A gene encoding phosphatidyl inositol-specific phospholipase C from Cryphonectria parasitica modulates the lac1 expression.
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.
Institute for Molecular Biology and Genetics, Basic Science Research Institute, Chonbuk National University, Jeonju, Chonbuk 561-756, Republic of Korea.
Hea-Jong
HJ
Chung
Myoung-Ju
MJ
Kim
Jin-Young
JY
Lim
Seung-Moon
SM
Park
Byeong-Jin
BJ
Cha
Young-Ho
YH
Kim
Moon-Sik
MS
Yang
Dae-Hyuk
DH
Kim
D000595:Amino Acid Sequence; D001203:Ascomycota / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D006128:Growth / Q000235:genetics; D042845:Laccase / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D009024:Morphogenesis / Q000235:genetics; D016254:Mutagenesis, Insertional; D025282:Mycelium / Q000235:genetics; D010858:Pigmentation / Q000235:genetics; D024301:Plant Bark / Q000382:microbiology; D010935:Plant Diseases / Q000382:microbiology; D017434:Protein Structure, Tertiary; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D010738:Type C Phospholipases / Q000235:genetics* / Q000378:metabolism*; D014774:Virulence / Q000235:genetics
D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D042845:Laccase; D010738:Type C Phospholipases