Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

Abstract Background The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs)—secreted, membrane-bound nanoparticles containing molecular cargo—being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. Results Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 and 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC–MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. Conclusion We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

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Extracellular Vesicles from the Apoplastic Fungal Wheat Pathogen Zymoseptoria Tritici

10.21203/rs.3.rs-40104/v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Erin H Hill ◽

Peter Solomon

Keyword(s):

Extracellular Vesicles ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Sequence Similarity ◽

Protein Composition ◽

Broth Culture ◽

Zymoseptoria Tritici ◽

Culture Filtrates ◽

In Vitro Investigation

Abstract Background: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs) – secreted, membrane-bound nanoparticles containing molecular cargo – being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes. Results: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 – 100 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs. Conclusion: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

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Extracellular vesicles from the apoplastic fungal wheat pathogen Zymoseptoria tritici

10.21203/rs.3.rs-40104/v2 ◽

2020 ◽

Author(s):

Erin H Hill ◽

Peter Solomon

Keyword(s):

Extracellular Vesicles ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Sequence Similarity ◽

Protein Composition ◽

Broth Culture ◽

In Planta ◽

Zymoseptoria Tritici ◽

Culture Filtrates

Abstract Background: The fungal pathogen Zymoseptoria tritici is a significant constraint to wheat production in temperate cropping regions around the world. Despite its agronomic impacts, the mechanisms allowing the pathogen to asymptomatically invade and grow in the apoplast of wheat leaves before causing extensive host cell death remain elusive. Given recent evidence of extracellular vesicles (EVs) – secreted, membrane-bound nanoparticles containing molecular cargo – being implicated in extracellular communication between plants and fungal pathogen, we have initiated an in vitro investigation of EVs from this apoplastic fungal wheat pathogen. We aimed to isolate EVs from Z. tritici broth cultures and examine their protein composition in relation to the soluble protein in the culture filtrate and to existing fungal EV proteomes.Results: Zymoseptoria tritici EVs were isolated from broth culture filtrates using differential ultracentrifugation (DUC) and examined with transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Z. tritici EVs were observed as a heterogeneous population of particles, with most between 50 – 250 nm. These particles were found in abundance in the culture filtrates of viable Z. tritici cultures, but not heat-killed cultures incubated for an equivalent time and of comparable biomass. Bottom-up proteomic analysis using LC-MS/MS, followed by stringent filtering revealed 240 Z. tritici EV proteins. These proteins were distinct from soluble proteins identified in Z. tritici culture filtrates, but were similar to proteins identified in EVs from other fungi, based on sequence similarity analyses. Notably, a putative marker protein recently identified in Candida albicans EVs was also consistently detected in Z. tritici EVs.Conclusion: We have shown EVs can be isolated from the devastating fungal wheat pathogen Z. tritici and are similar to protein composition to previously characterised fungal EVs. EVs from human pathogenic fungi are implicated in virulence, but the role of EVs in the interaction of phytopathogenic fungi and their hosts is unknown. These in vitro analyses provide a basis for expanding investigations of Z. tritici EVs in planta, to examine their involvement in the infection process of this apoplastic wheat pathogen and more broadly, advance understanding of noncanonical secretion in filamentous plant pathogens.

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Evidence of Selection for Fungicide Resistance in Zymoseptoria tritici Populations on Wheat in Western Oregon

Plant Disease ◽

10.1094/pdis-02-15-0214-re ◽

2016 ◽

Vol 100 (2) ◽

pp. 483-489 ◽

Cited By ~ 22

Author(s):

Laura E. Hayes ◽

Kathryn E. Sackett ◽

Nicole P. Anderson ◽

Michael D. Flowers ◽

Christopher C. Mundt

Keyword(s):

Fungicide Resistance ◽

Fungal Pathogen ◽

Plant Pathogens ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Zymoseptoria Tritici ◽

