Biosynthesis, chemical structure, and structure-activity relationship of orfamide lipopeptides produced by Pseudomonas protegens and related species

Orfamide type cyclic lipopeptides (CLPs) are biosurfactants produced by Pseudomonas and involved in lysis of oomycete zoospores, biocontrol of Rhizoctonia and insecticidal activity against aphids. In this study, we compared the biosynthesis, structural diversity, in vitro and in planta activities of orfamides produced by rhizosphere-derived Pseudomonas protegens and related Pseudornonas species. Genetic characterization together with chemical identification revealed that the main orfamide compound produced by the P. protegens group is orfamide A, while the related strains Pseudomonas sp. CMR5c and CMR12a produce orfamide B. Comparison of orfamide fingerprints led to the discovery of two new orfamide homologs (orfamide F and orfamide G) in Pseudornonas sp. CMR5c. The structures of these two CLPs were determined by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. Mutagenesis and complementation showed that orfamides determine the swarming motility of parental Pseudomonas sp. strain CMR5c and their production was regulated by luxR type regulators. Orfamide A and orfamide B differ only in the identity of a single amino acid, while orfamide B and orfamide G share the same amino acid sequence but differ in length of the fatty acid part. The biological activities of orfamide A, orfamide B, and orfamide G were compared in further bioassays. The three compounds were equally active against Magnaporthe oryzae on rice, against Rhizoctonia solani AG 4-HGI in in vitro assays, and caused zoospore lysis of Phytophthora and Pythium. Furthermore, we could show that orfamides decrease blast severity in rice plants by blocking appressorium formation in M. oryzae. Taken all together, our study shows that orfamides produced by P protegens and related species have potential in biological control of a broad spectrum of fungal plant pathogens

Tissue Culture and Refreshment Techniques for Improvement of Transformation in Local Tetraploid and Diploid Potato with Late Blight Resistance as an Example

Potato (Solanum tuberosum) is among the best producers of edible biomass in terms of yield per hectare and a variety of different regional cultivars are used as a staple commodity inmany countries. However, this crop is attacked by several diseases, with the worst being the late blight disease caused by Phytophthora infestans. Stacking of resistance (R) genes from wild Solanum relatives are interesting prospects for the sustainable control of late blight. Therefore, we optimized methods for the efficient generation and screening of R-gene-containing transformants in tetraploidand diploid hybrid potato genotypes. Using these methods, a high transformation efficiency was achieved for the transformation of tetraploid and diploid potato lines with a triple resistance (3R)gene construct. Transformation efficiencies were improved by optimizing several factors affecting regeneration, including the quality of the starting plant material, and the composition of the plant growth regulators used during selective regeneration. A refreshment protocol was designed to alleviatein vitro related stress in stock plants, which significantly improved the growth vigor and resulted in a 4- to 10-fold increase in transformation efficiency. Furthermore, long-term exposure to exogenous Indole-3-butyric acid that is usually used for the initiation of rootsin vitro, was found to cause aberrant morphological phenotypes in potato

Coregulation of the cyclic lipopeptides orfamide and sessilin in the biocontrol strain Pseudomonas sp. CMR12a

