Construction, Analysis And Validation Of Quantitative Phosphoproteome And Lysine Acetylome In Beauveria Bassiana

Beauveria bassiana is a filamentous entomopathogen that has been widely applied in biological control of agricultural and forest insect pests. The fungal pathogen usually survives in asexual cycle and is able to infect the broadest spectrum of insect hosts and adapt to their diverse habitats. The B. bassiana-based insecticides comprise active ingredients of intact cells, such as conidia, and upon application, are inevitably exposed to environmental stresses, such as high temperature, solar UV irradiation and applied pesticides, which are detrimental to fungal cells. Thus, it is necessary to explore mechanisms involved in the regulation of asexual development, cellular anti-stress responses and host infection. This sought to reveal a significance of phosphorylation and acetylation for the fungal life by constructing and analyzing quantitative proteome and phosphoproteomc mediated by the phosphatase Cdcl4 and the fungal lysine acetylome. The validity of the constructs was examined by multi-phenotypic analyses of deletion and phosphorylation site-directed mutants of a mitogen-activated protein kinase kinase (Ste7) in Fus3/Kssl pathway and of two O-mannosyltransferases (Pmtl and Pmt4) in B. bassiana. The results are summarized below.Proteomic and phosphoproteomic insights into a signaling hub role for Cdcl4 in asexual development and multiple stress responses in B. bassiana. Cdcl4 is a dual-specificity phosphatase that regulates nuclear behavior by dephosphorylating phosphotyrosine and phosphoserine/phospho-threonine in fungi. Previously, Cdc14 was shown to act as a positive regulator of cytokinesis, asexual development and multiple stress responses in B. bassiana, a fungal insect pathogen. This study seeks to gain deep insight into a pivotal role of Cdcl4 in the signaling network of B. bassiana by analyzing the Cdc14-mediated proteome and phosphoproteome generated by the 8-plex iTRAQ labeling and MS/MS analysis of peptides and phosphopeptides. Under normal conditions,154 proteins and 86 phosphorylation sites in 67 phosphoproteins were upregulated in Δcdc14 versus wild-type, whereas 117 proteins and 85 phosphorylation sites in 58 phosphoproteins were significantly downregulated. Co-cultivation of Δcdc14 with NaCl (1 M), H2O2 (3 mM) and Congo red (0.15 mg/mL) resulted in the upregulation/downregulation of 23/63,41/39 and 79/79 proteins and of 127/112,52/47 and 105/226 phosphorylation sites in 85/92,45/36 and 79/146 phosphoproteins, respectively. Bioinformatic analyses revealed that Cdcl4 could participate in many biological and cellular processes, such as carbohydrate metabolism, glycerophospholipid metabolism, the MAP Kinase signaling pathway, and DNA conformation, by regulating protein expression and key kinase phosphorylation in response to different environmental cues. These indicate that in B. bassiana, Cdcl4 is a vital regulator of not only protein expression but also many phosphorylation events involved in developmental and stress-responsive pathways.Characterization of the signaling kinase Ste7 and its phosporylation sites in the Fus3/Kssl pathway of B. bassiana. Ste7 acts as an intermediate kinase in the Fus3/Kssl pathway of B. bassiana and harbors six phorphorylation sites, including two (S216 and T220) present in the budding yeast and four (S362, S391, S440 and T485) identified from the Cdc14-mediated phosphoproteome. Deletion of ste7, dual mutation of S216A/T220A and single mutation of S362A caused not only similar severe defects in nutrient utilization, conidial germination, vegetative growth, aerial conidiation, submerged blastospore production and multiple stress tolerance but also a complete loss of virulence via the normal route penetration through insect cuticle. All the phenotypes were also differentially impaired by the single mutations S391A, S440A and T485A but restored to wild-type levels by targeted ste7 complementation. Transcripts of some phenotype-related genes were qunatified in all mutant strains versus wild-type. As a result, transcript levels of ten genes required for nutrient utilization (creA) and asexual development (flbA-D, fluG, brlA, abaA, wetA and vosA) were significantly or drastically reduced in Aste7 and most site-mutated strains, coinciding well with their defects in growth, asexual cycle and virulence. Five P-type N+/K+ ATPase genes (enal-5) were all drastically depressed in the mutant strains, well in agreement with their extremely high sensitivity to either NaCl or KC1 stress during growth. Additionally, greatly suppressed expression of cytosolic MnSOD gene (sod2) and two catalase genes (catA and catP) in Aste7 and the strains with mutated sites S216A/T220A and S362A were likely causative of their remarked defects in antioxidation, heat tolerance and UV-B resistance. Taken together, Ste7 and each of its Ser/Thr phosphorylation sites play important roles in regulating nutrient utilization, vegetative growth, asexual cycle, multiple stress responses and host infection. The phosphorylation of either S216/T220 or S362 was indispensible for the regulative role of Ste7 in the life of B. bassiana. The results highlight a validity of phosphoproteins and their phosphorylaitons sites identified from the Cdc14-mediated phosphoproteome.Construction and validation of lysine acetylome in B. bassiana. Lysine acetylation (Kac) is a ubiquitous, conserved posttranslational modification in all organisms but fungal acetylomic study has been restricted to only model yeast. In this study, a B. bassiana acetylome was successfully constructed by enrichment of Kac peptides with anti-Kac antibody by means of LC-MS/MS technique. We identified 464 Kac sites from 283 Kac proteins in he constructed acetylome. These Kac proteins are involved mainly in the processes of gene transcription, chromatin organization, protein folding, and sugar/lipid metabolism. Revealed by bioinformatic analysis, six Kac motifs were found in the fungal acetylome, and five (KacY, KacH, KacF, FxKac, KacxxxxK and KacW) of them were highly conserved because they exist in other organisms. However, the motif KacW and 17 Kac proteins were uniquely present in the fungal acetylome. Two O-mannosyltransferases (Pmtl and Pmt4) identified from the B. bassiana acetylome were selected to validate the acetylome by multi-phenotypic analyses of Apmtl, Δpmt4 and their Kac site-mutated strains. As a result, the site mutation K88R or K482R of Pmtl caused equal or more severe defects in conidiation, virulence and cell tolerance to oxidation and cell wall perturbation than the function loss of Pmtl. These phenotypes were better than or as stable in the K360R mutant of Pmt4 as wild-type although they were remarkably impaired in Δpmt4. Thus, the Kac modification of K88 and K482 in Pmtl was indispensable for the regulative role of Pmtl in normal conidiation, host infection and cellular responses to oxidative and cell wall perturbing stresses. The Kac modification of K360 in Pmt4 was also important for the maintenance of the fungal phenotypes. These results not only indicate an essentiality of Kac for the fungal life but also confirm the validity of the constructed B. bassiana acetylome.