scholarly journals The crystal structure of SnTox3 from the necrotrophic fungus Parastagonospora nodorum reveals a unique effector fold and insights into Kex2 protease processing of fungal effectors

2020 ◽  
Author(s):  
Megan A. Outram ◽  
Yi-Chang Sung ◽  
Daniel Yu ◽  
Bayantes Dagvadorj ◽  
Sharmin A. Rima ◽  
...  

SummaryPlant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3.We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi.The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici.We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence.

2021 ◽  
Vol 7 (2) ◽  
pp. 86
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann

Obligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence or pathogenicity. In this study, we present the Ustilago hordei–barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We generate U. hordei solopathogenic strains, which form infectious filaments without the presence of a compatible mating partner. Solopathogenic strains are suitable for heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization are analyzed using transmission electron microscopy, showing that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.


2020 ◽  
Author(s):  
Bilal Ökmen ◽  
Daniela Schwammbach ◽  
Guus Bakkeren ◽  
Ulla Neumann ◽  
Gunther Doehlemann

AbstractObligate biotrophic fungal pathogens, such as Blumeria graminis and Puccinia graminis, are amongst the most devastating plant pathogens, causing dramatic yield losses in many economically important crops worldwide. However, a lack of reliable tools for the efficient genetic transformation has hampered studies into the molecular basis of their virulence/pathogenicity. In this study, we present the U. hordei-barley pathosystem as a model to characterize effectors from different plant pathogenic fungi. We have generated U. hordei solopathogenic strains, which form infectious filaments without presence of compatible mating partner. Solopathogenic strains are suitable as heterologous expression system for fungal virulence factors. A highly efficient Crispr/Cas9 gene editing system is made available for U. hordei. In addition, U. hordei infection structures during barley colonization were analyzed by transmission electron microscopy, which shows that U. hordei forms intracellular infection structures sharing high similarity to haustoria formed by obligate rust and powdery mildew fungi. Thus, U. hordei has high potential as a fungal expression platform for functional studies of heterologous effector proteins in barley.


2021 ◽  
Vol 22 (6) ◽  
pp. 3147
Author(s):  
Marta Suarez-Fernandez ◽  
Ana Aragon-Perez ◽  
Luis Vicente Lopez-Llorca ◽  
Federico Lopez-Moya

Fungal LysM effector proteins can dampen plant host–defence responses, protecting hyphae from plant chitinases, but little is known on these effectors from nonpathogenic fungal endophytes. We found four putative LysM effectors in the genome of the endophytic nematophagous fungus Pochonia chlamydosporia (Pc123). All four genes encoding putative LysM effectors are expressed constitutively by the fungus. Additionally, the gene encoding Lys1—the smallest one—is the most expressed in banana roots colonised by the fungus. Pc123 Lys1, 2 and 4 display high homology with those of other strains of the fungus and phylogenetically close entomopathogenic fungi. However, Pc123 Lys3 displays low homology with other fungi, but some similarities are found in saprophytes. This suggests evolutionary divergence in Pc123 LysM effectors. Additionally, molecular docking shows that the NAcGl binding sites of Pc123 Lys 2, 3 and 4 are adjacent to an alpha helix. Putative LysM effectors from fungal endophytes, such as Pc123, differ from those of plant pathogenic fungi. LysM motifs from endophytic fungi show clear conservation of cysteines in Positions 13, 51 and 63, unlike those of plant pathogens. LysM effectors could therefore be associated with the lifestyle of a fungus and give us a clue of how organisms could behave in different environments.


Author(s):  
Bhimanagoud Kumbar ◽  
Shivananda Kandagalla ◽  
Bharath B R ◽  
Sharath B S ◽  
Riaz Mahmood

Aim And Objectives: Phytophthora infestans (Mont.) de Bary, fungal pathogen it causes late blight, which results in devastating economic loss among the Solanaceae. The bacillus lipopeptides shows the antagonistic activity against the many plant pathogens, among bacillus lipopeptides Iturin A reported as the antifungal gene. Hence, to understand the in silico antifungal activity, we have selected gene iturin A (AXN89987) produced by Bacillus spp to check the molecular dynamics study with the effector proteins of the P. infestanse. In this concern known effectors proteins of P. infestans were subjected to the protein-protein interaction and followed by simulation. Material and Method: Iturin A gene was amplified using the soil bacterium Bacillus subtilis with gene specific primers. Cloned into pTZ 57R/T vector and confirmed by sequencing. To get better insight the protein model was developed for Iturin A using Modeller 9.17, also for the effector proteins by using PDB structure of ID 4MRT (Phosphopantetheine Transferase Sfp) and 1QR0 (4'-phosphopantetheinyl moiety of coenzyme A) as template it shares the identity 72% and expected P-value: 3e-121 respectively. The model quality was assessed using ProSA and PROCHECK programs. Results: The potency of modelled protein against effector proteins of P. infestans were evaluated in silico using the HADDOCK server and the results showed the high affinity of Iturin A toward the effector protein Host ATG8 (PDB-5L83). Finally, the simulation was performed to the docked conformation of Iturin A with Host ATG8 to further understand the stability of the complex using Desmond program. Conclusion: Altogether the protein-protein interaction and simulation study propose a new methodology and intern it also attempted to uncover possible antifungal activity of iturin A against effector proteins of P. infestans.


Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 842
Author(s):  
Larisa Shcherbakova ◽  
Maksim Kartashov ◽  
Natalia Statsyuk ◽  
Tatyana Pasechnik ◽  
Vitaly Dzhavakhiya

Agricultural fungicides contaminate the environment and promote the spread of fungicide-resistant strains of pathogenic fungi. The enhancement of pathogen sensitivity to these pesticides using chemosensitizers allows the reducing of fungicide dosages without a decrease in their efficiency. Using Petri plate and microplate bioassays, 6-demethylmevinolin (6-DMM), a putative sensitizer of a microbial origin, was shown to affect both colony growth and conidial germination of Alternaria solani, A. alternata, Parastagonospora nodorum, Rhizoctonia solani, and four Fusarium species (F. avenaceum, F. culmorum, F. oxysporum, F. graminearum) forming a wheat root rot complex together with B. sorokiniana. Non- or marginally toxic 6-DMM concentrations suitable for sensitizing effect were determined by the probit analysis. The range of determined concentrations confirmed a possibility of using 6-DMM as a putative sensitizer for the whole complex of root rot agents, other cereal pathogens (A. alternata, P.nodorum), and some potato (R. solani, A. solani) and tomato (A. solani) pathogens. Despite the different sensitivities of the eight tested pathogens, 6-DMM lacked specificity to fungi and possessed a mild antimycotic activity that is typical of other known pathogen-sensitizing agents. The pilot evaluation of the 6-DMM sensitizing first confirmed a principal possibility of using it for the sensitization of B. sorokiniana and R. solani to triazole- and strobilurin-based fungicides, respectively.


2021 ◽  
Author(s):  
Evan John ◽  
Kar-Chun Tan ◽  
Richard Peter Oliver ◽  
Karam Singh

Plant-pathogenic fungi span diverse taxonomic lineages. Their host-infection strategies are often specialised and require the coordinated regulation of molecular virulence factors. Transcription factors (TFs) are fundamental regulators of gene expression, controlling development and virulence in plant pathogenic fungi. Recent research has established regulatory roles for several taxonomically conserved fungal TFs, but the evolution of specific virulence regulators is not well understood. This study sought to explore the representation of TFs across a taxonomically-diverse range of fungi, with a focus on plant pathogens. A significant trend was observed among the obligate, host-associated pathogens, which possess a reduced overall TF repertoire, alluding to a lack of pressure for maintaining diversity. A novel orthology-based analysis is then presented that refined TF classifications, traditionally based on the nature of the DNA-binding domains. Using this analysis, cases of TF over/underrepresentation across fungal pathogen lineages are systematically highlighted. Specific examples are then explored and discussed that included the TF orthologues of Ste12, Pf2 and EBR1, plus phytotoxic secondary-metabolite cluster regulators, which all presented novel and distinct evolutionary insights. Ultimately, as the examples presented demonstrate, this resource can be interrogated to guide functional studies that seek to characterise virulence-specific regulators and shed light on the factors underpinning plant pathogenicity.


mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Jamie McGowan ◽  
David A. Fitzpatrick

ABSTRACT The oomycetes are a class of microscopic, filamentous eukaryotes and include ecologically significant animal and plant pathogens. Oomycetes secrete large arsenals of effector proteins that degrade host cell components, manipulate host immune responses, and induce necrosis, enabling parasitic colonization. In this study, we catalogued the number and evolution of effectors in 37 oomycete species whose genomes have been completely sequenced. Large expansions of effector protein families in Phytophthora species, including glycoside hydrolases, pectinases, and necrosis-inducing proteins, were observed. Species-specific expansions were detected, including chitinases in Aphanomyces astaci and Pythium oligandrum. Novel effectors which may be involved in suppressing animal immune responses were identified in Ap. astaci and Py. oligandrum. Type 2 necrosis-inducing proteins with an unusual phylogenetic history were also located. This work represents an up-to-date in silico catalogue of the effector arsenal of the oomycetes based on the 37 genomes currently available. The oomycetes are a class of microscopic, filamentous eukaryotes within the stramenopiles-alveolate-Rhizaria (SAR) supergroup and include ecologically significant animal and plant pathogens. Oomycetes secrete large arsenals of effector proteins that degrade host cell components, manipulate host immune responses, and induce necrosis, enabling parasitic colonization. This study investigated the expansion and evolution of effectors in 37 oomycete species in 4 oomycete orders, including Albuginales, Peronosporales, Pythiales, and Saprolegniales species. Our results highlight the large expansions of effector protein families, including glycoside hydrolases, pectinases, and necrosis-inducing proteins, in Phytophthora species. Species-specific expansions, including expansions of chitinases in Aphanomyces astaci and Pythium oligandrum, were detected. Novel effectors which may be involved in suppressing animal immune responses in Ap. astaci and Py. insidiosum were also identified. Type 2 necrosis-inducing proteins with an unusual phylogenetic history were also located in a number of oomycete species. We also investigated the "RxLR" effector complement of all 37 species and, as expected, observed large expansions in Phytophthora species numbers. Our results provide in-depth sequence information on all putative RxLR effectors from all 37 species. This work represents an up-to-date in silico catalogue of the effector arsenal of the oomycetes based on the 37 genomes currently available. IMPORTANCE The oomycetes are a class of microscopic, filamentous eukaryotes and include ecologically significant animal and plant pathogens. Oomycetes secrete large arsenals of effector proteins that degrade host cell components, manipulate host immune responses, and induce necrosis, enabling parasitic colonization. In this study, we catalogued the number and evolution of effectors in 37 oomycete species whose genomes have been completely sequenced. Large expansions of effector protein families in Phytophthora species, including glycoside hydrolases, pectinases, and necrosis-inducing proteins, were observed. Species-specific expansions were detected, including chitinases in Aphanomyces astaci and Pythium oligandrum. Novel effectors which may be involved in suppressing animal immune responses were identified in Ap. astaci and Py. oligandrum. Type 2 necrosis-inducing proteins with an unusual phylogenetic history were also located. This work represents an up-to-date in silico catalogue of the effector arsenal of the oomycetes based on the 37 genomes currently available.