Resistance Evolution ◽

Regional Control ◽

Selection For ◽

Moderately Resistant

Plant pathogens pose a major challenge to maintaining food security in many parts of the world. Where major plant pathogens are fungal, fungicide resistance can often thwart regional control efforts. Zymoseptoria tritici, causal agent of Septoria tritici blotch, is a major fungal pathogen of wheat that has evolved resistance to chemical control products in four fungicide classes in Europe. Compared with Europe, however, fungicide use has been less and studies of fungicide resistance have been infrequent in North American Z. tritici populations. Here, we confirm first reports of Z. tritici fungicide resistance evolution in western Oregon through analysis of the effects of spray applications of propiconazole and an azoxystrobin + propiconazole mixture during a single growing season. Frequencies of strobilurin-resistant isolates, quantified as proportions of G143A mutants, were significantly higher in azoxystrobin-sprayed plots compared with plots with no azoxystrobin treatment at two different locations and were significantly higher in plots of a moderately resistant cultivar than in plots of a susceptible cultivar. Thus, it appears that western Oregon Z. tritici populations have the potential to evolve levels of strobilurin resistance similar to those observed in Europe. Although the concentration of propiconazole required to reduce pathogen growth by 50% values were numerically greater for isolates collected from plots receiving propiconazole than in control plots, this effect was not significant (P > 0.05).

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AMINO ACIDS AND PROTEINS IN CULTURE FILTRATES OF THE ANTHRACNOSE PATHOGEN FUNGUS COLLETOTRICHUM LINI

Vestnik of Ulyanovsk state agricultural academy ◽

10.18286/1816-4501-2020-4-121-127 ◽

2020 ◽

pp. 121-127

Author(s):

N. V. Proletova ◽

Keyword(s):

Amino Acids ◽

Culture Filtrate ◽

Nutrient Medium ◽

Culture Medium ◽

Protein Composition ◽

Entire Period ◽

Culture Filtrates ◽

Russian Research Institute ◽

Russian Research

The research was carried out on the basis of laboratory biotechnologies of All-Russian research institute of flax (Tver region) in 2010–2012, 2016. The aim of the work was to determine the amino acid and protein composition of culture filtrates of the anthracnose pathogen fungus Colletotrichum lini Manns et Bolley in order to adjust the concentration of selective agent in the nutrient medium when creating in vitro new flax genotypes resistant to anthracnose. It was established that the culture filtrates of strains 527 and 608 contain such amino acids as alanine, glycine, asparagine, cysteine, threonine, aspartic acid, glutamic acid, as well as arginine in strain 527 and traces of tyrosine and lysine in strain 608. It was established that the concentration of amino acids in EC of strain 527 was significantly higher than in culture filtrate of strain 608. It was shown that the toxicity of the culture filtrate depended on the degree of aggressiveness of the anthracnose pathogen strain – culture filtrate of a strongly aggressive strain is more toxic than the culture filtrate of a weakly aggressive strain. Studies have revealed that when cultivating the fungus-causative agent of anthracnose on a nutrient medium, as the mycelium of fungus grew, the concentrations of asparagine, alanine, aspartic and glutamic acids, and glycine decreased in the culture filtrates. It was established that the change in amount of proteins happened during the entire period of cultivation of the mycelium of fungus on a liquid nutrient medium. It is shown that accumulation and content of proteins in culture filtrates of strains of different aggressiveness occurs in different ways. The more aggressive strain is (639), which is more toxic, contains and accumulates more proteins in the culture medium during the entire period of growth and development the less aggressive strain is (419).

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Genome mining of the citrus pathogen Elsinoë fawcettii; prediction and prioritisation of candidate effectors, cell wall degrading enzymes and secondary metabolite gene clusters

10.1101/2019.12.19.882142 ◽

2019 ◽

Author(s):

Sarah Jeffress ◽

Kiruba Arun-Chinnappa ◽

Ben Stodart ◽

Niloofar Vaghefi ◽

Yu Pei Tan ◽

...

