Molecular Mechanism Of Autophagy Involved In Biocontrol Potential Of Beauveria Bassiana

Beauveria bassiana is a typical entomopathogenic fungus and plays an important role in the biocontrol of agricultural and forestry pests.Autophagy is a conserved self-degradation mechanism that governs a large array of cellular processes in filamentous fungi,including vegetative growth,cell differentiation,asexual or sexual development and pathogenesis.This study aims to update the understanding of autophagic events that are associated with their phenotypes crucial for biocontrol potential.This study provides a theoretical basis for improving the biological potential of B.bassiana.Autophagic and non-autophagic roles of BbATG8 ATG8 is the‘core'gene of autophagy pathway,which not only plays an important role in the process of autophagy,but also has some non-autophagy functions.In B.bassiana,the absence of BbATG8significantly affected the asexual development,tolerance to oxidative stress and virulence.These are mainly due to the autophagic function of BbATG8,while its non-autophagic function contributes to conidiation and virulence.In the?BbATG8,the conidial yield was reduced by 86.50%when compare to the wild type.In?BbATG8^ATG8-(GSI)strain(complementing?BbATG8 strain with ATG8 without C-terminal GSI),the conidia yield was 9.44%higher than that of?BbATG8.In the bioassay via cuticle infection,the virulence of?BbATG8^ATG8-(GSI)was recovered by 15.70%when compared with?BbATG8.In addition,in the wild type strain,over-expression of BbATG8 resulted in the enhanced pexophagy and mitophagy,which causing the significant impairments on asexual development,stress resistance and virulence.However,over-expression of BbATG8-(GSI)had no significant effect on fungal physiologies.This study showed that the significance of BbATG8 in B.bassiana is mainly to its autophagic function,and its protein level must be maintained in the whole lifecycle.Functional analysis of selective autophagy scaffold protein BbATG11Autophagy is a conserved degradation system in eukaryotic cells that includes non-selective and selective processes.Selective autophagy functions as a selective degradation mechanism for specific substrates in which autophagy-related protein 11(ATG11)acts as an essential scaffold protein.In B.bassiana,there is a unique ATG11family protein,which is designated as BbATG11.Disruption of BbATG11 resulted in significantly reduced conidial germination under starvation stress.The mutant?BbATG11 displayed enhanced sensitivity to oxidative stress and impaired asexual reproduction.The conidial yield was reduced by approximately 75%,and this defective phenotype could be repressed by increasing exogenous nutrients.The virulence of the?BbATG11 mutant strain was significantly impaired as indicated in topical and intra-hemocoel injection bioassays,with a greater reduction in topical infection.Notably,BbATG11 was involved in pexophagy and mitophagy,but these two autophagic processes appeared in different fungal physiological aspects.Both pexophagy and mitophagy were associated with nutrient shift,starvation stress and growth in the host hemocoel,but only pexophagy appeared in both oxidation-stressed cells and aerial mycelia.This study highlights that BbATG11 mediates pexophagy and mitophagy in B.bassiana and links selective autophagy to the fungal stress response,conidiation and virulence.Autophagy involved in conidial survival Asexual conidiation is crucial for fungal persistence and dispersal in environment,and conidia act as the active ingredients of fungal insecticides.Therefore,conidial yield and quality are determinant for biocontrol potential of entomopathogenic fungi.In this study,disruption of the‘core'autophagy gene BbATG8 severely impaired the survival rate of conidia,which is mainly involved autophagy function.In?BbATG8 mutant strain,conidia almost did not germinate on the nutrient medium after being stored for 28 days and lost the ability to infect the hosts.Also,deletion of BbATG1 and BbATG11 also significantly compromised the survival rate of conidia.Notably,BbATG1 and BbATG8 had more important influences on conidial survival than BbATG11.Further research revealed that the deletion of the aspartyl peptidase(BbAPE4)gene had a significant effect on the survival of conidia.Importantly,BbAPE4 trafficked into vacuole via direct interacting with BbATG8,which differing with the trafficking of yeast APE4 that is dependant on BbATG11.In this study,autophagy had been linked to the survival B.bassiana conidia post maturation,including selective and non-selective autophagy.Meanwhile,we found a BbATG11-independent selective transport mechanism in which ATG8 acts as a direct receptor.Functional analysis of BbAPE1A and BbAPE1B The cytoplasmic vacuole targeted transport(CVT)pathway is a selective transport pathway mediated by autophagy related proteins.Aminopeptidase I(APE1)is the major cargo protein of the CVT pathway in yeast.There are two typical APE1 homologous proteins in B.bassiana,named BbAPE1A and BbAPE1B.Under starvation stress,the deletion of BbAPE1A or BbAPE1B genes resulted in a decrease in conidial germination.BbAPE1A and BbAPE1B significantly contributed to fungal development and virulence.The single disruption of gene BbAPE1A or BbAPE1B reduced the blastospore production by 85.20%and 24.67%,respectively.BbAPE1A and BbAPE1B were transported into the vacuole in a quite different.Similar to yeast CVT pathway,the entry of BbAPE1A and BbAPE1B into vacuoles requires the autophagy gene BbATG1 and BbATG8.However,BbAPE1A required BbATG11,while BbAPE1B did not.Moreover,our results showed that BbAPE1B did not interact with BbATG8 directly.This study emphasizes that B.bassiana harbors two orthologs of yeast APE1,and they contribute to fungal physiologies via different trafficking pathways.BbATG8 interacts with Bb GIL in regulation of lipid homeostasis Lipid is a critical molecular component in filamentous fungi and plays an important role in mycelial growth,stress tolerance and virulence.In this study,B.bassiana GDSL lipase(Bb GIL)was functially characterized.Disruption of Bb GIL resulted in a significantly reduced conidial yield with reduction of 32.34%.The content of lipid droplets in the conidia was significantly increased,and the virulence of?Bb GIL was significantly delayed compared with the wild strain.When over-expressing Bb GIL in the wild type,the amount of lipid droplets was significantly reduced in mycelia;conidial yield was decreased by 90.69%;fungal virulence was significantly weakened.Bb GIL protein was mostly located in the cytoplasm during the normal growth stage,but almost all of them were transferred to the vacuole during starvation stress.Moreover,the entry of Bb GIL into the vacuoles was directly mediated by BbATG8.This study indicates that BbATG8interacts with Bb GIL to maintain the lipid homeostasis during conidiation,and to translocate the lipase to vacuoles under starvation...