AreA,a Pleiotropic Regulator,Controlling Nitrogen Metabolism,Stress Resistance And Virulence In Beauveria Bassiana

Fungi have the ability to utilize various compounds as nitrogen sources,but ammonia and glutamine have been recognized as the preferred nitrogen sources.The regulatory strategy that fungi utilize preferred nitrogen sources and limit the expression of nonpreferred nitrogen assimilation-related enzymes is called nitrogen metabolite repression,AreA is a key regulator of nitrogen metabolite repression in filamentous fungi.The nitrogen metabolite repression is relieved when there is no favorable nitrogen source or nitrogen is deficiency.For pathogenic fungi,the nutritional environment and the non-parasitic stage during infection are significantly different,regulation of nutrient metabolism is also one of the determining factors of fungal pathogenesis,and nitrogen metabolism plays an important role in the identification of host and infection of pathogenic fungi.Host is the only source of nutrient in the process of fungal infection and effective nutrient requisition from host is the prerequisite for successful infection and colonization.As one of the important insect pathogenic fungi,Beauveria bassiana infects and kills host insects.Host nutrient deprivation and exhaustion is main reason resulting the final death event.This work aims to explore the roles of AreA-homologous protein,BbAreA,in nutrition metabolism and pathogenicity of Beauveria bassiana by means of RNA antisense and transcriptome analysis,providing a new insight into the mechanism of fungal pathogens-hosts interaction.The main results are as follows:1.Sequence characters and expression features of BbAreA from Beauveria bassianaGenome sequence of BbAreA(BBA?02371)was isolated by searching the Beauveria bassiana genome database,and the open reading frame consists of 3,073 bp.Genome and c DNA sequence analysis revealed that BbAreA contains two introns.Domain analysis revealed that BbAreA contains two nuclear localization signals(245-262 AA and 639-656 AA)and one GATA-like zinc finger domain(594-641 AA).BbAreA was constitutively expressed under different nutrient conditions,particularly induced by insect hemolymph,suggesting that it might be involved in the nutrient assimilation and pathogenicity of Beauveria bassiana.2.Construction and screening of BbAreA-knockdown mutantsThree homologous fragments(850-2100 bp)with different lengths were designed according to the upstream and downstream sequences of BbAreA coding region,and no BbAreA-knockout mutant was obtained using homologous recombination,indicating that BbAreA might be an essential gene in Beauveria bassiana.Subsequently,on the basis of the partial 3'coding region and untranslated region(UTR)of BbAreA,the BbAreA-knockdown mutants were constructed by RNA antisense,among which the expression levels of the transformants,anti AreA-8 and anti AreA-25,were down-regulated by 49.24%and 60.79%compared with wild-type strain,respectively.The two ransformants were used to investigate the function.3.BbAreA is important for nitrogen metabolism and insect nutrients utilization in Beauveria bassianaKnockdown of BbAreA impaired nitrogen,particularly nitrate assimilation in Beauveria bassiana.There was no significant difference between BbAreA knockdown mutants and wild-type strain in terms of colony morphology and growth rate on SDAY medium,but BbAreA knockdown mutants showed a decreased growth rate on different nitrogen source media,and aerial hyphae on secondary nitrogen source(especially sodium nitrate)were barren.Further analysis revealed that the promoter regions of Nrt B,Nia D,Nii A and Nir A,corresponding to nitrate assimilation in Beauveria bassiana,contain 7-14 AreA binding sites,and their expression levels were reduced significantly in anti AreA-8 or anti AreA-25,compared with that in wild-type strain.Suggesting that BbAreA might regulate the expression of these genes through interacting with their promoters.Host is the only nutrient during infection of pathogenic microorganism,the ability of nutrient requisition from host is involved in fungal pathogenicity.Knockdown of BbAreA decreased the fungal protease activity,which contributes partially to the impaired growth under the conditions of insect nutrients.The results suggested that BbAreA might be related to the pathogenicity of Beauveria bassiana.4.BbAreA is necessary for the pathogenicity of Beauveria bassianaBioassay results indicated that knockdown of BbAreA decreased the virulence significantly in Beauveria bassiana,meanwhile affected the host immune response.Larvae injected