Regulation Mechanisms Of BbPacC And Bbmsn2 For Oosporein Production In Beauveria Bassiana

Beauveria bassiana,a class of widely used biocontrol fungi,during its’growth and infection,the fungi often synthesizes a kind of secondary metabolites for helpingthemselves to prevent various biological and abiotic stresses.Previous researches have indicate that after the death of infected insects,B.bassiana would produce a mount of oosporein which can inhibit bacterial proliferation.The produce of oosporein are aim to ensure the nutrition required for its’own growth.Oosporein is a red polyketide pigment,which is synthesized by a polyketide synthases-containing gene cluster（Op S）.Op S3 is a transcription factor that directly regulates the expression of other genes in this genecluster.Therefore,to clarify the regulation mechanism of oosporein,we need to explore the regulation mechanism of the Op S3,at present research,we haven’t know thespecific mechanism of oosporein synthesis.In previous works,we analyzed the promoter of OpS3,which found four potential binding sites of transcription factors BbPacC and Bbmsn2.Earlier work confirmed the positive regulation effect of BbPacC on Op S3 by a single hybrid carrier of yeast（Zhang Wenwen,2018）.Our studies have shown that,BbPacC and Bbmsn2 may synergistically regulate the oosporein’s production in liquid culture.The main purpose of this studywas to explore the potential interaction mechanism of oosporein biosynthesis in Beauveria bassiana,the obtained results are shown as below:1.BbPacC and Bbmsn2 regulate the production of oosporein coordinatelyIn this study,we analyze the regulation mechanism of BbPacC and Bbmsn2 on Op S3 by gene knockout and overexpression.We have constructed the two genesoverexpression and double knockout mutants,totally five strains,including msn2^OE,Δmsn2,Pac C^OEmsn2^OE,Pac C^OEΔmsn2 andΔPac CΔmsn2.⑴In liquid culture,the positive regulator BbPacC and the negative regulator Bbmsn2 regulate the biosynthesis of oosporein coordinately;The obtained strains were cultured in 0.5×SDB liquid medium for 3 days,we have observed the color of the liquid culture（the color of oosporein is red）and analyzed the content of oosporein,then find some patterns as below:（1）On the condition that BbPacC has been knockout,the related strain could not produce oosporein,and the overexpression of BbPacC can promote oosporein’s synthesis,which indicate BbPacC is a positive regulator in the biosynthesis of oosporein;（2）On the condition that Bbmsn2 has been knockout,the oosporein synthesis ability of the related strain has increased represently,while overexpression of Bbmsn2 reduce the production of oosporein,indicating Bbmsn2is a negative regulator in the biosynthesis of oosporein;（3）Although overexpression of BbPacC in wild type can increase the oosporein synthesis,while in this condition,overexpression the Bbmsn2 inhibits the synthesis of oosporein;（4）Although knocking out Bbmsn2 in the wild type can increase the production of oosporein,when the strain are lack of both Bbmsn2 and BbPacC it cannot produce oosporein.The above results indicate that BbPacC is a necessary positive regulator in thebiosynthesis of oosporein,and its’regulatory activity is inhibited by the negative regulator Bbmsn2.In different culture conditions,BbPacC and Bbmsn2 regulate the production of oosporein coordinately.After RT-PCR analysis of different strains cultured in liquid 0.5×SDB,we can clearly found the difference in color of the culture medium,as well as,the quantitative detection of oosporein were consistent with the expression changes level about Bb Op S1 and Bb Op S3.⑵The production of oosporein and related gene expression on dead worms infected by different strains;We detected the content of oosporein in dead Galleria mellonella,which were infected by all the type of the mutants and wild type of Beauveria bassiana respectively.The result showed that the content of oosporein was similar to the results of liquid culture.The Galleria mellonella that infected by strains Pac C^OE,Δmsn2,and Pac C^OEΔmsn2seems turn purple than those of infected by wild type and other strains.The determination of oosporein content showed the same result.2.In different culture conditions,The synthesis of oosporein mainly depends on the expression level of BbPacC and Bbmsn2.The previous study shows that the knockout of the zinc finger protein gene Bb Smr1negatively regulated the synthesis of oosporein.Both WT and?Bbsmr1 strain were cultured in 0.25×SDB medium for 3 days,the expression levels of BbPacC and Bbmsn2genes have showed that knocking out Bbsmr1 did not significantly change the expression of BbPacC gene,but significantly reduced the expression level of Bbmsn2.Therefore,Bbsmr1 may negatively regulate the synthesis of oosporein by affecting the expression level of Bbmsn2.In addition,Beauveria bassiana wild-type strains are likely to produce oosporein at higher p H condition.Expression analysis also showed that under low p H conditions,BbPacC expression levels were low and Bbmsn2 expression levels were high;but in high p H conditions,BbPacC expression levels were high and Bbmsn2 expression levels were low.Therefore,the difference in the expression levels of the two genes is responsible for the large amount of synthesis of oosporein by wild-type strains under high p H conditions.In addition,in order to investigate the expression changes of oosporein-related genes in different periods of time after the insect death,we extracted the dead bugs’RNA at 24h,48h,72h,and 96h after death to analyze the expression of Op S1,Bb Smr1,BbPacC,and Bbmsn2.The results showed that,the expression of Op S1 gradually decreased,while the expression of Bbmsn2 gradually increased,so that the increase of the expression level of Bbmsn2 suppressed the expression of Op S3,which proved that Bbmsn2 is negative regulatory factors of oosporein’s produce.The results shows that in the early time after the insect death,the expression levels of Bbsmr1 and Bbmsn2 were very low,so the expression of oosporein synthesis gene Op S1 was high;but 48h after the death of insects,the expression levels of Bbsmr1 and Bbmsn2 increased significantly.Although the expression level of BbPacC was also significantly up-regulated,due to the high expression level of Bbmsn2,the expression of oosporein synthesis gene were also inhibited.This result is consistent with the results which we have observed earlier,in the early time of insect death the level of oosporein synthesis was higher and as time goes,it decreased gradually.3.BbPacC positively regulates oosporein by directly binding to the OpS3 promoter.To prove whether BbPacC and Bbmsn2 are directly binding to the promoter of the oosporein synthesis regulatory gene Op S3,we expressed these proteins in E.coli.All the proteins are GST-tagged and were constructed by inserting gene（Bbmsn2,BbPacC）or gene fragment(BbPacC²⁵¹)into the MCS of p GEX-6p vectors.The purified protein were used for EMSA,the result shows there are no obvious binding phenomenon in the EMSA result.Considering the fusion proteins were produced in E.coli,it may affecting the correct structure of proteins,so we use another way to obtain those proteins.We use the total protein in different Beauveria bassiana strains for EMSA by cell disruption solution.The results have shown that the total protein extracted from BbPacC^OEand WT strains were binding to the Op S3 probe,and the result of total protein fromΔBbPacC strain shows no binding phenomenon.This result indicates that BbPacC can directly bind to the Op S3 promoter,thereby regulating its expression.This study explored the regulatory effects of BbPacC and Bbmsn2 on oosporein synthesis.BbPacC is a necessary regulatory factor for oosporein synthesis,but Bbmsn2can inhibit the positive regulatory activity of BbPacC;Bb Smr1 negatively regulates the expression of Bbmsn2,thereby negatively regulating oosporein synthesis.BbPacC positively regulates the Op S3 promoter by binding to the potential site directly.