scholarly journals A Functional Screen to Characterize the Secretomes of Eukaryotic Pathogens and Their Hosts In Planta

2006 ◽  
Vol 19 (12) ◽  
pp. 1368-1377 ◽  
Author(s):  
Sang-Jik Lee ◽  
Brendan S. Kelley ◽  
Cynthia M. B. Damasceno ◽  
Bonnie St. John ◽  
Byung-Soo Kim ◽  
...  
Keyword(s):  
Infected Plant ◽  
Effector Proteins ◽  
Secreted Proteins ◽  
Vector System ◽  
In Planta ◽  
Functional Screen ◽  
Genes Encoding ◽  
Experimental Approaches ◽  
Genome Scale ◽  
Infected Plant Material

Complex suites of proteins that are secreted by plants and phytopathogens into the plant apoplast play crucial roles in surveillance, assault, defense, and counter-defense. High-throughput genome-scale strategies are being developed to better understand the nature of these “secretomes” and the identity of pathogen-derived effector proteins that subvert plant defenses and promote pathogenicity. Although combined bioinformatic and experimental approaches recently have provided comprehensive coverage of secreted proteins from bacterial phytopathogens, far less is known about the secretomes and batteries of effectors of eukaryotic phytopathogens; notably fungi and oomycetes. The yeast secretion trap (YST) represents a potentially valuable technique to simultaneously target pathogen and host secretomes in infected plant material. A YST screen, using a new vector system, was applied to study the interaction between tomato (Solanum lycopersicum) and the oomycete Phytophthora infestans, revealing sets of genes encoding secreted proteins from both pathogen and host. Most of those from the oomycete had no identifiable function and were detectable in planta only during pathogenesis, underlining the value of YST as a tool to identify new candidate effectors and pathogenicity factors. In addition, the majority of the P. infestans proteins had homologs in the genomes of the related oomycetes P. sojae and P. ramorum.

An aspartic proteinase gene family in the filamentous fungus Botrytis cinerea contains members with novel features

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2475-2489 ◽  
Author(s):  
Arjen ten Have ◽  
Ester Dekkers ◽  
John Kay ◽  
Lowri H. Phylip ◽  
Jan A. L. van Kan
Keyword(s):  
Botrytis Cinerea ◽  
Culture Medium ◽  
Infected Plant ◽  
Aspartic Proteinase ◽  
Proteinase Activity ◽  
In Planta ◽  
Wheat Germ Extract ◽  
Fungal Plant Pathogen ◽  
Genes Encoding ◽  
Metalloproteinase Activity

Botrytis cinerea, an important fungal plant pathogen, secretes aspartic proteinase (AP) activity in axenic cultures. No cysteine, serine or metalloproteinase activity could be detected. Proteinase activity was higher in culture medium containing BSA or wheat germ extract, as compared to minimal medium. A proportion of the enzyme activity remained in the extracellular glucan sheath. AP was also the only type of proteinase activity in fluid obtained from B. cinerea-infected tissue of apple, pepper, tomato and zucchini. Five B. cinerea genes encoding an AP were cloned and denoted Bcap1–5. Features of the encoded proteins are discussed. BcAP1, especially, has novel characteristics. A phylogenetic analysis was performed comprising sequences originating from different kingdoms. BcAP1 and BcAP5 did not cluster in a bootstrap-supported clade. BcAP2 clusters with vacuolar APs. BcAP3 and BcAP4 cluster with secreted APs in a clade that also contains glycosylphosphatidylinositol-anchored proteinases from Saccharomyces cerevisiae and Candida albicans. All five Bcap genes are expressed in liquid cultures. Transcript levels of Bcap1, Bcap2, Bcap3 and Bcap4 are subject to glucose and peptone repression. Transcripts from all five Bcap genes were detected in infected plant tissue, indicating that at least part of the AP activity in planta originates from the pathogen.

scholarly journals Analysis of Epichloë festucae small secreted proteins in the interaction with Lolium perenne