2000 ◽  
Vol 13 (1) ◽  
pp. 122-143 ◽  
Author(s):  
Mahmoud A. Ghannoum

SUMMARY Microbial pathogens use a number of genetic strategies to invade the host and cause infection. These common themes are found throughout microbial systems. Secretion of enzymes, such as phospholipase, has been proposed as one of these themes that are used by bacteria, parasites, and pathogenic fungi. The role of extracellular phospholipase as a potential virulence factor in pathogenic fungi, including Candida albicans, Cryptococcus neoformans, and Aspergillus, has gained credence recently. In this review, data implicating phospholipase as a virulence factor in C. albicans, Candida glabrata, C. neoformans, and A. fumigatus are presented. A detailed description of the molecular and biochemical approaches used to more definitively delineate the role of phospholipase in the virulence of C. albicans is also covered. These approaches resulted in cloning of three genes encoding candidal phospholipases (caPLP1, caPLB2, and PLD). By using targeted gene disruption, C. albicans null mutants that failed to secrete phospholipase B, encoded by caPLB1, were constructed. When these isogenic strain pairs were tested in two clinically relevant murine models of candidiasis, deletion of caPLB1 was shown to lead to attenuation of candidal virulence. Importantly, immunogold electron microscopy studies showed that C. albicans secretes this enzyme during the infectious process. These data indicate that phospholipase B is essential for candidal virulence. Although the mechanism(s) through which phospholipase modulates fungal virulence is still under investigations, early data suggest that direct host cell damage and lysis are the main mechanisms contributing to fungal virulence. Since the importance of phospholipases in fungal virulence is already known, the next challenge will be to utilize these lytic enzymes as therapeutic and diagnostic targets.


2020 ◽  
Vol 21 (22) ◽  
pp. 8681
Author(s):  
Nicolò Orsoni ◽  
Francesca Degola ◽  
Luca Nerva ◽  
Franco Bisceglie ◽  
Giorgio Spadola ◽  
...  

As key players in biotic stress response of plants, jasmonic acid (JA) and its derivatives cover a specific and prominent role in pathogens-mediated signaling and hence are promising candidates for a sustainable management of phytopathogenic fungi. Recently, JA directed antimicrobial effects on plant pathogens has been suggested, supporting the theory of oxylipins as double gamers in plant-pathogen interaction. Based on these premises, six derivatives (dihydrojasmone and cis-jasmone, two thiosemicarbazonic derivatives and their corresponding complexes with copper) have been evaluated against 13 fungal species affecting various economically important herbaceous and woody crops, such as cereals, grapes and horticultural crops: Phaeoacremonium minimum, Neofusicoccum parvum, Phaeomoniella chlamydospora, Fomitiporia mediterranea, Fusarium poae, F. culmorum, F. graminearum, F. oxysporum f. sp. lactucae,F. sporotrichioides, Aspergillus flavus, Rhizoctonia solani,Sclerotinia spp. and Verticillium dahliae. The biological activity of these compounds was assessed in terms of growth inhibition and, for the two mycotoxigenic species A. flavus and F. sporotrichioides, also in terms of toxin containment. As expected, the inhibitory effect of molecules greatly varied amongst both genera and species; cis-jasmone thiosemicarbazone in particular has shown the wider range of effectiveness. However, our results show that thiosemicarbazones derivatives are more effective than the parent ketones in limiting fungal growth and mycotoxins production, supporting possible applications for the control of pathogenic fungi.


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