Deletion of Dip2 leads to increased cell wall stress resistance.

(A) A model depicting the possible role of Dip2 in regulating DAG species required for PKC activation which governs the CWI pathway in yeast by activating the downstream MAPK cascade. Activation of the MAPK cascade results in increased cell wall synthesis thereby strengthening the cell wall. Dip2 has been depicted in its three-domain architecture, harbouring DMAP-binding domain 1 (DBD1), tandem fatty acyl-AMP ligase-like domains (FLD1 and FLD2).

(B) Serial dilution assay for WT, two biological replicates of Δdip2 (Δdip2_Colony1 and Δdip2_Colony2; Δdip2_Colony1 has been used for further experiments). pDip2::Δdip2 represents Δdip2_Colony1 complemented with Dip2 expressed under its native promoter. Control plate contains synthetic complete (SC) media and cell wall (CW) stress plate has either congo red (CR) (100µg/mL) or calcofluor white (CFW) (50µg/mL). N=3.

(C) Colony forming units of WT and Δdip2 grown in SC media control and cell wall stress induced by CR (100µg/mL). Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n = 3; ***p < 0.001; ns = not significant). N=3.

(D) Representative western blot showing pSlt2 (56 kDa) levels in WT and Δdip2 compared to the WT treated with CFW for 30 min, used as a positive control. Bar graph showing quantification of fold change in the pSlt2 levels, normalised with total Slt2. Triose phosphate isomerase (Tpi1) (27 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n > 3; ***p < 0.001; ns = not significant.

(E) Serial dilution assay image in synthetic complete (SC) media control plate and in the presence of congo red (100µg/mL) for WT and Δdip2 compared with Δdip2 complemented with wildtype and catalytically inactive Dip2 mutants (Dip2D523A and Dip2L687A), expressed in a plasmid pYSM7, under its native promoter. N=3.

(F) Serial dilution assay for WT and Δdip2 grown in SD media control and in the presence of CR (100µg/mL) and cercosporamide (2µg/mL). N=3.

(G) Representative western blot for WT, Δdip2 and cercosporamide treated Δdip2 showing the levels of pslt2 (56 kDa). Quantification of pSlt2 levels for Δdip2 treated with cercosporamide (5µg/mL) compared to WT and Δdip2 control, normalised with total Slt2. Phosphoglycerate kinase (Pgk1) (45 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N=3; **p < 0.01; ns = not significant).

Dip2-regulated selective DAG subspecies are associated with the activation of PKC signalling.

(A) Lipidomic analysis of WT treated with CFW (50µg/mL) for 30 minutes. DAG intensities normalized to the total protein content in WT+CFW have been compared to the intensities from the same species in WT control sample. Data are represented as mean ± standard error of mean (SEM) (unpaired, two-tailed Student’s t-test; N=6). ****p < 0.0001; ***p < 0.001 ns = not significant.

(B) A flowchart for the liposome sedimentation assay explaining the incubation of liposomes with protein, followed by ultracentrifugation and western blotting for supernatant (unbound protein) and pellet (bound protein) fraction.

(C) Representative western blot for liposome sedimentation assay with different DAGs showing supernatant fraction (unbound protein) and pellet fraction (liposome bound protein), probed for C1 domain using anti-GFP antibody (43k Da). N > 3.

(D) Western blot probing for C1 domain bound to different concentrations of C36:0 DAG containing liposomes (pellet fraction) and the unbound protein (supernatant fraction) using anti GFP antibody. Structure of respective DAGs is shown on the right side. N > 3.

(E) Western blot probing for C1 domain at different concentrations of C34:1 DAG containing liposomes in both pellet and supernatant fractions. Structure of respective DAGs have been shown on the right side. N > 3.

(F) Bar graph showing quantification of percentage binding of C1-GFP with increasing concentrations of specific (C36:0) and non-specific (C34:1) DAGs. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; n=3; ***p < 0.001; ns = not significant), N=3.

PKC activation is independent of bulk DAG metabolism pathway.

(A-C) Lipidomic analysis of DAGs in Δdga1, Δlro1 and Δdgk1, compared to WT. Fold change values are mentioned below and are represented as green colour gradient. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; N = 5) ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns = not significant.

(D) Serial dilution assay for single knockouts of bulk DAG acting enzymes (Dga1, Lro1 and Dgk1) in the presence of CFW (50µg/mL), compared to SC media control plate. N=3.

(E) Representative western blot and quantification of pSlt2 (56 kDa) levels in deletion strains of DAG metabolizing enzymes. Fold change has been quantified with respect to the total Slt2. Triose phosphate isomerase (Tpi1) (27 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test); N=4; ****p < 0.0001; *p < 0.05; ns = not significant.

(F) DAG subspecies quantification using lipidomics for the double knockout of LRO1 and DGA1, compared to WT and Δdip2. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test); n = 6; ****p < 0.0001; ***p < 0.00; *p < 0.05; ns = not significant.

