SUMMARY
It has become increasingly apparent that the production of reactive oxygen species (ROS) by the NADPH oxidase (Nox) complex is vital for cellular differentiation and signalling in fungi. We ...cloned and characterized an AaNoxA gene of the necrotrophic fungus Alternaria alternata, which encodes a polypeptide analogous to mammalian gp91phox and fungal Noxs implicated in the generation of ROS. Genetic analysis confirmed that AaNoxA is responsible for the production of ROS. Moreover, deletion of AaNoxA in A. alternata resulted in an elevated hypersensitivity to hydrogen peroxide (H2O2), menadione, potassium superoxide (KO2), diamide and many ROS‐generating compounds. The results implicate the involvement of AaNoxA in cellular resistance to ROS stress. The impaired phenotypes strongly resemble those previously seen for the ap1 null mutant defective in a YAP1‐like transcriptional regulator and for the hog1 mutant defective in a HOG1‐like mitogen‐activated protein (MAP) kinase. The noxA null mutant was also hypersensitive to Nox inhibitors, nitric oxide (NO·) donors and NO· synthase inhibitors, implying a role of AaNoxA in the NO· signalling pathway. Expression of AaNoxA was activated by H2O2, menadione, KO2, NO· donors and l‐arginine (a substrate for NO· synthase). AaNoxA may be able to sense and respond to both ROS and nitric oxide. Moreover, AaNoxA is required for normal conidiation and full fungal virulence. AaNoxA promoted the expression of the AaAP1 and AaHOG1 genes in A. alternata. Inactivation of AaNoxA greatly reduced the transcriptional activation of AaAP1 in response to ROS stress. Thus, we conclude that the regulatory functions of AaNoxA conferring ROS resistance are modulated partially through the activation of the YAP1‐ and HOG1 MAP kinase‐mediated signalling pathways.
RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a ...genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms.
Summary
The fungal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) complex, which has been implicated in the production of low‐level reactive oxygen species (ROS), contains mainly ...NoxA, NoxB (gp91phox homologues) and NoxR (p67phox homologue). Here, we report the developmental and pathological functions of NoxB and NoxR in the tangerine pathotype of Alternaria alternata. Loss‐of‐function genetics revealed that all three Nox components are required for the accumulation of cellular hydrogen peroxide (H2O2). Alternaria alternata strains lacking NoxA, NoxB or NoxR also displayed an increased sensitivity to H2O2 and many ROS‐generating oxidants. These phenotypes are highly similar to those previously seen for the Δyap1 mutant lacking a YAP1 transcriptional regulator and for the Δhog1 mutant lacking a HOG1 mitogen‐activated protein (MAP) kinase, implicating a possible link among them. A fungal strain carrying a NoxA NoxB or NoxA NoxR double mutation was more sensitive to the test compounds than the strain mutated at a single gene, implicating a synergistic function among Nox components. The ΔnoxB mutant strain failed to produce any conidia; both ΔnoxA and ΔnoxR mutant strains showed a severe reduction in sporulation. Mutant strains carrying defective NoxB had higher chitin content than the wild‐type and were insensitive to calcofluor white, Congo red and the fungicides vinclozolin and fludioxonil. Virulence assays revealed that all three Nox components are required for the elaboration of the penetration process. The inability to penetrate the citrus host, observed for Δnox mutants, could be overcome by wounding and by reacquiring a dominant Nox gene. The A. alternata NoxR did not influence the expression of NoxB, but negatively regulated NoxA. Importantly, the expression of both YAP1 and HOG1 genes, whose products are involved in resistance to ROS, was down‐regulated in fungi carrying defective NoxA, NoxB or NoxR. Our results highlight the requirement of Nox in ROS resistance and provide insights into its critical role in regulating both YAP1 and HOG1 in A. alternata.
