Journal: bioRxiv

Article Title: PacC-dependent adaptation and modulation of host cellular pH controls hemibiotrophic invasive growth and disease development by the rice blast fungus

doi: 10.1101/2020.06.22.164590

Figure Lengend Snippet: (A) Expression of PAG1 in mycelium of WT P131, Δ pacC , NGP239, and NGP559. For the Q-PCR, the expression level of P131 cultured at pH 5.5 was arbitrarily set to 1. (B) PacC binds to GCCAAG motifs in the PAG1 promoter. Purified GST-PacC 222 protein was used to detect binding of putative PacC binding motifs. Probes 1 and 2 contain predicted PacC-binding motifs and were prepared by labelling with 32 P-dCTP and incubated with GST-PacC 222 for 30 min, before loading a native-PAGE gel. For competition experiments, 100x or 10x concentrations of un-labelled Probe 1 were mixed with the GST-PacC 222 protein for 30 min before incubation with 32 P-dCTP-labelled probes. (C) WT and Δ pag1 are same in colony growth on OTA plates. (D) WT and Δ pag1 are equivalent in conidiation. (E) Reduced virulence of Δ pag1 mutant compared to WT. (F) Infection assays of WT and Δ pag1 on barley epidermis. (G) Arrested biotrophic growth of Δ pag1 compared to WT. Bar = 25 μm.

Article Snippet: GST-fused PacC 559 , PacC 222 and PacC 80 proteins were individually expressed in the pGEX-4T-3 vector (Promega, USA) in E. coli BL21DE3 and purified, as described in supplementary data.

Techniques: Expressing, Cell Culture, Purification, Binding Assay, Incubation, Clear Native PAGE, Mutagenesis, Infection

Journal: bioRxiv

Article Title: PacC-dependent adaptation and modulation of host cellular pH controls hemibiotrophic invasive growth and disease development by the rice blast fungus

doi: 10.1101/2020.06.22.164590

Figure Lengend Snippet: (A) Infected plant cells are alkalinized during the early biotrophic growth of M. oryzae and then become acidified during the later necrotrophic growth. (B) During biotrophic growth, the PacC 559 and PacC 222 transcription factor isoforms localize to the nucleus, where PacC 559 acts as a transcriptional repressor to repress expression of genes associated with conidiation and necrotrophic growth, including PRG1 , HTF1 , and PIG1 while PacC 222 acts as a transcriptional activator to activate genes associated with biotrophic growth. (C) As host cells become acidified and lose viability, the PacC functional isoforms exit from the nucleus thereby de-repressing expression of genes related to necrotrophic growth and conidiation.

Article Snippet: GST-fused PacC 559 , PacC 222 and PacC 80 proteins were individually expressed in the pGEX-4T-3 vector (Promega, USA) in E. coli BL21DE3 and purified, as described in supplementary data.

Techniques: Infection, Expressing, Functional Assay

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Previously undescribed mutant pacC alleles characterized in this work

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Mutagenesis, Sequencing

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Plasmids used in this study

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Plasmid Preparation

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: Two-hybrid interaction of PalA with PacC. Yeast strain CTY10-5d was used, and proteins were expressed from plasmids listed in Table ​Table2.2. (A) GAD-PacC fusions contain the indicated PacC residues. The shaded bar indicates the DNA binding domain (DBD). Arrows mark the approximate position of the signaling-protease (∼493 to 500) (8) and processing-protease (∼252 to 254) (25) cleavage sites. Values are the average β-galactosidase activity of four transformants. Standard errors were <14%. In control experiments, GAD protein fusions did not interact with LexA (<0.4 U). ND, not determined. (B) Western analysis of protein extracts from transformants expressing LexA-PalA and the indicated GAD-PacC protein fusions which were detected with anti-HA antibodies. WT, wild type.

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: Binding Assay, Activity Assay, Western Blot, Expressing

Journal:

Article Title: YPXL/I Is a Protein Interaction Motif Recognized by Aspergillus PalA and Its Human Homologue, AIP1/Alix

doi: 10.1128/MCB.23.5.1647-1655.2003

Figure Lengend Snippet: In vitro binding of PalA to PacC by using pull-down assays. (A) GST fusion proteins bound to glutathione-Sepharose 4B beads (Pharmacia) used in the binding assays (10% of the total) were separated by SDS-PAGE (10% polyacrylamide) and stained with Coomassie blue. (B) Beads loaded with GST alone (lanes 4 to 6) or with GST fusions to PalA (lanes 7 to 9), PacC(529-678) (lanes 10 to 12), or PacC (169-410) (lanes 13 and 14) were incubated with in vitro-synthesized [35S]PacC (wild type, double-mutant Y455D-Y662N, or mutant L340S, as indicated). After being washed, bound proteins were boiled in sample buffer, separated by SDS-PAGE, and analyzed by autoradiography (top) and by Coomassie staining (bottom). Lanes marked Input contain in vitro-synthesized PacC (wild type [lane 1], double-mutant Y455D-Y662N [lane 2] and mutant L340S [lane 3]) used for binding experiments (20% of the total reaction mixture). Protein markers are in kilodaltons.

Article Snippet: N-terminally His-tagged PacC proteins (wild-type, double-mutant Y455D-Y662N, and mutant L340S) were synthesized in vitro and labeled with [ 35 S]methionine (1,000 Ci/mmol) by using the Promega TNT coupled transcription-translation system and appropriate templates (Table ).

Techniques: In Vitro, Binding Assay, SDS Page, Staining, Incubation, Synthesized, Mutagenesis, Autoradiography