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1.
Biochem Biophys Res Commun ; 587: 36-41, 2022 01 08.
Article in English | MEDLINE | ID: mdl-34864393

ABSTRACT

LncRNAs are widely involved in various biological processes of plants. Recent evidences indicated that lncRNAs could act as competing endogenous RNAs (ceRNAs) to adsorb complementary miRNAs in a type of target mimicry, thereby indirectly regulating the target genes of miRNAs. In this study, a lncRNA, lncRNA08489 was identified to be the ceRNA of miR482e-3p in tomato plants. The expression patterns of lncRNA08489 and miR482e-3p showed opposite trends after tomato plants infected with Phytophthora infestans. In tomato leaves overexpressing lncRNA08489 (OE08489), the expression level of miR482e-3p decreased and its target gene, NBS-LRR increased. After infection with P. infestans, the resistance of OE08489 plants was stronger than that of the wild type, and the reactive oxygen species (ROS) scavenging ability of OE08489 plants was significantly improved. Taken together, these results indicated that lncRNA08489 acted as a ceRNA to decoy miR482e-3p and regulate the expression of NBS-LRR to enhance tomato resistance through ROS-scavenging system.


Subject(s)
MicroRNAs/genetics , Phytophthora infestans/pathogenicity , Plant Diseases/genetics , RNA, Long Noncoding/genetics , RNA, Plant/genetics , Solanum lycopersicum/genetics , Base Pairing , Base Sequence , Disease Resistance/genetics , Gene Expression Regulation, Plant , Solanum lycopersicum/immunology , Solanum lycopersicum/microbiology , MicroRNAs/immunology , Phytophthora infestans/growth & development , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity/genetics , Plant Leaves/genetics , Plant Leaves/immunology , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/immunology , RNA, Long Noncoding/immunology , RNA, Plant/immunology , Reactive Oxygen Species/immunology , Reactive Oxygen Species/metabolism
2.
mSphere ; 6(3): e0042721, 2021 06 30.
Article in English | MEDLINE | ID: mdl-34077259

ABSTRACT

Phenazine-producing Pseudomonas spp. are effective biocontrol agents that aggressively colonize the rhizosphere and suppress numerous plant diseases. In this study, we compared the ability of 63 plant-beneficial phenazine-producing Pseudomonas strains representative of the worldwide diversity to inhibit the growth of three major potato pathogens: the oomycete Phytophthora infestans, the Gram-positive bacterium Streptomyces scabies, and the ascomycete Verticillium dahliae. The 63 Pseudomonas strains are distributed among four different subgroups within the P. fluorescens species complex and produce different phenazine compounds, namely, phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid, and 2-hydroxphenazine. Overall, the 63 strains exhibited contrasted levels of pathogen inhibition. Strains from the P. chlororaphis subgroup inhibited the growth of P. infestans more effectively than strains from the P. fluorescens subgroup. Higher inhibition was not associated with differential levels of phenazine production nor with specific phenazine compounds. The presence of additional biocontrol-related traits found in P. chlororaphis was instead associated with higher P. infestans inhibition. Inhibition of S. scabies by the 63 strains was more variable, with no clear taxonomic segregation pattern. Inhibition values did not correlate with phenazine production nor with specific phenazine compounds. No additional synergistic biocontrol-related traits were found. Against V. dahliae, PCN producers from the P. chlororaphis subgroup and PCA producers from the P. fluorescens subgroup exhibited greater inhibition. Additional biocontrol-related traits potentially involved in V. dahliae inhibition were identified. This study represents a first step toward harnessing the vast genomic diversity of phenazine-producing Pseudomonas spp. to achieve better biological control of potato pathogens. IMPORTANCE Plant-beneficial phenazine-producing Pseudomonas spp. are effective biocontrol agents, thanks to the broad-spectrum antibiotic activity of the phenazine antibiotics they produce. These bacteria have received considerable attention over the last 20 years, but most studies have focused only on the ability of a few genotypes to inhibit the growth of a limited number of plant pathogens. In this study, we investigated the ability of 63 phenazine-producing strains, isolated from a wide diversity of host plants on four continents, to inhibit the growth of three major potato pathogens: Phytophthora infestans, Streptomyces scabies, and Verticillium dahliae. We found that the 63 strains differentially inhibited the three potato pathogens. These differences are in part associated with the nature and the quantity of the phenazine compounds being produced but also with the presence of additional biocontrol-related traits. These results will facilitate the selection of versatile biocontrol agents against pathogens.


