RESUMEN
The SWI/SNF complex is guided to the promoters of designated genes by its co-operator to activate transcription in a timely and appropriate manner to govern development, pathogenesis, and stress responses in fungi. Nevertheless, knowledge of the complexes and their co-operator in phytopathogenic fungi is still fragmented. We demonstrate that the heat shock transcription factor SsHsf1 guides the SWI/SNF complex to promoters of heat shock protein (hsp) genes and antioxidant enzyme genes using biochemistry and pharmacology. This is accomplished through direct interaction with the complex subunit SsSnf5 under heat shock and oxidative stress. This results in the activation of their transcription and mediates histone displacement to maintain reactive oxygen species (ROS) homeostasis. Genetic results demonstrate that the transcription module formed by SsSnf5 and SsHsf1 is responsible for regulating morphogenesis, stress tolerance, and pathogenicity in Sclerotinia sclerotiorum, especially by directly activating the transcription of hsp genes and antioxidant enzyme genes counteracting plant-derived ROS. Furthermore, we show that stress-induced phosphorylation of SsSnf5 is necessary for the formation of the transcription module. This study establishes that the SWI/SNF complex and its co-operator cooperatively regulate the transcription of hsp genes and antioxidant enzyme genes to respond to host and environmental stress in the devastating phytopathogenic fungi.
Asunto(s)
Ascomicetos , Proteínas de Unión al ADN , Factores de Transcripción , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Especies Reactivas de Oxígeno , Antioxidantes , Virulencia , Proteínas de Choque Térmico/metabolismo , HomeostasisRESUMEN
Soybean sclerotinia stem rot (SSR) is a disease caused by Sclerotinia sclerotiorum that causes incalculable losses in soybean yield each year. Considering the lack of effective resistance resources and the elusive resistance mechanisms, we are urged to develop resistance genes and explore their molecular mechanisms. Here, we found that loss of GmSWEET15 enhanced the resistance to S. sclerotiorum, and we explored the molecular mechanisms by which gmsweet15 mutant exhibit enhanced resistance to S. sclerotiorum by comparing transcriptome. At the early stage of inoculation, the wild type (WT) showed moderate defense response, whereas gmsweet15 mutant exhibited more extensive and intense transcription reprogramming. The gmsweet15 mutant enriched more biological processes, including the secretory pathway and tetrapyrrole metabolism, and it showed stronger changes in defense response, protein ubiquitination, MAPK signaling pathway-plant, plant-pathogen interaction, phenylpropanoid biosynthesis, and photosynthesis. The more intense and abundant transcriptional reprogramming of gmsweet15 mutant may explain how it effectively delayed colonization by S. sclerotiorum. In addition, we identified common and specific differentially expressed genes between WT and gmsweet15 mutant after inoculation with S. sclerotiorum, and gene sets and genes related to gmsweet15_24 h were identified through Gene Set Enrichment Analysis. Moreover, we constructed the protein-protein interaction network and gene co-expression networks and identified several groups of regulatory networks of gmsweet15 mutant in response to S. sclerotiorum, which will be helpful for the discovery of candidate functional genes. Taken together, our results elucidate molecular mechanisms of delayed colonization by S. sclerotiorum after loss of GmSWEET15 in soybean, and we propose novel resources for improving resistance to SSR.
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Sclerotinia sclerotiorum is a necrotrophic phytopathogenic fungus that produces sclerotia. Sclerotia are essential components of the survival and disease cycle of this devastating pathogen. In this study, we analyzed comparative transcriptomics of hyphae and sclerotia. A total of 1959 differentially expressed genes, 919 down-regulated and 1040 up-regulated, were identified. Transcriptomes data provide the possibility to precisely comprehend the sclerotia development. We further analyzed the differentially expressed genes (DEGs) in sclerotia to explore the molecular mechanism of sclerotia development, which include ribosome biogenesis and translation, melanin biosynthesis, autophagy and reactivate oxygen metabolism. Among these, the autophagy-related gene SsAtg1 was up-regulated in sclerotia. Atg1 homologs play critical roles in autophagy, a ubiquitous and evolutionarily highly conserved cellular mechanism for turnover of intracellular materials in eukaryotes. Therefore, we investigated the function of SsAtg1 to explore the function of the autophagy pathway in S. sclerotiorum. Deficiency of SsAtg1 inhibited autophagosome accumulation in the vacuoles of nitrogen-starved cells. Notably, ΔSsAtg1 was unable to form sclerotia and displayed defects in vegetative growth under conditions of nutrient restriction. Furthermore, the development and penetration of the compound appressoria in ΔSsAtg1 was abnormal. Pathogenicity analysis showed that SsAtg1 was required for full virulence of S. sclerotiorum. Taken together, these results indicate that SsAtg1 is a core autophagy-related gene that has vital functions in nutrient utilization, sclerotia development and pathogenicity in S. sclerotiorum.
