The zinc cluster transcription factor Rha1 is a positive filamentation regulator in Candida albicans.

Zinc cluster transcription factors (TFs) are essential fungal regulators of gene expression. In the pathogen Candida albicans, the gene orf19.1604 encodes a zinc cluster TF regulating filament development. Hyperactivation of orf19.1604, which we have named RHA1 for Regulator of Hyphal Activity, generates wrinkled colony morphology under nonhyphal growth conditions, triggers filament formation, invasiveness, and enhanced biofilm formation and causes reduced virulence in the mouse model of systemic infection. The strain expressing activated Rha1 shows up-regulation of genes required for filamentation and cell-wall-adhesion-related proteins. Increased expression is also seen for the hyphal-inducing TFs Brg1 and Ume6, while the hyphal repressor Nrg1 is downregulated. Inactivation of RHA1 reduces filamentation under a variety of filament-inducing conditions. In contrast to the partial effect of either single mutant, the double rha1 ume6 mutant strain is highly defective in both serum- and Spider-medium-stimulated hyphal development. While the loss of Brg1 function blocks serum-stimulated hyphal development, this block can be significantly bypassed by Rha1 hyperactivity, and the combination of Rha1 hyperactivity and serum addition can generate significant polarization even in brg1 ume6 double mutants. Thus, in response to external signals, Rha1 functions with other morphogenesis regulators including Brg1 and Ume6, to mediate filamentation.

Corrigendum to "Loss of Arp1, a putative actin-related protein, triggers filamentous and invasive growth and impairs pathogenicity in Candida albicans" (Computational and Structural Biotechnology Journal, 2020, 18: 4002-4015).

[This corrects the article DOI: 10.1016/j.csbj.2020.11.034.].

DNA damage checkpoint and repair: From the budding yeast Saccharomyces cerevisiae to the pathogenic fungus Candida albicans.

Cells are constantly challenged by internal or external genotoxic assaults, which may induce a high frequency of DNA lesions, leading to genome instability. Accumulation of damaged DNA is severe or even lethal to cells and can result in abnormal proliferation that can cause cancer in multicellular organisms, aging or cell death. Eukaryotic cells have evolved a comprehensive defence system termed the DNA damage response (DDR) to monitor and remove lesions in their DNA. The DDR has been extensively studied in the budding yeast Saccharomyces cerevisiae. Emerging evidence indicates that DDR genes in the pathogenic fungus Candida albicans show functional consistency with their orthologs in S. cerevisiae, but may act through distinct mechanisms. In particular, the DDR in C. albicans appears critical for resisting DNA damage stress induced by reactive oxygen species (ROS) produced from immune cells, and this plays a vital role in pathogenicity. Therefore, DDR genes could be considered as potential targets for clinical therapies. This review summarizes the identified DNA damage checkpoint and repair genes in C. albicans based on their orthologs in S. cerevisiae, and discusses their contribution to pathogenicity in C. albicans.

Hexokinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera.

Acetyl-CoA, the precursor of sex pheromone biosynthesis in Helicoverpa armigera, is generated from glycolysis. As the first speed-limited enzyme in glycolysis, Hexokinase (HK) plays an important role in acetyl-CoA production. However, the function of HK in sex pheromone production remains unclear. This study employed H. armigera as material to explore the role of HK in sex pheromone production. Results demonstrated that the transcription profile of HaHK in female moth pheromone glands (PGs) was consistent with the release fluctuation of sex pheromone. Interference of HaHK prevented the increase of acetyl-CoA content induced by PBAN. Therefore, knockdown of HaHK in female PGs caused significant decreases in (Z)-11-hexadecenal (Z11-16:Ald) production, female capability to attract males, and mating rate. Furthermore, sugar feeding (5% sugar) increased the transcription and enzymatic activity of HK. Pheromone biosynthesis activating neuropeptide (PBAN) signal phospho-activated HaHK in PGs and Sf9 cells via protein kinase A (PKA), as shown by pharmacological inhibitor analysis. In general, our study confirmed that PBAN/cAMP/PKA signal activated HaHK, in turn promoted glycolysis to ensure the supply of acetyl-CoA, and finally facilitated sex pheromone biosynthesis and subsequent mating behavior.

Transcriptome Analysis of Ostrinia furnacalis Female Pheromone Gland: Esters Biosynthesis and Requirement for Mating Success.

