RESUMO
The eukaryotic cytoskeleton is a complex scaffold consisting of actin filaments, intermediate filaments, and microtubules. Although fungi and plants lack intermediate filaments, their dynamic structural network of actin filaments and microtubules regulates cell shape, division, polarity, and vesicular trafficking. However, the specialized functions of the cytoskeleton during plant-fungus interactions remain elusive. Recent reports demonstrate that the plant cytoskeleton responds to signal cues and pathogen invasion through remodeling, thereby coordinating immune receptor trafficking, membrane microdomain formation, aggregation of organelles, and transport of defense compounds. Emerging evidence also suggests that cytoskeleton remodeling further regulates host immunity by triggering salicylic acid signaling, reactive oxygen species generation, and pathogenesis-related gene expression. During host invasion, fungi undergo systematic cytoskeleton remodeling, which is crucial for successful host penetration and colonization. Furthermore, phytohormones act as an essential regulator of plant cytoskeleton dynamics and are frequently targeted by fungal effectors to disrupt the host's growth-defense balance. This review discusses recent advances in the understanding of cytoskeleton dynamics during plant-fungus interactions and provides novel insights into the relationship between phytohormones and cytoskeleton remodeling upon pathogen attack. We also highlight the importance of fungal cytoskeleton rearrangements during host colonization and suggest directions for future investigations in this field.
Assuntos
Citoesqueleto , Fungos , Interações Hospedeiro-Patógeno , Plantas , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Plantas/microbiologia , Plantas/metabolismo , Plantas/imunologia , Interações Hospedeiro-Patógeno/fisiologia , Fungos/fisiologia , Reguladores de Crescimento de Plantas/metabolismo , Reguladores de Crescimento de Plantas/fisiologia , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologiaRESUMO
In eukaryotes, targeted protein degradation (TPD) typically depends on a series of interactions among ubiquitin ligases that transfer ubiquitin molecules to substrates leading to degradation by the 26S proteasome. We previously identified that the bacterial effector protein SAP05 mediates ubiquitin-independent TPD. SAP05 forms a ternary complex via interactions with the von Willebrand Factor Type A (vWA) domain of the proteasomal ubiquitin receptor Rpn10 and the zinc-finger (ZnF) domains of the SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) and GATA BINDING FACTOR (GATA) transcription factors (TFs). This leads to direct TPD of the TFs by the 26S proteasome. Here, we report the crystal structures of the SAP05-Rpn10vWA complex at 2.17 Å resolution and of the SAP05-SPL5ZnF complex at 2.20 Å resolution. Structural analyses revealed that SAP05 displays a remarkable bimodular architecture with two distinct nonoverlapping surfaces, a "loop surface" with three protruding loops that form electrostatic interactions with ZnF, and a "sheet surface" featuring two ß-sheets, loops, and α-helices that establish polar interactions with vWA. SAP05 binding to ZnF TFs involves single amino acids responsible for multiple contacts, while SAP05 binding to vWA is more stable due to the necessity of multiple mutations to break the interaction. In addition, positioning of the SAP05 complex on the 26S proteasome points to a mechanism of protein degradation. Collectively, our findings demonstrate how a small bacterial bimodular protein can bypass the canonical ubiquitin-proteasome proteolysis pathway, enabling ubiquitin-independent TPD in eukaryotic cells. This knowledge holds significant potential for the creation of TPD technologies.
Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Proteínas de Transporte/metabolismo , Ligação Proteica , Eucariotos/metabolismoRESUMO
The Spot Blotch (SB) caused by hemibiotrophic fungal pathogen Bipolaris sorokiniana is one of the most devastating wheat diseases leading to 15-100% crop loss. However, the biology of Triticum-Bipolaris interactions and host immunity modulation by secreted effector proteins remain underexplored. Here, we identified a total of 692 secretory proteins including 186 predicted effectors encoded by B. sorokiniana genome. Gene Ontology categorization showed that these proteins belong to cellular, metabolic and signaling processes, and exhibit catalytic and binding activities. Further, we functionally characterized a cysteine-rich, B. sorokiniana Candidate Effector 66 (BsCE66) that was induced at 24-96 hpi during host colonization. The Δbsce66 mutant did not show vegetative growth defects or stress sensitivity compared to wild-type, but developed drastically reduced necrotic lesions upon infection in wheat plants. The loss-of-virulence phenotype was rescued upon complementing the Δbsce66 mutant with BsCE66 gene. Moreover, BsCE66 does not form homodimer and conserved cysteine residues form intra-molecular disulphide bonds. BsCE66 localizes to the host nucleus and cytosol, and triggers a strong oxidative burst and cell death in Nicotiana benthamiana. Overall, our findings demonstrate that BsCE66 is a key virulence factor that is necessary for host immunity modulation and SB disease progression. These findings would significantly improve our understanding of Triticum-Bipolaris interactions and assist in the development of SB resistant wheat varieties.