Subject(s)
Bacteria/drug effects , Phenazines/pharmacology , Pseudomonas/chemistry , Pseudomonas/genetics , Solanum tuberosum/microbiology , Ascomycota/drug effects , Ascomycota/growth & development , Bacteria/classification , Bacteria/pathogenicity , Biological Control Agents/chemistry , Biological Control Agents/metabolism , Genetic Variation , Genome, Bacterial , Phenazines/chemistry , Phenazines/metabolism , Phytophthora infestans/drug effects , Phytophthora infestans/growth & development , Pseudomonas/classification , Streptomyces/drug effects , Streptomyces/growth & development
3.
Molecules ; 26(6)2021 Mar 21.
Article in English | MEDLINE | ID: mdl-33800987

ABSTRACT

Phytochemical investigation of the methanolic extract obtained from the aerial parts of Lagochilus setulosus (Lamiaceae) afforded the new compound 1-methoxy-3-O-ß-glucopyranosyl-α-l-oliose (1) together with five known glycosides, namely sitosterol-3-O-ß-glucoside (2), stigmasterol-3-O-ß-glucoside (3), pinitol (4), 6ß-hydroxyl-7-epi-loganin (5), and chlorotuberoside (6). The structures of these compounds were elucidated by extensive spectroscopic analyses, especially HR-MS, 1D and 2D NMR spectroscopy. The in vitro cytotoxic activity of the methanolic extract and the isolated compounds was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and crystal violet (CV) staining assays. In addition, the antifungal activities of the components were evaluated against Botrytis cinerea, Septoria tritici, and Phytophthora infestans. The anthelmintic potential was determined against Caenorhabditis elegans nematodes. Neither the extract nor the isolated compounds showed promising activity in all the bioassays.


Subject(s)
Anthelmintics , Antifungal Agents , Glycosides , Lamiaceae/chemistry , Plant Extracts/chemistry , Animals , Anthelmintics/chemistry , Anthelmintics/isolation & purification , Anthelmintics/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/isolation & purification , Antifungal Agents/pharmacology , Ascomycota/growth & development , Botrytis/growth & development , Caenorhabditis elegans/growth & development , Glycosides/chemistry , Glycosides/isolation & purification , Glycosides/pharmacology , Phytophthora infestans/growth & development
4.
Microbiol Res ; 247: 126724, 2021 Jun.
Article in English | MEDLINE | ID: mdl-33640575

ABSTRACT

Tagatose is a rare sugar metabolised by a limited number of microorganisms that inhibits a large spectrum of phytopathogens. In particular, tagatose inhibited Phytophthora infestans growth and negatively affected mitochondrial processes. However, the possible effects of tagatose on P. infestans metabolism have not yet been investigated. The aim of this study was to analyse the impact of this rare sugar on the sugar metabolism in P. infestans, in order to better understand its mode of action. Tagatose inhibited the growth of P. infestans with a precise reprogramming of the carbohydrate metabolism that involved a decrease of glucose, glucose-1-phosphate and mannose content and ß-glucosidase activity. The combination of tagatose with common sugars led to three different responses and highlighted antagonistic interactions. In particular, glucose partially attenuated the inhibitory effects of tagatose, while fructose fully impaired tagatose-mediated growth inhibition and metabolite changes. Moreover, sucrose did not attenuate tagatose effects, suggesting that the inhibition of sucrose catabolism and the alteration of glucose-related pathways contributed to the growth inhibition caused by tagatose to P. infestans. The interactions of tagatose with the common sugar metabolism were found to be a key mode of action against P. infestans growth, which may represent the basis for the further development of tagatose as an eco-friendly fungicide.


Subject(s)
Carbohydrate Metabolism , Hexoses/metabolism , Phytophthora infestans/growth & development , Phytophthora infestans/metabolism , Fungicides, Industrial/pharmacology , Glucose , Glucosephosphates , Hexoses/pharmacology , Mannose/metabolism , Phytophthora infestans/drug effects , Plant Diseases , Sucrose , beta-Glucosidase/metabolism
5.
J Appl Microbiol ; 131(1): 375-391, 2021 Jul.
Article in English | MEDLINE | ID: mdl-33249672