Keyword(s):

Secondary Metabolite ◽

Plant Pathogens ◽

Virulence Genes ◽

Sequence Similarity ◽

Leptosphaeria Maculans ◽

Gene Clusters ◽

Cell Wall Degrading Enzymes ◽

Zymoseptoria Tritici ◽

Biosynthesis Gene Cluster ◽

Sequencing Project

Abstract:Elsinoë fawcettii, a necrotrophic fungal pathogen, causes citrus scab on numerous citrus varieties around the world. Known pathotypes of E. fawcettii are based on host range; additionally, cryptic pathotypes have been reported and more novel pathotypes are thought to exist. E. fawcettii produces elsinochrome, a non-host selective toxin which contributes to virulence. However, the mechanisms involved in potential pathogen-host interactions occurring prior to the production of elsinochrome are unknown, yet the host-specificity observed among pathotypes suggests a reliance upon such mechanisms. In this study we have generated a whole genome sequencing project for E. fawcettii, producing an annotated draft assembly 26.01 Mb in size, with 10,080 predicted gene models and low (0.37%) coverage of transposable elements. The assembly showed evidence of AT-rich regions, potentially indicating genomic regions with increased plasticity. Using a variety of computational tools, we mined the E. fawcettii genome for potential virulence genes as candidates for future investigation. A total of 1,280 secreted proteins and 203 candidate effectors were predicted and compared to those of other necrotrophic (Botrytis cinerea, Parastagonospora nodorum, Pyrenophora tritici-repentis, Sclerotinia sclerotiorum and Zymoseptoria tritici), hemibiotrophic (Leptosphaeria maculans, Magnaporthe oryzae, Rhynchosporium commune and Verticillium dahliae) and biotrophic (Ustilago maydis) plant pathogens. Genomic and proteomic features of known fungal effectors were analysed and used to guide the prioritisation of 77 candidate effectors of E. fawcettii. Additionally, 378 carbohydrate-active enzymes were predicted and analysed for likely secretion and sequence similarity with known virulence genes. Furthermore, secondary metabolite prediction indicated nine additional genes potentially involved in the elsinochrome biosynthesis gene cluster than previously described. A further 21 secondary metabolite clusters were predicted, some with similarity to known toxin producing gene clusters. The candidate virulence genes predicted in this study provide a comprehensive resource for future experimental investigation into the pathogenesis of E. fawcettii.

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In-Vitro Efficacy of Trichoderma viride Against Sclerotium rolfsii and Macrophomina phaseolina

Notulae Scientia Biologicae ◽

10.15835/nsb447818 ◽

2012 ◽

Vol 4 (4) ◽

pp. 39-44 ◽

Cited By ~ 3

Author(s):

Khirood DOLEY ◽

Paramjit Kaur JITE

Keyword(s):

Fungal Pathogen ◽

Crop Production ◽

Plant Pathogens ◽

Trichoderma Viride ◽

Sclerotium Rolfsii ◽

Macrophomina Phaseolina ◽

Dual Culture ◽

Human Beings ◽

Trichoderma Species

The fungal pathogen causes serious widespread losses to agricultural crops worldwide. Therefore, economy of countries may worsen especially of developing countries. In addition, harmful chemical pesticides which are being used today for increasing crop production creates very serious health hazardous problems to human beings and ecosystem as a whole. The antagonistic potential of Trichoderma species which has been long known to control various soil-borne fungal pathogens in biological way may be utilized. The faster growth rates with which it competes with fungal pathogen mainly brings upon their antagonistic characteristics. An investigation was carried out in laboratory condition towards biological efficacy of T. viride on potato dextrose agar (PDA) medium for the bio-control of soil-borne plant pathogens Sclerotium rolfsii and Macrophomina phaseolina in in-vitro condition. The dual culture technique was followed in which T. viride showed significant antifungal activities towards both the pathogens. T. viride significantly inhibited the mycelial radial growth of S. rolfsii by 75% and M. phaseolina by 71.42%. The results showed variable mycelial growth rate for all fungal isolates which was determined after 6 days of incubation in which T. viride showed minimum of 4.00 days to completely cover the petri-plates and S. rolfsii showed 4.33 days whereas M. phaseolina showed 6.33 days. Thus, T. viride showed encouraging results regarding their biopesticidal and biofungicidal potential against plant pathogens which may be endorsed to substitute harmful chemical supplements that exists in modern day agricultural practices.

