A new identified suppressor of Cdc7p/SepH kinase, PomA, regulates fungal asexual reproduction via affecting phosphorylation of MAPK-HogA

Abstract

The septation initiation network (SIN), composed of a conserved SepH (Cdc7p) kinase cascade, plays an essential role in fungal cytokinesis/septation and conidiation for asexual reproduction, while the mitogen-activated protein kinase (MAPK) pathway depends on successive signaling cascade phosphorylation to sense and respond to stress and environmental factors. In this study, a SepH suppressor-PomA in the filamentous fungus A. nidulans is identified as a negative regulator of septation and conidiation such that the pomA mutant is able to cure defects of sepH1 in septation and conidiation and overexpression of pomA remarkably suppresses septation. Under the normal cultural condition, SepH positively regulates the phosphorylation of MAPK-HogA, while PomA reversely affects this process. In the absence of PbsB (MAPKK, a putative upstream member of HogA), PomA and SepH are unable to affect the phosphorylation level of HogA. Under the osmostress condition, the induced phosphorylated HogA is capable of bypassing the requirement of SepH, a key player for early events during cytokinesis but not for MobA/SidB, the last one in the core SIN protein kinase cascade, indicating the osmotic stimuli-induced septation is capable of bypassing requirement of SepH but unable to bypass the whole SIN requirement. Findings demonstrate that crosstalk exists between the SIN and MAPK pathways. PomA and SepH indirectly regulate HogA phosphorylation through affecting HogA-P upstream kinases.

Fig 1. Septation and conidiation were partly rescued in S11 and S53 compared to the sepH1 mutant at 42 °C.

(A) Colony morphologies of the wild type (R21), sepH1, S11 and S53 strains cultured on YUU at 30 or 42 °C for 2 days. (B) Conidiophores for the conidia of the wild type (R21), sepH1, S11 and S53 strains. Bars, 10 μm. (C) Comparison of septum formation in hyphal cells of the wild type (R21), sepH1, S11 and S53 strains cultured on liquid medium YUU at 42 °C for 20 h. Bars, 10 μm. (D) Colony morphologies of the wild type (R21), sin11 and sin53 strains cultured on YUU at 30 or 42 °C for 2 days. (E) Morphological comparison of hyphal cells labeled with an RFP-histone H2A tag showing nuclei distribution and stained with CFW showing septa in relative strains cultured in liquid medium YUU at 42 °C for 7.5 h. Bars, 10 μm.

Fig 2. Single nucleotide polymorphism comparison and identification of the pomA mutation in S11 and S53.

(A) (B) SNP comparison by next-generation sequencing for the whole genomes of S11 and S53. (C) Domain analysis of PomA with SMART (http://smart.embl-heidelberg.de/). (D) Mutations in the cloned pomA gene from S11 and S53 identified by sequencing.

Fig 3. PomA mutation or deletion suppressed sepH1 defects in septation and conidiation.

(A) Colony morphologies of the wild type (TN02A7), sepH1, ΔpomA, PomA^L1265S, sepH1 ΔpomA and sepH1 PomA^L1265S strains cultured on YUU at 30 or 42 °C for 2 days. (B) Septa comparison in hyphal cells stained with CFW in the indicated strains cultured in liquid rich media YUU at 42 °C for 20 h. Arrows indicate the locations of septa. Bars, 10 μm. (C) Localization of GFP-PomA under control of the alcA conditional promoter when strain ZXA14 was cultured with liquid minimal media PGRT. Bars, 10 μm. (D) Colony morphologies of the indicated strains cultured on YAG medium or YAG medium supplemented with 1 M KCl, 1 M NaCl, calcofluor white (CFW) (50 μg/ml), Congo red (CR) (100 μg/ml) and caspofungin (1.25 μg/ml) at 37 °C for 2 days. (E) Germling morphological comparison by differential interference contrast (DIC) images of the parental wild type (TN02A7) and ΔpomA strains cultured in liquid media YUU for 8 h. Bars, 10 μm. (F) Morphological comparison of hyphal cells labeled with an RFP-histone H2A tag showing nuclei distribution and strained with CFW showing septa in the related strains cultured in liquid medium YUU at 37 °C for 7 h. Bars, 10 μm. (G) Comparison of formed septa in hyphal cells stained with CFW in the parental wild type (TN02A7) and OE::pomA strains cultured in liquid media YUU at 37 °C for 10 h. Bars, 10 μm.

Fig 4. Quantitative phosphoproteomics comparison between the septa-restored strain sepH1ΔpomA (ZXA10) and the septa-abolished control strain sepH1 revealed that the deletion of pomA activated the HOG pathway.

(A) Total number of detected phosphorylation sites, peptides and proteins and (B) fold changes of phosphorylated proteins. (C) GO terms enriched for all phosphorylated proteins and (D) KEGG pathways enriched in phosphorylated proteins with more than 1.2-fold changes. (E) Visual analysis of the increased phosphorylated proteins in the MAPK pathway (labeled in red).

Fig 5. PomA and SepH reversely affected the phosphorylation of HogA.

(A) The phosphorylation level of wild type (TN02A7) and sepH1 strains cultured in liquid minimal media PGRUU treated with or without 1 M NaCl for 10 min at 42 °C for 24 h. (B) Diagram showing strategies of HogA full-length deletion and deleted for predicted phosphorylation sites (171–173) of HogA. (C) Comparison of hyphal cells stained with CFW for the sepH1, sepH1 ΔhogA and sepH1 HogA-ΔP with or without treatment of 1 M NaCl or 1 M KCl at 42 °C. Arrows indicate the locations of septa. Bars, 10 μm. (D) Western blot analysis showing the expression level of P-HogA in the pomA null mutant and the pomA site-directed strain cultured in liquid minimal media PGRUU at 37 °C or (F) sepH1 relative strains at 42 °C for 24 h. (E) Western blot analysis showing the expression level of P-HogA in the alc(p)::GFP-pomA strain cultured in de-repressed media MMPGR at 37 °C for 24 h or transferred to repressed media YAG for 30 min.

Fig 6. Suppressed sepH1 defects by deletion AnkA in septation and a cross-link working model between the SIN and the MAPK.

(A) Colony morphologies of the wild type (TN02A7), sepH1, ΔankA sepH1 and ΔankA strains cultured on YUU at 30 or 42 °C for 2 days. (B) Septa comparison in hyphal cells stained with CFW in the indicated strains cultured in liquid rich media YUU at 42 °C for 20 h. Arrows indicate the locations of septa. Bars, 10 μm. (C) A schematic model for the MAPK and the SIN-SepH-MobA/SidB pathways regulated by PomA-AnkA regulation during septation and conidiation. Green arrows indicate induce and labels for red lines indicate inhibit.