| To understand the metabolic effect of aaIT gene on transgenic Beauveriabassiana, this study investigated differential metabolites of transgenic and wide-typeisolates using HPLC-MS-based metabolomics. In addition, we used the same methodto identify the biomarkers of germination and virulence of B. bassiana, the result willcontribute to the screening rate of B. bassiana isolates and products quality control.1The metabolic effects of exogenous gene on transgenic B. bassianaB. bassiana is widely used to control a variety of insect species, but itsapplication has been limited by slow kill and sensitive to environmental conditions.The use of genetically modified B. bassiana with the scorpion neurotoxin aaIT genecan distinctly increase its virulence, whereas the metabolic influence of thisexogenous gene on B. bassiana is unknown till now. Thus, we first used liquidchromatography-mass spectrometry (LC-MS) coupled with multivariate statisticalanalysis to study the global metabolic profiling of mycelia and conidia fromtransgenic and wild-type B. bassiana. Principal component analysis (PCA) andorthogonal projection on latent structure-discriminant analysis (OPLS-DA) revealedclear discrimination of mycelia and conidia from transgenic and wild-type strains,which indicated that the metabolism of B. bassiana is strongly affected by exogenousgene. Hierarchical clustering analysis (HCA) further corroborated significantmetabolic differences between transgenic and wild-type strains. The discriminatingvariations included ergothioneine, glyoxylate, pyruvic acid, fumarate, betaine,carnitine, acetylcarnitine, fatty acids, glycerophospholipids, trehalose, beauverolides,glutamic acid, sphinganine, dimethyl guanosine, etc, of which most important is thedecrease of fatty acids and glycerophospholipids. Pathway analysis found that mostchanged metabolites in mycelia are related to oxidative stress and energy metabolism,for example, a decrease of glycerophospholipids, fatty acids as well as an increase ofoxylipins in transgenic mycelia are due to β-oxidation of fatty acids and the reactionof adding an O2molecule to fatty acids, respectively; the biosynthesis of glyoxylate isfacilitated by isocitrate lyase which may be stimulated by acetyl-CoA generated fromβ-oxidation of fatty acids; carnitine shuttle helps acetyl-CoA cross mitochondrialmembranes to enter TCA cycle for the synthesis of proteins (AaIT and beauverolides)and other metabolites; in addition, trehalose is increased as a result ofgluconeogenesis metabolism is accelerated for the pyruvate carboxylase may be induced by excessive acetyl-CoA. These metabolic changes revealed enhancedoxidative reactions in cells. Correspondingly, some antioxidant metabolites, such asergothioneine and trehalose showed higher levels in transgenic mycelia, whichsuggested that fungal cells are under oxidative stress and cellular protective reactionhas been induced. The alterations of sugars, lipids and carnitine in transformantsuggested that the energy metabolism of B. bassiana was interfered by exogenousgene. Energy metabolism provides driving force for material metabolism in biologicalsystem, it could be inferred that the global metabolic regulation in transgenic strains iscaused by the perturbation of energy metabolism. In contrast to mycelia, althoughglycerophospholipids are decreased in engineered conidia, other compounds related tooxidative stress, such as carnitine, oxylipin, trehalose, mannitol and so on, are eithernot significantly changed or even reduced in conidia, which indicated that theoxidative stress is generated in the processes of transcription and translation, so thereis no significant oxidative stress response when exogenous gene is quiescent inconidia.2Identification of biomarkers of germination and virulence of B.bassianaB. bassiana is widely applied in the control of agricultural pests, the sporegermination rate and virulence to insect are important indicators to estimate itsapplication value. In this study, spore germination rate and virulence of7B. bassianaisolates to Euproctis pseudoconspersa were determined, and LC-MS-basedmetabolomic analysis was applied to identify biomarkers of germination rate andvirulence of mycelia and conidia extracts. The results showed that carnitine, hercynine,acetylcarnitine, α,α-trehalose; Octa-Me, arg-arg-gln, PE(18:2/0:0), PC(18:3/0:0) andPC(18:2/0:0)) are higher in the mycelia of highly virulent isolates. Conidia of highgermination rate were characterized by the higher levels of2,3-dimethylmaleate,acetylcarnitine, propionyl-carnitine and PC(18:2/0:0). Histamine, V-PYRRO/NO,carnitine, acetylcarnitine, propionyl-carnitine, butyryl carnitine, PE(18:2/0:0),PC(16:1/0:0) and PC(18:3/0:0) were higher in the conidia of highly virulent isolates.Furthermore, content comparison of insecticidal peptides, such as beauverolides，beauvericins and bassianolide in mycelia showed that the content of a single peptide isnot responsible for virulence. however, the contents of nine peptides were higher inBb1898indicated that they might exert synergetic effects. This study found that thecommon biomarkers of virulence and germination are acyl carnitine and phospholipid which play roles in maintaining appressorium turgor and providing energy forpenetrating the host cuticle. Thus, the ability of adhesion and penetration is key to thevirulence of B. bassiana, and this is the first report that acyl carnitine andphospholipid are involved in conidia germination. Furthermore, this study alsoidentified other biomarkers, such as hercynine and α,α-trehalose; Octa-Me in highvirulent mycelia,2,3-dimethylmaleate in high germinative conidia, histamine andV-PYRRO/NO in high virulent conidia, their functional mechanisms deserve furtherinvestigations. |