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Corn and Soybean Host Root Endophytic Fungi with Toxicity Toward the Soybean Cyst Nematode

Phytopathology ◽

10.1094/phyto-07-19-0243-r ◽

2020 ◽

Vol 110 (3) ◽

pp. 603-614 ◽

Cited By ~ 4

Author(s):

Noah Strom ◽

Weiming Hu ◽

Deepak Haarith ◽

Senyu Chen ◽

Kathryn Bushley

Keyword(s):

Soybean Cyst Nematode ◽

Plant Pathogens ◽

Sequence Similarity ◽

Fungal Endophytes ◽

Cyst Nematode ◽

Malt Extract ◽

Ammonium Tartrate ◽

Internal Transcribed Spacer Region ◽

Culture Filtrates ◽

Soybean Roots

Although fungal endophytes are commonly investigated for their ability to deter microbial plant pathogens, few studies have examined the activity of fungal root endophytes against nematodes. The soybean cyst nematode (SCN; Heterodera glycines), the most severe yield-limiting pathogen of soybean (Glycine max), is commonly managed through rotation of soybean with corn (Zea mays), a nonhost of the SCN. A total of 626 fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which soybean and corn had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous monoculture. Fungal isolates were grouped into 401 morphotypes, which were clustered into 108 operational taxonomic units (OTUs) based on 99% sequence similarity of the full internal transcribed spacer region. Morphotype representatives within each OTU were grown in malt extract broth and in a secondary metabolite-inducing medium buffered with ammonium tartrate, and their culture filtrates were tested for nematicidal activity against SCN juveniles. A majority of OTUs containing isolates with nematicidal culture filtrates were in the order Hypocreales, with the genus Fusarium being the most commonly isolated nematicidal genus from corn and soybean roots. Less commonly isolated taxa from soybean roots included the nematophagous fungi Hirsutella rhossiliensis, Metacordyceps chlamydosporia, and Arthrobotrys iridis. Root endophytic fungal diversity in soybean was positively correlated with SCN density, suggesting that the SCN plays a role in shaping the soybean root endophytic community.

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A Maize (Zea mays L.) BIK1-Like Receptor-Like Cytoplasmic Kinase Contributes to Disease Resistance

Plant Molecular Biology Reporter ◽

10.1007/s11105-021-01299-2 ◽

2021 ◽

Author(s):

Weiran Li ◽

Chao-Jan Liao ◽

Burt H. Bluhm ◽

Tesfaye Mengiste ◽

Charles P. Woloshuk

Keyword(s):

Zea Mays ◽

Protein Kinase ◽

Pseudomonas Syringae ◽

Plant Resistance ◽

Fungal Pathogen ◽

Sequence Similarity ◽

Bacterial Pathogen ◽

Protein Kinase Activity ◽

Ear Rot

AbstractReceptor-like cytoplasmic kinases (RLCKs) form a large subfamily of proteins in plants. RLCKs are known to regulate plant immunity to bacterial and fungal pathogens. In this study, we analyzed the genome-wide complement of maize RLCK genes and conducted detailed studies on one maize RLCK. The maize genome encodes 192 RLCKs that largely mirror the RLCK family in other plants. Previous studies implicated Arabidopsis BOTRYTIS INDUCED KINASE1 (BIK1) and TOMATO PROTEIN KINASE 1b (TPK1b) in plant resistance to the bacterial pathogen Pseudomonas syringae and the fungal pathogen Botrytis cinerea. A novel maize RLCK, Zea Mays BIK1-LIKE KINASE 1 (ZmBLK1), was identified based on sequence similarity to the tomato and Arabidopsis RLCKs. We demonstrated that ZmBLK1 displays protein kinase activity in vitro and the protein localizes to the plasma membrane. Importantly, expression of ZmBLK1 partially rescued the growth and disease phenotypes of the Arabidopsis bik1 mutant plants. The expression of ZmBLK1 was induced in maize at 12 h after inoculation with Clavibacter michiganensis subsp. nebraskensis (CMN), the bacterial pathogen causing Goss’s wilt. Interestingly, overexpression of ZmBLK1 in transgenic maize increased resistance to CMN but did not impact resistance to Aspergillus ear rot caused by the fungal pathogen Aspergillus flavus and the associated aflatoxin contamination. These findings support our hypothesis that ZmBLK1 contributes to plant resistance to bacterial pathogens likely by modulating events early after pathogen infection, implying that the protein may interact with other membrane proteins early in the immune response pathway.

