scholarly journals VdNPS, a Nonribosomal Peptide Synthetase, Is Involved in Regulating Virulence in Verticillium dahliae

2020 ◽  
Vol 110 (8) ◽  
pp. 1398-1409
Author(s):  
Xiumei Luo ◽  
Tingting Tian ◽  
Xue Tan ◽  
Yixuan Zheng ◽  
Chengjian Xie ◽  
...  
Keyword(s):  
Verticillium Dahliae ◽  
Durable Resistance ◽  
Functional Characterization ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Pathogenesis Related ◽  
Pathogenesis Related Gene ◽  
Type V ◽  
Species Production

Nonribosomal peptide synthetases (NPS) are known for the biosynthesis of antibiotics, toxins, and siderophore production. They are also a virulence determinant in different phytopathogens. However, until now, the functional characterization of NPS in Verticillium dahliae has not been reported. Deletion of the NPS gene in V. dahliae led to the decrease of conidia, microsclerotia, and pathogenicity. ΔVdNPS strains were tolerant to H2O2, and the genes involved in H2O2 detoxification, iron/copper transport, and cytoskeleton were differentially expressed in ΔVdNPS. Interestingly, ΔVdNPS strains exhibited hypersensitivity to salicylic acid (SA), and the genes involved in SA hydroxylation were up-regulated in ΔVdNPS compared with wild-type V. dahliae under SA stress. Additionally, during infection, ΔVdNPS induced more pathogenesis-related gene expression, higher reactive oxygen species production, and stronger SA-mediated signaling transduction in host to overcome pathogen. Uncovering the function of VdNPS in pathogenicity could provide a reliable theoretical basis for the development of cultivars with durable resistance against V. dahliae-associated diseases.

scholarly journals Verticillium dahliae VdTHI20, Involved in Pyrimidine Biosynthesis, Is Required for DNA Repair Functions and Pathogenicity

2020 ◽  
Vol 21 (4) ◽  
pp. 1378
Author(s):  
Tengfei Qin ◽  
Wei Hao ◽  
Runrun Sun ◽  
Yuqing Li ◽  
Yuanyuan Wang ◽  
...  
Keyword(s):  
Verticillium Dahliae ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Pathogenesis Related ◽  
Mutant Strains ◽  
Transgenic Lines ◽  
Tobacco Seedlings ◽  
Pathogenesis Related Gene ◽  
Green Fluorescent ◽  
Thiamine Biosynthesis

Verticillium dahliae (V. dahliae) infects roots and colonizes the vascular vessels of host plants, significantly reducing the economic yield of cotton and other crops. In this study, the protein VdTHI20, which is involved in the thiamine biosynthesis pathway, was characterized by knocking out the corresponding VdTHI20 gene in V. dahliae via Agrobacterium tumefaciens-mediated transformation (ATMT). The deletion of VdTHI20 resulted in several phenotypic defects in vegetative growth and conidiation and in impaired virulence in tobacco seedlings. We show that VdTHI20 increases the tolerance of V. dahliae to UV damage. The impaired vegetative growth of ΔVdTHI20 mutant strains was restored by complementation with a functional copy of the VdTHI20 gene or by supplementation with additional thiamine. Furthermore, the root infection and colonization of the ΔVdTHI20 mutant strains were suppressed, as indicated by green fluorescent protein (GFP)-labelling under microscope observation. When the RNAi constructs of VdTHI20 were used to transform Nicotiana benthamiana, the transgenic lines expressing dsVdTHI20 showed elevated resistance to V. dahliae. Together, these results suggest that VdTHI20 plays a significant role in the pathogenicity of V. dahliae. In addition, the pathogenesis-related gene VdTHI20 exhibits potential for controlling V. dahliae in important crops.

scholarly journals A Nonribosomal Peptide Synthetase Gene (mgoA) of Pseudomonas syringae pv. syringae Is Involved in Mangotoxin Biosynthesis and Is Required for Full Virulence

2007 ◽  
Vol 20 (5) ◽  
pp. 500-509 ◽  
Author(s):  
Eva Arrebola ◽  
Francisco M. Cazorla ◽  
Diego Romero ◽  
Alejandro Pérez-García ◽  
Antonio de Vicente
Keyword(s):  
Pseudomonas Syringae ◽  
Chromosomal Region ◽  
Open Reading Frames ◽  
Modular Architecture ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Large Gene ◽  
Peptide Synthetase ◽  
Apical Necrosis ◽  
Reading Frames

