Fig 1.
Mutations in A. thaliana SNUPO genes reduce the reproductive success of the oomycete downy mildew pathogen Hpa.
(a + b) Bar charts represent the mean number of sporangiophores ± s.e.m on infected cotyledons of A. thaliana wild-type (Col-0) or the indicated mutants 4 dpi with Hpa isolate Noco2. n = 14–63 (c) Bar charts represent the mean number of spores ± s.e.m per mg fresh weight isolated from A. thaliana wild-type (Col-0) or the indicated mutants 4 dpi with Hpa isolate Noco2. n = 9. Stars indicate significant differences to Col-0 (Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction; ′, p = 0.066 *, p < 0.05; **, p < 0.01; ***, p < 0.001). Each experiment was performed at least three times independently.
Fig 2.
The morphology of Hpa haustoria is altered in A. thaliana SNUPO mutants. Boxplots represent the percentage of multilobed haustoria among total haustoria on A. thaliana wild-type (Col-0), the indicated mutants and transgenic homozygous T3 complementation lines (pollux co, pPOLLUX:POLLUX; sec13 co, pSEC13:SEC13; shrk1 co, pUbi:ShRK1; shrk2 co, pUbi:ShRK2; LjSYMRK, pUbi:Lotus japonicus SYMRK-mOrange) 5 dpi (a + b), or 4, 5 and 7 dpi with Hpa isolate Noco2 (d). For each genotype, at least ten independent stretches of hyphae per leaf have been analysed on at least 5 leaves. On average, 1100 haustoria have been analysed per genotype. (c) Representative pictures of intracellular haustoria are displayed. Bars = 25 μm. (e) Upper panel: Light microscopy of Technovit section of A. thaliana wild-type (Col-0) or the shrk1 x shrk2 double mutant 7 dpi with Hpa isolate Noco2. Bars, 10 μm. Middle panel: Confocal light scanning microscopy of leaves of A. thaliana wild-type (Col-0) or the shrk1 x shrk2 double mutant stained with aniline-blue 5 dpi with Hpa isolate Noco2. For every haustorium, the entry point can be identified by the formation of the usually bright callose neck. Bars, 25 μm. Lower panel: Confocal light scanning microscopy of leaves of A. thaliana wild-type (Col-0) expressing RPW8.2-YFP, which localises to the perihaustorial membrane 7 dpi with Hpa isolate Noco2. *, round haustoria; +, multilobed haustoria; arrows, hyphae. Bars, 10 μm. Black circles, data points outside 1.5 IQR of the upper/lower quartile; bold black line, median; box, Interquartile Range (IQR); whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. Stars indicate significant differences to Col-0 (Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Each experiment was performed at least three times independently.
Fig 3.
Mutations in A. thaliana SNUPO genes do not impair the reproductive success of the fungal powdery mildew pathogen E. cruciferarum.
Bar charts represent the mean number of conidiophores ± s.e.m on leaves of the indicated mutants relative to the wild-type (Col-0) 5 dpi with E. cruciferarum. At least ten colonies per leaf on at least 6 leaves have been counted. No significant differences to Col-0 were detected (Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
Fig 4.
Activation of defence responses in response to Hpa infection is unaltered in A. thaliana SNUPO mutants.
(a) Expression of PR1, ERF1 or PDF1.2a relative to the housekeeping genes TIP41-like and PP2A in mock-treated samples (black circles) or in Hpa-infected samples (empty circles) was determined in three biological replicates for each genotype by qRT-PCR. Stars indicate significant differences to Col-0 (Dunnett’s Test with Bonferroni correction; *, p < 0.05). (b) Boxplots represent the mean intensity of callose deposition in leaves of A. thaliana wild-type (Col-0) or the indicated mutants 4 dpi with Hpa isolate Noco2. n = 27–72. Circles, data points outside 1.5 IQR of the upper/lower quartile; bold black line, median; box, IQR; whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. No significant differences to Col-0 were detected (Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001). Representative pictures of haustoria-associated callose deposition at the neck band of Hpa haustoria growing on the indicated mutants are shown on the right. Bars, 25 μm.
Fig 5.
Cell death responses are unaltered in A. thaliana SNUPO mutants. Differential interference contrast microscopy of A. thaliana wild-type (Col-0) and CSG mutant leaves stained with trypan-blue lactophenol 4 dpi with Hpa isolate Noco2.
First column, non-infected without cell death; second column, non-infected with random cell death; third column, infected with cell death in, or adjacent to, haustoria-containing cells; fourth column, infected without cell death. Bars, 25 μm. Boxplots represent the number of random (upper graph) or Hpa-associated (lower graph) cell death spots per leaf on A. thaliana wild-type (Col-0) or the indicated mutants 5 dpi with Hpa isolate Noco2. On each leaf four to ten spots have been analysed and a total of five leaves per genotype were investigated. Circles, data points outside 1.5 IQR of the upper/lower quartile; bold black line, median; box, IQR; whiskers, lowest/highest data point within 1.5 IQR of the lower/upper quartile. Stars indicate significant differences to Col-0 (Wilcoxon-Mann-Whitney test with Bonferroni-Holm correction; *, p < 0.05; **, p < 0.01; ***, p < 0.001).
Fig 6.
Expression of flg22-inducible genes as well as growth of the bacterial pathogen Pseudomonas syringae are not affected in A. thaliana SNUPO mutants.
(a) Expression of FRK1 and ERF1 relative to the housekeeping genes TIP41-like and PP2A in mock-treated samples (black circles) or in flg22-treated samples (empty circles) was determined in three biological replicates for each genotype by qRT-PCR. The fls2 mutant served as negative control. Stars indicate significant differences to Col-0 (Dunnett’s Test with Bonferroni correction; *, p < 0.01; **, p < 0.001). (b) Bacterial density of Pto DC3000 or Pto DC3000 ΔAvrPto/PtoB on A. thaliana wild-type (Col-0) or the indicated mutants was determined as colony forming units per cm2 2 dpi. Data represent means ± s.d. of six replicate measurements/genotype/data point. Results from one representative of at least four independent experiments are shown.