Characterization Of Thiol/Disulfidee Related Redox-system And Two Eisosome Proteins In Beauveria Bassiana

Beauveria bassiana is a classic filamentous entomopathogen that has been widely applied in insect pest control. The fungal biocontrol potential often declines upon exposure to outdoor stresses, such as high temperature, solar UV irradiation and applied chemical fungicides or herbicides. After entry into insect haemocoel, fungal cells are also exposed to osmotic stress in the host haemolymph and oxidative stress generated from the host immunity defense. Thus, exploring molecular mechanisms involved in fungal responses to multiple stressful cues is crucial to understanding the fungal potential against arthropod pests. Thioredoxin-thioredoxin reductase (TRX-TRR) system and glutaredoxin-glutathione reductase (GRX-GLR) system are two important antioxidant systems, protecting cells from the damage of reactive oxygen species (ROS), which results from outdoor stresses or insect immunity defense. However, the two systems have not been explored in fungal entomopathogens. One goal of this study is to characterize TRX-TRR and GRX-GLR systems in B. bassiana and to probe an interaction of both and of each with the antioxidant systems of superoxide dismutases (SODs), catalases (CATs) and peroxidases (PODs). Moreover, cell menbrane is critical for cellular function and fungal survival. Eisosome is an important cell membrane structure that acts on cellular conformation, response and transfer of internal/external signals, and nutritional uptake. Another goal of this study is to characterize two eisosme proteins Pill and Lspl, which is generally unexplored in filamentous fungi. Main results are summarized as follows.Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity and iron homeostasis of B. bassiana. Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized B. bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5 and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some partners under normal conditions. Particularly, grx3 compensated the absence of grxl, grx2, grx5 or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most partners in response to menadione. Consequently, the redox state was disturbed in Ag/r more than in Agrx3 but not in Agrxl/2/5. SODs were more active in normal AgrxJ-3 cultures but less in Agrx5 or Aglr response to menadione. Total CAT activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Aglr than of Agrx3. Among the mutants, Agrx3 showed slightly increased sensitivity to menadione or H2O2; Aglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing l-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe3+-inclusive medium perhaps due to elevated transcripts of two Fe3+transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Agrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other partners in the Glr-dependent redox system of the fungal entomopathogen.Subcellular localizaion of six TRXs and their antioxidant activity and contributions to biological control potential in B. bassiana. Thioredoxins (Trx) can detoxify sulfide or act as electron donors in the reduction of disulfide and dithiol to protect yeast cells from ROS damage but remain poorly explored in filamentous fungi. Here we show more Trx homologs in Beauveria bassiana than in many other fungi and examine their functions. This filamentous entomopathogen has six Trx homologs, including four (Txrl^4) evidently localized in cytoplasm, one (Trx5) in nuclear membrane and another (Trx6) in midtochondria. Deletion of each trx had no effect on radial growth on rich or minimal medium but resulted in remarkable transcriptional up-regulation of other partners for compensation. Compared with wild-type, only Atrx2 was significantly more sensitive to menadione whereas none of six Atrx mutants was responsive to other oxidants including H2O2. Intriguingly, Atrx2 showed uniquely a significant increase in total Trx activity in normal cultures but a remarkable decrease in total SOD activity in the cultures grown normally or co-cultivated with menadione. The ratio of reduced/oxidized glutathione accumulated in hyphal cells stressed with menadione decreased to only 0.4 in Atrx2 from-1.0 observed in wild-type and other mutants. The six Atrx mutants displayed one or more phenotypic changes associated with the fungal biocontrol potential, including conidiation, and germination, thermotolerance, UV-B resistance and virulence of their conidia. All the changes were restored by trx complementation. Taken together, the greater Trx diversity evolutionarily gained by B. bassiana could help it to maintain cellular redox homeostasis and infect insect hosts in diverse habitats.Distinct roles of two cytoplasmic TRRs in the redox system, cysteine synthesis and host infection of B. bassiana. Two thioredoxin reductases (Trrl/2) are known to play overlapping roles in yeast thioredoxin (Trx) redox system but generally unexplored in filamentous fungi, which possess multiple Trx homologues. This study seeks to characterize the functions of Trrl and Trr2 inBeauveria bassiana, a filamentous fungal insect pathogen, and to probe Trx targets of each. Both Trrl and Trr2 were evidently localized in B. bassiana cytoplasm, unlike one yeast homologue in cytoplasm and another in mitochondria. Deletion of trrl resulted in transcriptional upregulation of more trx genes than that of trr2. In Atrrl, increased Trx activity and severe cysteine auxotrophy concurred with drastically reduced activities of peroxidases and superoxide dismutases, under normal or oxidative conditions despite unaffected catalase activity. Such changes were absent in Atrr2 and completely restored by complementation of trrl/2 or overexpression of trxl/6 in Atrrl, but not restored at all by overexpression of trx2/3/4/5 or trr2 in Atrrl. All these mutants showed similar trends of changes in antioxidant response, conidiation, germination, thermotolerance, UV-B resistance and virulence. Conclusively, Trrl could reduce Trx2-5 and dominated the redox state affecting profoundly the potential of B. bassiana against arthropod pests. Trr2 could reduce Trx 1/6 but worked only when Trrl was absent.Pill and Lspl, two peripherally localizated eisososme proteins, are vital for the autophagy and pathogenicity of B. bassiana. Pill and Lspl are two crucial eisosome proteins that localize in cell membrane and play vital roles in model yeast but are poorly understood in filamentous fungi. We characterized Pill and Lspl homologues in B. bassiana. Both Pill and Lspl were evidently co-localized in cell periphery membrane or subcortex but their subcellular localization was independent of each other, unlike the yeast Pill localization that is prerequisite for Lspl localization. Deletion of lspl in B. bassiana resulted in largely decresed transcripts of almost all 21 autophgy genes and hence no autophagosome formed in vacuoles. In contrast, deletion of pill led to transcriptional upregulation in most autophage genes and no change in a few, accompanied with the formation of an extremely large autophagosome filling each vacuole in hyphal cells. Double pill/lspl deletions genes resulted in upregulated or unchanged transcripts of most autophage genes and hence restored autophagosome to wild-type size in each vacuole. The three deletion mutants showed differential changes in conidiation, thickened cell walls, utilization of carbon sources, and cellular responses to rapamycin, cell wall perturbation, high osmolariry, oxidation, high temperature and UV-B irradiation. Intriguingly, median lethal action of Apill, â–³lspl and Apillâ–³lspl against Galleria mellonella larvae was delayed by 1.6-, 2.5- and 3.2-fold via standardized cuticle infection, and by 1.2-,1.3- and 2.0-fold through haemocoel injection, respectively. Taken together, subcortex-localized Pill and Lspl regulate the fungal autophagy antagonistically and are indispensible for the fungal pathogenicity.