ABSTRACT

AIMS: Compare and characterize Chaetomium strains with special regard to their potentialities as biocontrol agents. METHODS AND RESULTS: Twelve strains of the fungal genus Chaetomium from diverse ecological niches were identified as belonging to six different species. Large differences were observed between the strains with regard to temperature requirements for mycelial growth and pigmentation of culture filtrates. Culture filtrates and ethyl acetate extracts were assayed for fungicidal effects against important phytopathogens both on agar media and in multiwell plates. The samples from Chaetomium globosum were particularly active against Botrytis cinerea, Pyrenophora graminea and Bipolaris sorokiniana, while those from C. cochliodes and C. aureum were inhibitory towards Phytophthora infestans, and P. infestans and Fusarium culmorum respectively. To narrow down the active principle, the most promising extracts were separated by preparative HPLC and the resulting fractions tested in bioassays. Chaetoglobosins were identified as active compounds produced by C. globosum. CONCLUSIONS: The bioassays revealed C. aureum and C. cochliodes as promising candidates for use in biocontrol. Both showed remarkably good activity against the prominent plant pathogen P. infestans. SIGNIFICANCE AND IMPACT OF THE STUDY: We provide the first systematic study comparing six different Chaetomium species with regard to their use as biocontrol agents.


Subject(s)
Antibiosis , Antifungal Agents/pharmacology , Biological Control Agents/pharmacology , Chaetomium/physiology , Fungi/growth & development , Antifungal Agents/analysis , Ascomycota/drug effects , Ascomycota/growth & development , Biological Control Agents/analysis , Botrytis/drug effects , Botrytis/growth & development , Chaetomium/growth & development , Fungi/drug effects , Fusarium/drug effects , Fusarium/growth & development , Indole Alkaloids/analysis , Indole Alkaloids/pharmacology , Phenotype , Phytophthora infestans/drug effects , Phytophthora infestans/growth & development
6.
Sci Rep ; 10(1): 22326, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33339950

ABSTRACT

To characterize the molecular mechanisms underlying life-stage transitions in Phytophthora infestans, we initiated a chemical genetics approach by screening for a stage-specific inhibitor of morphological development from microbial culture extracts prepared mostly from actinomycetes from soil in Japan. Of the more than 700 extracts, one consistently inhibited Ph. infestans cyst germination. Purification and identification of the active compound by ESI-MS, 1H-NMR, and 13C-NMR identified ß-rubromycin as the inhibitor of cyst germination (IC50 = 19.8 µg/L); ß-rubromycin did not inhibit growth on rye media, sporangium formation, zoospore release, cyst formation, or appressorium formation in Ph. infestans. Further analyses revealed that ß-rubromycin inhibited the germination of cysts and oospores in Pythium aphanidermatum. A chemical genetic approach revealed that ß-rubromycin stimulated the expression of RIO kinase-like gene (PITG_04584) by 60-fold in Ph. infestans. Genetic analyses revealed that PITG_04584, which lacks close non-oomycete relatives, was involved in zoosporogenesis, cyst germination, and appressorium formation in Ph. infestans. These data imply that further functional analyses of PITG_04584 may contribute to new methods to suppress diseases caused by oomycetes.


Subject(s)
Phytophthora infestans/genetics , Plant Diseases/genetics , Protein Serine-Threonine Kinases/genetics , Spores, Fungal/genetics , Amino Acid Sequence/genetics , Phytophthora infestans/growth & development , Plant Diseases/microbiology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Quinones/pharmacology , Spores, Fungal/pathogenicity
7.
J Agric Food Chem ; 68(31): 8163-8171, 2020 Aug 05.
Article in English | MEDLINE | ID: mdl-32790355

ABSTRACT

We tested the ability of 14 strains of Trichoderma to emit volatile compounds that decreased or stopped the growth of Phytophthora infestans. Volatile organic compounds (VOCs) emitted from Trichoderma strains designated T41 and T45 inhibited the mycelial growth of P. infestans grown on a laboratory medium by 80 and 81.4%, respectively, and on potato tubers by 93.1 and 94.1%, respectively. Using the DNA sequence analysis of the translation elongation factor region, both Trichoderma strains were identified as Trichoderma atroviride. VOCs emitted by the strains were analyzed, and 39 compounds were identified. The most abundant compounds were 3-methyl-1-butanol, 6-pentyl-2-pyrone, 2-methyl-1-propanol, and acetoin. Electron microscopy of the hyphae treated with T. atroviride VOCs revealed serious morphological and ultrastructural damages, including cell deformation, collapse, and degradation of cytoplasmic organelles. To our knowledge, this is the first report describing the ability of Trichoderma VOCs to suppress the growth of the late blight potato pathogen.


