Characterization Of Superoxide Dismutases, And Developmental Activators And Related Transcription Factors In Beauveria Bassiana

Beauveria bassiana is a filamentous fungal entomopathogen that has been widely applied for biological control of instect and mite pests worldwide. Unicellular conidia are active ingredients of numerous fungal insecticide based on B.bassiana.Upon field application, formulated conidia are inevitably exposed to environmental stresses, such as high t;emperature,solar UV irradiation, and applied chemical flingicides and herbicides,which are inducers of reactive oxygen species（ROS） harmful 1 to fungal cells. Thus,the biological control potential of a candidate strain is dependent not only on conidiationcapacity and conidial quality but also on virulence and multistress tolerance. Superoxide dismutases（SODs） are very important antioxidant enzymes teat constUue a primary source of celluar defenses against ROS damage. Asexual development for conidial production and maturation is genetically controlled by central developmente 1 pathway in filamentous flmgi but teis pathway has not been explored in fungal insect pathogens. This sidy sought to elucidate roles of teree uncharacterized SODs in l:he antioxidant and anti-UV responses and host infection of B,bassiana,and to probe how three developmental activators in tihe central pateway and five transcripton factors up- or downstream of tee pateway regulatetee fungal conidiation capacity and conidial quality. The results are summarized below.Distinct contributions of one Fesod and two Cu/ZnSODs to antioxidation, UV tolerance and virulence of .The insect pathogente bassiana harbors five distinct SODs, including cytosolic MnSOD 2) and mhochondrial MnSOD od3)which dominate intracelluar SOD activity and conl;ribute additively to antioxidation and virulence. Here we show the functions of a cytosolic Cu/ZnSOD（Sodl）, a mitochondrial FeSOD （Sod4） and a cell wall-anchored Cu/ZnSOD （Sod5）. The latter two are poorly known despite exi(Sence in many filamentous fxmgi, and Iheir subcellular localization was well COnfirmed with fused Sod4::eGFP and Sod5::eGFP expressed in transgenic cells stained with specific s1;ains. To1:al SOD activity was decreased by in increased by 11₂0 in three knockdown mutants tmt unaffected in when co-cultivated with menadone or H202. Surprisingly, knockdown of whose deletion was lethal, caused more decrease of total catalase activity（69-75%） than 50^/7过eletion（27—33%）, COntxasting to n0 change in AsodJ. Transcript levels of other SODpartners and five catalases also changed tee miuants much an in Asoc/7 an5, As a COnsequence of global effect, A5(%/7 showed most di"astic increase of intracellular ROS level in response to both oxidants,Howed by tee mutants and AJl the mutants displayed differentially increased sensitivity to either oxidant,accompanied with deerased tolerance to UV-AAJV irradiations,attenuated vimlence but no response to high osmolarity,cell wall stress and high temperatuK, Taken together wUh previously charaerized Sod2 and Sod3, our resuhs provide full insight into tee SOD family, unveiling inteiactions of SODs catalases in 1:he fungal antioxidant reaction.Control of asexual cycle, conidial maturation and cemidial quality by the devdopmente 1 activates rlA,AbaA, WetA and VosA in te to/a(Ste Many(Slamentous fungi produce only conidia as means to dispersal and survival Zro orte Here we show thaUhe developmental iegulators BrlA, AbaA, WetA and VosA are inspensible for conidiophore devdopment,COnidiation and matuiation in te Abassianaful/(SG.Deletion of each target gene drastically altered 1:emporal transcription patterns of not only three other partners but upstream transcription factors（FluG and FteB—E）. Consequently,and Zkibbassiana cannot fbmi any conidiophore and aerial COnidium or produce unicellular blast;ospores under submerged conditions. Conidiation was reduced by 98% in and 88% in and also suffered severe defects in blastospore production.In hyphal cells, autophagic bodies disappeared entirely in the hyphai vacuoles of and Aa&te4, largely reduced in AweL4, but appeared normal!y in wil-type and Avas Mature conidia of suffered more changes in size, density, hydrophobicky and cell wall composition than of while less hehalose accurrmlated in aged Avosyi cukure.Cemidia of te(Se two mutants showed (Sten defects in viability, 1;hermotoleiance,UV-B resistsnce and viru!ence. Taken together, the four regulators cbassianantrol sequential events in asexual cycle transcriptional interaction and/or autophagic regulation and hence contribute greatly to conidial yield and/or quality in 6bassianasana.Characterization of Flb family transcription factors upstrea of the cental developenta! pathway in te AassZa/ifif. FteB, FteC, FteD and nbE ate imcharacterized transcription factors upstream of the central pathway in 6osbassiana（Sbassiana.Single /76 deletions resuhed in elastic changes in Ixanscriptional expression of a key activator gene 1the activation of the central pathway,during 7-day norma） cultivation.The transcriptwas depressed by 90-99% in B and 82-94% in A/teC consistently during,50% in/SflbD on days 5 and 6, and more 1:han 50% in on days 4 and 5 and less 1;han 50 during the first three days. Moreover, one deletion also led to drastic alterations intempoi’al transcription patterns of three other genes at the critical stages of vegetative growth and/or conidiophores development under normal culhiie conditions. These changes indicate that the four factors infract one another at transcriptional level and hence directly or indirectly activat;ettie4 expression fbr 1:he activation of l:he central pathway and l:he initiation of conidiophore development. Consequently, conidiation capacity was mostly reduced by 91% in iSflbC compared with wild-type, and by 42%, 30% and 25% in iSflbE,AflbD and AflbB respectively. The four fib deletion mutants also showed different degrees of multi-phenotypic defects, including reduced capability of their using environmerUal carbon/nitrogen sources, delayed conidial germination, decreased conidial size and complexity（density）, alt:ered cell wall composition,attenuated virulence, and increased sensitivity te oxidation,cell wall perturbation,high osmolarity and high temperatute,Allteese changes were resbi’ed by each targe1;ed complementation. Taken t;ogether,FteB,FlbG, FlbD and FlbE not 0nly activate the BrlA expression in a direct or indirct maimer but are differentially involved in nutritional utilization, multistress responses and insect host infection.