GH16 and GH81 family β-(1,3)-glucanases inAspergillus fumigatusare essential for conidial cell wall morphogenesis

Abstract

The fungal cell wall is a rigid structure because of fibrillar and branched β-(1,3)-glucan linked to chitin. Softening of the cell wall is an essential phenomenon during fungal morphogenesis, wherein rigid cell wall structures are cleaved by glycosylhydrolases. During the search for glycosylhydrolases acting on β-(1,3)-glucan, we identified seven genes in the Aspergillus fumigatus genome coding for potential endo-β-(1,3)-glucanase. ENG1 (previously characterized and named ENGL1, Mouyna et al., ), belongs to the Glycoside-Hydrolase 81 (GH81) family, while ENG2 to ENG7, to GH16 family. ENG1 and four GH16 genes (ENG2-5) were expressed in the resting conidia as well as during germination, suggesting an essential role during A. fumigatus morphogenesis. Here, we report the effect of sequential deletion of AfENG2-5 (GH16) followed by AfENG1 (GH81) deletion in the Δeng2,3,4,5 mutant. The Δeng1,2,3,4,5 mutant showed conidial defects, with linear chains of conidia unable to separate while the germination rate was not affected. These results show, for the first time in a filamentous fungus, that endo β-(1,3)-glucanases are essential for proper conidial cell wall assembly and thus segregation of conidia during conidiation.

Aspergillus fumigatusexoβ(1‐3)glucanases family GH55 are essential for conidial cell wall morphogenesis

“…Several others putative exoβ(1‐3)glucanase have been identified in the A. fumigatus genome, like the GH3 and the GH5 families that could be responsible of the residual β(1‐3)glucanase activities observed in the undecuple glucanase mutants. Our RNAseq analysis data confirmed that several genes belonging to these two families were highly expressed (Mouyna et al, ; Valsecchi et al, ). Their analysis should allow us to better understand the role of the β(1‐3)glucanases during the global maturation process of A. fumigatus conidia.…”

“…Gene expression levels of the AfEXG genes have been investigated by RNAseq analysis during conidial germination. Transcripts were isolated from dormant (T0h), swollen (T4h), and germinating conidia (T8h; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81956; Mouyna et al, ). As shown in Figure a, EXG5 and EXG9 are more specifically expressed in dormant conidia, in contrast to EXG6 , EXG7 , EXG8 , and EXG10 that are constitutively expressed at all stages.…”

“…Moreover, like the Δexg5,6,7,8,9,10 mutant, ΔwetA showed smooth and ovoid conidia (Sewall et al, ; Tao & Yu, ). In contrast, the deletion of the Δeng1‐5 (family GH16/GH81) did not lead to an ovoid form for the conidia (Mouyna et al, ), suggesting that the GH55 family could play a role upstream of the endoβ(1‐3)glucanase in the maturation of conidia. According to our TEM results, β(1‐3)glucanases GH55 have shown their essentiality not only in the establishment of a tight junction between conidia but also in the establishment of cell wall roughness, which are important for the airborne conidia to hang a surface in order to find nutrients and start to germinate.…”

“…To make single and multiple gene deletion, we transformed A. fumigatus Ku80Δ strain to obtain the GH55 deletion family and the quintuple mutant GH16/GH81 (Mouyna et al, ) to obtain the undecuple GH55/GH81/GH16 mutant using the β‐Rec/six‐Specific Recombination System (Hartmann et al, ). One kb of the 5′ and 3′ flanking regions of each EXG gene were amplified by polymerase chain reaction from A. fumigatus genomic DNA using primers (Forw1/Rev1 and Forw2/Rev2; Table S5).…”

“…In A. fumigatus , the GH81 family has only one coding member, an endoβ(1‐3)glucanase Eng1p (previously characterised and named Engl1p; Fontaine, Hartland, Beauvais, Diaquin, & Latge, ; Mouyna et al, ), whereas GH16 subfamily of endoβ(1‐3)glucanases have five members (ENG2 to ENG7; Hartl, Gastebois, Aimanianda, & Latgé, ). The sequential deletion of genes from these two families resulted in a lack of conidial separation showing for the first time that endoβ(1‐3)glucanases were essential in the assembly and maturation of the conidial wall (Mouyna et al, ).…”