Subject(s)
Fungicides, Industrial/pharmacology , Phytophthora infestans/drug effects , Plant Diseases/microbiology , Solanum tuberosum/microbiology , Trichoderma/chemistry , Volatile Organic Compounds/pharmacology , Fungicides, Industrial/chemistry , Fungicides, Industrial/metabolism , Phytophthora infestans/growth & development , Plant Tubers/microbiology , Trichoderma/metabolism , Volatile Organic Compounds/chemistry , Volatile Organic Compounds/metabolism
8.
PLoS One ; 15(1): e0221604, 2020.
Article in English | MEDLINE | ID: mdl-31961875

ABSTRACT

Phytophthora infestans (Mont.) de Bary, a hemibiotrophic oomycete, has caused severe epidemics of late blight in tomato and potato crops around the world since the Irish Potato Famine in the 1840s. Breeding of late blight resistant cultivars is one of the most effective strategies to overcome this disruptive disease. However, P. infestans is able to break down host resistance and acquire resistance to various fungicides, possibly because of the existence of high genetic variability among P. infestans isolates via sexual and asexual reproduction. Therefore, to manage this disease, it is important to understand the genetic divergence of P. infestans isolates. In this study, we analyzed the genomes of P. infestans isolates collected from Egypt and Japan using various molecular approaches including the mating type assay and genotyping simple sequence repeats, mitochondria DNA, and effector genes. We also analyzed genome-wide single nucleotide polymorphisms using double-digest restriction-site associated DNA sequencing and whole genome resequencing (WGRS). The isolates were classified adequately using high-resolution genome-wide approaches. Moreover, these analyses revealed new clusters of P. infestans isolates in the Egyptian population. Monitoring the genetic divergence of P. infestans isolates as well as breeding of resistant cultivars would facilitate the elimination of the late blight disease.


Subject(s)
Genes, Mating Type, Fungal/genetics , High-Throughput Nucleotide Sequencing , Phytophthora infestans/genetics , Plant Diseases/microbiology , DNA, Mitochondrial/genetics , Fungicides, Industrial/pharmacology , Genotype , Solanum lycopersicum/microbiology , Microsatellite Repeats/genetics , Phytophthora infestans/growth & development , Plant Diseases/genetics , Sequence Analysis, DNA , Solanum tuberosum/microbiology
9.
Appl Microbiol Biotechnol ; 103(15): 6187-6194, 2019 Aug.
Article in English | MEDLINE | ID: mdl-31175427

ABSTRACT

Purpureocillium lilacinum is a promising commercial agent for controlling plant-parasitic nematodes and plant pathogens. Leucinostatins are a family of lipopeptides produced by P. lilacinum that are synthesized, modified, and regulated by a gene cluster consisting of 20 genes. Sequence analyses have indicated that lcsL, a gene in the lcs cluster, is a putative bZIP transcription factor. In this study, the CRISPR-Cas9 system was introduced to increase the efficiency of homologous recombination for the disruption of lcsL. The expression of genes in the cluster was significantly reduced in lcsL disruption mutants, and the output of leucinostatins was decreased to undetectable levels. In the lcsL overexpression strain, the expression of genes in the cluster and the yield of leucinostatins were all increased. The antagonism of both the wild type and mutant against Phytophthora infestans was also consistent with the gene expression and the output of leucinostatins. These results indicate that the gene lcsL is crucial for the regulating the synthesis of leucinostatins.


Subject(s)
Biosynthetic Pathways/genetics , Gene Expression Regulation, Fungal , Hypocreales/metabolism , Multigene Family , Peptides/metabolism , Transcription Factors/metabolism , Transcription, Genetic , Antimicrobial Cationic Peptides , CRISPR-Associated Protein 9 , Clustered Regularly Interspaced Short Palindromic Repeats , Gene Deletion , Gene Knockout Techniques , Homologous Recombination , Hypocreales/genetics , Phytophthora infestans/drug effects , Phytophthora infestans/growth & development , Transcription Factors/genetics
10.
Dokl Biochem Biophys ; 484(1): 6-8, 2019 May.
Article in English | MEDLINE | ID: mdl-31012001

ABSTRACT

The results of the study of the structure and function of harpin-like peptides (alpha-harpinins) of the EcAMP group from the barnyard grass (E. crusgalli) seeds and the possibility of their involvement in the innate immunity to biotic stresses are presented.


Subject(s)
Antimicrobial Cationic Peptides/chemistry , Antimicrobial Cationic Peptides/pharmacology , Echinochloa/chemistry , Phytophthora infestans/growth & development , Plant Proteins/chemistry , Plant Proteins/pharmacology , Protein Structure, Secondary , Structure-Activity Relationship
11.
J Agric Food Chem ; 67(5): 1360-1370, 2019 Feb 06.
Article in English | MEDLINE | ID: mdl-30640452

