Functional Analyses Of Autophagy-related Genes In Nematode-trapping Fungus Arthrobotrys Oligospora

Arthrobotrys oligospora is a representative species of nematode-trapping(NT)fungi,which can develop the typical three-dimensional adhesive networks that called traps for nematode predation.In the early stage of trap formation,the hyphae induced by nematodes that formed one or two-dimensional adhesive networks,and then form three-dimensional sticky networks in the mature stage of trap formation.Moreover,the nitrogen nutrition(amino acid,peptide and urea)was essential for trap formation.Autophagy is a highly conserved pathway in eukaryotes,when cells response to different stress that induce the process of autophagy to degrade organelles and protein aggregates,recycling of these components to cells against the environmental stresses,such as starvation.The autophagy was precisely regulated by autophagy-related genes(proteins).In this paper,four homologous genes of autophagy-related genes(AoATG1,AoATG4,AoATG5,and AoATG13)were characterized in A.oligospora by multiple methods such as gene knockout and GFP-flag technology,phenotype analysis,transcriptome,and metabolome.Our results provide an essential experimental evidence of the autophagy involved in growth,development,and cell differentiation of A.oligospora and other NT fungi.The main results as follows:Ⅰ.AoATG1,AoATG4,and AoATG5 genes impaired the mycelia growth and the development of aerial hyphae.Four autophagy-related genes(AoATG1,AoATG4,AoATG5,and AoATG13)were retrieved from A.oligospora by blasted analysis in Gen Bank.The generation of the deletion constructs was conducted using the homologous recombination method.In this study,the ΔAoATG1,ΔAoATG4,andΔAoATG5 mutants reduced growth rate of hyphae and reduced density of the aerial hyphae in different medium in comparison with the wild-type(WT)strain.Ⅱ.AoATG1,AoATG4,and AoATG5 genes involved in conidiation,conidial germination and the expression of sporulation-relative genes.Deletion of AoATG1,AoATG4,and AoATG5 caused a decrease in conidia yield and conidial germination.Moreover,the relative transcript level(RTL)of sporulation-related genes flu G,aba A,brl A and wet A was significantly decreased in the ΔAoATG1,ΔAoATG4,and ΔAoATG5 mutants.Ⅲ.AoATG4 and AoATG5 genes weaken the cell nucleus number.The cell nucleus was treated using DAPI staining solution,the number of cell nucleus was significantly decreased in ΔAoATG4 and ΔAoATG5 mutants compared to the WT strain.In addition,according to the transcriptome of the WT and ΔAoATG5 mutant,many differentially expressed genes(DEGs)were enriched in DNA biosynthesis,repair,and ligation under nitrogen starvation.And several genes associated to DNA biosynthesis,repair,and ligation,such as h1,ner,prbs,rad3,rad25,spo11,and tfiik,their RTLs were remarkably decreased in ΔAoATG5 mutants.Ⅳ.AoATG1,AoATG4,AoATG5,and AoATG13 genes play a crucial role in autophagy.In nitrogen starvation condition,using functionally expressed GFP-AoATG8 fusion proteins and TEM to detect the autophagosome formation and autophagic process in the WT and mutant strains,GFP-ATG8 signals were observed in the vacuoles of the hyphae and traps of WT,whereas no GFP-ATG8 signals were observed in vacuoles of mutants.These results revealed that the transport and degradation of GFP-ATG8 delivered to vacuoles was blocked in the mutants.Ⅴ.AoATG1,AoATG4,and AoATG5 genes involved in trap formation and pathogenicity.Compared with WT strain,the trap production was remarkably reduced and the nematocidal activity of mutants was significantly impaired.The morphology of traps was furtherly observed,many traps produced by the ΔAoATG4,ΔAoATG4,andΔAoATG5 are unmatured traps containing mycelial loops from one to three.Moreover,the RTLs of the autophagy genes such as ATG1,ATG8,ATG9,and ATG10 were significantly upregulated in ΔAoATG5 mutants under nitrogen starvation.Ⅵ.AoATG4 and AoATG5 affected the stress resistance.Mycelia growth on media containing osmotic,oxidative and cell wall synthesis-disturbed reagents were compared between the WT and mutants.The relative growth inhibition rates of ΔAoATG4 andΔAoATG5 mutants showed change in different degrees,and the sensitivity of ΔAoATG4 mutants was increased to menadione and Congo red.Ⅶ.AoATG1,AoATG4 and AoATG5 genes are involved in secondary metabolism.Using the metabolome analysis,the peak area of many compounds was remarkably altered in ΔAoATG1,ΔAoATG4,and ΔAoATG5 mutants compared to the WT strain,especially the peak area of arthrobotrisin was significantly increased than that in the WT strain.Ⅷ.Comparison of transcription profiles between the WT,ΔAoATG4,andΔAoATG5 mutants.The DEGs mainly enriched in autophagy,vesicle-mediated transport,endosomal transport,protein ubiquitination and ribosome systhesis in ΔAoATG4 andΔAoATG5 mutants under nitrogen starvation condition.At the early stage of trap formation(12 h),the DEGs of WT vs ΔAoATG5 mainly enriched in oxidation-reductio process,oxidoreductase activity,small molecule metabolic process,chitinase activity,organic substance biosynthetic process,cellular biosynthetic process and transmembrane transport.At the later stage of trap formation(36 h),DEGs of WT vsΔAoATG5 enriched in molecular-function,catalytic activity,peroxidase activity,oxidation-reductio process and oxidoreductase activity.At the stage of trap mature(24h),the downregulated DEGs of WT vs ΔAoATG4 mainly enriched in carbohydrate metabolic process,cellular amino acid metabolic process,peptide biosynthetic process,oxidation-reductio process and ribosome were downregulated;in contract,the upregulated DEGs mainly enriched in DNA ligation,unsaturated fatty acid biosynthetic process,chitinase activity and peroxidase activity.The main innovation as follows:Ⅰ.Four autophagy-related genes AoATG1,AoATG4,AoATG5,and AoATG13 were characterized in A.oligospora for the first time,they all involved in the regulation of autophagosome formation and autophagic process.Especially,the autophagic process was investigated using GFP-Atg8,resulted in blockage of autophagic process in traps cell by deletion of AoATG4 and AoATG5 genes.These results provide a basis for further elucidating the mechanism of autophagy regulating trap development.Ⅱ.This study revealed that AoATG4 and AoATG5 weaken the cell nucleus number development in filamentous fungi.According to transcriptome analysis,the DEGs associated to cell nucleus development were enriched in DNA biosynthesis,repair,and ligation under nitrogen starvation,and the expression of these DEGs was significantly upregulated in the ΔAoATG5 mutant.These results provide a basis for further elucidating the mechanism of autophagy-related genes regulating cell nucleus number development.Ⅲ.This study revealed that AoATG1,AoATG4,and AoATG5 involved in the regulation of trap formation.According to transcriptome analysis at the different stages of trap development,several processes may involve in trap formation were identified,such as chitinase activity and peroxidase activity.This research provides a basis for investigating the developmental mechanism of trap morphology.