Fig 2: EXO70H4 contributes to bacterium-induced callose deposits, but not ROS burst. a Callose deposits area was measured in each genotype after four- to five-week-old plants were syringe-infiltrated water as a mock control or with 1 × 108 CFU.mL- 1 of Pst DC3118, STm 14,028 s, or E. coli O157:H7. Results are shown as average of three to four biological replicate (n = 18 to 37 ± SE) and the experiment was repeated twice with similar results. Statistical difference between the means (wild type versus mutant plant) was calculated using the two-tailed Student’s t-test (** = p < 0.01; *** = p < 0.001; ns = non-significant). The pictures on the left are representative of each plant genotype per treatment. b Leaf discs of four- to five-week-old plants were incubated with control solution or 1 × 108 CFU.mL- 1 of boiled Pst DC3118, STm 14,028 s, or E. coli O157:H7. Results are shown as the average of luminescence per genotype in three independent biological replicates (n = 21 to 37 ± SE). Luminescence was recorded over 60 min using a Synergy™ H1 microplate reader and the Biotek Gen5 (Biotek) software. RLU = Relative Light Units

Fig 3: Spt4 phosphorylation by Hog1 is required for cell growth in the presence of osmostress.Wild type Spt4 (wt), hog1? and the indicated spt4 mutant strains were grown to mid-log exponential phase in YPD and then diluted 1:10 in YPD, 1.4 M NaCl or 1.4 M KCl before starting the assay. Growth curves over 21 hours were analyzed using a Synergy H1 Hybrid Reader (BioTek). Data are the means ± SD of three technical replicates from a representative experiment. Growth curves are represented on a semi-log plot.

Fig 4: A spermine analog, DenSpm, inhibited HSV-1 infection in vitro. (A) Schematic representing target site for DenSpm action in the spermine synthesis pathway. (B) MTT assay showing dose-dependent viability in HCE cells after DenSpm treatment. (C) A thin-layer chromatography plate showed spermine detection in HCE cells with or without DenSpm treatment during HSV-1 or mock infected conditions. Letter M shows marker spot for standard spermine. (D) The quantitative levels of spermine in experimental conditions were measured with zone elution fluorescence assay (Ex 365, Em 505) with BioTek synergy H1 microplate reader. (E) HCE cells were pretreated with DenSpm or vehicle control, DMSO for 72 h followed by an infected with HSV-1 KOS at 0.1 MOI. At 2 hpi, the cells were again treated with DenSpm or DMSO. Representative fluorescence micrograph of HCE cells indicates HSV-1 infection. Green and red fluorescence represent the transcription of immediate early and true-late viral genes, respectively. Images were captured using Biotek Lionheart FX automated microscope under a 10× objective. Scale bar, 200 µm. (F) Quantification of fluorescence imaging using Biotek image analysis software. Graph showing expression of transcripts of (G) HSV-1 ICP-0, (H) HSV-1 gD, (I) HSV-1 latency-associated transcript enhancer (LAT-E), (J) HSV-1 latency-associated transcript major (LAT-M) as determined by RT-qPCR (K) Representative micrograph of immunoblots for HSV-1 ICP0, gB and GAPDH. (L) Graph showing generation of mature virus particles (PFU/mL) from DenSpm treated and infected HCE cells with respective concentrations (µM). Significance was determined by one-way ANOVA with Sidak multiple comparisons. Student t test was performed for statistical analysis (a = 0.05). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. ns, not significant; NT, no treatment.

Fig 5: H4-S47D phosphomimetic mutant exhibits transcriptional and growth defects upon osmostress. (A) Fluorescence of the quadruple-Venus (qV) reporter driven by ALD3 promoter was measured by flow cytometry before (control; gray) and after osmostress (0.4 M NaCl for 45 min, blue) in wild-type (WT) and the mutant H4-S47A and H4-S47D strains. Dotted black lines indicate the level of transcription of the WT in response to stress. Fluorescence intensity under stress was quantified with respect to the WT strain and the percentages are indicated. (B) WT and the indicated histone mutant strains were subjected to osmostress (0.4 M NaCl) for the indicated length of time. Total mRNA was assayed by northern blot with radiolabeled probes for ALD3 and HSP12 (stress-responsive genes) and ENO1 (as loading control). RNA quantification is expressed in percentage as the ratio of mRNA levels normalized by ENO1 and taking the value of maximum gene expression of the WT strain as 100% reference. (C) Volcano plot showing significantly altered gene expression in WT strain upon stress (0.4 M NaCl, 15 min). Dashed vertical lines mark the imposed log2 fold change threshold of 1 (up-/down-regulation), whereas the dashed horizontal line indicates an adjusted P-value of 0.05. (D) Differences in the transcriptional stress-activated and stress-repressed response defined in (C) between H4-S47D mutant and WT strains. Horizontal lines in the boxplots indicate the median, dashed gray lines indicate a relative absolute fold change of 1. The indicated P-value is based on a Wilcoxon-test, while the highlighted numbers represent genes significantly altered by the mutant in response to osmostress compared to non-stress conditions (blue dots). (E, F) Cell growth analysis in response to osmostress. WT and the indicated histone mutant strains were grown to mid-log exponential phase in YPD, serially diluted (2-fold), spotted onto YPD plates with and without 1.2 M NaCl and grown at 30°C for 3–5 days (E). Exponential phase cell cultures were diluted in YPD or YPD 1.2 M NaCl and grown at 30°C with constant agitation for 40 h. Cell density (OD660) was measured using a Synergy H1 Hybrid Reader (BioTek). Growth curve data are the means ± SD of a minimum of three technical replicates from a representative experiment (F).