ABSTRACT

Oxathiapiprolin is one of the best active fungicides discovered for oomycetes control. To develop a fungicide candidate with a broad spectrum of activity, 22 new piperidinylthiazole derivatives were designed and synthesized. Compound 5l showed the best activity against Pseudoperonospora cubensis (Berk. et Curt.) Rostov and Phytophthora infestans in vivo with 100% and 80% of inhibition, respectively, at 1 mg/L, and 72.87% of field efficacy against P. cubensis at 1 g ai/667 m2 validated these results. Compound 5i exhibited a broad spectrum of excellent activity against Sclerotinia sclerotiorum with EC50 = 0.30 mg/L (>10 times more active than oxathiapiprolin and azoxystrobin in vitro), good activity against Botrytis cinerea, Cercospora arachidicola, and Gibberella zeae with EC50 of 14.54, 5.57, and 14.03 mg/L in vitro and against P. cubensis and P. infestans with 60% and 30% inhibition rates, respectively, at 1 mg/L in vivo. Mode of action studies by RNA sequencing analysis discovered oxysterol-binding protein (OSBP), chitin synthase (CHS1), and (1,3)-ß-glucan synthase (FKS2) as the potent target of 5i against S. sclerotiorum. Quenching studies validated that OSBP was the same target of both 5i and oxathiapiprolin; it was quenched by both of them. Our studies discovered isothiazole-containing piperidinylthiazole as an OSBP target-based novel lead for fungicide development.


Subject(s)
Fungicides, Industrial/chemistry , Fungicides, Industrial/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , Ascomycota/drug effects , Ascomycota/growth & development , Botrytis/drug effects , Botrytis/growth & development , Cucumis sativus/microbiology , Drug Discovery , Solanum lycopersicum/microbiology , Phytophthora infestans/drug effects , Phytophthora infestans/growth & development , Plant Diseases/microbiology , Structure-Activity Relationship
12.
Mol Plant Microbe Interact ; 32(1): 76-85, 2019 01.
Article in English | MEDLINE | ID: mdl-30048603

ABSTRACT

Phytophthora infestans causes the devastating potato late blight disease, which is widely controlled with fungicides. However, the debate about chemical control is fueling a promotion toward alternative methods. In this context, the enhancement of natural plant immunity could be a strategy for more sustainable protection. We previously demonstrated that a concentrated culture filtrate (CCF) of P. infestans primes defense reactions in potato. They are genotype-dependent and metabolites produced decrease pathogen growth in vitro but not in vivo on tubers. Induced potato defenses are assumed to affect P. infestans life history traits depending on strains. This assumption was studied in vivo through induced leaflets on a susceptible genotype inoculated with four P. infestans strains differing for lesion growth rate. This study combines both defenses mechanistic analysis and ecological observations. Defense-gene expressions were thus assessed by quantitative reverse transcription-polymerase chain reaction; pathogen development was simultaneously evaluated by measuring necrosis, quantifying mycelial DNA, and counting sporangia. The results showed that CCF pretreatment reduced the pathogenicity differences between slow- and fast-growing strains. Moreover, after elicitation, PR-1, PR-4, PAL, POX, and THT induction was strain-dependent. These results suggest that P. infestans could develop different strategies to overcome plant defenses and should be considered in biocontrol and epidemic management of late blight.


Subject(s)
Disease Resistance , Phytophthora infestans , Solanum tuberosum , Disease Resistance/genetics , Genotype , Phytophthora infestans/growth & development , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Immunity/genetics , Plant Tubers/microbiology , Solanum tuberosum/genetics , Solanum tuberosum/immunology
13.
Mol Plant Microbe Interact ; 32(5): 515-526, 2019 May.
Article in English | MEDLINE | ID: mdl-30480479

ABSTRACT

Sporangia of Phytophthora infestans from pure cultures on agar plates are typically used in lab studies, whereas sporangia from leaflet lesions drive natural infections and epidemics. Multiple assays were performed to determine if sporangia from these two sources are equivalent. Sporangia from plate cultures showed much lower rates of indirect germination and produced much less disease in field and moist-chamber tests. This difference in aggressiveness was observed whether the sporangia had been previously incubated at 4°C (to induce indirect germination) or at 21°C (to prevent indirect germination). Furthermore, lesions caused by sporangia from plates produced much less sporulation. RNA-Seq analysis revealed that thousands of the >17,000 P. infestans genes with a RPKM (reads per kilobase of exon model per million mapped reads) >1 were differentially expressed in sporangia obtained from plate cultures of two independent field isolates compared with sporangia of those isolates from leaflet lesions. Among the significant differentially expressed genes (DEGs), putative RxLR effectors were overrepresented, with almost half of the 355 effectors with RPKM >1 being up- or downregulated. DEGs of both isolates include nine flagellar-associated genes, and all were down-regulated in plate sporangia. Ten elicitin genes were also detected as DEGs in both isolates, and nine (including INF1) were up-regulated in plate sporangia. These results corroborate previous observations that sporangia produced from plates and leaflets sometimes yield different experimental results and suggest hypotheses for potential mechanisms. We caution that use of plate sporangia in assays may not always produce results reflective of natural infections and epidemics.


