RELATe enables genome-scale engineering in fungal genomics

Li, Zhongming; Kim, Kwang Sik

doi:10.1126/sciadv.abb8783

Cited by 11 publications

(8 citation statements)

References 48 publications

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“…The CNAG_05141 Δ mutant had minor sensitivity to EMS compared to the wild-type control, and was not sensitive to UV damage. Based on AlphaFold and FoldSeek analysis, this protein contained a predicted EamA-like transporter domain [ 73 ] and had structural homology to the Nipal2 and Nipa1 transporter proteins. Potentially, loss of this transporter may lead to a higher intracellular concentration of EMS resulting in increased cell death.…”

Section: Resultsmentioning

confidence: 99%

CryptoCEN: A Co-Expression Network for Cryptococcus neoformans reveals novel proteins involved in DNA damage repair

O’Meara,

Rapala,

Nichols

et al. 2024

PLoS Genet

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Elucidating gene function is a major goal in biology, especially among non-model organisms. However, doing so is complicated by the fact that molecular conservation does not always mirror functional conservation, and that complex relationships among genes are responsible for encoding pathways and higher-order biological processes. Co-expression, a promising approach for predicting gene function, relies on the general principal that genes with similar expression patterns across multiple conditions will likely be involved in the same biological process. For Cryptococcus neoformans, a prevalent human fungal pathogen greatly diverged from model yeasts, approximately 60% of the predicted genes in the genome lack functional annotations. Here, we leveraged a large amount of publicly available transcriptomic data to generate a C. neoformans Co-Expression Network (CryptoCEN), successfully recapitulating known protein networks, predicting gene function, and enabling insights into the principles influencing co-expression. With 100% predictive accuracy, we used CryptoCEN to identify 13 new DNA damage response genes, underscoring the utility of guilt-by-association for determining gene function. Overall, co-expression is a powerful tool for uncovering gene function, and decreases the experimental tests needed to identify functions for currently under-annotated genes.

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Section: Resultsmentioning

confidence: 99%

CryptoCEN: A Co-Expression Network for Cryptococcus neoformans reveals novel proteins involved in DNA damage repair

O’Meara,

Rapala,

Nichols

et al. 2024

PLoS Genet

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“…albicans ( S1 Table ). As a previous independent study named CNAG_02402 WDR1 [ 25 ], we denoted it as WDR1 instead of WDR7 .…”

Section: Resultsmentioning

confidence: 99%

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

Choi,

Lee

et al. 2023

PLoS Pathog

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V-ATPase, which comprises 13–14 subunits, is essential for pH homeostasis in all eukaryotes, but its proper function requires a regulator to assemble its subunits. While RAVE (regulator of H+-ATPase of vacuolar and endosomal membranes) and Raboconnectin-3 complexes assemble V-ATPase subunits in Saccharomyces cerevisiae and humans, respectively, the function of the RAVE complex in fungal pathogens remains largely unknown. In this study, we identified two RAVE complex components, Rav1 and Wdr1, in the fungal meningitis pathogen Cryptococcus neoformans, and analyzed their roles. Rav1 and Wdr1 are orthologous to yeast RAVE and human Rabconnectin-3 counterparts, respectively, forming the hybrid RAVE (hRAVE) complex. Deletion of RAV1 caused severe defects in growth, cell cycle control, morphogenesis, sexual development, stress responses, and virulence factor production, while the deletion of WDR1 resulted in similar but modest changes, suggesting that Rav1 and Wdr1 play central and accessary roles, respectively. Proteomics analysis confirmed that Wdr1 was one of the Rav1-interacting proteins. Although the hRAVE complex generally has V-ATPase-dependent functions, it also has some V-ATPase-independent roles, suggesting a unique role beyond conventional intracellular pH regulation in C. neoformans. The hRAVE complex played a critical role in the pathogenicity of C. neoformans, and RAV1 deletion attenuated virulence and impaired blood-brain barrier crossing ability. This study provides comprehensive insights into the pathobiological roles of the fungal RAVE complex and suggests a novel therapeutic strategy for controlling cryptococcosis.

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“…At present, large-scale and high-throughput genetic screens based on CRISPR-Cas9 have been achieved in mammalian cells through the transformation of sgRNA libraries which is commercially available [ 32 , 33 ]. In the studies of pathogenic fungi, Li et al, 2010, exploited a similar method called restriction/ligation coupled with Agrobacterium-mediated transformation (RELATe), which generated sgRNA libraries from Cryptococcus neoformans genomic DNA and covered 98% of the protein-coding genes [ 34 ]. In C. albicans , the genetic screen based on CRISPR-Cas9 and sgRNA libraries has not been reported, which indicated that the construction of sgRNA libraries, high-efficient transformation, recovery, and sequencing of sgRNA should be further explored.…”

Section: Methods Used In the Mutant Library Construction In ...mentioning

confidence: 99%

Application of the Mutant Libraries for Candida albicans Functional Genomics

Wang¹,

Liu²,

Liu³

et al. 2022

IJMS

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Candida albicans is a typical opportunistic pathogen in humans that causes serious health risks in clinical fungal infections. The construction of mutant libraries has made remarkable developments in the study of C. albicans molecular and cellular biology with the ongoing advancements of gene editing, which include the application of CRISPR-Cas9 and novel high-efficient transposon. Large-scale genetic screens and genome-wide functional analysis accelerated the investigation of new genetic regulatory mechanisms associated with the pathogenicity and resistance to environmental stress in C. albicans. More importantly, sensitivity screening based on C. albicans mutant libraries is critical for the target identification of novel antifungal compounds, which leads to the discovery of Sec7p, Tfp1p, Gwt1p, Gln4p, and Erg11p. This review summarizes the main types of C. albicans mutant libraries and interprets their applications in morphogenesis, biofilm formation, fungus–host interactions, antifungal drug resistance, and target identification.

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RELATe enables genome-scale engineering in fungal genomics

Cited by 11 publications

References 48 publications

CryptoCEN: A Co-Expression Network for Cryptococcus neoformans reveals novel proteins involved in DNA damage repair

CryptoCEN: A Co-Expression Network for Cryptococcus neoformans reveals novel proteins involved in DNA damage repair

The hybrid RAVE complex plays V-ATPase-dependent and -independent pathobiological roles in Cryptococcus neoformans

Application of the Mutant Libraries for Candida albicans Functional Genomics

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