2018 ◽  
Author(s):  
Barry Scott ◽  
Berit Hassing ◽  
David Winter ◽  
Carl Mesarich ◽  
Carla Eaton ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Vital Role ◽  
Effector Proteins ◽  
Secreted Proteins ◽  
In Planta ◽  
Dynamic Features ◽  
Symbiotic Interaction ◽  
Extracellular Medium ◽  
Epichloë Festucae ◽  
Small Secreted Proteins

Epichloë festucae is an endophyte of the agriculturally important perennial ryegrass. This species systemically colonises the aerial tissues of this host where its growth is tightly regulated thereby maintaining a mutualistic symbiotic interaction. Recent studies have suggested that small secreted proteins, termed effectors, play a vital role in the suppression of host defence responses. To date only a few effectors with important roles in mutualistic interactions have been described. Here we make use of the fully assembled E. festucae genome and EffectorP to generate a suite of 141 effector candidates. These were analysed with respect to their genome location and expression profiles in planta and in several symbiosis-defective mutants. We found an association between effector candidates and a class of transposable elements known as MITEs, but no correlation with other dynamic features of the E. festucae genome, such as transposable element-rich regions. Three effector candidates and a small GPI-anchored protein were chosen for functional analysis based on their high expression in planta compared to in culture and their differential regulation in symbiosis defective E. festucae mutants. All three candidate effector proteins were shown to possess a functional signal peptide and two could be detected in the extracellular medium by western blotting. Localization of the effector candidates in planta suggests that they are not translocated into the plant cell, but rather, are localized in the apoplastic space or are attached to the cell wall. Deletion and overexpression of the effector candidates, as well as the putative GPI-anchored protein, did not affect the plant growth phenotype or restrict growth of E. festucae mutants in planta. These results indicate that these proteins are either not required for the interaction at the observed life stages or that there is redundancy between effectors expressed by E. festucae.

scholarly journals Arabidopsis Genes Essential for Seedling Viability: Isolation of Insertional Mutants and Molecular Cloning

Genetics ◽  
2001 ◽  
Vol 159 (4) ◽  
pp. 1765-1778
Author(s):  
Gregory J Budziszewski ◽  
Sharon Potter Lewis ◽  
Lyn Wegrich Glover ◽  
Jennifer Reineke ◽  
Gary Jones ◽  
...  
Keyword(s):  
Large Scale ◽  
Protein Translocation ◽  
Gene Families ◽  
Mutant Phenotype ◽  
Lethal Mutant ◽  
A Genome ◽  
Genes Encoding ◽  
High Level ◽  
Mutant Lines ◽  
Genome Scale

Abstract We have undertaken a large-scale genetic screen to identify genes with a seedling-lethal mutant phenotype. From screening ~38,000 insertional mutant lines, we identified >500 seedling-lethal mutants, completed cosegregation analysis of the insertion and the lethal phenotype for >200 mutants, molecularly characterized 54 mutants, and provided a detailed description for 22 of them. Most of the seedling-lethal mutants seem to affect chloroplast function because they display altered pigmentation and affect genes encoding proteins predicted to have chloroplast localization. Although a high level of functional redundancy in Arabidopsis might be expected because 65% of genes are members of gene families, we found that 41% of the essential genes found in this study are members of Arabidopsis gene families. In addition, we isolated several interesting classes of mutants and genes. We found three mutants in the recently discovered nonmevalonate isoprenoid biosynthetic pathway and mutants disrupting genes similar to Tic40 and tatC, which are likely to be involved in chloroplast protein translocation. Finally, we directly compared T-DNA and Ac/Ds transposon mutagenesis methods in Arabidopsis on a genome scale. In each population, we found only about one-third of the insertion mutations cosegregated with a mutant phenotype.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals Identification and Genomic Characterization of Pathogenic Bacillus altitudinis from Common Pear Trees in Morocco

Agronomy ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 1344
Author(s):  
Naima Lemjiber ◽  
Khalid Naamani ◽  
Annabelle Merieau ◽  
Abdelhi Dihazi ◽  
Nawal Zhar ◽  
...  
Keyword(s):  
16S Rrna Gene Sequencing ◽  
Genomic Islands ◽  
Rrna Gene ◽  
In Planta ◽  
Bacterial Strains ◽  
Bacillus Altitudinis ◽  
Genes Encoding ◽  
Bacillus Spp ◽  
Pear Trees ◽  
Rrna Gene Sequencing

