Abstract
Septa of filamentous ascomycetes are perforated by septal pores that allow communication between individual hyphal compartments. Upon injury, septal pores are plugged rapidly by Woronin bodies (WBs), thereby preventing extensive cytoplasmic bleeding. The mechanism by which WBs translocate into the pore is not known, but it has been suggested that wound‐induced cytoplasmic bleeding “flushes” WBs into the septal opening. Alternatively, contraction of septum‐associated tethering proteins may pull WBs into the septal pore. Here, we investigate WB dynamics in the wheat pathogen Zymoseptoria tritici. Ultrastructural studies showed that 3.4 ± 0.2 WBs reside on each side of a septum and that single WBs of 128.5 ± 3.6 nm in diameter seal the septal pore (41 ± 1.5 nm). Live cell imaging of green fluorescent ZtHex1, a major protein in WBs, and the integral plasma membrane protein ZtSso1 confirms WB translocation into the septal pore. This was associated with the occasional formation of a plasma membrane “balloon,” extruding into the dead cell, suggesting that the plasma membrane rapidly seals the wounded septal pore wound. Minor amounts of fluorescent ZtHex1‐enhanced green fluorescent protein (eGFP) appeared associated with the “ballooning” plasma membrane, indicating that cytoplasmic ZtHex1‐eGFP is recruited to the extending plasma membrane. Surprisingly, in ~15% of all cases, WBs moved from the ruptured cell into the septal pore. This translocation against the cytoplasmic flow suggests that an active mechanism drives WB plugging. Indeed, treatment of unwounded and intact cells with the respiration inhibitor carbonyl cyanide m‐chlorophenyl hydrazone induced WB translocation into the pores. Moreover, carbonyl cyanide m‐chlorophenyl hydrazone treatment recruited cytoplasmic ZtHex1‐eGFP to the lateral plasma membrane of the cells. Thus, keeping the WBs out of the septal pores, in Z. tritici, is an ATP‐dependent process.
Highlights
The Pezizomycetes are the largest class within the ascomycetes
We show that cell injury creates a pressure gradient, which is consistent with cytoplasmic flow-driven movement of Woronin body (WB) into the septal pore
A sub-population of the WBs moves against the flow from the ruptured cell into the septal pore, suggesting an active mechanism of WB-based pore plugging. In agreement with this notion, we report that reduced cellular ATP levels trigger movement of WBs into the septal pore in intact hyphae
Summary
The Pezizomycetes are the largest class within the ascomycetes. This fungal group includes important human and plant pathogens, such as Aspergillus fumigatus and Zymoseptoria tritici, the causal agent of Septoria tritici blotch in wheat. This hyphal architecture bears the risk that wounding of individual cells causes extensive cytoplasmic bleeding and catastrophic damage to the entire hypha To meet this challenge, the pezizomycetes have developed an efficient protection mechanism, based on the rapid closure of septal pores by Woronin bodies (WBs; Jedd & Pieuchot, 2012). Quantitative electron microscopy studies revealed that a single WB closes the septum after wounding, whilst other WBs remain largely unaffected This was taken as an argument against a pressure driven mechanism of pore sealing by WBs (Markham & Collinge, 1987). In agreement with this notion, we report that reduced cellular ATP levels trigger movement of WBs into the septal pore in intact hyphae
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