Gβ-like CpcB plays a crucial role for growth and development of Aspergillus nidulans and Aspergillus fumigatus.

Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented

BEVDepth: Acquisition of Reliable Depth for Multi-View 3D Object Detection

In this research, we propose a new 3D object detector with a trustworthy depth estimation, dubbed BEVDepth, for camera-based Bird's-Eye-View~(BEV) 3D object detection. Our work is based on a key observation -- depth estimation in recent approaches is surprisingly inadequate given the fact that depth is essential to camera 3D detection. Our BEVDepth resolves this by leveraging explicit depth supervision. A camera-awareness depth estimation module is also introduced to facilitate the depth predicting capability. Besides, we design a novel Depth Refinement Module to counter the side effects carried by imprecise feature unprojection. Aided by customized Efficient Voxel Pooling and multi-frame mechanism, BEVDepth achieves the new state-of-the-art 60.9% NDS on the challenging nuScenes test set while maintaining high efficiency. For the first time, the NDS score of a camera model reaches 60%. Codes have been released

Summary of <i>cpcB</i>.

<p>(A∼B) <i>cpcB</i> mRNA levels during the life cycle of <i>A. nidulans</i> (A) and <i>A. fumigatus</i> (B). Conidia (asexual spores) are indicated as C. The numbers indicate the time (hours) after incubation in liquid MMG (Vegetative), and on solid MMG under conditions inducing conidiation (Asexual) or sexual development (Sexual). The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and each band was normalized by the amount of 18S rRNA (<i>cpcB</i>/18S rRNA) in the relevant lane. Ethidium bromide staining of ribosomal RNAs was used as a loading control. (C) Alignment of <i>A. nidulans</i> (Ani) CpcB, <i>A. fumigatus</i> (Afu) CpcB, <i>A. flavus</i> (Afl) CpcB with Gib2 of <i>C. neoformans var. grubii</i> (Cgr; accession: AY907679) and <i>C. neoformans var. neoformans</i> (Cne; accession: AY907680). ClustalW (<a href="http://align.genome.jp/" target="_blank">http://align.genome.jp/</a>) and BoxShade 3.21 (<a href="http://www.ch.embnet.org/software/BOX_form.html" target="_blank">http://www.ch.embnet.org/software/BOX_form.html</a>) were used for the alignment and presentation. (D) A phylogenetic tree of CpcB-like proteins identified in various fungal species including <i>A. clavatus</i>, <i>A. flavus</i>, <i>A. fumigatus</i>, <i>A. nidulans</i>, <i>A. niger</i>, <i>A. oryzae</i>, <i>A. terreus (A = Aspergillus)</i>, <i>C. neoformans</i>, <i>Neurospora crassa</i>, <i>Penicillium chrysogenum</i>, <i>Penicillium marneffei</i>, <i>Schizophyllum commune</i>, <i>Schizosaccharomyces japonicus</i> and <i>Talaromyces stipitatus</i>. The putative CpcB proteins were retrieved from NCBI BlastX (<a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi" target="_blank">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a>) using <i>A. nidulans</i> CpcB. A phylogenetic tree of 14 putative CpcB homologues was generated by the TreeTop software (<a href="http://genebee.msu.su/services/phtree_reduced.html" target="_blank">http://genebee.msu.su/services/phtree_reduced.html</a>) using the alignment data from ClustalW. The phylogenetic tree is constructed based on the matrix of pairwise distances between sequences. Numbers indicate the computed distances given the residue substitution weights from the alignment data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070355#pone.0070355-Brodskii1" target="_blank">[59]</a>.</p

Requirement of CpcB for sexual development in <i>A. nidulans</i>.

<p>(A) Photographs of the point-inoculated colonies of WT (TNJ36), Δ<i>cpcB</i> (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of <i>A. nidulans</i> grown on MMG for 7 days (top panels), and the close-up views of the colonies (bottom panels) captured by a Zeiss Axioplan 2 stereomicroscope are shown. Cleistothecia are marked as CT. (B) Morphology of cleistothecia formed by the three strains. Images were captured by a Zeiss Axioplan 2 stereomicroscope. (C) Quantitative analyses of cleistothecia (CLS) per cm² produced by the three strains grown on MMG for 7 days. (D) Northern blot analyses for levels of <i>veA</i> and <i>vos</i>A transcripts after sexual developmental induction of <i>A. nidulans</i> WT (FGSC4) and Δ<i>cpcB</i> (RJMP1.59-8) strains. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of RNA bands using ImageJ and normalized by the amounts of 18S rRNA (<i>veA</i>/18S rRNA or <i>vosA</i>/18S rRNA) in each sample. Ethidium bromide staining of ribosomal RNAs was used as a loading control. Numbers indicate the time post sexual developmental induction.</p

Phenotype of the Δ<i>flbA</i>Δ<i>cpcB</i> and Δ<i>rgsA</i>Δ<i>cpcB</i> mutants.

