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The Farnesyltransferase ?-Subunit Ram1 Regulates Sporisorium scitamineum Mating, Pathogenicity and Cell Wall Integrity.

ABSTRACT: The basidiomycetous fungus Sporisorium scitamineum causes a serious sugarcane smut disease in major sugarcane growing areas. Sexual mating is essential for infection to the host; however, its underlying molecular mechanism has not been fully studied. In this study, we identified a conserved farnesyltransferase (FTase) ? subunit Ram1 in S. scitamineum. The ram1? mutant displayed significantly reduced mating/filamentation, thus of weak pathogenicity to the host cane. The ram1? mutant sporidia showed more tolerant toward cell wall stressor Congo red compared to that of the wild-type. Transcriptional profiling showed that Congo red treatment resulted in notable up-regulation of the core genes involving in cell wall integrity pathway in ram1? sporidia compared with that of WT, indicating that Ram1 may be involved in cell wall integrity regulation. In yeast the heterodimeric FTase is responsible for post-translational modification of Ras (small G protein) and a-factor (pheromone). We also identified and characterized two conserved Ras proteins, Ras1 and Ras2, respectively, and a MAT-1 pheromone precursor Mfa1. The ras1?, ras2? and mfa1? mutants all displayed reduced mating/filamentation similar as the ram1? mutant. However, both ras1? and ras2? mutants were hypersensitive to Congo red while the mfa1? mutant was the same as wild-type. Overall our study displayed that RAM1 plays an essential role in S. scitamineum mating/filamentation, pathogenicity, and cell wall stability.

SUBMITTER: Sun S

PROVIDER: S-EPMC6517510 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The Farnesyltransferase β-Subunit Ram1 Regulates Sporisorium scitamineum Mating, Pathogenicity and Cell Wall Integrity.

Sun Shuquan S Deng Yizhen Y Cai Enping E Yan Meixin M Li Lingyu L Chen Baoshan B Chang Changqing C Jiang Zide Z

Frontiers in microbiology 20190508

The basidiomycetous fungus Sporisorium scitamineum causes a serious sugarcane smut disease in major sugarcane growing areas. Sexual mating is essential for infection to the host; however, its underlying molecular mechanism has not been fully studied. In this study, we identified a conserved farnesyltransferase (FTase) β subunit Ram1 in S. scitamineum. The ram1Δ mutant displayed significantly reduced mating/filamentation, thus of weak pathogenicity to the host cane. The ra ...[more]

PMID: 31134021

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Project description:Sporisorium scitamineum is the fungal pathogen causing severe sugarcane smut disease that leads to massive economic losses globally. S. scitamineum invades host cane by dikaryotic hyphae, formed after sexual mating of two haploid sporidia of opposite mating type. Therefore, mating/filamentation is critical for S. scitamineum pathogenicity, while its molecular mechanisms remain largely unknown. The AGC (cyclic AMP [cAMP]-dependent protein kinase 1 [protein kinase A {PKA}], cGMP-dependent protein kinase [PKG], and protein kinase C [PKC]) kinase family is a group of serine/threonine (Ser/Thr) protein kinases conserved among eukaryotic genomes, serving a variety of physiological functions, including cell growth, metabolism, differentiation, and cell death. In this study, we identified an AGC kinase, named SsAgc1 (for S. scitamineum Agc1), and characterized its function by reverse genetics. Our results showed that SsAgc1 is critical for S. scitamineum mating/filamentation and pathogenicity, and oxidative stress tolerance under some circumstances. Transcriptional profiling revealed that the SsAgc1 signaling pathway may control expression of the genes governing fungal mating/filamentation and tryptophan metabolism, especially for tryptophol production. We showed that tryptophan and tryptophol could at least partially restore ssagc1Δ mating/filamentation. Overall, our work revealed a signaling pathway mediated by AGC protein kinases to regulate fungal mating/filamentation, possibly through sensing and responding to tryptophol as signal molecules.IMPORTANCE The AGC signaling pathway represents a conserved distinct signaling pathway in regulation of fungal differentiation and virulence, while it has not been identified or characterized in the sugarcane smut fungus Sporisorium scitamineum In this study, we identified a PAS domain-containing AGC kinase, SsAgc1, in S. scitamineum Functional analysis revealed that SsAgc1 plays a regulatory role on the fungal dimorphic switch.