Subject(s)
Phytophthora infestans/physiology , Solanum lycopersicum , Sporangia/physiology , Transcriptome , Solanum lycopersicum/parasitology , Phytophthora infestans/genetics , Phytophthora infestans/growth & development , Sporangia/genetics , Sporangia/growth & development
14.
Sci Rep ; 9(1): 18778, 2019 12 30.
Article in English | MEDLINE | ID: mdl-31889050

ABSTRACT

Plant diseases are a major cause for yield losses and new strategies to control them without harming the environment are urgently needed. Plant-associated bacteria contribute to their host's health in diverse ways, among which the emission of disease-inhibiting volatile organic compounds (VOCs). We have previously reported that VOCs emitted by potato-associated bacteria caused strong in vitro growth inhibition of the late blight causing agent Phytophthora infestans. This work focuses on sulfur-containing VOCs (sVOCs) and demonstrates the high in planta protective potential of S-methyl methane thiosulfonate (MMTS), which fully prevented late blight disease in potato leaves and plantlets without phytotoxic effects, in contrast to other sVOCs. Short exposure times were sufficient to protect plants against infection. We further showed that MMTS's protective activity was not mediated by the plant immune system but lied in its anti-oomycete activity. Using quantitative proteomics, we determined that different sVOCs caused specific proteome changes in P. infestans, indicating perturbations in sulfur metabolism, protein translation and redox balance. This work brings new perspectives for plant protection against the devastating Irish Famine pathogen, while opening new research avenues on the role of sVOCs in the interaction between plants and their microbiome.


Subject(s)
Phytophthora infestans/growth & development , Plant Diseases/microbiology , Solanum tuberosum/microbiology , Sulfur/metabolism , Volatile Organic Compounds/metabolism , Plant Diseases/parasitology , Plant Leaves/metabolism , Plant Proteins/metabolism , Solanum tuberosum/metabolism , Solanum tuberosum/parasitology
15.
Mol Microbiol ; 110(4): 562-575, 2018 11.
Article in English | MEDLINE | ID: mdl-30194883

ABSTRACT

MADS-box transcription factors play significant roles in eukaryotes, but have not yet been characterized in oomycetes. Here, we describe a MADS-box protein from Phytophthora infestans, which causes late blight of potato. P. infestans and most other oomycetes express a single MADS-box gene. PiMADS is not transcribed during vegetative growth, but is induced early during asexual sporulation. Its mRNA levels oscillate in response to light, which suppresses sporulation. The protein was not detected in nonsporulating mycelia, but was found in sporulating mycelia and spores. Both mRNA and protein levels decline upon spore germination. A similar expression pattern as well as nuclear localization was observed when the protein was expressed with a fluorescent tag from the native promoter. Gene silencing triggered by a construct expressing 478 nt of MADS sequences indicated that PiMADS is required for sporulation but not hyphal growth or plant colonization. A comparison of wild type to a silenced strain by RNA-seq indicated that PiMADS regulates about 3000 sporulation-associated genes, and acts before other genes previously shown to regulate sporulation. Analysis of the silenced strain also indicated that the native gene was not transcribed while the transgene was still expressed, which contradicts current models for homology-dependent silencing in oomycetes.


Subject(s)
MADS Domain Proteins/genetics , Mycelium/metabolism , Phytophthora infestans/growth & development , Phytophthora infestans/genetics , Spores, Protozoan/growth & development , Spores, Protozoan/genetics , Gene Expression Regulation , Gene Silencing , Genome, Protozoan/genetics , Phytophthora infestans/metabolism , Plant Diseases/parasitology , Solanum tuberosum/parasitology , Spores, Protozoan/metabolism , Transcription Factors/metabolism
16.
J Microbiol Methods ; 152: 80-85, 2018 09.
Article in English | MEDLINE | ID: mdl-30075235