using conidia suspension of BbAreA knockdown mutants did not produce apparent melanisation response as compared with that treated with wild-type strain,with lower phenoloxidase(PO)activity of hemolymph in the period of 24-60 h post injection.Data derived from RNA-seq of hyphal bodies at 48 h post injection indicated that expression levels of the cluster genes of Op S1 to Op S7 were upregulated in wild-type strain vs anti AreA-8,indicating knockdown of BbAreA enhanced expression of the cluster genes in vivo.Here,we inferred that knockdown of BbAreA activated the expression of Op S cluster in advanced,which enhanced oosporein production,resulting the decreased PO activity in vivo.It was also consistent with phenomenon of visible red pigments produced on cadavers earlier(even not died larvae).5.BbAreA inhibits Beauveria bassiana hyphal body proliferation in the host hemocoelIn terms to evading the host immune response(nodulation and encapsulation)and hemocyte concentration,no obvious difference observed between wild-type strain and BbAreA knockdown mutants.However,knockdown of BbAreA delayed the producing of free-living hyphal bodies in hemoceol,resulting less number of hyphal bodies in vivo.Hyphal body is similar to or also called blastospore in respect to its morphology and development.In vitro(PDB),blastospore concentration of anti AreA-8 or anti AreA-25 was lower 2-times than that of wild-type strain,although the concentrations of all showed increased gradually during the course of 84 h to 144 h.Meanwhile,BbAreA knockdown mutants also showed lower morphology transition as compared with wild-type strain.Thus,knockdown of BbAreA impaired morphology transition and proliferation of hyphal bodies in insect hemecoal cavity,resulting decreased virulence in Beauveria bassiana.6.BbAreA influences the stress responses in Beauveria bassianaBbAreA is involved in fungal adaptation to osmotic and oxidative stresses in Beauveria bassiana.The knockdown of BbAreA led to a significant decrease in the tolerance to osmotic stresses(Na Cl,KCl,Sorbitol),but a significant increase in the tolerance to oxidants(H₂O₂,MND,t-BHP).The results of q PCR and transcriptome datas showed that the expression levels of the aldosterone reductase gene Bbakr1 in anti AreA-8 and anti AreA-25 were significantly lower than that in wild-type strain.There are 17AreA binding sites in the Bbakr1 promoter sequence.Furthermore,sequence analysis indicated there are 17 AreA binding sites in the promote region of Bbakr1,suggesting that BbAreA regulated Bbakr1 expression through interacting with conserved sequences.Expression of Bbakr1 downregulated might be one of the main reasons resulting sensitivity of BbAreA knockdown mutants to osmostresses.However,contrary to Bbakr1deletion mutant(Wang et al.,2018),anti AreA-8 and anti AreA-25 showed antioxidative phenotype,which is not clear yet.BbAreA acts as a negative regulator of secondary metabolite production in Beauveria bassiana.Previous bioassay results indicated that BbAreA was involved in oosporin production in insect.It was also noted that anti AreA-8 and anti AreA-25 could produce obvious yellow pigments in mycelia and blastospores when cultured in PDB.Mass spectrometry analysis showed that the main product of yellow extracts was tenellin,and the production of which in anti AreA-8 and anti AreA-25 was much higher than that in wild-type strain.The cluster corresponding to Tenellin synthesis in Beauveria bassiana contains four core genes,ten A,ten B,ten C and ten S.Expression levels of all these genes were significantly higher in BbAreA knockdown mutants than that in wild-type strain.Previous studies have proved that Tenellin could act as an iron chelator to prevent iron-generated reactive oxygen species toxicity in Beauveria bassiana(Jirakkakul et al.,2015),although it is not required in the fungal pathogenesis(Eley et al.,2007).It might be partially corresponding to antioxidative phenotype resulted by knockdown of BbAreA.7.BbAreA interacts with other metabolic pathways in Beauveria bassianaData derived from RNA-seq of hyphal bodies revealed that knockdown of BbAreA disturbed metabolism of amino acid,fatty acid,energy and peroxisome of Beauveria bassiana in vivo,suggesting the crosstalk among different nutrient metabolism.It was concluded that BbAreA acts as a pleiotropic regulator in nutrient metabolism,adverse tolerance,secondary metabolite production and pathogenesis in Beauveria bassiana.BbAreA contributes to the virulence via modulating metabolism network,morphology transition and proliferation of free-living hyphal bodies in vivo.