Female moths use sex pheromones to attract males, and corresponding regulatory mechanism underlying sex pheromone biosynthesis is species-dependent. However, the detailed mechanism involved in sex pheromone biosynthesis in Ostrinia furnacalis has not yet been fully addressed. In the present study, transcriptome sequencing of O. furnacalis pheromone glands screened a serials of candidate genes involved in sex pheromone biosynthesis. Our analysis showed that sex pheromone release in O. furnacalis females arrives its peak at the 2nd scotophase, consistent with its mating behavior. Pheromone biosynthesis-activating neuropeptide (PBAN) was confirmed to regulate sex pheromone biosynthesis, and Ca2+ is the secondary messenger of PBAN signaling in O. furnacalis. The functional analysis of candidate genes demonstrated that the decreased mRNA levels or activities of calcineurin (CaN) and acetyl-CoA carboxylase (ACC) led to significant decrease in sex pheromone production and female capability to attract males, as demonstrated by RNAi-mediated knockdown and pharmacological inhibitor assay. Most importantly, the activities of CaN and ACC depend on the activation of PBAN/PBANR/Ca2+. Furthermore, fatty-acyl reductase 14 was involved in PBAN-mediated sex pheromone biosynthesis. Altogether, our results demonstrated that PBAN regulates sex pheromone biosynthesis through PBANR/Ca2+/CaN/ACC pathway to promote sex pheromone biosynthesis in O. furnacalis and provided a reference for non-model organism to study neuropeptide signal transduction.

Pyruvate Kinase Is Required for Sex Pheromone Biosynthesis in Helicoverpa armigera.

Pyruvate kinase (PYK) is a speed-limited enzyme of glycolysis that catalyzes the formation of pyruvate, and plays an important role in acetyl-CoA synthesis. The acetyl-CoA is the precursor of sex pheromone biosynthesis in Helicoverpa armigera. However, the role of PYK in sex pheromone biosynthesis remains elusive. Here, PYK in H. armigera (HaPYK) was found to be highly expressed in the pheromone glands (PGs). The developmental expression profile of HaPYK was consistent with the fluctuation of sex pheromone release. Function analysis revealed that the knockdown of HaPYK led to a decrease in the levels of pyruvic acid and acetyl-CoA in PGs, which in turn caused a significant decrease in cis-11-hexadecenal (Z11-16: Ald) production, female capability to attract males, and mating frequency. Further study demonstrated that sugar feeding (5% sugar) increased the transcription and enzyme activity of HaPYK, thereby facilitating sex pheromone biosynthesis. Moreover, pheromone biosynthesis activating neuropeptide (PBAN) upregulated HaPYK activity through protein kinase C (PKC), as shown by PKC-specific inhibitor analysis. Altogether, our results revealed that PBAN activated HaPYK by Ca2+/PKC, thereby regulating the synthesis of pyruvate and subsequent acetyl-CoA, ensuring the supply of sex pheromone precursor, and finally facilitating sex pheromone biosynthesis and mating behavior.

New insights on the effects of spinosad on the development of Helicoverpa armigera.

Helicoverpa armigera (cotton bollworm) is one of the most destructive pests worldwide. Due to resistance to Bacillus thuringiensis and conventional insecticides, an effective management strategy to control this pest is urgently needed. Spinosad, a natural pesticide, is considered an alternative; however, the mechanism underlying the developmental effects of sublethal spinosad exposure remains elusive. In this study, the mechanism was examined using an insect model of H. armigera. Results confirmed that exposure to sublethal spinosad led to reduced larval wet weight, delayed larval developmental period, caused difficulty in molting, and deformed pupae. Further investigation demonstrated that exposure to sublethal spinosad caused a significant decrease in 20E titer and increase in JH titer, thereby leading to the discordance between 20E and JH titers, and consequently alteration in the expression levels of HR3 and Kr-h1. These results suggested that sublethal spinosad caused hormonal disorders in larvae, which directly affect insect development. Our study serves as a reference and basis for the toxicity evaluation of spinosad on molting and pupation in insect metamorphosis, which may contribute to identifying targets for effective control of cotton bollworm.

Loss of Arp1, a putative actin-related protein, triggers filamentous and invasive growth and impairs pathogenicity in Candida albicans.

The polymorphous cellular shape of Candida albicans, in particular the transition from a yeast to a filamentous form, is crucial for either commensalism or life-threatening infections of the host. Various external or internal stimuli, including serum and nutrition starvation, have been shown to regulate filamentous growth primarily through two classical signaling pathways, the cAMP-PKA and the MAPK pathways. Genotoxic stress also induces filamentous growth, but through independent pathways, and little is known about negative regulation during this reversible morphological transition. In this study, we established that ARP1 in C. albicans, similar to its homolog in S. cerevisiae, has a role in nuclei separation and spindle orientation. Deletion of ARP1 generated filamentous and invasive growth as well as increased biofilm formation, accompanied by up-regulation of hyphae specific genes, such as HWP1, UME6 and ALS3. The filamentous and invasive growth of the ARP1 deletion strain was independent of transcription factors Efg1, Cph1 and Ume6, but was suppressed by deleting checkpoint BUB2 or overexpressing NRG1. Deletion of ARP1 impaired the colonization of Candida cells in mice and also attenuated virulence in a mouse model. All the data suggest that loss of ARP1 activates filamentous and invasive growth in vitro, and that it positively regulates virulence in vivo, which provides insight into actin-related morphology and pathogenicity in C. albicans.

