Scientific report on EMBO fellowship. ASTF 6-2010. Olga A. Kudryavtseva
The object of research is a short living filamentous fungus Podospora anserina. The main aim of the fellowship was to clarify mechanisms of lifespan prolongation of isolates as a consequence of shake flack (submerged or liquid) cultivation. Genetic and molecular analyses of chosen long living and immortal P. anserina isolates let us put forward the following hypothesis. Shake flack serial cultivation of P. anserina (passage into fresh medium was done every 3 days) is a system with marked selection pressure, enabling effectively select mycelial subpopulations with new genetic characteristics.
1. Immortal isolate V2-s1-IV(2) had been obtained after two cycles of short term shake flack cultivation. The first cycle included 4 passages and let us obtain long living isolate V-s1-IV(3), then this isolate was exposed additionally to 3 passages in liquid culture (the second cycle). Peaces of mycelium isolated from the second submerged culture were immortal.
Southern blot hybridization revealed mitochondrial DNA modification in immortal isolate V2-s1-IV(2). Total DNA of V2-s1-IV(2) was restricted by enzyme HaeIII and hybridized with radioactive probe Eco4. This isolate has deletion covered the gene cox1. Such a deletion in P. anserina means inactivation of main respiratory chain and respiration via alternative oxidase (AOX). Concerning our isolate V2-s1-IV(2) the link between alternative respiration and cox1 deletion was completely confirmed. V2-s1-IV(2) was resistant to antimycin A (inhibitor of main respiratory chain) and showed high level of AOX expression comparable with AOX expression in other available immortal respiratory mutants of P. anserina – nuclear mutant cox5 and mitochondrial mutant mex16. Respiration of V2-s1-IV(2) protoplasts was absolutely resistant to KCN (inhibitor of main respiratory chain) and sensitive to SHAM (AOX inhibitor). Other characteristics of V2-s1-IV(2) mutant were similar to cox5 and mex16: growth rate was 2-2.5 times lower then wild type, it was female sterile, amplification of α-senDNA (main factor of senescence in P. anserina) was not observed, respiration level was 2 times higher and ROS production was 35% lower then in wild type strains of P. anserina.
In theory mutants V2-s1-IV(2) and cox5 growing together with P. anserina strains type S on the same Petri dish and contact with them must form barrage (special structure characterized by programmed death of apical cells and stoppage of colony growth). In fact, we observed opposite situation: these mutants not only had no barrage with S strain but S filaments could grow between V2-s1-IV(2) and cox5 filaments and occupy their growth area. Probably, the reason of barrage absence is deficiency of energy in respiratory mutants. Another fact conflicting with common P. anserina knowledge is that during crossing respiratory mitochondrial mutant V2-s1-IV(2) with wild type strain immortal progeny carrying respiratory mutation were obtained. It was thought before mitochondria can not be transmitted during crossing of P. anserina. But in our experiments frequency of mitochondria transmission was 1.7 %. New facts require more detailed research.
2. Long living P. anserina isolates V-GFP1-43(1), V-GFP1-51(1), V-GFP1-XXXVIII(4), V-s1-22(1,2)light and V-s1-46(4,5) had been obtained after long term shake flack cultivation. It was needed from 22 to 51 passages respectively to obtain isolates of this group. In contrast to immortal isolate V2-s1-IV(2) described above, long living isolates characterized by high rate of growth (approximately as P. anserina wild type), normal respiration (they were sensitive to antimycin A) and absence of rearrangements into region Eco4 of mitochondrial DNA.
The most remarkable phenotypic features of long living isolates are absence of main pigment melanin and special flat structure of aerial mycelium. These two features were found to be inherited. Meiotic progeny obtained as a result of crossing wild type P. anserina strains with V-GFP1-43(1), V-GFP1-51(1), V-GFP1-XXXVIII(4), V-s1-22(1,2)light or V-s1-46(4,5) showed both wild type phenotype and phenotype of long living isolate. Moreover more then 50% of progeny had new phenotypes – neither wild type no phenotype of long living isolate. Four groups of new phenotypes were divided in progeny. We analyzed both monokaryotic and heterokaryotic progeny. So long living isolates used as parents have a mixture of mutations of different kinds. The most important that all these mutations appeared during long term shake flack cultivation of P. anserina.
Unfortunately we have not managed to identify mutations that were charged with lifespan prolongation in long lived isolates. All 69 progeny which continuous growth was maintained occurred to be short lived.
3. Another important characteristic of analyzed isolates both immortal V2-s1-IV(2) and long living V-GFP1-43(1), V-GFP1-51(1), V-GFP1-XXXVIII(4), and V-s1-46(4,5) was low production or absence of microconidia. In P. anserina microconidia can not germinate and serve for fertilization only. In current papers microconidia of P. anserina are described as a single factor of fertilization. But our isolates in spite of microconidia lacking were male fertile. We found the period of development in wild type strains of P. anserina when there are no microconidia and showed that fertilization without microconidia is possible in P. anserina. No doubt another factor of mail fertility in P. anserina mutants and wild strains were vegetative filaments. Fertilization by not specialized vegetative filaments is known for many ascomycetic fungi but never took into account during experiments with widely used model ascomycetic fungus P. anserina.
The loss of microconidia during long term shake flack cultivation may be explained as a side effect of multiple genome reorganization that were essential for adaptation to liquid conditions of cultivations – new and stressful for the fungus P. anserina.
Our study may be applied into biotechnological area. P. anserina serial cultivation under submerged condition is an example of filamentous fungi strains changing by the method of evolutionary engineering. Such a method is now intensively being studied in point of unicellular microorganisms. Nowadays there is only one publication reported improvement of filamentous fungus (Metarhizium anisopliae) strain by long term submerged cultivation (Crecy E., Jaronski S., Lyons B., Lyons T.J., Keyhani N.O. Directed evolution of a filamentous fungus for thermotolerance // BMC Biotechnology. 2009. Vol. 9, № 74, http://www.biomedcentral.com/1472-6750/9/74).
