Preliminary Study On Conditions And Mechanisms Of Amino Acids Induced Adventitious Organs

Nematode-trapping fungi play important roles in maintaining nematode population density in natural environments, and they are important materials for biological control of plant disease caused by nematodes. Trapping devices (traps) are the important tools for fungi to capture nematodes, and the formation of traps by nematode-trapping fungi is an important indicator of their switch from the saprophytic to the predacious lifestyles. Previous studies found that several amino acids can induce the nematode-trapping fungi to produce traps, while little is known about the molecular basis of amino acids regulate the trap formation in the nematode-trapping fungi. In this study, the fungus Arthrobotrys oligospora, a typical strain of nematode-trapping fungi, was used to screen amino acids which could induce the trap formation effectively, and identify the candidate genes involved in the regualtion of trap formation in A. oligospora under induction conditions by amino acids using real time PCR analyses, and reveal the biological roles of candidate genes in A. oligospora using gene knockout.The main findings of this study were presented as follows:1. Trap formation of A. oligospora induced by amino acids and conditions optimizationSpores of A. oligospora were suspended using different kinds and concentrations of the amino acids solution, and spread to water agar plates and incubated at28℃for one week, the formation and the number of traps of A. oligospora was observed and calculated. The results showed that tested amino acids can induce the trap formation of A. oligospora, and different kinds of amino acids can induce the A. oligospora to produce the most traps when the concentration of amino acid at0.05g/L. Meanwhile, three amino acids (valine, leucine and glutamic acid) can induce the A. oligospora produce more traps, and the induced effect of valine is the best.2. Amino acids induce the Arthrobotrys brochopaga to produce trapsMeanwhile, the traps of A. brochopaga were induced by amino acids, and ten kinds of amino acids including valine can induce the A. brochopaga to produce traps. Similarly,0.05g/L of amino acids can induce the A. brochopaga to produce the most traps. In addition, leucine and glutamic can induce the A. brochopaga to produce more traps, and the effect of leucine is the best.3. Identify the candidate genes involved in the regualtion of trap formation in A. oligospora under induction conditions by amino acidsBased on the A. oligospora genomic and transcriptomic data, partial genes involved in nitrogen utilization and the G protein-mediated signal transduction pathways were selected. The expression levels of these genes were analyzed using real time PCR in induced and non-induced conditions. Real time PCR results showed that many genes showed a larger amount of change, indicating that these genes may involved in the regulation of trap formation during induction process by amino acids. By comparison the data of real time PCR, several genes/proteins may be involved in the regulation of trap formation in A. oligospora during induction process by amino acids were chosen, such as GPCR (AOL_s00079g307and AOL_s00054g626), G protein subunits (AOL_s00083g167), Phospholipase C (AOL_s00007g83and AOL_s00007g547), Gcn2(AOL_s00215g639) and Tor (AOL_s00043g12). Two genes, AOL_s00043g12(Tor) and AOL_s00215g639(Gcn2), both associated with nitrogen metabolism genes, were chosen as target genes for study the function in A. oligospora by gene knockout.4. Construction of AoTor and AoGcn2Knockout vectors and transformationThe specific primers were designed and amplified the5’and3’ends homologous sequences of AoTor and AoGcn2, and the marker gene (Hygromycin resistance gene hph). The plasmid pRS426was linearized, and was electroporated to yeast and E. coli competent cells, the knockout vectors of AoTor and AoGcn2had been constructed. Meanwhile, the positive tranformant of gene AoGcn2has been screened.5. Phenotypic characteristics of wild type strain and△Pex strains.The△Pex5and△Pex7strains were constructed by homologous recombination in our previous study. In this study, three mutants (△Pex5-51,△Pex5-X and△Pex7-59) were compared with the wild type in growth rate, sporophore morphology, conidiation, trap formation, extracellaluar proteases production and stress resistance. The results showed that△Pex5and△Pex7strains are more or less associated with those biological processes. Meanwhile, all mutants are significantly lower than wild-type in sporulation. Especially, the△Pex5strains abolished to produce conidia and traps.The novelties of this study were described as follows:1) A new method was developed for trap formation in nematode-trapping fungi A. oligospora and A. brochopaga using amino acids. Several amino acids were screened to induce the trap formation effectively in A. oligospora and A. brochopaga, and the optimal conditions for trap formation were determined.2) The candidate genes involved in the regualtion of trap formation in A. oligospora were chosen using real time PCR analysis. The knockout vectors of the AOL_s00043g12(AoTor) and AOL_s00215g639(AoGcn2) were constructed, and the positive transformant of gene AoGcn2has been screened.