Chung Hea-Jong, Kim Myoung-Ju, Lim Jin-Young, Park Seung-Moon, Cha Byeong-Jin, Kim Young-Ho, Yang Moon-Sik, Kim Dae-Hyuk
Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
Fungal Genet Biol. 2006 May;43(5):326-36. doi: 10.1016/j.fgb.2005.12.009. Epub 2006 Mar 15.
Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is known to downregulate the fungal laccase1 (lac1), the modulation of which is tightly governed by the inositol triphosphate (IP(3)) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), to investigate the regulation of lac1 expression and to better characterize fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd beta-strand and the alpha-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a delta-type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. Accordingly, reduced virulence of the cplc1-null mutant as compared to the wild-type was observed, which can be ascribed to the growth defect. However, other PLC-associated characteristics including temperature sensitivity and osmosensitivity did not differ from the wild-type strain. Northern blot analysis revealed no accumulation of the lac1 gene transcript due to the disruption of the cplc1 gene. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for normal mycelial growth rate and colony morphology, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.
已知栗疫病菌寄生隐孢壳菌的低病毒感染会下调真菌漆酶1（lac1），在无病毒菌株中，其调节受肌醇三磷酸（IP(3)）和钙第二信使系统严格控制。我们克隆了编码磷脂酰肌醇特异性磷脂酶C（PLC）的基因cplc1，以研究lac1表达的调控，并更好地表征低病毒对真菌基因的调控。cplc1基因的序列分析表明，蛋白质产物包含X和Y结构域，这是在所有已知PLC中发现的两个保守区域，在X结构域的第2条β链和α螺旋之间有133个氨基酸的延伸。此外，基因结构似乎与δ型PLC的高度相似。cplc1基因的破坏导致生长缓慢，产生的菌落特征是气生菌丝少且颜色深橙色。因此，观察到与野生型相比，cplc1缺失突变体的毒力降低，这可归因于生长缺陷。然而，其他与PLC相关的特征，包括温度敏感性和渗透敏感性，与野生型菌株没有差异。Northern印迹分析显示，由于cplc1基因的破坏，lac1基因转录本没有积累。用酿酒酵母的PLC1基因对cplc1缺失突变体进行功能互补恢复了lac1表达，这表明克隆的基因编码PLC活性。本研究表明，cplc1基因是正常菌丝生长速率和菌落形态所必需的，并且它调节lac1表达，而lac1表达也受低病毒调节。尽管在各种简单真核生物中已经鉴定出几个PLC基因，但本研究中对cplc1基因的缺失分析似乎是丝状真菌中PLC功能分析的首次报道。
