Comprehensive Interactome Analysis for the Sole Adenylyl Cyclase Cyr1 of Candida albicans.

Cyr1, the sole adenylyl cyclase of the fungal pathogen Candida albicans, is a central component of the cAMP/protein kinase A signaling pathway that controls the yeast-to-hypha transition. Cyr1 is a multivalent sensor and integrator of various external and internal signals. To better understand how these signals are relayed to Cyr1 to regulate its activity, we sought to establish the interactome of Cyr1 by using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to identify the proteins that coimmunoprecipitated with Cyr1. The method identified 36 proteins as candidates for authentic Cyr1-interacting partners, together with two known Cyr1-binding proteins, Cap1 and Act1. Fourteen identified proteins belonged to three functional groups, including actin regulation, cell wall components, and mitochondrial activities, that are known to play important roles in cell morphogenesis. To validate the proteomics data, we used biochemical and genetic methods to characterize two cell wall-related proteins, Mp65 and Sln1. First, coimmunoprecipitation confirmed their physical association with Cyr1. Second, deleting either MP65 or SLN1 resulted in severe defects in filamentation on serum plates. This study establishes the first Cyr1 interactome and uncovers a potential role for cell wall proteins in directly regulating Cyr1 activity to determine growth forms in C. albicans. IMPORTANCE A critical virulence trait of the human fungal pathogen Candida albicans is its ability to undergo the yeast-to-hypha transition in response to diverse environmental and cellular stimuli. Previous studies suggested that the sole adenylyl cyclase of C. albicans, Cyr1, is a multivalent signal sensor and integrator synthesizing cAMP to activate the downstream hypha-promoting events through the cAMP/protein kinase A pathway. To fully understand how Cyr1 senses and processes multiple stimuli to generate appropriate signal outputs, it was necessary to identify and characterize Cyr1-interacting partners. This study employed SILAC-based quantitative proteomic approaches and identified 36 Cyr1-associated proteins, many having functions associated with hyphal morphogenesis. Coimmunoprecipitation verified two cell surface proteins, Mp65 and Sln1. Furthermore, genetic and phenotypic analyses demonstrated the cAMP-dependent roles of these two proteins in determining hyphal growth. Our study establishes the first Cyr1 interactome and uncovers new Cyr1 regulators that mediate cell surface signals to influence the growth mode of C. albicans.

Genome assembly and chemogenomic profiling of National Flower of Singapore Papilionanthe Miss Joaquim 'Agnes' reveals metabolic pathways regulating floral traits.

Singapore's National Flower, Papilionanthe (Ple.) Miss Joaquim 'Agnes' (PMJ) is highly prized as a horticultural flower from the Orchidaceae family. A combination of short-read sequencing, single-molecule long-read sequencing and chromatin contact mapping was used to assemble the PMJ genome, spanning 2.5 Gb and 19 pseudo-chromosomal scaffolds. Genomic resources and chemical profiling provided insights towards identifying, understanding and elucidating various classes of secondary metabolite compounds synthesized by the flower. For example, presence of the anthocyanin pigments detected by chemical profiling coincides with the expression of ANTHOCYANIN SYNTHASE (ANS), an enzyme responsible for the synthesis of the former. Similarly, the presence of vandaterosides (a unique class of glycosylated organic acids with the potential to slow skin aging) discovered using chemical profiling revealed the involvement of glycosyltransferase family enzymes candidates in vandateroside biosynthesis. Interestingly, despite the unnoticeable scent of the flower, genes involved in the biosynthesis of volatile compounds and chemical profiling revealed the combination of oxygenated hydrocarbons, including traces of linalool, beta-ionone and vanillin, forming the scent profile of PMJ. In summary, by combining genomics and biochemistry, the findings expands the known biodiversity repertoire of the Orchidaceae family and insights into the genome and secondary metabolite processes of PMJ.

Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress.

Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.