ABSTRACT

Phytophthora infestans is one of the most notorious pathogen among Phytophthora species causing potato late blight disease. Stable and long-term preservation of this pathogen is essential for biological research and fungicide screening. The aim of this study was to find a suitable long-term preservation method for P. infestans. We adjusted the storage temperature, made a slight modification to the rye seed method, and compared the influence of four preservation methods (the mineral oil method, the sterile water method, the rye seed method, and the modified rye seed method) on survival, growth and virulence of four isolates of P. infestans. The results showed that all four methods maintained high viability of the tested P. infestans isolates, but the two rye seed methods were the best ways to maintain 100% viability of the P. infestans isolates without contamination. The four preservation methods did not significantly influence growth or morphological characteristics of the P. infestans isolates. The impacts of the four methods on the virulence of the four P. infestans isolates were isolate-specific. For isolates YF3 and 64093, all four methods were suitable for maintaining their virulence. Whilst for isolate HQK8-3, the rye seed and sterile water methods were more suitable to maintain its virulence than the other two methods. For isolate 32835, storage under mineral oil was the best method for maintaining its virulence. In view of these results, it is recommended P. infestans should be stored by several different storage methods to ensure the safety and stability of the isolates.


Subject(s)
Phytophthora infestans/growth & development , Preservation, Biological/methods , Microbial Viability , Mycelium/growth & development , Phytophthora infestans/cytology , Phytophthora infestans/isolation & purification , Phytophthora infestans/pathogenicity , Plant Diseases/microbiology , Preservation, Biological/economics , Solanum tuberosum , Virulence
17.
Elife ; 72018 06 22.
Article in English | MEDLINE | ID: mdl-29932422

ABSTRACT

During plant cell invasion, the oomycete Phytophthora infestans remains enveloped by host-derived membranes whose functional properties are poorly understood. P. infestans secretes a myriad of effector proteins through these interfaces for plant colonization. Recently we showed that the effector protein PexRD54 reprograms host-selective autophagy by antagonising antimicrobial-autophagy receptor Joka2/NBR1 for ATG8CL binding (Dagdas et al., 2016). Here, we show that during infection, ATG8CL/Joka2 labelled defense-related autophagosomes are diverted toward the perimicrobial host membrane to restrict pathogen growth. PexRD54 also localizes to autophagosomes across the perimicrobial membrane, consistent with the view that the pathogen remodels host-microbe interface by co-opting the host autophagy machinery. Furthermore, we show that the host-pathogen interface is a hotspot for autophagosome biogenesis. Notably, overexpression of the early autophagosome biogenesis protein ATG9 enhances plant immunity. Our results implicate selective autophagy in polarized immune responses of plants and point to more complex functions for autophagy than the widely known degradative roles.


Subject(s)
Autophagy/genetics , Host-Pathogen Interactions , Phytophthora infestans/genetics , Plant Diseases/genetics , Plant Proteins/genetics , Solanum tuberosum/genetics , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/immunology , Autophagosomes/immunology , Autophagosomes/parasitology , Autophagy/immunology , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/immunology , Carrier Proteins/genetics , Carrier Proteins/immunology , Gene Expression Regulation , Membrane Proteins/genetics , Membrane Proteins/immunology , Phytophthora infestans/growth & development , Phytophthora infestans/pathogenicity , Plant Cells/immunology , Plant Cells/parasitology , Plant Diseases/immunology , Plant Diseases/parasitology , Plant Immunity/genetics , Plant Proteins/immunology , Protein Binding , Signal Transduction , Solanum tuberosum/immunology , Solanum tuberosum/parasitology
18.
BMC Genomics ; 19(1): 474, 2018 Jun 19.
Article in English | MEDLINE | ID: mdl-29914352

ABSTRACT

BACKGROUND: Phytophthora infestans is responsible for late blight, one of the most important potato diseases. Phenazine-1-carboxylic acid (PCA)-producing Pseudomonas fluorescens strain LBUM223 isolated in our laboratory shows biocontrol potential against various plant pathogens. To characterize the effect of LBUM223 on the transcriptome of P. infestans, we conducted an in vitro time-course study. Confrontational assay was performed using P. infestans inoculated alone (control) or with LBUM223, its phzC- isogenic mutant (not producing PCA), or exogenically applied PCA. Destructive sampling was performed at 6, 9 and 12 days and the transcriptome of P. infestans was analysed using RNA-Seq. The expression of a subset of differentially expressed genes was validated by RT-qPCR. RESULTS: Both LBUM223 and exogenically applied PCA significantly repressed P. infestans' growth at all times. Compared to the control treatment, transcriptomic analyses showed that the percentages of all P. infestans' genes significantly altered by LBUM223 and exogenically applied PCA increased as time progressed, from 50 to 61% and from to 32 to 46%, respectively. When applying an absolute cut-off value of 3 fold change or more for all three harvesting times, 207 genes were found significantly differentially expressed by PCA, either produced by LBUM223 or exogenically applied. Gene ontology analysis revealed that both treatments altered the expression of key functional genes involved in major functions like phosphorylation mechanisms, transmembrane transport and oxidoreduction activities. Interestingly, even though no host plant tissue was present in the in vitro system, PCA also led to the overexpression of several genes encoding effectors. The mutant only slightly repressed P. infestans' growth and barely altered its transcriptome. CONCLUSIONS: Our study suggests that PCA is involved in P. infestans' growth repression and led to important transcriptomic changes by both up- and down-regulating gene expression in P. infestans over time. Different metabolic functions were altered and many effectors were found to be upregulated, suggesting their implication in biocontrol.


