Functional Analysis Of Inhibitor Of Apoptosis Protein Gene Cpbir1 In Cryphonectria Parasitica

Apoptosis is an essential part during growth and development, cell differentiation and morphogenesis of muticellular organisms. The inhibitor of apoptosis proteins (IAPs) family not only take part in inhibition of apoptosis, but also participate in the process of cell division, reception of signaling, regulation of immune response, heavy metal metabolism and morphogenesis et al., suggesting that the functions of IAPs are extremely complex and important in organisms. IAPs have been studied in virus, yeast, mammalian, nematode and fruit fly, whereas, to our knowledge, there was no report on the disruption of IAP gene in filamentous fungus. In this paper, the function of IAP gene from Cryphonectria parasitica, the causal agent of chestnut blight, was studied.A putative gene encoding an IAP protein, named Cpbir1, was identified in C. parasitica. Bioinformatics analysis indicated that the CpBir1 contains two typical BIR domains, and has a close evolutionary relationship with Birlp in yeast and Survivin in Homo sapiens. Cpbir1 was confirmed to unique in C. parasitica genome by Southern blotting. Then Cpbir1 was disrupted by gene replacement and the phenotypes of deletion mutantΔCpbir1 were characterized.ΔCpbir1 showed a reduction in the formation of aerial hyphae, an increase in the pigmentation, a decrease of the growth rate and ceased growth after a period of cultivation. At the microscopic level, the hyphae of Cpbir1 disruptant exhibited abnormal branching and swellings. Disruption of Cpbir1 also resulted in abolishing conidial production and almost losing virulence completely. CpBir1 localized to the nucleus in juvenile cultures, but as well as cytoplasm in old cultures. Quantitative real-time PCR analysis showed that CpBirl was dramatically down-regulated by hypovirus infection, and H2O2 was able to induce CpBir1 up-regulated in wild-type strain.ΔCpbir1 was more sensitive to oxidative stress. Consistently, overexpression of CpBir1 increased resistance to oxidative stress. Rhodaminel23 staining assay showed that accumulation of reactive oxygen species increased in hyphae ofΔCpbir1. Cpbir1 deletion mutant showed reduced resistance to Cu2+ and Na+ stresses. The activity of laccase didn't decrease but the activity of polygalacturonase reduced remarkably in Cpbir1 mutant strain. Disruption of Cpbirl changed frequence of virus transmision between some pairs of vegetative compatibility types. Our results suggested that Cpbir1 disrupted was an obstacle to transmit virus to Cpbir1 deletion mutant. On the contrary, Cpbir1 disrupted hypovirulent srain possessed similar or more capability to donate virus to other virus-free isolates compared with normal hypovirulent strain.In conclusion, CpBir1 is involved in anti-aging, anti-apoptosis and anti-stress processes of C. parasitica, and plays a crucial role in conidiogenesis and virulence. Cpbir1 transcript level is affected by hypovirus infection. Conversely, CpBir1 can also regulate hypovirus transmission.