<p>Photographs of colonies of WT (FGSC4), Δ<i>cpcB</i> (RJMP1.59-8), Δ<i>flbA</i> (QK1), and Δ<i>flbA</i>Δ<i>cpcB</i> (QK3) strains (A); and Δ<i>rgsA</i> (QK2) and Δ<i>rgsA</i>Δ<i>cpcB</i> (QK4) strains (B) grown on solid MMG for 3 days are shown. The two left panels show the point-inoculated strains (top and bottom), and the two right panels show close-up views of single and double mutant colonies shown in the left panels. Photomicrographs were taken by Zeiss Axioplan 2 stereomicroscope. Note the highly restricted growth of the double mutants (A and B), and the lack of conidiation in the Δ<i>flbA</i> and Δ<i>flbA</i> Δ<i>cpcB</i> mutant (A).</p

Requirement of CpcB for proper growth and conidiation in <i>A. nidulans</i> and <i>A. fumigatus</i>.

<p>(A) Photographs of the point-inoculated colonies of WT control (TNJ36), Δ<i>cpcB</i> (RJMP1.59-8) and complemented (C’; RJMP1.59-8C) strains of <i>A. nidulans</i> grown on solid MMG for 4 days (top panels) and the close-up views of the colonies (bottom panels) are shown in left panel. Photographs of the colonies of WT (AF293), Δ<i>AfucpcB</i> (Af293.1-7) and complemented (C’; Af293.1-7C) strains of <i>A. fumigatus</i> grown on solid MM+0.5%YE for 4 days (top panels), and the close-up views of the colonies (bottom panels) are shown in right panel. (B) Quantitative analyses of the diameter of colonies and conidiation levels in the designated strains. (C) Northern blot analyses for levels of <i>brl</i>A transcript in the WT (FGSC4) and Δ<i>AnicpcB</i> (RJMP1.59-8) strains of <i>A. nidulans</i>, and in WT (Af293) and Δ<i>AfucpcB</i> (Af293.1-7) strains of <i>A. fumigatus.</i> The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and normalized by the amounts of 18S rRNA (<i>brlA</i>/18S rRNA) in each sample. Ethidium bromide staining of rRNA was used as a loading control.</p

The roles of CpcB in conidial germination and hyphal growth.

<p>(A∼B) Quantitative analyses of conidial germination and hyphal growth post conidial germination of WT (filled bar; TNJ36), Δ<i>cpcB</i> (blank bar; RJMP1.59-8) and complemented (C) (shaded bar; RJMP1.59-8C) <i>A. nidulans</i> strains on MMG (A); and of WT (AF293), Δ<i>AfucpcB</i> (Af293.1-7) and complemented (C’; Af293.1-7C) <i>A. fumigates</i> strains on MMG (B). (C) Photographs of the three <i>A. nidulans</i> strains grown on solid MMG for 1 day (top panels) and the close-up views (bottom panels). Note the lack of conidiophores in Δ<i>cpcB</i> strain. (D) Photographs of the three <i>A. fumigates</i> strains of grown on solid MMG for 1 day (top panels), and the close-up views (bottom panels). Note the differences in the size and number of conidiophores.</p

CpcB is not required for ST biosynthesis.

<p>(A) TLC of ST produced after 2 days of liquid submerged culture (MM+0.5%YE) of WT (FGSC4), Δ<i>cpcB</i> (RJMP1.59-8, RJMP1.59-24) and complemented (RJMP1.59-8C1, RJMP1.59-8C5) <i>A. nidulans</i> strains. The relative intensity (mean±SD) of ST produced by each strain was determined by ImageJ and normalized by the amount of the ST standard (spot/ST). (B) TLC of GT produced upon 2 days of liquid submerged culture (MM+0.5%YE) of WT (Af293), Δ<i>AfucpcB</i> (Af293.1-4, Af293.1-6, Af293.1-7) and complemented (Af293.1-7C4, Af293.1-7C5, Af293.1-7C6) <i>A. fumigatus</i> strains. The relative intensity (mean±SD) of GT produced by each strain was determined by ImageJ and normalized by the amount of the GT standard (spot/GT). (C) Northern blot for levels of <i>stcU</i> and <i>aflR</i> transcripts in WT (FGSC4) and Δ<i>cpcB</i> (RJMP1.59-8) of <i>A. nidulans</i>. The relative band intensities (mean±SD) of Northern blot were determined by densitometric scanning of mRNA bands using ImageJ and normalized by the amounts of 18S rRNA (<i>aflR</i>/18S rRNA or <i>stcU</i>/18S rRNA) in each sample. Ethidium bromide staining of ribosomal RNAs was used as a loading control.</p