Study On The Function Of Ubiquitination-related Genes In Arthrobotrys Oligospora

The ubiquitin-proteasome pathway（UPP）is an essential way for organisms to degrade proteins.The research on UPP is mainly concentrated in plants and mammals,and few in filamentous fungi.The reports documented that ubiquitination-related genes are involved in the biological processes of fungal mycelium growth,stress tolerance,spore production and pathogenicity.Nematode-trapping fungus is a crucial control factor for nematodes population in nature.The formation of trapping devices marks a lifeswitch of such fungi.We have previously shown that NH₃ is a signal for the creation of trap structures,and the transcriptome data linked some ubiquitin-proteasome related genes to regulate NH₃-induced traps formation.Here,we use the fungus Arthrobotrys oligospora ATCC 24927 as a research subject;and collected the genes AOL_s00078g94（E2 ubiquitin conjugating enzyme）,AOL_s00215g519（RING-type E3 ubiquitin ligases）,AOL_s00215g178（RING-type E3 ubiquitin ligases）and AOL_s00004g417（HECT type E3 ubiquitin ligases）as UPP pathway-related genes in A.oligospora through the methods of BLAST comparison,online analysis.The amino acid sequences analysis of the above four genes revealed that the ubiquitination-involved proteins in A.oligospora are conserved in fungi;especially highly in the nematode-trapping fungi Arthrobotrys flagrans and Dactylellina haptotyla CBS 200.50 harboring higher homology:UBCc（AOL_s00078g94）is99.34%and 89.40%,respectively;U-box（AOL_s00215g519）is 94.35%and 86.85%,respectively;Zinc finger（AOL_s00215g178）is 74.03%and 44.50%respectively;HECTc（AOL_s00004g417）is 92.20%and 80.85%,respectively.These data indicated that the ubiquitination regulation is a highly conservative evolution between nematode-trapping fungi.Four mutants of ubiquitination-related genes were successfully obtained by homologous recombination.Western blot results showed that the mutantsΔ78g94,Δ4g417 andΔ215g178 affected the degradation of ubiquitinated protein,and the level of ubiquitination was significantly reduced.Phenotype testing revealed that on basal medium the growth rate of the mutants of RING-type E3 ubiquitin ligases AOL_s00215g519 and AOL_s00215g178 was not significantly different from that of the wildtype.However,the relative growth rate in hyperosmotic medium and high concentration of oxidant-added medium is significantly higher than that of WT,and the sensitivity is reduced;as well as the resistance of the antifungal drug Dimetachlone is increased,and spores production by the mutant on CMY medium is reduced by 47.28±2.28%and 21.95±2.43%,respectively.Besides,nematode-induced traps of these two mutants have not been significantly affected to the wildtype,while exposure to ammonia,the traps ofΔ215g519 decreased by 23.9%against the wildtype.The relative growth rate of the mutants from HECT-type E3 ubiquitin ligases AOL_s00004g417 on Congo red medium that is resistant to cell wall synthesis,is significantly higher than that of the wildtype;and the resistance to Dimetachlone is enhanced;Under nematode induction,the number of traps were reduced by14.36%compared with WT,traps induced by ammonia,increased by 27.47%to the WT;the sporulation on the CMY medium was reduced by 31.74±1.94%.The relative growth rate of the mutant of E2ubiquitin-conjugating enzyme AOL_s00078g94 on anti-cell wall synthesis and hyperosmotic medium was significantly increased,and the sensitivity was reduced;aerial mycelia increased,and the ability of spores production is not substantially different from that of wildtype,and the resistance to Dimetachlone is diminished.Under the induction of nematodes and ammonia,the number of traps produced by the mutant strains were reduced by 34.23%and 33.72%compared with WT,respectively.The results prove that the ubiquitination-related genes are involved in biological processes such as stress,traps formation,hypha growth,drug resistance and spore production in A.oligospora.Snf7 is a component of the ubiquitination-modified protein degradation of ESCRT in the process of eukaryotic cells participating in endocytosis.The homologous recombination method was used to obtain the AOL_s00054g689 mutant strain in A.oligospora,and their phenotypes were assayed.Phenotypic analysis revealed that the growth rate of the mutant was slower than that of the WT,and the edge of the colony was irregular,suggesting that Snf7 may be related to the polar growth of fungi.In the0.3 M NaCl hyperosmotic medium,the relative growth rate of the mutant strain was higher than that of WT,the sensitivity was reduced,and the spore yield was decreased by 29.99±2.46%.The number of nematodes and ammonia induced traps decreased with time by 21.97%and 21.52%,respectively,compared with the WT.In summary,the regulation of ubiquitination of nematode-trapping fungi is highly conserved during fungal evolution.Related genes involved in ubiquitination may be involved in the formation of trps,hypha growth,stress and spore production in A.oligospora.