Bacterial burn is one of the major diseases affecting pear trees worldwide, with serious impacts on producers and economy. In Morocco, several pear trees (Pyrus communis) have shown leaf burns since 2015. To characterize the causal agent of this disease, we isolated fourteen bacterial strains from different parts of symptomatic pear trees (leaves, shoots, fruits and flowers) that were tested in planta for their pathogenicity on Louise bonne and Williams cultivars. The results showed necrotic lesions with a significant severity range from 47.63 to 57.77% on leaves of the Louise bonne cultivar inoculated with isolate B10, while the other bacterial isolates did not induce any disease symptom. 16S rRNA gene sequencing did not allow robust taxonomic discrimination of the incriminated isolate. Thus, we conducted whole-genome sequencing (WGS) and phylogenetic analyzes based on gyrA, gyrB and cdaA gene sequences, indicating that this isolate belongs to the Bacillus altitudinis species. This taxonomic classification was further confirmed by the Average Nucleotide Identity (ANI) and the in silico DNA-DNA hybridization (isDDH) analyzes compared to sixty-five Bacillus spp. type strains. The genome was mined for genes encoding carbohydrate-active enzymes (CAZymes) known to play a role in the vegetal tissue degradation. 177 candidates with functions that may support the in planta phytopathogenicity results were identified. To the best of our knowledge, this is the first data reporting B. altitudinis as agent of leaf burn in P. communis in Morocco. Our dataset will improve our knowledge on spread and pathogenicity of B. altitudinis genotypes that appears as emergent phytopathogenic agent, unveiling virulence factors and their genomic location (i.e., within genomic islands or the accessory genome) to induce trees disease.

scholarly journals A Deadly Cargo: Gene Repertoire of Cytotoxic Effector Proteins in the Camelidae

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 304
Author(s):  
Ján Futas ◽  
Jan Oppelt ◽  
Pamela Anna Burger ◽  
Petr Horin
Keyword(s):  
Cytotoxic T Cells ◽  
Effector Proteins ◽  
Target Cells ◽  
Gene Repertoire ◽  
Interspecific Differences ◽  
Genes Encoding ◽  
Bactrian Camels ◽  
Pore Forming Proteins ◽  
Sequence Similarities ◽  
Cytotoxic Effector

Cytotoxic T cells and natural killer cells can kill target cells based on their expression and release of perforin, granulysin, and granzymes. Genes encoding these molecules have been only poorly annotated in camelids. Based on bioinformatic analyses of genomic resources, sequences corresponding to perforin, granulysin, and granzymes were identified in genomes of camelids and related ungulate species, and annotation of the corresponding genes was performed. A phylogenetic tree was constructed to study evolutionary relationships between the species analyzed. Re-sequencing of all genes in a panel of 10 dromedaries and 10 domestic Bactrian camels allowed analyzing their individual genetic polymorphisms. The data showed that all extant Old World camelids possess functional genes for two pore-forming proteins (PRF1, GNLY) and six granzymes (GZMA, GZMB, GZMH, GZMK, GZMM, and GZMO). All these genes were represented as single copies in the genome except the GZMH gene exhibiting interspecific differences in the number of loci. High protein sequence similarities with other camelid and ungulate species were observed for GZMK and GZMM. The protein variability in dromedaries and Bactrian camels was rather low, except for GNLY and chymotrypsin-like granzymes (GZMB, GZMH).

scholarly journals The Yeast Signal Sequence Trap Identifies Secreted Proteins of the Hemibiotrophic Corn Pathogen Colletotrichum graminicola

2008 ◽  
Vol 21 (10) ◽  
pp. 1325-1336 ◽  
Author(s):  
Jorrit-Jan Krijger ◽  
Ralf Horbach ◽  
Michael Behr ◽  
Patrick Schweizer ◽  
Holger B. Deising ◽  
...  
Keyword(s):  
Cell Walls ◽  
Leaf Extract ◽  
Signal Sequence ◽  
Stem Rot ◽  
Colletotrichum Graminicola ◽  
In Planta ◽  
Chain Reaction ◽  
Genes Encoding ◽  
Polymerase Chain