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The necrotrophic fungus Sclerotinia sclerotiorum is a devastating pathogen. S. sclerotiorum can cause Sclerotinia stem rot in more than 600 species of plants, which results in serious economic losses every year. Chitin is one of the most important polysaccharides in fungal cell walls. Chitin and ß-Glucan form a scaffold that wraps around the cell and determines the vegetative growth and pathogenicity of pathogens. UDP-GlcNAc is a direct precursor of chitin synthesis. During the synthesis of UDP-GlcNAc, the conversion of GlcNAc-6P to GlcNAc-1P that is catalyzed by AGM1 (N-acetylglucosamine-phosphate mutase) is a key step. However, the significance and role of AGM1 in phytopathogenic fungus are unclear. We identified a cytoplasm-localized SsAGM1 in S. sclerotiorum, which is homologous to AGM1 of Saccharomyces cerevisiae. We utilized RNA interference (RNAi) and overexpression to characterize the function of SsAGM1 in S. sclerotiorum. After reducing the expression of SsAGM1, the contents of chitin and UDP-GlcNAc decreased significantly. Concomitantly, the gene-silenced transformants of SsAGM1 slowed vegetative growth and, importantly, lost the ability to produce sclerotia and infection cushion; it also lost virulence, even on wounded leaves. In addition, SsAGM1 was also involved in the response to osmotic stress and inhibitors of cell wall synthesis. Our results revealed the function of SsAGM1 in the growth, development, stress response, and pathogenicity in S. sclerotiorum.
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The plant pathogenic fungus Sclerotinia sclerotiorum can survive on a wide range of hosts and cause significant losses on crop yields. FKH, a forkhead box (FOX)-containing protein, functions to regulate transcription and signal transduction. As a transcription factor (TF) with multiple biological functions in eukaryotic organisms, little research has been done on the role of FKH protein in pathogenic fungi. SsFkh1 encodes a protein which has been predicted to contain FOX domain in S. sclerotiorum. In this study, the deletion mutant of SsFkh1 resulted in severe defects in hyphal development, virulence, and sclerotia formation. Moreover, knockout of SsFkh1 lead to gene functional enrichment in mitogen-activated protein kinase (MAPK) signaling pathway in transcriptome analysis and SsFkh1 was found to be involved in the maintenance of the cell wall integrity (CWI) and the MAPK signaling pathway. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that SsFkh1 interacts with SsMkk1. In addition, we explored the conserved MAPK signaling pathway components, including Bck1, Mkk1, Pkc1, and Smk3 in S. sclerotiorum. ΔSsmkk1, ΔSspkc1, ΔSsbck1, and ΔSssmk3knockout mutant strains together with ΔSsmkk1 com, ΔSspkc1 com, ΔSsbck1 com, and ΔSssmk3 com complementation mutant strains were obtained. The results indicated that ΔSsmkk1, ΔSspkc1, ΔSsbck1, and ΔSssmk3 displayed similar phenotypes to ΔSsfkh1 in sclerotia formation, compound appressorium development, and pathogenicity. Taken together, SsFkh1 may be the downstream substrate of SsMkk1 and involved in sclerotia formation, compound appressorium development, and pathogenicity in S. sclerotiorum.
