: 10.1038/s41467-018-04149-0

Figure Lengend Snippet: Rsp3 blocks the antifungal activity of maize secreted mannose binding protein AFP1. a Mannose binding of AFP1 and mutant AFP1 variants. AFP1-His, AFP1*-His (S34A, R115A, and E126A) and AFP1**-His (S34A, R115A, E126A, N144A, Q227A, and E238A) purified from N. benthamiana were incubated with mannose-agarose beads. Bound proteins were analyzed by western blot using anti-His-HRP antibodies. Cmu1-His expressed and purified from E. coli BL21 served as negative control. The experiment was preformed three times and one representative experiment is shown. Full blots are shown in Supplementary Fig. . b AFP1 has antifungal activity. The indicated strains were grown to an OD 600 of 0.6 and subsequently diluted to OD 600 = 0.001. Cells were incubated for 3 h with either AFP1-His or AFP1**-His protein. The cell suspensions were spotted twice on a PD agar plate and incubated at 28 °C for 2 days until colonies appeared. The experiment was performed in three biological replicates and one representative experiment is shown. The full photograph is shown in Supplementary Fig. . c Quantification of the antifungal activity of AFP1. The survival of the indicated U. maydis cells treated with AFP1 or AFP1** was quantified by counting colony forming units (CFUs) from b . Data represent mean ± sd of the three biological replicates. p -values were calculated by Student’s t -test, and significant differences ( p < 0.05) are indicated by +. d SYTOX Orange staining reveals cell death inducing ability of AFP1. SG200Δrsp3 was incubated with either AFP1-His or AFP1**-His for 3 h before staining with SYTOX Orange. SYTOX Orange-stained cells were visualized by epifluorescence microscopy. e The experiment shown in d was done in three biological replicates. About 250 cells were evaluated in each experiment. The percentage of cells stained with SYTOX Orange is indicated. Values represent mean ± sd

Article Snippet: Beads with bound proteins were boiled and subjected to western blotting analysis using anti-His antibodies conjugated to horseradish peroxidase (HRP) (1:8000 dilution, Qiagen Cat#34460).

Techniques: Activity Assay, Binding Assay, Mutagenesis, Purification, Incubation, Western Blot, Negative Control, Staining, Epifluorescence Microscopy