Subject(s)
Phytophthora infestans/genetics , Pseudomonas fluorescens/metabolism , Transcriptome , Biological Control Agents , Gene Expression Profiling , Phenazines/metabolism , Phytophthora infestans/growth & development , Phytophthora infestans/metabolism , Sequence Analysis, RNA
19.
Fungal Genet Biol ; 116: 73-82, 2018 07.
Article in English | MEDLINE | ID: mdl-29704555

ABSTRACT

The oomycete Phytophthora infestans is a notorious plant pathogen with potato and tomato as its primary hosts. Previous research showed that the heterotrimeric G-protein subunits Gα and Gß have a role in zoospore motility and virulence, and sporangial development, respectively. Here, we present analyses of the gene encoding a Gγ subunit in P. infestans, Pigpg1. The overall similarity of PiGPG1 with non-oomycete Gγ subunits is low, with only the most conserved amino acids maintained, but similarity with its homologs in other oomycetes is high. Pigpg1 is expressed in all life stages and shows a similar expression profile as the gene encoding the Gß subunit, Pigpb1. To elucidate its function, transformants were generated in which Pigpg1 is silenced or overexpressed and their phenotypes were analyzed. Pigpg1-silenced lines produce less sporangia, which are malformed. Altogether, the results show that PiGPG1 is crucial for proper sporangia development and zoosporogenesis. PiGPG1 is a functional Gγ, and likely forms a dimer with PiGPB1 that mediates signaling.


Subject(s)
GTP-Binding Protein gamma Subunits/physiology , Phytophthora infestans/growth & development , Sporangia/growth & development , Conserved Sequence , GTP-Binding Protein gamma Subunits/genetics , Mycelium/metabolism , Phytophthora infestans/genetics , RNA Interference , Sporangia/genetics , Spores/metabolism
20.
Plant Physiol ; 177(1): 398-410, 2018 05.
Article in English | MEDLINE | ID: mdl-29588335

ABSTRACT

Plant pathogens deliver effectors to manipulate processes in their hosts, creating a suitable environment for invasion and proliferation. Yet, little is known about the host proteins that are targeted by effectors from filamentous pathogens. Here, we show that stable transgenic expression in potato (Solanum tuberosum) and transient expression in Nicotiana benthamiana of the arginine-any amino acid-leucine-arginine effector Pi17316 enhances leaf colonization by the late blight pathogen Phytophthora infestans Expression of Pi17316 also attenuates cell death triggered by the pathogen-associated molecular pattern Infestin1 (INF1), indicating that the effector suppresses pattern-triggered immunity. However, this effector does not attenuate cell death triggered by a range of resistance proteins, showing that it specifically suppresses INF1-triggered cell death (ICD). In yeast two-hybrid assays, Pi17316 interacts directly with the potato ortholog of VASCULAR HIGHWAY1-interacting kinase (StVIK), encoding a predicted MEK kinase (MAP3K). Interaction in planta was confirmed by coimmunoprecipitation and occurs at the plant plasma membrane. Virus-induced gene silencing of VIK in N. benthamiana attenuated P. infestans colonization, whereas transient overexpression of StVIK enhanced colonization, indicating that this host protein acts as a susceptibility factor. Moreover, VIK overexpression specifically attenuated ICD, indicating that it is a negative regulator of immunity. The abilities of Pi17316 to enhance P. infestans colonization or suppress ICD were compromised significantly in NbVIK-silenced plants, demonstrating that the effector activity of Pi17316 is mediated by this MAP3K. Thus, StVIK is exploited by P. infestans as a susceptibility factor to promote late blight disease.


Subject(s)
Phytophthora infestans/physiology , Plant Diseases/microbiology , Plant Proteins/metabolism , Solanum tuberosum/enzymology , Solanum tuberosum/microbiology , Virulence Factors/metabolism , Cell Death , Cell Membrane/metabolism , Green Fluorescent Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phytophthora infestans/growth & development , Phytophthora infestans/pathogenicity , Protein Binding , Nicotiana/microbiology , Virulence
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