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

ABSTRACT FUNCTIONAL ANALYSIS OF INHIBITOR OF APOPTOSIS PROTEIN GENE CPBIR1 IN CRYPHONECTRIA PARASITICA ABSTRACT Apoptosis is an essential part during growth and development, cell differentiation andmorphogenesis of muticellular organisms. The inhibitor of apoptosis proteins (IAPs) familynot only take part in inhibition of apoptosis, but also participate in the process of celldivision, reception of signaling, regulation of immune response, heavy metal metabolismand morphogenesis et al., suggesting that the functions of IAPs are extremely complex andimportant in organisms. IAPs have been studied in virus, yeast, mammalian, nematode andfruit fly, whereas, to our knowledge, there was no report on the disruption of IAP gene infilamentous 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 CpBirl contains two typical BIRdomains, and has a close evolutionary relationship with Birlp in yeast and Survivin inHomo sapiens. Cpbir1 was confirmed to unique in C. parasitica genome by Southernblotting. Then Cpbir1 was disrupted by gene replacement and the phenotypes of deletionmutantΔCpbir1 were characterized.ΔCpbir1 showed a reduction in the formation of aerialhyphae, an increase in the pigmentation, a decrease of the growth rate and ceased growthafter a period of cultivation. At the microscopic level, the hyphae of Cpbirl disruptantexhibited abnormal branching and swellings. Disruption of Cpbir1 also resulted inabolishing conidial production and almost losing virulence completely. CpBirl localized tothe nucleus in juvenile cultures, but as well as cytoplasm in old cultures. Quantitativereal-time PCR analysis showed that CpBirl was dramatically down-regulated by hypovirusinfection, and H2O2 was able to induce CpBirl up-regulated in wild-type strain.ΔCpbirl was more sensitive to oxidative stress. Consistently, overexpression of CpBirl increased resistance to oxidative stress. Rhodamine123 staining assay showed that accumulation of reactive oxygen species increased in hyphae ofΔCpbirl. Cpbirl 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 Cpbirl mutant strain. Disruption of Cpbirl changed frequence of virus transmision between some pairs of vegetative compatibility types. Our results suggested that Cpbirl disrupted was an obstacle to transmit virus to Cpbirl deletion mutant. On the contrary, Cpbirl disrupted hypovirulent srain possessed similar or more capability to donate virus to other virus-free isolates compared with normal hypovirulent strain.In conclusion, CpBirl is involved in anti-aging, anti-apoptosis and anti-stress processes of C. parasitica, and plays a crucial role in conidiogenesis and virulence. Cpbirl transcript level is affected by hypovirus infection. Conversely, CpBirl can also regulate hypovirus transmission.