α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. Aspergillus nidulans has two α-1,3-glucan synthase genes, agsA and agsB. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing agsA (agsAOE) or agsB (agsBOE), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and agsBOE strains formed tightly aggregated hyphal pellets, whereas agsAOE hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the agsAOE and agsBOE strains. The peak molecular mass of α-1,3-glucan from the agsAOE strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and agsBOE (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: agsAOE, 41.6 ± 5.8 kDa; agsBOE, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain–fused GFP (AGBD-GFP). α-1,3-Glucan of the agsBOE cells was clearly located in the outermost layer, whereas weak labeling was detected in the agsAOE cells. However, the agsAOE cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the amyG gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of A. nidulans.

Filamentous fungi generally form aggregated hyphal pellets in liquid culture. We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did not form hyphal pellets and their hyphae were fully dispersed, and we suggested that α-1,3-glucan functions in hyphal aggregation. However, Aspergillus oryzae α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we hypothesized that another factor responsible for forming hyphal pellets remains in these mutants. Here, we identified an extracellular matrix polysaccharide galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, suggesting that hydrogen bond formation by this group is important for aggregation. Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many filamentous fungi and thus may function in hyphal aggregation in these fungi. We also demonstrated that production of a recombinant polyesterase, CutL1, was higher in the AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal aggregation factors of filamentous fungi may increase productivity in the fermentation industry.

226 words 2 Text, 5,353 words 3 3Abstract Filamentous fungi generally form aggregated hyphal pellets in liquid culture. 1 We previously reported that α-1,3-glucan-deficient mutants of Aspergillus nidulans did 2 not form hyphal pellets and their hyphae were fully dispersed, and we suggested that 3 α-1,3-glucan functions in hyphal aggregation. Yet, Aspergillus oryzae 4 α-1,3-glucan-deficient (AGΔ) mutants still form small pellets; therefore, we 5 hypothesized that another factor responsible for forming hyphal pellets remains in these 6 mutants. Here, we identified an extracellular matrix polysaccharide 7 galactosaminogalactan (GAG) as such a factor. To produce a double mutant of A. oryzae 8 (AG-GAGΔ), we disrupted the genes required for GAG biosynthesis in an AGΔ mutant. 9 Hyphae of the double mutant were fully dispersed in liquid culture, suggesting that 10 GAG is involved in hyphal aggregation in A. oryzae. Addition of partially purified GAG 11 fraction to the hyphae of the AG-GAGΔ strain resulted in formation of mycelial pellets. 12 Acetylation of the amino group in galactosamine of GAG weakened GAG aggregation, 13 suggesting that hydrogen bond formation by this group is important for aggregation. 14 Genome sequences suggest that α-1,3-glucan, GAG, or both are present in many 15 filamentous fungi and thus may function in hyphal aggregation in these fungi. We also 16 demonstrated that production of a recombinant polyesterase, CutL1, was higher in the 17 AG-GAGΔ strain than in the wild-type and AGΔ strains. Thus, controlling hyphal 18 aggregation factors of filamentous fungi may increase productivity in the fermentation 19 industry. 20 21 22Importance Production using filamentous fungi is an important part of the 1 fermentation industry, but hyphal aggregation in these fungi in liquid culture limits 2 productivity compared with that of yeast or bacterial cells. We found that 3 galactosaminogalactan and α-1,3-glucan both function in hyphal aggregation in 4 Aspergillus oryzae, and that the hyphae of a double mutant deficient in both 5 polysaccharides become fully dispersed in liquid culture. We also revealed the relative 6 contribution of α-1,3-glucan and galactosaminogalactan to hyphal aggregation. 7 Recombinant protein production was higher in the double mutant than in the wild-type 8 strain. Our research provides a potential technical innovation for the fermentation 9 industry that uses filamentous fungi, as regulation of the growth characteristics of A.10 oryzae in liquid culture may increase productivity. 11 12 13

The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried out in silico screening by threedimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis of MLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress in M. grisea. We discuss the relationships between PKC and melanin biosynthesis. IMPORTANCE A candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified by in silico screening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae. Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of the AGS1 mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization in M. grisea, and this melanization, which is required for pathogenicity of M. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of 4HNR, 3HNR, and