Physiological Mechanisms For Beauveria Bassiana To Detoxify Lipotoxicity Caused By Polyunsaturated Fatty Acid Linoleic Acid

Entomopathogenic fungi（EPF）have been widely developed into mycoinsecticides for integrated pest management program（IPM）,in which unsaturated FAs（UFA）have been considered as bioactive fungicides widely used in control of phytopathogenic fungi.Thus,the compatibility of mycoinsecticides with fungicides has attracted more and more attention.In addition,fungal resistance to UFAs is essential for its interaction with host defense.However,the mechanisms underlying fungal resistance to polyunsaturated fatty acid（PUFA）remain largely unknown.In this study,Beauveria bassiana was used as a representative of EPF to explore fungal responses to linoleic acid（LA）.Lipid droplet protein BbLar1 contributes to fungal tolerance to LA stress Genome-wide expression revealed the transcriptomic responses of fungal cells to LA in a stress-intensity dependent manner.Enrichment analyses indicated that the up-regulated differentially expressed genes（DEGs）associated with metabolism included many genes involved in lipid/fatty acid metabolism,oxidation of fatty acid,cheolesterol homeostasis,and so on.Notably,a lipid-droplet protein（BbLar1）maintains the intracellular homeostasis of fatty acids and is crucial to fungal tolerance to LA stress,which significantly contributes to fungal compatibility with PUFA when infecting the insect hosts.Additionally,BbLar1 links the lipid droplets to global expression profiles in B.bassiana under LA stress.Our investigations provide an initial framework for exploring more mechanistic details about the fungal resistance to PUFA,which improving the efficacy of insect pathogenic fungi.Transcription factor BbLir1 in response to LA stress Among the LA stressed fungal transcriptome,a Zn（II）₂Cys₆transcription factor gene was differentially up-regulated and designated as BbLIR1.Functional analyses indicated BbLir1significantly contributes to fungal tolerance to LA lipotoxicity.Chromatin immunoprecipitation sequencing（Ch IP-seq）was used to explore the down-stream targets of BbLir1.Electrophoretic mobility shift assay（EMSA）was used to determine the direct interaction BbLir1 with four potential target genes.Gene functional analyses a 3-hydroxyacyl-Co A dehydrogenase（BbHad,BBA＿01357）was significantly required for fungal resistance to LA stress.This study highlights a LA-differentially-induced transcription factor for the first time in filamentous fungi and a pathway involved in fungal elimination of lipotoxicity.These findings provide a fundamental framework for explore the mechanisms involved in fungal interaction with PUFA.Bbbcs1 is involved in fungal responses to LA stress by mediating mitochondrial membrane potential In yeasts,bcs1 is a mitochondrial AAA protein（ATPase associated with diverse cellular activities）and required for biogenesis of the complex III in mitochondrial electron transfer chain.However,the presence and biological roles of bcs1 remain largely unknown in the filamentous fungi.In present study,genome-wide identification revealed that there were six BCS1-domain containing proteins（Bbbcs1a through f）in the filamentous insect pathogenic fungus B.bassiana,five of which（except for Bbbcs1f）were functionally analyzed.Phenotypic evaluation revealed that only Bbbcs1b and Bbbcs1c contributed to fungal physiologies,and they localized to nuclei and mitochondria,respectively.Hence,Bbbcs1c is considered as the ortholog of yeast bcs1 in B.bassiana.Ablation of Bbbcs1c did not affect biogenesis of mitochondria,but its loss significantly attenuated mitochondrial functionality（e.g.,ATP synthesis and mitochondrial targeting of proteins）significantly.ΔBbbcs1c mutant displayed the impaired phenotypes in vegetative growth,stress response,development,and virulence.Notably,ΔBbbcs1c mutant displayed the increased sensitivity to linoleic acid（LA）stress and lost the intracellular fatty acid homeostasis.The Bbbcs1c loss compromised the mitochondrial membrane potential,and LA stress exacerbated this damage.These findings indicate that Bbbcs1c is a functional homolog of yeast bcs1 in B.bassiana and links mitochondrial functionality to unique lifestyle in the entomopathogenic fungi.Functional resolution of the BbPEX1,BbPEX6 and BbPEX26 genes in the peroxisome receptor export system In filamentous fungi,recycling of receptors responsible for protein targeting to peroxisomes depends on the receptor export system（RES）,which consists of peroxins Pex1,Pex6,and Pex26.This study seeks to functionally characterize these peroxins in the entomopathogenic fungus B.bassiana.BbPex1,BbPex6,and BbPex26 are associated with peroxisomes and interact with each other.The loss of these peroxins did not completely abolish the peroxisome biogenesis.Three peroxins were all absolutely required for PTS1 pathway;however,only BbPex6 and BbPex26 were required for protein translocation via PTS2 pathway.Three gene disruption mutants displayed the similar phenotypic defects in assimilation of nutrients（e.g.,fatty acid,protein,and chitin）,stress response（e.g.,oxidative and osmotic stress）,and virulence.Notably,all disruptant displayed significantly enhanced sensitivity to linoleic acid,a polyunsaturated fatty acid.This study reinforces the essential roles of the peroxisome in the lifecycle of entomopathogenic fungi and highlights peroxisomal roles in combating the host defense system.Functional analyses of peroxisomal biogenesis factor 11 In fungi and mammals,Pex11 was identified as the first protein to control peroxisome proliferation and membrane elongation,regulating the volume and abundance of peroxisomes.Genome-wide identification of B.bassiana revealed three proteins containing the PEX11 structural domain,named BbPex11a,BbPex11b and BbPex11c.Functional analyses showed that only BbPex11b contributed to utilization of fatty acids,development,and virulence in B.bassiana.In addition,deletion of BbPEX11b leads to a reduction in the abundance and larger size of the peroxisome.Peroxisomal biogenesis factor BbPex11b contributes to fungal tolerance to LA-induced toxicity and compatibility with PUFA when fungus infecting their hosts.Thus,peroxisomal proliferation is also essential for fungal survival under the stress caused by PUFA.In summary,this study revealed several physiological pathways of B.bassiana in response to the stress caused by PUFA.At the organelle level,we revealed that peroxisomes,mitochondria and lipid droplets were involved in fungal response to PUFA stress.At the physiological level,we revealed that the catabolism of fatty acid,mitochondrial membrane potential homeostasis and lipid droplet homeostasis play important roles in fungal tolerance to lipid toxicity.These results not only improve our understanding of molecular pathogenesis of entomopathogenic fungi,but also provide a theoretical basis for improving the biocompatibility of mycoinsecticides with fatty acid-derived fungicides.Additionally,these findings also provide a reference for revealing the more comprehensive mechanisms of filamentous fungi in response to polyunsaturated fatty acid stress.