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A tradeoff between tolerance and resistance to a major fungal pathogen in elite wheat cultivars

10.1101/354357 ◽

2018 ◽

Author(s):

Alexey Mikaberidze ◽

Bruce A. McDonald

Keyword(s):

Fungal Pathogen ◽

Plant Pathogens ◽

Plant Diseases ◽

List Type ◽

Fruiting Bodies ◽

Wheat Cultivars ◽

Zymoseptoria Tritici ◽

Green Area ◽

Digital Phenotyping ◽

The Relationship

SummaryTolerance and resistance represent two strategies that hosts evolved to protect themselves from pathogens. Tolerance alleviates the reduction in host fitness due to infection without reducing a pathogen’s growth, while resistance reduces pathogen growth. We investigated tolerance of wheat to the major fungal pathogen Zymoseptoria tritici in 335 elite wheat cultivars.We used a novel digital phenotyping approach that included 11,152 infected leaves and counted 2,069,048 pathogen fruiting bodies.We discovered a new component of tolerance that is based on the relationship between the green area remaining on a leaf and the number of pathogen fruiting bodies. We found a negative correlation between tolerance and resistance among intolerant cultivars, presenting the first compelling evidence for a tradeoff between tolerance and resistance to plant pathogens. Surprisingly, the tradeoff arises due to limits in the host resources available to the pathogen and not due to metabolic constraints, contrary to what ecological theory suggests.The mechanism underlying this tradeoff may be relevant for many plant diseases in which the amount of host resources available to the pathogen can limit the pathogen population. Our analysis indicates that European wheat breeders may have selected for tolerance instead of resistance to an important pathogen.

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Isolation and Identification of Lipopeptide-Producing Bacillus velezensis Strains from Wheat Phyllosphere with Antifungal Activity against the Wheat Pathogen Zymoseptoria tritici

Agronomy ◽

10.3390/agronomy12010095 ◽

2021 ◽

Vol 12 (1) ◽

pp. 95

Author(s):

Rémi Platel ◽

Mélodie Sawicki ◽

Qassim Esmaeel ◽

Béatrice Randoux ◽

Pauline Trapet ◽

...

Keyword(s):

Antifungal Activity ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Bacterial Strains ◽

Bacillus Velezensis ◽

Zymoseptoria Tritici ◽

Isolation And Identification ◽

Culture Filtrates ◽

The Difference

Septoria tritici blotch, caused by the fungal pathogen Zymoseptoria tritici, is a highly significant disease on wheat crops worldwide. The objective of the present study was to find out new bacterial strains with bio-antimicrobial activity against Z. tritici. Two phyllospheric bacteria (S1 and S6) were isolated from wheat ears and identified as Bacillus velezensis strains according to 16S rRNA Sanger sequencing. Antagonistic assays performed with either living strains or cell-free culture filtrates showed significant in vitro antifungal activities against Z. tritici. For the culture filtrates, the half-maximal inhibitory dilution and the minimal inhibitory dilution were 1.4% and 3.7% for the strain S1, and 7.4% and 15% for the strain S6, respectively. MALDI—ToF analysis revealed that both strains synthesize cyclic lipopeptides but from different families. Interestingly, only strain S1 produces putative bacillomycin D. Such differential lipopeptide production patterns might explain the difference observed between the antifungal activity of the culture filtrates of the two strains. This study allows the identification of new lipopeptide-producing strains of B. velezensis with a high potential of application for the biocontrol of Z. tritici.

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