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Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

ABSTRACT: The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ?-1,6-branched ?-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ?-1,3-glucans under turgor pressure in vivo.

SUBMITTER: Zhou J

PROVIDER: S-EPMC6643254 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

Zhou Jiangsheng J Kang Liqin L Liu Cuicui C Niu Xin X Wang Xiaojun X Liu Hailong H Zhang Wenming W Liu Zhonghua Z Latgé Jean-Paul JP Yuan Sheng S

Applied and environmental microbiology 20190718 15

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chit ...[more]

PMID: 31126941

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Project description:This study reports that a high concentration of the endo-?-1,3-glucanase ENG (200??g ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ?-glucosidase BGL2 (1,000??g ml-1) did not; however, in combination, low concentrations of ENG (25??g ml-1) and BGL2 (260??g ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ?-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60?min; the stipe cell wall extension induced by a high concentration of ?-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ?-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ?-1,6 branched ?-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ?-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ?-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ?-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ?-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ?-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ?-glucans during stipe elongation growth.

Project description:Coprinopsis cinerea is a model mushroom particularly suited for the study of fungal fruiting body development and the evolution of multicellularity in fungi. While microRNAs (miRNAs) have been extensively studied in animals and plants for their essential roles in post-transcriptional regulation of gene expression, miRNAs in fungi are less well characterized and their potential roles in controlling mushroom development remain unknown. To identify miRNA-like RNAs (milRNAs) in C. cinerea and explore their expression patterns during the early developmental transition of mushroom development, small RNA libraries of vegetative mycelium and primordium were generated and putative milRNA candidates were identified following the standards of miRNA prediction in animals and plants. Two out of 22 novel predicted milRNAs, cci-milR-12c and cci-milR-13e-5p, were validated by northern blot and stem-loop reverse transcription real-time PCR. Cci-milR-12c was differentially expressed whereas the expression levels of cci-milR-13e-5p were similar in the two developmental stages. Target prediction of the validated milRNAs resulted in genes associated with fruiting body development, including pheromone, hydrophobin, cytochrome P450, and protein kinase. Essential genes for miRNA biogenesis, including three coding for Dicer-like (DCL), one for Argonaute (AGO), one for AGO-like and one for quelling deficient-2 (QDE-2) proteins, were also identified in the C. cinerea genome. Phylogenetic analysis showed that the DCL and AGO proteins of C. cinerea were more closely related to those in other basidiomycetes and ascomycetes than to those in animals and plants. Taken together, our findings provided the first evidence for milRNAs in the model mushroom and their potential roles in regulating fruiting body development. New information on the evolutionary relationship of milRNA biogenesis proteins across kingdoms has also provided new insights for guiding further functional and evolutionary studies of miRNAs.

Dataset Information

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

Publications

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom <i>Coprinopsis cinerea</i>.

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