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Autophagy is a highly conserved degrading process that is crucial for cell growth and development in eukaryotes, especially when they face starvation and stressful conditions. To evaluate the functions of Atg4 and Atg8 in mycelial growth, asexual and sexual development, and virulence in Cochliobolus heterostrophus, ΔChatg4 and ΔChatg8 mutants were generated by gene replacement. Strains deleted for ChATG4 and ChATG8 genes showed significant changes in vegetative growth and development of conidia and ascospores compared with the wild-type strain. The autophagy process was blocked and virulence was reduced dramatically in ΔChatg4 and ΔChatg8 mutants. In addition, deletion of ChATG4 and ChATG8 disordered Cdc10 subcellular localization and formation of septin rings. The direct physical interaction between ChAtg4 and ChAtg8 was detected by yeast two-hybrid assay, and ChAtg4-GFP was dispersed throughout the cytoplasm, although GFP-ChAtg8 appeared as punctate structures. All phenotypes were restored in complemented strains. Taken together, these findings indicate that ChATG4 and ChATG8 are crucial for autophagy to regulate fungal growth, development, virulence, and localization of septin in C. heterostrophus.
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Ascomicetos , Septinas , Ascomicetos/fisiología , Autofagia , Bipolaris , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Enfermedades de las Plantas/microbiología , Septinas/genética , Esporas Fúngicas/genética , Virulencia/genéticaRESUMEN
Sclerotinia sclerotiorum, the notorious necrotrophic phytopathogenic fungus with wide distribution, is responsible for sclerotium disease in more than 600 plant species, including many economic crops such as soybean, oilseed rape, and sunflower. The compound appressorium is a crucial multicellular infection structure that is a prerequisite for infecting healthy tissues. Previously, the Forkhead-box family transcription factors (FOX TFs) SsFoxE2 and SsFKH1 were shown to play a key regulatory role in the hyphae growth, sexual reproduction, and pathogenicity of S. sclerotiorum. However, little is known about the roles of SsFoxE3 regulating growth and development and pathogenicity. Here, we report SsFoxE3 contributes to sclerotium formation and deletion of SsFoxE3 leads to reduced formation of compound appressoria and developmental delays. Transcripts of SsFoxE3 were greatly increased during the initial stage of infection and SsFoxE3 deficiency reduced virulence on the host, while stabbing inoculation could partially restore pathogenicity. The SsFoxE3 mutant showed sensitivity to H2 O2 , and the expression of reactive oxygen species detoxification and autophagy-related genes were reduced. Moreover, expression of SsAtg8 was also decreased during the infection process of the SsFoxE3 mutant. Yeast 1-hybrid tests suggested that SsFoxE3 interacted with the promoter of SsAtg8. Disruption of SsAtg8 resulted in a phenotype similar to that of the SsFoxE3 mutant. Comparative analysis of the level of autophagy in the wild type and SsFoxE3 mutant showed that N starvation-induced autophagy was reduced in the SsFoxE3 mutant. Taken together, our findings indicate that SsFoxE3 plays an important role in compound appressorium formation and is involved in transcriptional activation of SsAtg8 during infection by S. sclerotiorum.
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Ascomicetos , Factores de Transcripción Forkhead , Fenotipo , VirulenciaRESUMEN
Higher plants and some algae convert the absorbed light into chemical energy through one of the most important organelles, chloroplast, for photosynthesis and store it in the form of organic compounds to supply their life activities. However, more and more studies have shown that the role of chloroplasts is more than a factory for photosynthesis. In the process of light conversion to chemical energy, any damage to the components of chloroplast may affect the photosynthesis efficiency and promote the production of by-products, reactive oxygen species, that are mainly produced in the chloroplasts. Substantial evidence show that chloroplasts are also involved in the battle of plants and microbes. Chloroplasts are important in integrating a variety of external environmental stimuli and regulate plant immune responses by transmitting signals to the nucleus and other cell compartments through retrograde signaling pathways. Besides, chloroplasts can also regulate the biosynthesis and signal transduction of phytohormones, including salicylic acid and jasmonic acid, to affect the interaction between the plants and microbes. Since chloroplasts play such an important role in plant immunity, correspondingly, chloroplasts have become the target of pathogens. Different microbial pathogens target the chloroplast and affect its functions to promote their colonization in the host plants.