Project description:Sugarcane is an important sugar crop and energy crop worldwide. Sugarcane smut caused by Sporisorium scitamineum is a serious fungal disease that occurs worldwide, seriously affecting the yield and quality of sugarcane. It is essential to reveal the molecular pathogenesis of S. scitamineum to explore a new control strategy of sugarcane smut. Based on transcriptome sequencing data of two S. scitamineum strains Ss16 and Ss47, each with a different pathogenicity, our laboratory screened out the SsCI80130 gene predicted to encode squalene monooxygenase. In this study, we obtained the knockout mutants (ΔSs80130+ and ΔSs80130-) and complementary mutants (COM80130+ and COM80130-) of this gene by the polyethylene glycol-mediated (PEG-mediated) protoplast transformation technology, and then performed a functional analysis of the gene. The results showed that the deletion of the SsCI80130 gene resulted in the increased content of squalene (substrate for squalene monooxygenase) and decreased content of ergosterol (the final product of the ergosterol synthesis pathway) in S. scitamineum. Meanwhile, the sporidial growth rate of the knockout mutants was significantly slower than that of the wild type and complementary mutants; under cell-wall stress or oxidative stress, the growth of the knockout mutants was significantly inhibited. In addition, the sexual mating ability and pathogenicity of knockout mutants were significantly weakened, while the sexual mating ability could be restored by adding exogenous small-molecular signal substance cAMP (cyclic adenosine monophosphate) or tryptophol. It is speculated that the SsCI80130 gene was involved in the ergosterol biosynthesis in S. scitamineum and played an important role in the sporidial growth, stress response to different abiotic stresses (including cell wall stress and oxidative stress), sexual mating/filamentation and pathogenicity. Moreover, the SsCI80130 gene may affect the sexual mating and pathogenicity of S. scitamineum by regulating the ergosterol synthesis and the synthesis of the small-molecular signal substance cAMP or tryptophol required for sexual mating. This study reveals for the first time that the gene encoding squalene monooxygenase is involved in regulating the sexual mating and pathogenicity of S. scitamineum, providing a basis for the molecular pathogenic mechanism of S. scitamineum.

Project description:Sugarcane is an important sugar crop. Sugarcane smut, caused by Sporisorium scitamineum, is a worldwide sugarcane disease with serious economic losses and lack of effective control measures. Revealing the molecular pathogenesis of S. scitamineum is very helpful to the development of effective prevention and control technology. Deubiquitinase removes ubiquitin molecules from their binding substrates and participates in a variety of physiological activities in eukaryotes. Based on the transcriptome sequencing data of two isolates (Ss16 and Ss47) of S. scitamineum with different pathogenicities, SsCI33130, a gene encoding an OTU1-deubiquitin enzyme, was identified. The positive knockout mutants and complementary mutants of the SsCI33130 gene were successfully obtained through polyethylene glycol-mediated protoplast transformation technology. In order to study the possible function of this gene in pathogenicity, phenotypic comparison of the growth, morphology, abiotic stress, sexual mating, pathogenicity, and gene expression levels of the knockout mutants, complementary mutants, and their wild type strains were conducted. The results demonstrated that the gene had almost no effect on abiotic stress, cell wall integrity, growth, and morphology, but was related to the sexual mating and pathogenicity of S. scitamineum. The sexual mating ability and pathogenicity between the knockout mutants or between the knockout mutant and wild type were more significantly reduced than between the wild types, the complementary mutants, or the wild types and complementary mutants. The sexual mating between the knockout mutants or between the knockout mutant and wild type could be restored by the exogenous addition of small-molecule signaling substances such as 5 mM cyclic adenosine monophosphate (cAMP) or 0.02 mM tryptophol. In addition, during sexual mating, the expression levels of tryptophol and cAMP synthesis-related genes in the knockout mutant combinations were significantly lower than those in the wild type combinations, while the expression levels in the complementary mutant combinations were restored to the level of the wild type. It is speculated that the SsCI33130 gene may be involved in the development of sexual mating and pathogenicity in S. scitamineum by regulating the synthesis of the small-molecule signaling substances (cAMP or tryptophol) required during the sexual mating of S. scitamineum, thereby providing a molecular basis for the study of the pathogenic mechanisms of S. scitamineum.

Dataset Information

The Farnesyltransferase ?-Subunit Ram1 Regulates Sporisorium scitamineum Mating, Pathogenicity and Cell Wall Integrity.

Publications

The Farnesyltransferase β-Subunit Ram1 Regulates <i>Sporisorium scitamineum</i> Mating, Pathogenicity and Cell Wall Integrity.

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