(G) Serial dilution assay image for the cell wall stress sensitivity of Δdga1Δlro1, compared to WT and Δdip2, in the presence of CFW (50µg/mL), compared to SC media control plate. N=3. SC media control image is reused in Figure 3-figure supplement 2.

(H) Representative western blot for pSlt2 (56 kDa) estimation in Δdga1Δlro1, compared to total Slt2 and a loading control Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) (36 kDa). Fold change has been quantified with respect to the total Slt2. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test); N=3; **p < 0.01; ns = not significant.

Psi1-Plc1-Dip2 triad axis regulates selective DAG subspecies to modulate PKC signalling.

(A) Schematic showing various pathways (green arrows) that feed into the production of DAGs in the presence of various enzymes (shown in blue) and different chemical inhibitors (red) blocking the respective pathways.

(B) Quantification of selective DAGs in WT and Δdip2 samples treated with two different concentrations, i.e., 0.5 and 1 µM of U73122. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; N = 5). ****p < 0.0001; ***p < 0.001; ns = not significant.

(C) Specific DAG quantification using lipidomics for deletion of Δplc1 and Δplc1Δdip2. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; n = 6). ****p < 0.0001; ***p < 0.001 ns = not significant. N=6.

(D) Representative western blot image and quantification of pSlt2 (56 kDa) levels for indicated samples. Fold change has been quantified with respect to the total Slt2. Gapdh (36 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N>4; ****p < 0.0001; **p < 0.01; ns = not significant.

(E) Lipidomic analysis of specific DAGs in double knockout of PSI1 and DIP2, compared with WT and Δpsi1. Data are represented as mean ± SEM (unpaired, two-tailed Student’s t-test; **p < 0.01; *p < 0.05; ns = not significant. N = 5).

(F) Representative western blot image and quantification of pSlt2 (56 kDa) levels for indicated samples. Two data points for controls (WT and Δdip2) are same as that of Figure 4D. Fold change has been quantified with respect to the total Slt2. Phosphoglycerate kinase (Pgk1) (45 kDa) has been used as a loading control. Data are represented as mean ± SD (unpaired, two-tailed Student’s t-test; N>3; ****p < 0.0001; ***p < 0.001; ns = not significant.

(G) Schematic showing Psi1-Plc1-Dip2 axis to regulate Pkc1 signalling where Psi1 remodels phosphoinositides (PI) to enrich it with C36:0 and C36:1 containing acyl chain, which is channelled to the selective DAGs via Plc1 and in turn activate Pkc1.

Dip2 and PKC share a parallel history of co-emergence coevolution across Opisthokonta.

(A) Emergence and distribution of DAG effector proteins across tree of life. Green circles represent presence, while red circles represent absence of effector proteins. Yellow circle represents the primitive form of PKC. List of DAG effectors is taken from a previous study (Colon-Gonzalez & Kazanietz, 2006).

(B) Phylogenetic profiling of Dip2 and PKC shows their co-emergence in Opisthokonta. Inset shows presence of Dip2 and PKC in different fungal branches (Ocana-Pallares et al., 2022). Green circle represents presence, red represents absence, while yellow circle represents a primitive form of PKC harbouring only C1 and kinase domain.

(C) Interspecies coevolution plot (using patristic distance) between fungal Dip2 and PKC using three different phylogenetic algorithms, with the correlation coefficient value represented by r. Number of organisms and significance levels are indicated.

(D) Interspecies correlation coefficient values (r) for Dip2 compared with PKC and control proteins Gapdh and Pgk using various algorithms for calculating phylogenetic distances. r value is calculated for Gapdh and Pgk the same way. The table contains the exact correlation coefficient values for all the above-mentioned protein pairs using different algorithms.

(E) Coevolution analysis (using genetic distance) between Dip2 and PKC along with their individual comparison with control protein Gapdh. Number of organisms and significance levels are indicated.

(F) Correlation coefficient values represented as bar graph for Dip2 compared with a set of housekeeping genes and lipid metabolizing proteins.

A graphical representation depicting the emergence of Dip2-PKC axis and the evolution of selective DAG-based PKC signalling in Opisthokonta.

Dip2 is recruited parallel to PKC in the tree of life at the root of Opisthokonta evolution ∼1.2 billion years ago. This Dip2-PKC axis remains conserved across Fungi and Metazoan. Two distinct DAG pools are sourced from de novo or salvage pathways and acyl chain remodelling of PIs, leading to metabolic and signalling DAG pool, respectively. Remodelled phosphoinositides (PI, PIP, PIP2) by Psi1 are enriched with 18:0 acyl chains and channelled to corresponding PIP2, which forms respective DAG species (C36:0, C36:1) upon hydrolysis by Plc1. These selective DAG subspecies act as secondary messenger for PKC signalling and therefore regulate the CWI pathway in yeast. Dip2 maintains the levels of these selective DAGs by facilitating their conversion to TAGs, thereby creating a diversification of DAG function into metabolic and signalling pathways.