The ability of the necrotrophic fungus Alternaria alternata to detoxify reactive oxygen species (ROS) is crucial for pathogenesis to citrus. We report regulation of siderophore-mediated iron ...acquisition and ROS resistance by the NADPH oxidase (NOX), the redox activating yes-associated protein 1 (YAP1) regulator, and the high-osmolarity glycerol 1 (HOG1) mitogen-activated protein kinase (MAPK). The A. alternata nonribosomal peptide synthetase (NPS6) is essential for the biosynthesis of siderophores, contributing to iron uptake under low-iron conditions. Fungal strains impaired for NOX, YAP1, HOG1 or NPS6 all display increased sensitivity to ROS. Exogenous addition of iron at least partially rescues ROS sensitivity seen for NPS6, YAP1, HOG1, and NOX mutants. Importantly, expression of the NPS6 gene and biosynthesis of siderophores are regulated by NOX, YAP1 and HOG1, supporting a functional link among these regulatory pathways. Although iron fully rescues H2O2 sensitivity seen in mutants impaired for the response regulator SKN7, neither expression of NPS6 nor biosynthesis of siderophores is controlled by SKN7. Our results indicate that the acquisition of environmental iron has profound effects on ROS detoxification.
Most pandemics of recent decades can be traced to RNA viruses, including HIV, SARS, influenza, dengue, Zika, and SARS-CoV-2. These RNA viruses impose considerable social and economic burdens on our ...society, resulting in a high number of deaths and high treatment costs. As these RNA viruses utilize an RNA genome, which is important for different stages of the viral life cycle, including replication, translation, and packaging, studying how the genome folds is important to understand virus function. In this review, we summarize recent advances in computational and high-throughput RNA structure-mapping approaches and their use in understanding structures within RNA virus genomes. In particular, we focus on the genome structures of the dengue, Zika, and SARS-CoV-2 viruses due to recent significant outbreaks of these viruses around the world.
Candida albicans cells homozygous at the mating-type locus stochastically undergo the white-to-opaque switching to become mating-competent. This switching is regulated by a core circuit of ...transcription factors organized through interlocking feedback loops around the master regulator Wor1. Although a range of distinct environmental cues is known to induce the switching, the pathways linking the external stimuli to the central control mechanism remains largely unknown. By screening a C. albicans haploid gene-deletion library, we found that SAC7 encoding a GTPase-activating protein of Rho1 is required for the white-to-opaque switching. We demonstrate that Sac7 physically associates with Rho1-GTP and the constitutively active Rho1
mutant impairs the white-to-opaque switching while the inactive Rho1
mutant promotes it. Overexpressing WOR1 in both sac7Δ/Δ and rho1
cells suppresses the switching defect, indicating that the Sac7/Rho1 module acts upstream of Wor1. Furthermore, we provide evidence that Sac7/Rho1 functions in a pathway independent of the Ras/cAMP pathway which has previously been positioned upstream of Wor1. Taken together, we have discovered new regulators and a signaling pathway that regulate the white-to-opaque switching in the most prevalent human fungal pathogen C. albicans.
Recent advances in RNA structurome Xu, Bingbing; Zhu, Yanda; Cao, Changchang ...
Science China. Life Sciences/Science China. Life sciences,
07/2022, Letnik:
65, Številka:
7
Journal Article
Recenzirano
Odprti dostop
RNA structures are essential to support RNA functions and regulation in various biological processes. Recently, a range of novel technologies have been developed to decode genome-wide RNA structures ...and novel modes of functionality across a wide range of species. In this review, we summarize key strategies for probing the RNA structurome and discuss the pros and cons of representative technologies. In particular, these new technologies have been applied to dissect the structural landscape of the SARS-CoV-2 RNA genome. We also summarize the functionalities of RNA structures discovered in different regulatory layers-including RNA processing, transport, localization, and mRNA translation-across viruses, bacteria, animals, and plants. We review many versatile RNA structural elements in the context of different physiological and pathological processes (e.g., cell differentiation, stress response, and viral replication). Finally, we discuss future prospects for RNA structural studies to map the RNA structurome at higher resolution and at the single-molecule and single-cell level, and to decipher novel modes of RNA structures and functions for innovative applications.