Assuntos
Ascomicetos , Bipolaris , Virulência/genética , Triticum/microbiologia , Cisteína/genética , Doenças das Plantas/microbiologiaRESUMO
Flavonoids are important plant pigments and defense compounds; understanding the transcriptional regulation of flavonoid biosynthesis may enable engineering crops with improved nutrition and stress tolerance. Here, we characterize R2R3-MYB domain subgroup 7 transcription factor CaMYB39, which regulates flavonol biosynthesis primarily in chickpea trichomes. CaMYB39 overexpression in chickpea was accompanied by a change in flux availability for the phenylpropanoid pathway, particularly flavonol biosynthesis. Lines overexpressing CaMYB39 showed higher isoflavonoid levels, suggesting its role in regulating isoflavonoid pathway. CaMYB39 transactivates the transcription of early flavonoid biosynthetic genes (EBG). FLAVONOL SYNTHASE2, an EBG, encodes an enzyme with higher substrate specificity for dihydrokaempferol than other dihydroflavonols explaining the preferential accumulation of kaempferol derivatives as prominent flavonols in chickpea. Interestingly, CaMYB39 overexpression increased trichome density and enhanced the accumulation of diverse flavonol derivatives in trichome-rich tissues. Moreover, CaMYB39 overexpression reduced reactive oxygen species levels and induced defense gene expression which aids in partially blocking the penetration efficiency of the fungal pathogen, Ascochyta rabiei, resulting in lesser symptoms, thus establishing its role against deadly Ascochyta blight (AB) disease. Overall, our study reports an instance where R2R3-MYB-SG7 member, CaMYB39, besides regulating flavonol biosynthesis, modulates diverse pathways like general phenylpropanoid, isoflavonoid, trichome density, and defense against necrotrophic fungal infection in chickpea.
Assuntos
Cicer , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Cicer/genética , Cicer/metabolismo , Flavonoides , Flavonóis , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismoRESUMO
Ascochyta blight (AB) caused by the filamentous fungus Ascochyta rabiei is a major threat to global chickpea production. The mechanisms underlying chickpea response to A. rabiei remain elusive to date. Here, we investigated the comparative transcriptional dynamics of AB-resistant and -susceptible chickpea genotypes upon A. rabiei infection, to understand the early host defense response. Our findings revealed that AB-resistant plants underwent rapid and extensive transcriptional reprogramming compared with a susceptible host. At the early stage (24 h postinoculation [hpi]), mainly cell-wall remodeling and secondary metabolite pathways were highly activated, while differentially expressed genes related to signaling components, such as protein kinases, transcription factors, and hormonal pathways, show a remarkable upsurge at 72 hpi, especially in the resistant genotype. Notably, our data suggest an imperative role of jasmonic acid, ethylene, and abscisic acid signaling in providing immunity against A. rabiei. Furthermore, gene co-expression networks and modules corroborated the importance of cell-wall remodeling, signal transduction, and phytohormone pathways. Hub genes such as MYB14, PRE6, and MADS-SOC1 discovered in these modules might be the master regulators governing chickpea immunity. Overall, we not only provide novel insights for comprehensive understanding of immune signaling components mediating AB resistance and susceptibility at early Cicer-Ascochyta interactions but, also, offer a valuable resource for developing AB-resistant chickpea. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Assuntos
Cicer , Cicer/genética , Cicer/microbiologia , Transcriptoma/genética , Doenças das Plantas/microbiologiaRESUMO
Old yellow enzymes (OYEs) play a critical role in antioxidation, detoxification and ergot alkaloid biosynthesis processes in various organisms. The yeast- and bacteria-like OYEs have been structurally characterized earlier, however, filamentous fungal pathogens possess a novel OYE class, that is, class III, whose biochemical and structural intricacies remain unexplored to date. Here, we present the 1.6 Å X-ray structure of a class III member, OYE 6 from necrotrophic fungus Ascochyta rabiei (ArOYE6), in flavin mononucleotide (FMN)-bound form (PDB ID-7FEV) and provide mechanistic insights into their catalytic capability. We demonstrate that ArOYE6 exists as a monomer in solution, forms (ß/α)8 barrel structure harbouring non-covalently bound FMN at cofactor binding site, and utilizes reduced nicotinamide adenine dinucleotide phosphate as its preferred reductant. The large hydrophobic cavity situated above FMN, specifically accommodates 12-oxo-phytodienoic acid and N-ethylmaleimide substrates as observed in ArOYE6-FMN-substrate ternary complex models. Site-directed mutations in the conserved catalytic (His196, His199 and Tyr201) and FMN-binding (Lys249 and Arg348) residues render the enzyme inactive. Intriguingly, the ArOYE6 structure contains a novel C-terminus (369-445 residues) made of three α-helices, which stabilizes the FMN binding pocket as its mutation/truncation lead to complete loss of FMN binding. Moreover, the loss of the extended C-terminus does not alter the monomeric nature of ArOYE6. In this study, for the first time, we provide the structural and biochemical insights for a fungi-specific class III OYE homologue and dissect the oxidoreductase mechanism. Our findings hold broad biological significance during host-fungus interactions owing to the conservation of this class among pathogenic fungi, and would have potential implications in the pharmacochemical industry.