Pseudomonas syringae pv. syringae, which causes the bacterial apical necrosis of mango, produces the antimetabolite mangotoxin. We report here the cloning, sequencing, and identity analysis of a chromosomal region of 11.1 kb from strain P. syringae pv. syringae UMAF0158, which is involved in mangotoxin biosynthesis. This chromosomal region contains six complete open reading frames (ORFs), including a large gene (ORF5) with a modular architecture characteristic of nonribosomal peptide synthetases (NRPS) named mgoA. A Tn 5 mutant disrupted in mgoA was defective in mangotoxin production, revealing the involvement of the putative NRPS gene in the biosynthesis of mangotoxin. This derivative strain impaired in mangotoxin production also showed a reduction in virulence as measured by necrotic symptoms on tomato leaflets. Mangotoxin production and virulence were restored fully in the NRPS mutant by complementation with plasmid pCG2-6, which contains an 11,103-bp chromosomal region cloned from the wildtype strain P. syringae pv. syringae UMAF0158 that includes the putative NPRS gene (mgoA). The results demonstrate that mgoA has a role in the virulence of P. syringae pv. syringae. The involvement of an NRPS in the production of an antimetabolite toxin from P. syringae inhibiting ornithine acetyltransferase activity is proposed.

Presence of peptide synthetase gene transcripts and accumulation of ergopeptines in Claviceps purpurea and Neotyphodium coenophialum

1998 ◽  
Vol 44 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Seanna L Annis ◽  
Daniel G Panaccione
Keyword(s):  
Tall Fescue ◽  
Claviceps Purpurea ◽  
Neotyphodium Coenophialum ◽  
Experimental Conditions ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Different Ages ◽  
Gene Transcripts ◽  
Functional Analyses

The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hybridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as in ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.Key words: Claviceps purpurea, endophyte, ergot, ergotamine, Neotyphodium coenophialum.

scholarly journals Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases

2015 ◽  
Vol 51 (12) ◽  
pp. 2262-2265 ◽  
Author(s):  
Sho Konno ◽  
Fumihiro Ishikawa ◽  
Takehiro Suzuki ◽  
Naoshi Dohmae ◽  
Michael D. Burkart ◽  
...  
Keyword(s):  
Natural Product ◽  
Active Site ◽  
Nonribosomal Peptide Synthetase ◽  
Protein Labeling ◽  
Nonribosomal Peptide ◽  
Nonribosomal Peptide Synthetases ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
A Domains

Active site-directed proteomic probes coupled to the 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality selectively target nonribosomal peptide synthetase (NRPS) adenylation (A) domains in natural product producer proteomes by ligand-directed protein labeling.

scholarly journals Inhibition of Grape Crown Gall by Agrobacterium vitis F2/5 Requires Two Nonribosomal Peptide Synthetases and One Polyketide Synthase

2016 ◽  
Vol 29 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Desen Zheng ◽  
Thomas J. Burr
Keyword(s):  
Crown Gall ◽  
Polyketide Synthase ◽  
Wild Type ◽  
Tissue Necrosis ◽  
Agrobacterium Vitis ◽  
Nonribosomal Peptide ◽  
Crown Gall Disease ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Polyketide Synthase Gene

Agrobacterium vitis nontumorigenic strain F2/5 is able to inhibit crown gall disease on grapevines. The mechanism of grape tumor inhibition (GTI) by F2/5 has not been fully determined. In this study, we demonstrate that two nonribosomal peptide synthetase (NRPS) genes (F-avi3342 and F-avi5730) and one polyketide synthase gene (F-avi4330) are required for GTI. Knockout of any one of them resulted in F/25 losing GTI capacity. We previously reported that F-avi3342 and F-avi4330 but not F-avi5730 are required for induction of grape tissue necrosis and tobacco hypersensitive response. F-avi5730 is predicted to encode a single modular NRPS. It is located in a cluster that is homologous to the siderophore vicibactin biosynthesis locus in Rhizobium species. Individual disruption of F-avi5730 and two immediate downstream genes, F-avi5731 and F-avi5732, all resulted in reduced siderophore production; however, only F-avi5730 was found to be required for GTI. Complemented F-avi5730 mutant (ΔF-avi5730+) restored a wild-type level of GTI activity. It was determined that, over time, populations of ΔF-avi4330, ΔF-avi3342, and ΔF-avi5730 at inoculated wound sites on grapevine did not differ from those of ΔF-avi5730+ indicating that loss of GTI was not due to reduced colonization of wound sites by mutants.