Citrus brown spot disease is caused by the necrotrophic fungus Alternaria alternata. Its pathogenic capability has been thought to depend exclusively on the production of host-selective ACT toxin. ...However, circumvention of plant defenses is also likely to be important for the disease process. To investigate the fungal response to host-generated reactive oxygen species (ROS), we cloned and characterized the AaAP1 gene of A. alternata, which encodes a polypeptide resembling yeast YAP1-like transcriptional activators implicated in cellular responses to stress. Expression of the AaAP1 gene in a wild-type strain was primarily induced by H2O2 or ROS-generating oxidants. Using a loss-of-function mutation in the AaAP1 gene, we demonstrated an essential requirement for oxidative tolerance during the host invasion step. Mutants lacking AaAP1 showed increased sensitivity to H2O2 and loss of fungal pathogenicity. The ΔAaAP1 null mutant did not cause any visible necrotic lesions on wounded or unwounded leaves of citrus cv. Minneola. Compared with the wild type, the null mutant displayed lower catalase, peroxidase, and superoxide dismutase activities. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of AaAP1. Upon exposure to H2O2, the AaAP1::sGFP (synthetic green fluorescent protein) fusion protein became localized in the nucleus. Inoculation of the mutant with NADPH oxidase inhibitors partially restored fungal pathogenicity. Our results highlight the global regulatory role of a YAP1 homolog in response to oxidative stress in A. alternata and provide insights into the critical role of ROS detoxification in the pathogenicity of A. alternata.
The necrotrophic fungal pathogen
Alternaria alternata
causes brown spot diseases in many citrus cultivars. The FUS3 and SLT2 mitogen-activated protein kinases (MAPK)-mediated signaling pathways have ...been shown to be required for conidiation. Exogenous application of cAMP to this fungal pathogen decreased conidia formation considerably. This study determined whether a cAMP-activated protein kinase A (PKA) is required for conidiation. Using loss-of-function mutations in PKA catalytic and regulatory subunit-coding genes, we demonstrated that PKA negatively regulates conidiation. Fungal mutants lacking PKA catalytic subunit gene (
PKA
cat
) reduced growth, lacked detectable PKA activity, and produced higher amounts of conidia compared to wild-type. Introduction of a functional copy of
PKA
cat
into a null mutant partially restored PKA activity and produced wild-type level of conidia. In contrast, fungi lacking PKA regulatory subunit gene (
PKA
reg
) produced detectable PKA activity, exhibited severe growth reduction, formed swelling hyphal segments, and produced no mature conidia. Introduction of the
PKA
reg
gene to a regulatory subunit mutant restored all phenotypes to wild type.
PKA
reg
-null mutants induced fewer necrotic lesions on citrus compared to wild-type, whereas
PKA
cat
mutant displayed wild-type virulence. Overall, our studies indicate that PKA and FUS3-mediated signaling pathways apparently have very different roles in the regulation of conidia production and
A. alternata
pathogenesis in citrus.
Successful pathogenesis by the necrotrophic pathogen
Alternaria alternata in citrus has been thought to be dependent on the production of host-specific toxins. This study determined whether the ...fungal pathogen can modulate reactive oxygen species (ROS) in the host plant by cloning and characterizing an
RLAP1 gene in the rough lemon pathotype of
A. alternata, which encodes a polypeptide that resembles many YAP1-like transcription activators of yeasts and fungi. Expression of the
RLAP1 gene in a wild-type strain was responsive to many ROS-generating compounds. Mutants disrupted at the
RLAP1 locus were hypersensitive to H
2O
2 and ROS-generating oxidants, showed reduced vegetative growth, were less effective in detoxifying H
2O
2, were non-pathogenic to rough lemon, and yet were unaltered in conidial formation or toxin production. When inoculated through wound sites, the mutants failed to incite any lesions. However, co-inoculation of the mutants with an NADPH oxidase inhibitor apocynin or diphenylene iodonium slightly restored fungal pathogenicity; introduction of the
RLAP1 gene to a null mutant fully restored all phenotypes to wild type. Upon activation by H
2O
2, RLAP1::sGFP fusion protein is localized to the nucleus. Overall, the results reveal the essential role of effective detoxification via an RLAP1-mediated pathway in oxidative stress response and pathogenicity in
A. alternata. It is proposed that the function of RLAP1 in
A. alternata is to protect the pathogen from the toxicity of ROS generated by the citrus host.
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