Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing, People's Republic of China.
Jiangsu Key Laboratory for Microbes and Microbial Functional Genomics, Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, College of Life Science, Nanjing Normal University, Nanjing, People's Republic of China
Appl Environ Microbiol. 2019 Jul 18;85(15). doi: 10.1128/AEM.00532-19. Print 2019 Aug 1.
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 were associated with the release of -acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from , ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released -acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other 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 ; 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. 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 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 β-1,6-branched β-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 β-1,3-glucans under turgor pressure .
菌柄的延伸生长是担子菌类的一个特征但尚未被很好理解的形态发生事件。我们发现，伸展天然菌柄细胞壁与释放 N-乙酰氨基葡萄糖和壳二糖以及几丁质酶活性有关。在所有从 中检测到的几丁质酶中，有两种几丁质酶 ChiE1 和 ChiIII 可以重建热失活菌柄壁的延伸并释放 N-乙酰氨基葡萄糖和壳二糖。有趣的是，ChiE1 和 ChiIII 都可以水解不溶性结晶几丁质粉末，而其他几丁质酶则不能，这表明菌柄细胞壁的结晶几丁质成分是 ChiE1 和 ChiIII 的作用靶标。ChiE1 或 ChiIII 重建的热失活菌柄壁在 pH4.5 时显示出最大的延伸活性，与天然菌柄壁延伸的最佳 pH 值一致；ChiE1 或 ChiIII 重建的热失活菌柄壁延伸活性与菌柄伸长生长区域有关；并且 ChiE1 和 ChiIII 的组合在低作用浓度下显示出重建热失活菌柄壁延伸的协同作用。场发射扫描电子显微镜 (FESEM) 图像显示，酸诱导的天然延伸菌柄细胞壁和 ChiE1 或 ChiIII 重建的热失活菌柄细胞壁的内表面显示出部分断裂的平行微原纤维结构；然而，在中性 pH 缓冲液或热失活诱导的未延伸菌柄细胞壁中未观察到这些横向排列的微原纤维断裂。ChiE1 和 ChiIII 的双重敲低导致菌柄伸长、菌丝生长和天然菌柄的热敏感细胞壁延伸减少。这些结果表明，几丁质酶水解机制是菌柄细胞壁延伸的基础。担子菌类果实发育的一个显著特征是菌柄伸长生长，主要是由于菌褶细胞的伸长。一些科学家认为，菌柄伸长是细胞壁多糖酶水解的结果，而另一些科学家则提出菌柄伸长可能是细胞壁多糖之间氢键非水解破坏的结果。在这里，我们展示了模型蘑菇中菌柄细胞壁伸长的几丁质酶水解机制的直接证据，该机制与植物细胞壁延伸的扩张素非水解机制不同。我们推测，在生长中的菌柄细胞壁中，平行的几丁质微原纤维被β-1,6-支链β-1,3-葡聚糖系链，几丁质酶的断裂导致这些微原纤维的分离，从而增加它们之间的间距，以便在膨压下插入新合成的几丁质和β-1,3-葡聚糖。