Structures of a dimodular nonribosomal peptide synthetase reveal conformational flexibility

Science ◽  
2019 ◽  
Vol 366 (6466) ◽  
pp. eaaw4388 ◽  
Author(s):  
Janice M. Reimer ◽  
Maximilian Eivaskhani ◽  
Ingrid Harb ◽  
Alba Guarné ◽  
Martin Weigt ◽  
...  
Keyword(s):  
Condensation Reaction ◽  
Conformational Flexibility ◽  
Scattering Data ◽  
Nonribosomal Peptide ◽  
X Ray ◽  
Peptide Synthetases ◽  
X Ray Scattering ◽  
Peptide Synthetase ◽  
Covariation Analysis ◽  
Full Core

Nonribosomal peptide synthetases (NRPSs) are biosynthetic enzymes that synthesize natural product therapeutics using a modular synthetic logic, whereby each module adds one aminoacyl substrate to the nascent peptide. We have determined five x-ray crystal structures of large constructs of the NRPS linear gramicidin synthetase, including a structure of a full core dimodule in conformations organized for the condensation reaction and intermodular peptidyl substrate delivery. The structures reveal differences in the relative positions of adjacent modules, which are not strictly coupled to the catalytic cycle and are consistent with small-angle x-ray scattering data. The structures and covariation analysis of homologs allowed us to create mutants that improve the yield of a peptide from a module-swapped dimodular NRPS.

scholarly journals Substrate recognition by nonribosomal peptide synthetase multi-enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1629-1636 ◽  
Author(s):  
Sylvie Lautru ◽  
Gregory L. Challis
Keyword(s):  
Molecular Basis ◽  
Substrate Selectivity ◽  
Substrate Selection ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Peptide Synthetase ◽  
Commercially Important ◽  
Potential Impact ◽  
Bacteria And Fungi ◽  
The Individual

Nonribosomal peptide synthetases (NRPSs) are giant multi-domain enzymes that catalyse the biosynthesis of many commercially important peptides produced by bacteria and fungi. Several studies over the last decade have shown that many of the individual domains within NRPSs exhibit significant substrate selectivity, which impacts on our ability to engineer NRPSs to produce new bioactive microbial peptides. Adenylation domains appear to be the primary determinants of substrate selectivity in NRPSs. Much progress has been made towards an empirical understanding of substrate selection by these domains over the last 5 years, but the molecular basis of substrate selectivity in these domains is not yet well understood. Perhaps surprisingly, condensation domains have also been reported to exhibit moderate to high substrate selectivity, although the generality of this observation and its potential impact on engineered biosynthesis experiments has yet to be fully elucidated. The situation is less clear for the thioesterase domains, which seem in certain cases to be dedicated to the hydrolysis/cyclization of their natural substrate, whereas in other cases they are largely permissive.

scholarly journals Virulence, Host-Selective Toxin Production, and Development of Three Cochliobolus Phytopathogens Lacking the Sfp-Type 4′-Phosphopantetheinyl Transferase Ppt1

2015 ◽  
Vol 28 (10) ◽  
pp. 1130-1141 ◽  
Author(s):  
Nur Ain Izzati Mohd Zainudin ◽  
Bradford Condon ◽  
Lieselotte De Bruyne ◽  
Christof Van Poucke ◽  
Qing Bi ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Toxin Production ◽  
Lysine Biosynthesis ◽  
Phosphopantetheinyl Transferase ◽  
Wild Type ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Asexual Sporulation ◽  
Peptide Synthetase ◽  
Sexual Crosses

The Sfp-type 4′-phosphopantetheinyl transferase Ppt1 is required for activation of nonribosomal peptide synthetases, including α-aminoadipate reductase (AAR) for lysine biosynthesis and polyketide synthases, enzymes that biosynthesize peptide and polyketide secondary metabolites, respectively. Deletion of the PPT1 gene, from the maize pathogen Cochliobolus heterostrophus and the rice pathogen Cochliobolus miyabeanus, yielded strains that were significantly reduced in virulence to their hosts. In addition, ppt1 mutants of C. heterostrophus race T and Cochliobolus victoriae were unable to biosynthesize the host-selective toxins (HST) T-toxin and victorin, respectively, as judged by bioassays. Interestingly, ppt1 mutants of C. miyabeanus were shown to produce tenfold higher levels of the sesterterpene-type non-HST ophiobolin A, as compared with the wild-type strain. The ppt1 strains of all species were also reduced in tolerance to oxidative stress and iron depletion; both phenotypes are associated with inability to produce extracellular siderophores biosynthesized by the nonribosomal peptide synthetase Nps6. Colony surfaces were hydrophilic, a trait previously associated with absence of C. heterostrophus Nps4. Mutants were decreased in asexual sporulation and C. heterostrophus strains were female-sterile in sexual crosses; the latter phenotype was observed previously with mutants lacking Nps2, which produces an intracellular siderophore. As expected, mutants were albino, since they cannot produce the polyketide melanin and were auxotrophic for lysine because they lack an AAR.

scholarly journals Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa

2003 ◽  
Vol 185 (9) ◽  
pp. 2848-2855 ◽  
Author(s):  
David F. Ackerley ◽  
Tom T. Caradoc-Davies ◽  
Iain L. Lamont
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Three Dimensional ◽  
Protein Product ◽  
Synthetase Activity ◽  
Nonribosomal Peptide ◽  
Peptide Synthetases ◽  
Three Dimensional Modeling ◽  
Peptide Synthetase

ABSTRACT Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. l-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including d-threonine, l-serine, and l-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases.

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