Fig 1.
Pathogenicity screening of C. higginsianum T-DNA insertion mutants.
(A) Selective PDA hygromycin plate six days after Agrobacterium tumefaciens-mediated transformation (ATMT) of C. higginsianum with pPK2. (B) Arabidopsis thaliana Col-0 plant directly after droplet inoculation with C. higginsianum. (C, D, E) Typical symptom development on Col-0 leaves 6 days after droplet infection (500 conidia/droplet) with wild type C. higginsianum strain CY5535 (C) and T-DNA insertion mutants showing reduced pathogenicity (D) or no symptoms (E). (F) Mock inoculation with water.
Table 1.
Phenotypes of C. higginsianum pathogenicity mutants.
Fig 2.
Phenotypes of selected vir mutants.
(A) Failure of group I mutant vir-4 to form appressoria on 1,16-hexadecanediol coated petri dishes. (B) vir-1 mutant on trypan blue stained A. thaliana leaf four days after infection. (C) Appressoria of group II mutant vir-22 on coated petri dishes. (D) vir-22 on trypan blue stained leaves four days after infection. (E) Growth of vir-13 on potato dextrose agar (PDA, after four days) and Czapek Dox plates (MM, after six days) compared to wild type C. higginsianum. (F) Trypan blue stained A. thaliana leaves four days after infection with vir-13. (G) Trypan blue stained A. thaliana leaves four days after infection with vir-11. (H) Appressoria formation of vir-28 on coated petri dishes. (I, J) Formation of primary hyphae on trypan blue stained A. thaliana leaves four days after infection with two group III mutants (I: vir-42; J: vir-40). Scale bar = 25 μm.
Table 2.
T-DNA insertion sites in C. higginsianum ATMT mutants.
Fig 3.
T-DNA insertion sites of selective vir mutants.
Location of T-DNA-insertions and typical phenotypes of vir-10 (A, B, C), vir-53 (D, E, F), vir-76 (G, H, I), and vir-84 (J, K, L). T-DNA sequences isolated by Genome Walker PCR are illustrated as solid rectangles. Predicted parts of the T-DNA that were not directly sequenced are shown as dotted lines. Genes are named corresponding to Table 2 or by their Gene ID (Colletotrichum Sequencing Project, Broad Institute of Harvard and MIT, http://www.broadinstitute.org/). Typical phenotypes of the respective vir mutants shown as light microscopic images of trypan blue stained leaves 4 days after infection (C, F, I, L) and the corresponding macroscopic symptom development (B, E, H). (K) Appressoria formation of vir-84 after 15 h on coated petri dishes. Scale bar = 25 μm.
Fig 4.
Verification of tagged mutants by targeted knockout.
(A, B) Trypan blue stained A. thaliana leaves four days after spray infection with C. higginsianum wild type (WT) or ΔChku80 strain (CY6021). (C, F, I) Schematic representation of T-DNA insertions in genomic loci of three pathogenicity mutants (vir-52, vir-2, vir-27). The respective homology regions used for targeted gene knockout by homologous recombination in the ΔChku80 strain are illustrated as dotted lines. The T-DNA border sequences identified by Genome Walker PCR are illustrated in dark gray. (D, E) Light microscopy of trypan blue stained A. thaliana Col-0 leaves four days after spray infection with vir-52 and ΔChlys1. (G, H) Czapek Dox minimal medium without adenine or supplemented with 55 μg/ml adenine four days after inoculation with wild type (WT), ΔChku80, vir-2 or ΔChade2 strains. (J, K) Light microscopy images of trypan blue stained leaves four days after spray infection with vir-27 or ΔChste12. Scale bar = 25 μm.
Fig 5.
Construction and phenotype of ΔChpma2 mutants.
(A) Schematic overview of the ChPMA2 locus and the knockout plasmid pCK3349 used for transformation of C. higginsianum (B, C) Macroscopic symptoms of A. thaliana leaves four days after spray infection with C. higginsianum ΔChpma2 mutants (B) and the parental ΔChku80 strain (C, control). (D, E) Trypan blue stained leaves four days after infection with ΔChpma2 (D) and the parental ΔChku80 strain (E). (F, G) Growth of ΔChpma2 strains compared to the parental strain on PDA medium after 3 days (F) and on minimal medium after five days (G). (H, I) Aniline blue staining of A. thaliana leaves three days after infection with ΔChpma2 and the parental strain (control). (J, K) In vitro appressoria of ΔChpma2 and the parental strain. (L, M) Penetration of dialysis tubes by the ΔChpma2 mutant (L) and the parental strain (M). Scale bar = 50 μm.
Fig 6.
Appressorial turgor pressure in ΔChpma2 mutants.
Appressoria were formed on coated petri dishes. After 24 h, the water was replaced with PEG-6000 solutions ranging from 100 mg/ml to 500 mg/ml. The percentage of collapsed appressoria was determined after 10 minutes of incubation in PEG by phase-contrast microscopy. (A) Typical microscopic images of parental ΔChku80 (control) and ΔChpma2 appressoria untreated (H2O) or treated with 400 mg/ml PEG-6000 (+ PEG-6000). (B) Percentage of collapsed appressoria of ΔChpma2 and the parental strain at the corresponding PEG-6000 concentration. Data corresponds to three independent experiments with at least 100 cells counted for each concentration. Scale bar = 15 μm.
Fig 7.
Expression of ChPMA1 and ChPMA2.
(A) Quantitative RT-PCR analysis of transcript abundance for ChPMA1 and ChPMA2 in conidia, in vitro appressoria (AP), mycelium grown 48 h or 72 h in liquid modified Mathur’s medium (M) and during infection of A. thaliana after three or four days (3 dpi, 4 dpi). Data was normalized against alpha-tubulin (CH063_01222) expression and plotted relative to alpha-tubulin (average from three biological replicates). (B) Normalized ChPMA2 expression plotted relative to ChPMA2 level in conidia. (C, D) Confocal images of C. higginsianum transformed with the promoter fusions Ppma2-mCherry (pCK3880) or Ppma1-GFP (pCK3973) after 0 to 32 h of incubation on coated ibidi μ-dishes (C) or after one and three days of spray infection of A. thaliana (D). The images are depicted as overlays of bright-field channel with mCherry or GFP-channel (C) or with an additional chloroplast autofluorescence channel in blue (D). Scale bar = 10 μm.
Fig 8.
Fungal H+-transporting P-type ATPases.
(A) Phylogenetic analysis of plasma membrane H+-ATPases. Sequences were aligned with ClustalW and the tree was generated by Geneious treebuilding (Jukes-Cantor; Neighbor-joining) with AHA2 from A. thaliana as outgroup. Bootstrap values (1000 replicates) are indicated as percentage at the right side of the nodes. (B) Schematic illustration of the domain structure of H+-P-type ATPases as found in AHA2 (A: actuator domain, P: phosphorylation domain, N: nucleotide binding domain). Regions specific for ChPma2-like proteins are depicted in red. The green asterisk marks regulatory phosphorylation sites in yeast Pma1. The variable C-terminus is illustrated in red for ChPma2-like proteins, in green for Pma1-like proteins and in black for the autoinhibitory region of Arabidopsis AHA2. (C/D) Amino acid sequence alignment of Pma1 and Pma2 proteins from Colletotrichum higginsianum (Ch), Colletotrichum graminicola (Cg), Magnaporthe oryzae (Mo), Saccharomyces cerevisiae (Sc) and Arabidopsis thaliana (AHA2). (C) Cytoplasmic region between transmembrane domains two and three. (D) C-terminal region. Potential regulatory serine and threonine residues are marked with green asterisks.