The Physiological Mechanisms Involved In Membrane Homeostasis And Iron Acquisition Regulated By The BZIP-type Transcription Factor BbHapX In Beauveria Bassiana

Beaueria bassiana is an entomogenous filamentous pathogen with a host range and excellent biocontrol potential.Whether in the in vitro environment or parasitic in the host,a series of mechanisms have evolved by reason of the adaptation to the environment and the development for niche.In an iron-stressed environment,fungi have evolved a conservative iron absorption and utilization system to help them survive the threat of environmental changes.In the process of species evolution,membrane homeostasis is one of the necessary conditions for cells to maintain the normal operation of the organism,and it is particularly important for fungal species.Not only they have to balance the mechanical pressure from the inside(cytoplasm)and the outside(cell wall),but also accurately complete the assembly and deployment of lipids and proteins.This study found that the downstream target genes of the bZIP transcription factor BbHapX respond to iron starvation regulation and the functions of membrane homeostasis regulation.BbHapX maintains plasma membrane homeostasis and cell membrane function of fungal conidia In pathogenic filamentous fungi,conidial germination not only is fundamental for propagation in the environment but is also a critical step of infection.In B.bassiana,we genetically characterized the role of the basic leucine zipper(bZIP)transcription factor HapX(BbHapX)in conidial nutrient reserves and pathogen-host interaction.Ablation of BbHapX resulted in an almost complete loss of virulence in the topical inoculation and intrahemocoel injection assays.Comparative transcriptomic analysis revealed that BbHapX is required for fatty acid(FA)/lipid metabolism,and biochemical analyses indicated that BbHapX loss caused a significant reduction in conidial FA contents.Exogenous oleic acid could partially or completely restore the impaired phenotypes of the ΔBbHapX mutant,including germination rate,membrane integrity,vegetative growth and virulence.BbHapX mediates fungal iron acquisition which is not required for desaturation of stearic acid.Additionally,inactivation of the Δ9-fatty acid desaturase gene(BbOle1)generated defects those of the ΔBbHapX mutant;oleic acid also had significant restorative effects on the defective phenotypes of the ΔBbOle1 mutant.EMSA revealed that BbHapX directly regulated the expression of BbOle1.Lipidomic analyses indicated that both BbHapX and BbOle1 contributed to the homeostasis of phospholipids with nonpolar tails derived from oleic acid;therefore,exogenous phospholipids could significantly restore membrane integrity.These data reveal that the HapX-Ole1 pathway contributes to conidial fatty acid/lipid reserves and that there are important links between the lipid biology and membrane functionality involved in the early stages of infection caused by B.bassiana.BbLip1 initiates the infection cycle by regulating the homeostasis of cell membrane lipids Lipases are an indispensable class of enzymes that support the growth and development of organisms.Not only they catalyze biochemical reactions,but also play an important role in maintaining lipid homeostasis and cell metabolism.Lipase is responsible for the catalytic degradation of triacylglycerols stored in lipid droplets into free fatty acids and triglycerides.In this study,the lack of BbLip1 not only directly ledto physiological defects in the growth and virulence of B.bassiana,but also caused a significant reduction(50%)in oleic acid content.When exogenous oleic acid was added,the defects of BbLip1 were partially restored.Lipidomic analysis showed that disruption of BbLip1 directly caused the imbalance of the ratio of free fatty acids and storage lipids in the conidia of B.bassiana.EMSA indicated that BbHapX was required for transcriptional activation of BbLip1.These results suggested the hydrolase BbLip1 is involved in endogenous lipid metabolism under the control of transcription factor BbHapX,which affecting cytomembrane homeostasis in fungal development,stress tolerance and virulence.BbLec1 is involved in the homeostasis of the cell membrane and wall Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology.B.bassiana has a lectin-like protein containing a Fruit Body＿domain.BbLec1 could bind to chitobiose and chitin in fungal cell wall.BbLec1 proteins displayed the potential of interacting between their own and translocated into eisosomes.Further,BbLec1 interacted with the eisosome protein PliA,and the interdependence between these two proteins was essential for stabilizing the eisosome architecture.To test the BbLec1 roles in B.bassiana,we constructed the gene disruption and complementation mutants.Notably,the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity.In addition,disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress.Finally,ΔBbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain.Taken together,BbLec1,as an eisosome component,maintains the architecture and functionality of eisosome that is critical for virulence of filamentous insect pathogenic fungus B.bassiana.The physiological roles of the BbCFEM family proteins in response to iron starvation Iron,as a trace element necessary for most prokaryotes and all eukaryotes,is essential for maintaining the cellular growth and metabolism.The CFEM(Cysteinerich Fungal Extracellular Membrane)protein family is characterized with conserved eight cysteine residues in fungi.In B.bassiana,there were 12 protein belong to CFEM protein family 11 of which were functionally characterized.All tested genes were involved in fungal acquisition of iron during vegetative and pathogenic growth,their necessities varied with ambient iron availabilities.In addition,BbCFEM family genes contributed to fungal competition with other entomopathogenic and saprophytic fungi.These results suggested that BbCFEM family proteins significantly contribute to iron acquisition,which facilitating fungal pathogenicity and survival in environment.In a word,Beauveria bassiana BbHapX maintains the mechanism of conidia membrane homeostasis and cell membrane function,BbLip1 participates in the catabolism of lipid droplets to maintain cell membrane lipid homeostasis,the lectin protein BbLec1 participates in the mechanism of cell membrane wall homeostasis,and the BbCFEM family Physiological mechanism in response to iron starvation regulation.Research reveals:(1)The bZIP transcription factor HapX acts as a master regulator involved in the virulence of conidia and regulates fatty acid/lipid metabolism;reveals that the HapX-Ole1 pathway is involved in fatty acid/lipid accumulation related to conidia maturation This partly clarifies the function of the HapX-Ole1 pathway to maintain membrane homeostasis;(2)B.bassiana fatty acid hydrolase gene BbLip1 participates in lipid droplet metabolism and endogenous lipid breakdown through BbHapX transcriptional activation,and Contributed to plasma membrane homeostasis;(3)confirmed that B.bassiana BbLec1 is closely related to cell wall function,cell membrane integrity development and virulence;(4)discovered the compensatory effect of the BbCFEM family in response to iron starvation regulation;elaboration The necessity of BbCFEM family genes in host-host interaction and competition among different fungal species is discussed.This confirmed the conservative function of the BbCFEM family of B.bassiana in the regulation of iron starvation.Those results provide a basis for analyzing the biological functions of genes involved in iron starvation regulation and membrane homeostasis downstream of BbHapX,and also provide a theoretical basis for the evolution of gene functions of filamentous fungi in complex and diverse environments,and enrich the interaction between fungi and hosts.The theoretical understanding of the role has important theoretical significance.