| The appearance of multidrug-resistant nematodes that withstands all available classes of anthehnintics and the scarcity of environmental friendly nematicidal drug make plant parasitic nematodes a global problem for agriculture. Screening for novel, high efficient and broad-spectrum nematidal compounds from the microorganism and identification of its mode of action has become an important strategy for developing environmental nematicides. In this study, a bioactivity-screening in nematophagous fungi Pochonia chlamydosporia gave birth to the nematidal aurovertin D with high yield and high activity. The toxicity mechanism of aurovertin D were investigataed in the animal model Caenorhabditis elegans, and the mutation conferring resistance to aurovertin D in C. elegans was also identified. The main findings are as follows:1. Three high toxic strains YMF1.00615,1.00613 and 1.00111 were obtained through bioactivity screening of Potato Dextrose Broth (PDB) of 17 P. chlamydosporia strains? Abundant aurovertins (more than 300 μg/mL) were detected in the bright yellow PDB fermentation broth of these three strains, and aurovertin D was identified as the major bioactive component through quantitive bioassay. Exposure to aurovertin D at sublethal concentration led to high moratlity, decreased pharyngeal pumping rate and brood size, delayed larval development, shorten lifespan in C. elegans. Aurovertin D showed high toxicity on Meloidogyne incognita J2 which was twice more than that on C. elegans.2. Microarray analysis revealed expression of genes related to detoxification, stress, innate immunity was largely upregulated in response to aurovertin D treatment.3. DAF-16/FOXO transcription factor was involved in the defense response against aruovertin D. The bioactivity assay on daf-16(mu86) and its negative regulator daf-2(e1370) mutant show that daf-16(mu86) was sensitive to aurovertin D and the daf-2(e1370) was resistant to aurovertin D.4. Mutants which were resistant to aurovertin D toxicity were obtained by F2 genetic screening.60,000 wild-type N2 genomes were randomly mutated by ethyl methyl sulfone, and the followed F2 genetic screen provided four mutants M1, M2, M3, M4. Aurovertin D-resistant phenotype in M1 may be controlled by a single dominant gene.5. Mutations conferring resistance to aurovertin D in Ml were identified. M1 were crossed with CB4856, which generate the recombination of the differencial single nucleotides in F2 offsprings. We analyzed F2 progeney for SNP and thereby mapped the mutation to 2.97 cM-1.57 cM interval on chromosome Ⅲ. Meanwhile, the genome of Ml was sequenced, and the variants in 2.97cM-1.57 cM interval were confirmed. Finally, the mutation was identified as R471→H in th β subunit of F1Fo-ATPase.6. The amino acid residue equivalent to C. elegans F1Fo-ATPase βArg471 is uaually arginine in other species of nematodes. Out of 15 parasitic nematodes for which the sequence is currently known, arginine is present at this position in 11.In this study, the bioactivity screen in nematophous fungi P. chlamydosporia recommended aurovertin D as the namticidal compound. From a nonclonal genetic screen in C. elegans we isolated 4 mutants resistant to aurovertin D. In one single-gene dominant mutant, a point mutation in R471→H in the β subunit of F1Fo-ATPase protects nematode from aurovertin D-induced injury. In addition, arginine ususally located in the position equivalent to βArg471 in parasitic nematode. These foundings will provide insights for developing novel, broad-spectrum nematicidal agents from the fungi. |
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