Mechanism For Trap Formation Of Arthrobotrys Oligopor Induced By Nitrate

The nematode-trapping fungus Arthrobotrys oligospora is a potential biocontrol agent of plant-parasitic nematodes, and a model fungus to study the interaction of nematode-trapping fungi and nematodes. A. oligospora can form three-dimensional networks (traps) to capture and kill nematodes. The capacity of the fungus capturing nematodes is associated with the amount of traps. The molecular mechanism of trap formation is elaborate. Previous researches showed that trap formation could be induced by nitrate and other unpreferred nitrogen sources. Therefore, it is valuable to study the relationship of nitrate metabolism and trap formation. Fungi can uptake and reduce nitrate to ammonium, and this process is regulated by multiple transcription factors. In this thesis, we choose niaD, niiA, nirA and nrtB, which are key enzymes or transporter in nitrate assimilation, to study their function in trap formation. Here, we made mutants of the four genes and compared with the wild type strain for their phenotypic, physiological and biochemical properties, the utilization of different nitrogen and so on.The main results were described as follows:1. We obtain four recombinant plasmids for homologous recombination:nitrate transporter nrtB(AOl_s00004g610), nitrate reductase niaD(AOl_s00210g163), nitrite reductase niiA(AOl_s00210g162) and nitrate assimilation pathway specific transcription factor nirA(AOl_s00004g609).2. Four genes were successfully knockout in A. oligospora. Comparisons were performed with wild type strain in growth rate, spore morphology, nematode-capturing ability and found that all of them can grow on hygromycin medium stably, the growth rate and spores are similar to that of the wild type strain. The abilities to capture nematodes of the△NiaD and△NiiA strains are decreased, but the△NirA and△NrtB strain did not change significantly.3. Diverse nitrogen sources can be used by A. oligospora, such as the preferred nitrogen source ammonium and unpreferred nitrogen sources like nitrate, nitrite, urea, and arginine. The growth of the mutants is not changed when growing on media containing ammonium, urea or Arg. All the stains cannot form trapping device under induction of nematodes on ammonium medium. As a result of lacking relevant genes,△NiaD、△NiiA、 and△NirA could not grow on nitrate and nitrite medium.4. A. oligospora can produce trapping device under induction of nitrate. In the same situation the amounts of traps of mutants are much less than that of the wild strain. Especially, no nematode-traps can be found in the△NiiA strain. Furthermore, add nitrate in medium can significant improve the number of three-dimensional networks under induction of nematodes. With increasing nitrate concentration,the quantity of three-dimensional networks increases, and this trend is end until up to20mM.Innovations in this study:1. Four critical genes related to nitrate assimilation in A. oligospora were knocked out by homologous recombination. We compared these mutants with wild type strain in phenotypic, physiological and biochemical properties, as well as the utilization of different nitrogen sources.2. The ability to use different nitrogen is not same for mutants, we also find that adding nitrate in medium can significant improve the number of three-dimensional networks under induction of nematodes, and help A. oligospora to capture and kill nematodes.