Assuntos
Alcaloides de Claviceps , NADPH Desidrogenase , Cristalografia por Raios X , Etilmaleimida , Mononucleotídeo de Flavina/química , NADP , NADPH Desidrogenase/química , Oxirredutases/metabolismo , Substâncias RedutorasRESUMO
Stomata offer an effortless opportunity for pathogens to enter host plants and exploit that resource. Upon pathogen attack, stomatal closure is a commonly observed response to prevent microbial invasion. A recent study by Zou et al. shows that stomatal closure following exposure to microbe-associated molecular patterns (MAMPs) is mediated by altered actin dynamics in an MPK3/6 phosphorylation- and VLN3-dependent manner.
Assuntos
Proteínas de Arabidopsis , Estômatos de Plantas , Proteínas de Arabidopsis/metabolismo , Fosforilação , Estômatos de Plantas/fisiologiaRESUMO
Fungal phytopathogens pose a serious threat to global crop production. Only a handful of strategies are available to combat these fungal infections, and the increasing incidence of fungicide resistance is making the situation worse. Hence, the molecular understanding of plant-fungus interactions remains a primary focus of plant pathology. One of the hallmarks of host-pathogen interactions is the overproduction of reactive oxygen species (ROS) as a plant defense mechanism, collectively termed the oxidative burst. In general, high accumulation of ROS restricts the growth of pathogenic organisms by causing localized cell death around the site of infection. To survive the oxidative burst and achieve successful host colonization, fungal phytopathogens employ intricate mechanisms for ROS perception, ROS neutralization, and protection from ROS-mediated damage. Together, these countermeasures maintain the physiological redox homeostasis that is essential for cell viability. In addition to intracellular antioxidant systems, phytopathogenic fungi also deploy interesting effector-mediated mechanisms for extracellular ROS modulation. This aspect of plant-pathogen interactions is significantly under-studied and provides enormous scope for future research. These adaptive responses, broadly categorized into "escape" and "exploitation" mechanisms, are poorly understood. In this review, we discuss the oxidative stress response of filamentous fungi, their perception signaling, and recent insights that provide a comprehensive understanding of the distinct survival mechanisms of fungal pathogens in response to the host-generated oxidative burst.
Assuntos
Fungos/fisiologia , Interações Hospedeiro-Patógeno , Estresse Oxidativo , Doenças das Plantas/microbiologia , Plantas/metabolismo , Plantas/microbiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Efficient transformation system for genetic improvement is essential in Crocus sativus, as it lacks sexual reproduction. This is the first report wherein an efficient protocol is developed for the transformation of Crocus sativus L. by Agrobacterium rhizogenes strain ARqua1 with a transformation efficiency of 78.51%. The ARqua1 strain harboring both Ri plasmid and binary vector plasmid pSITE-4NB, and marker genes for red fluorescent protein (RFP) and a ß-glucuronidase (GUS) reporter gene were used for selection. Transformation was confirmed by RFP signal, GUS reporter assay and polymerase chain reaction (PCR) analysis of the test samples after 21 days post inoculation. These results confirm the establishment of protocol for hairy root transformation in C. sativus that can be further used for gene transfer or gene editing in Crocus for its genetic improvement.
RESUMO
Necrotrophic pathogens experience host-generated oxidative stress during pathogenesis. They overcome such hostile environment by intricate mechanisms which are largely understudied. In this article, reference-based transcriptome analysis of a devastating Ascochyta Blight (AB) disease causing chickpea pathogen Ascochyta rabiei was explored to get insights into survival mechanisms under oxidative stress. Here, expression profiling of mock-treated and menadione-treated fungus was carried out by RNA-Seq approach. A significant number of genes in response to oxidative stress were overrepresented, suggestive of a robust and coordinated defense system of A. rabiei. A total 73 differentially expressed genes were filtered out from both the transcriptomes, among them 64 were up-regulated and 9 were found down-regulated. The gene ontology and KEGG mapping were conducted to comprehend the possible regulatory roles of differentially expressed genes in metabolic networks and biosynthetic pathways. Transcript profiling, KEGG pathway and gene ontology-based enrichment analysis revealed 12 (16.43%) stress responsive factors, 25 (34.24%) virulence associated genes, 10 (13.69%) putative effectors and 28 (38.35%) important interacting proteins associated with various metabolic pathways. In addition, genes with differential expression were further explored for underlying putative pathogenicity factors. We identified five genes ST47_g10291, ST47_g9396, ST47_g10294, ST47_g4395, and ST47_g7191 that were common to stress and fungal pathogenicity. The factors recognized in this work can be used to establish molecular tools to explain the regulatory gene networks engaged in stress response of fungal pathogens and disease management.