HapX, an Indispensable bZIP Transcription Factor for Iron Acquisition, Regulates Infection Initiation by Orchestrating Conidial Oleic Acid Homeostasis and Cytomembrane Functionality in Mycopathogen Beauveria bassiana

Abstract

In pathogenic filamentous fungi, conidial germination not only is fundamental for propagation in the environment but is also a critical step of infection. In the insect mycopathogen Beauveria bassiana, we genetically characterized the role of the basic leucine zipper (bZIP) transcription factor HapX (BbHapX) in conidial nutrient reserves and pathogen-host interaction. Ablation of BbHapX resulted in an almost complete loss of virulence in the topical inoculation and intrahemocoel injection assays. Comparative transcriptomic analysis revealed that BbHapX is required for fatty acid (FA)/lipid metabolism, and biochemical analyses indicated that BbHapX loss caused a significant reduction in conidial FA contents. Exogenous oleic acid could partially or completely restore the impaired phenotypes of the ΔBbHapX mutant, including germination rate, membrane integrity, vegetative growth, and virulence. BbHapX mediates fungal iron acquisition which is not required for desaturation of stearic acid. Additionally, inactivation of the Δ9-fatty acid desaturase gene (BbOle1) generated defects similar to those of the ΔBbHapX mutant; oleic acid also had significant restorative effects on the defective phenotypes of the ΔBbOle1 mutant. A gel retarding assay revealed that BbHapX directly regulated the expression of BbOle1. Lipidomic analyses indicated that both BbHapX and BbOle1 contributed to the homeostasis of phospholipids with nonpolar tails derived from oleic acid; therefore, exogenous phospholipids could significantly restore membrane integrity. These data reveal that the HapX-Ole1 pathway contributes to conidial fatty acid/lipid reserves and that there are important links between the lipid biology and membrane functionality involved in the early stages of infection caused by B. bassiana. IMPORTANCE Conidial maturation and germination are highly coupled physiological processes in filamentous fungi that are critical for the pathogenicity of mycopathogens. Compared to the mechanisms involved in conidial germination, those of conidial reserves during maturation are less understood. The insect-pathogenic fungus Beauveria bassiana, as a representative species of filamentous fungi, is important for applied and fundamental research. In addition to its conserved roles in fungal adaptation to iron status, the bZIP transcription factor HapX acts as a master regulator involved in conidial virulence and regulates fatty acid/lipid metabolism. Further investigation revealed that the Δ9-fatty acid desaturase gene (Ole1) is a direct downstream target of HapX. This study reveals the HapX-Ole1 pathway involved in the fatty acid/lipid accumulation associated with conidial maturation and provides new insights into the startup mechanism of infection caused by spores from pathogenic fungi.

A Small Cysteine-Free Protein Acts as a Novel Regulator of Fungal Insect-Pathogenic Lifecycle and Genomic Expression

“…S3C ) but similar to nonshifting signals of the ole1 DNA reacting with gradient thioredoxin (Trx1) samples as a negative control ( Fig. S3D ) ( 48 ). These data indicate an inability for CFP to bind the promoter regions of potential target genes for their transcription mediation.…”

“…The gels stained with Coomassie brilliant blue were rinsed repeatedly in washing buffer and visualized for signal of the protein bound to DNA. The signals of the gradient CFP samples in binding each DNA extract were compared with those of the ole1 (BBA_07664) DNA extract bound by gradient protein samples (0.5 to 9 μg) of the bZIP transcription factor HapX ( EJP64582 ) as a positive control rather than by the samples of the thioredoxin Trx1 ( EJP68440 ) as a negative control ( 48 ).…”

Proteomic and Phosphoryproteomic Investigations Reveal that Autophagy-Related Protein 1, a Protein Kinase for Autophagy Initiation, Synchronously Deploys Phosphoregulation on the Ubiquitin-Like Conjugation System in the Mycopathogen Beauveria bassiana

“…BbAtg1 was purified through immunoprecipitation with anti-FLAG M2 magnetic beads (M8823; Sigma-Aldrich) and anti-HA agarose (26181; Thermo Scientific, IL). BbAtg3 and its mutated forms were prepared in a bacterial expression system as described previously ( 54 ). Their coding sequences were amplified using the primer pair P25/P26 ( Table S1 ) and cloned into the BamHI and XhoI sites in the pET28a expression vector (Novagen) ( 54 ).…”

Transcription Activator Swi6 Interacts with Mbp1 in MluI Cell Cycle Box-Binding Complex and Regulates Hyphal Differentiation and Virulence in Beauveria bassiana

“…Beauveria bassiana is a filamentous entomopathogenic fungus that naturally parasitizes insects, and therefore has a great potential for pest management [ 2 ]. Conidia attach to the host cuticle and germinate into invasive mycelia via mobilizing endogenous nutrients, which initiates the infection process [ 3 , 4 ]. Once entering into the insect hemocoel, B. bassiana develops into yeast-like cells (in vivo hyphal bodies) via dimorphic transition.…”