State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China.
Fungal Genet Biol. 2011 Oct;48(10):956-65. doi: 10.1016/j.fgb.2011.06.007. Epub 2011 Jul 7.
Coniothyrium minitans is an important sclerotial parasite of the fungal phytopathogen, Sclerotinia sclerotiorum. Previously, we constructed a T-DNA insertional library, and screened for many conidiation-deficient mutants from this library. Here, we report a T-DNA insertional mutant ZS-1T21882 that completely lost conidiation. In mutant ZS-1T21882, the T-DNA was integrated into a gene (CmPrat-1) which encodes phosphoribosylamidotransferase (PRAT, EC 2.4.2.14), an enzyme catalyzing the first committed step in de novo purine nucleotide synthesis. Gene replacement and complementation experiments confirmed that phosphoribosylamidotransferase is essential for conidiation of C. minitans. Mutant ZS-1T21882 did not grow on modified Czapek-Dox broth (MCD), but it grew well on MCD amended with IMP or AMP. The conidial production of this mutant was dependent on the dosage of IMP amended. At low concentrations, such as 0.1 mM and 0.25 mM, the mutant produced very few pycnidia, while up to 0.75 mM or higher, the conidiation of this mutant was restored completely. cAMP could not restore the conidiation of mutant ZS-1T21882 when amended into MCD, but could when amended into PDA. Neither GMP nor cGMP could restore the conidiation in MCD or in PDA. Our findings suggest that phosphoribosylamidotransferase is essential for conidiation of C. minitans via adenosine related molecules. Furthermore, when dual cultured with its host, this mutant produced conidia in the host mycelium and on the sclerotia of S. sclerotiorum, but not in dead mycelium or on dead sclerotia, suggesting that C. minitans is likely to able to obtain adenosine or related components from its host during parasitization.
康氏长蠕孢菌是真菌病原菌核盘菌的一种重要的菌核寄生菌。此前，我们构建了 T-DNA 插入文库，并从该文库中筛选了许多产孢缺陷突变体。在这里，我们报告了一个 T-DNA 插入突变体 ZS-1T21882，该突变体完全丧失了产孢能力。在突变体 ZS-1T21882 中，T-DNA 插入到一个基因（CmPrat-1）中，该基因编码磷酸核糖基氨基转移酶（PRAT，EC 2.4.2.14），该酶催化从头合成嘌呤核苷酸合成的第一步。基因替换和互补实验证实，磷酸核糖基氨基转移酶是康氏长蠕孢菌产孢所必需的。突变体 ZS-1T21882不能在改良的察氏培养基（MCD）上生长，但在添加 IMP 或 AMP 的 MCD 上生长良好。该突变体的产孢量依赖于添加的 IMP 剂量。在低浓度（如 0.1 mM 和 0.25 mM）下，突变体产生的分生孢子很少，而在 0.75 mM 或更高浓度下，该突变体的产孢完全恢复。当添加到 MCD 中时，cAMP 不能恢复突变体 ZS-1T21882 的产孢，但当添加到 PDA 中时可以。GMP 或 cGMP 都不能在 MCD 或 PDA 中恢复产孢。我们的研究结果表明，磷酸核糖基氨基转移酶通过与腺苷相关的分子对康氏长蠕孢菌的产孢是必需的。此外，当与宿主双重培养时，该突变体在宿主菌丝和核盘菌的菌核上产生分生孢子，但不在死亡的菌丝或菌核上产生分生孢子，这表明康氏长蠕孢菌在寄生过程中可能能够从宿主中获得腺苷或相关成分。