Nucleotide Excision Repair Protein Rad23 Regulates Cell Virulence Independent of Rad4 in Candida albicans.

In the pathogenic yeast Candida albicans, the DNA damage response contributes to pathogenicity by regulating cell morphology transitions and maintaining survival in response to DNA damage induced by reactive oxygen species (ROS) in host cells. However, the function of nucleotide excision repair (NER) in C. albicans has not been extensively investigated. To better understand the DNA damage response and its role in virulence, we studied the function of the Rad23 nucleotide excision repair protein in detail. The RAD23 deletion strain and overexpression strain both exhibit UV sensitivity, confirming the critical role of RAD23 in the nucleotide excision repair pathway. Genetic interaction assays revealed that the role of RAD23 in the UV response relies on RAD4 but is independent of RAD53, MMS22, and RAD18RAD4 and RAD23 have similar roles in regulating cell morphogenesis and biofilm formation; however, only RAD23, but not RAD4, plays a negative role in virulence regulation in a mouse model. We found that the RAD23 deletion strain showed decreased survival in a Candida-macrophage interaction assay. Transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) data further revealed that RAD23, but not RAD4, regulates the transcription of a virulence factor, SUN41, suggesting a unique role of RAD23 in virulence regulation. Taking these observations together, our work reveals that the RAD23-related nucleotide excision pathway plays a critical role in the UV response but may not play a direct role in virulence. The virulence-related role of RAD23 may rely on the regulation of several virulence factors, which may give us further understanding about the linkage between DNA damage repair and virulence regulation in C. albicansIMPORTANCECandida albicans remains a significant threat to the lives of immunocompromised people. An understanding of the virulence and infection ability of C. albicans cells in the mammalian host may help with clinical treatment and drug discovery. The DNA damage response pathway is closely related to morphology regulation and virulence, as well as the ability to survive in host cells. In this study, we checked the role of the nucleotide excision repair (NER) pathway, the key repair system that functions to remove a large variety of DNA lesions such as those caused by UV light, but whose function has not been well studied in C. albicans We found that Rad23, but not Rad4, plays a role in virulence that appears independent of the function of the NER pathway. Our research revealed that the NER pathway represented by Rad4/Rad23 may not play a direct role in virulence but that Rad23 may play a unique role in regulating the transcription of virulence genes that may contribute to the virulence of C. albicans.

Supplemental Sugar Is Required for Sex Pheromone Biosynthesis in Mythimna separata.

Supplemental nutrients of adult moths maximize moth fitness and contribute to the pollination of many plants. Previous reports have revealed that sugar feeding promotes to sex pheromone biosynthesis by increasing the haemolymph trehalose concentration in mating moths. Here, Mythimna separata adults were employed as a model to investigate the effect of sugar feeding on sex pheromone biosynthesis. Results showed that in virgin females, sugar feeding markedly increased the concentrations of trehalose, pyruvic acid, and acyl-CoA in pheromone glands (PGs), which in turn led to an increase in sex pheromone titer, female ability to attract males and successfully mating frequency in sugar-fed females. Consistently, sugar-fed females laid more eggs than water-fed females. Furthermore, the refeeding of starved females also caused significantly increase in the concentrations of trehalose, pyruvic acid, and acyl-CoA in PGs, thus facilitating a significant increase in sex pheromone production. Most importantly, RNAi-mediated knockdown of trehalase (leading to PG starvation) resulted in an increase in trehalose content, and decrease in the concentrations of pyruvic acid, and acyl-CoA in PGs, which in turn led to a decrease of sex pheromone titer, female ability to attract males and successful mating efficacy. Altogether, results revealed a mechanism by which sugar feeding contributed to trehalose utilization in PGs, promoted to significantly increased sex pheromone precursor by increasing the concentrations of pyruvic acid and acyl-CoA, and facilitated to sex pheromone biosynthesis and successful mating.

Calcineurin-Modulated Antimicrobial Peptide Expression Is Required for the Development of Helicoverpa armigera.

Helicoverpa armigera is a universal pest around the world that has been extensively used as a model organism for agricultural pests. Calcineurin (CAN) is an important Ca2+-dependent phosphatase that is participated in various biological pathways. Here, we revealed that CAN inhibition significantly arrested H. armigera larval development by reducing larvae weight, prolonging development time and reducing pupate rates. Furthermore, CAN serves as an immune activator and regulates antimicrobial peptide (AMP; cecropin D, attacin, and gloverin) expression by binding with relish transcript factor in H. armigera. This study provides a potential target to control H. armigera by using synergistic agents for pesticides or plant-mediated RNA interference technology.