The hemibiotroph Colletotrichum graminicola is the causal agent of stem rot and leaf anthracnose on Zea mays. Following penetration of epidermal cells, the fungus enters a short biotrophic phase, followed by a destructive necrotrophic phase of pathogenesis. During both phases, secreted fungal proteins are supposed to determine progress and success of the infection. To identify genes encoding such proteins, we constructed a yeast signal sequence trap (YSST) cDNA-library from RNA extracted from mycelium grown in vitro on corn cell walls and leaf extract. Of the 103 identified unigenes, 50 showed significant similarities to genes with a reported function, 25 sequences were similar to genes without a known function, and 28 sequences showed no similarity to entries in the databases. Macroarray hybridization and quantitative reverse-transcriptase polymerase chain reaction confirmed that most genes identified by the YSST screen are expressed in planta. Other than some genes that were constantly expressed, a larger set showed peaks of transcript abundances at specific phases of pathogenesis. Another set exhibited biphasic expression with peaks at the biotrophic and necrotrophic phase. Transcript analyses of in vitro-grown cultures revealed that several of the genes identified by the YSST screen were induced by the addition of corn leaf components, indicating that host-derived factors may have mimicked the host milieu.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
Vector System ◽  
In Planta ◽  
Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

2021 ◽  
Author(s):  
David A Baltrus ◽  
Qian Feng ◽  
Brian H Kvitko
Keyword(s):  
Pseudomonas Syringae ◽  
Evolutionary Dynamics ◽  
Effector Proteins ◽  
In Planta ◽  
Genomic Context ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Genome Context ◽  
The Impact ◽  
Multiple Type

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

scholarly journals An ancient antimicrobial protein co-opted by a fungal plant pathogen for in planta mycobiome manipulation

2021 ◽  
Author(s):  
Nick Snelders ◽  
Gabriella Petti ◽  
Grardy van den Berg ◽  
Michael Seidl ◽  
Bart Thomma
Keyword(s):  
Life Stage ◽  
Antibacterial Properties ◽  
Plant Evolution ◽  
Effector Proteins ◽  
Plant Colonization ◽  
Antimicrobial Protein ◽  
In Planta ◽  
Antimicrobial Proteins ◽  
Pathogenic Microbes ◽  
Plant Microbiome

Microbes typically secrete a plethora of molecules to promote niche colonization. Soil-dwelling microbes are well-known producers of antimicrobials that are exploited to outcompete microbial co-inhabitants. Also plant pathogenic microbes secrete a diversity of molecules into their environment for niche establishment. Upon plant colonization, microbial pathogens secrete so-called effector proteins that promote disease development. While such effectors are typically considered to exclusively act through direct host manipulation, we recently reported that the soil-borne fungal xylem-colonizing vascular wilt pathogen Verticillium dahliae exploits effector proteins with antibacterial properties to promote host colonization through the manipulation of beneficial host microbiota. Since fungal evolution preceded land plant evolution, we now speculate that a subset of the pathogen effectors involved in host microbiota manipulation evolved from ancient antimicrobial proteins of terrestrial fungal ancestors that served in microbial competition prior to the evolution of plant pathogenicity. Here, we show that V. dahliae has co-opted an ancient antimicrobial protein as effector, named VdAMP3, for mycobiome manipulation in planta. We show that VdAMP3 is specifically expressed to ward off fungal niche competitors during resting structure formation in senescing mesophyll tissues. Our findings indicate that effector-mediated microbiome manipulation by plant pathogenic microbes extends beyond bacteria and also concerns eukaryotic members of the plant microbiome. Finally, we demonstrate that fungal pathogens can exploit plant microbiome-manipulating effectors in a life-stage specific manner, and that a subset of these effectors has evolved from ancient antimicrobial proteins of fungal ancestors that likely originally functioned in manipulation of terrestrial biota.

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