Cyclic lipopeptides (CLPs) are synthesized by nonribosomal peptide synthetases (NRPS), which are often flanked by LuxR-type transcriptional regulators. Pseudomonas sp. CMR12a, an effective biocontrol strain, produces two different classes of CLPs namely sessilins and orfamides. The orfamide biosynthesis gene cluster is flanked up- and downstream by LuxR-type regulatory genes designated ofaR1 and ofaR2, respectively, whereas the sessilin biosynthesis gene cluster has one LuxR-type regulatory gene which is situated upstream of the cluster and is designated sesR. Our study investigated the role of these three regulators in the biosynthesis of orfamides and sessilins. Phylogenetic analyses positioned OfaR1 and OfaR2 with LuxR regulatory proteins of similar orfamide-producing Pseudomonas strains and the SesR with that of the tolaasin producer, Pseudomonas tolaasii. LC-ESI-MS analyses revealed that sessilins and orfamides are coproduced and that production starts in the late exponential phase. However, sessilins are secreted earlier and in large amounts, while orfamides are predominantly retained in the cell. Deletion mutants in ofaR1 and ofaR2 lost the capacity to produce both orfamides and sessilins, whereas the sesR mutant showed no clear phenotype. Additionally, RT-PCR analysis showed that in the sessilin cluster, a mutation in either ofaR1 or ofaR2 led to weaker transcripts of the biosynthesis genes, sesABC, and putative transporter genes, macA1B1. In the orfamide cluster, mainly the biosynthesis genes ofaBC were affected, while the first biosynthesis gene ofaA and putative macA2B2 transport genes were still transcribed. A mutation in either ofaR1, ofaR2, or sesR genes did not abolish the transcription of any of the other two

Utilization of protoplasts to facilitate gene editing in plants: schemes for in vitro shoot regeneration from tissues and protoplasts of potato and rapeseed: implications of bioengineering such as gene editing of broad-leaved plants

Schemes for efficient regeneration and recovery of shoots from in vitro tissues or single cells, such as protoplasts, are only available for limited numbers of plant species and genotypes and are crucial for establishing gene editing tools on a broader scale in agriculture and plant biology. Growth conditions, including hormone and nutrient composition as well as light regimes in key steps of known regeneration protocols, display significant variations, even between the genotypes within the same species, e.g., potato (Solanum tuberosum). As fresh plant material is a prerequisite for successful shoot regeneration, the plant material often needs to be refreshed for optimizing the growth and physiological state prior to genetic transformation. Utilization of protoplasts has become a more important approach for obtaining transgene-free edited plants by genome editing, CRISPR/Cas9. In this approach, callus formation from protoplasts is induced by one set of hormones, followed by organogenesis, i.e., shoot formation, which is induced by a second set of hormones. The requirements on culture conditions at these key steps vary considerably between the species and genotypes, which often require quantitative adjustments of medium compositions. In this mini-review, we outline the protocols and notes for clonal regeneration and cultivation from single cells, particularly protoplasts in potato and rapeseed. We focus mainly on different hormone treatment schemes and highlight the importance of medium compositions, e.g., sugar, nutrient, and light regimes as well as culture durations at the key regeneration steps. We believe that this review would provide important information and hints for establishing efficient regeneration strategies from other closely related and broad-leaved plant species in general

Genetically modified (GM) late blight-resistant potato and consumer attitudes before and after a field visit

Late blight, caused by Phytophthora infestans, is the most devastating disease in potato production. Here, we show full late blight resistance in a location with a genetically diverse pathogen population with the use of GM potato stacked with three resistance (R) genes over three seasons. In addition, using this field trials, we demonstrate that in-the-field intervention among consumers led to change for more favorable attitude generally toward GM crops

Clinical evaluation of dengue and identification of risk factors for severe disease: protocol for a multicentre study in 8 countries

Background: The burden of dengue continues to increase globally, with an estimated 100 million clinically apparent infections occurring each year. Although most dengue infections are asymptomatic, patients can present with a wide spectrum of clinical symptoms ranging from mild febrile illness through to severe manifestations of bleeding, organ impairment, and hypovolaemic shock due to a systemic vascular leak syndrome. Clinical diagnosis of dengue and identification of which patients are likely to develop severe disease remain challenging. This study aims to improve diagnosis and clinical management through approaches designed a) to differentiate between dengue and other common febrile illness within 72 h of fever onset, and b) among patients with dengue to identify markers that are predictive of the likelihood of evolving to a more severe disease course. Method/Design: This is a prospective multi-centre observational study aiming to enrol 7–8000 participants aged ≥ 5 years presenting with a febrile illness consistent with dengue to outpatient health facilities in 8 countries across Asia and Latin America. Patients presenting within 72 h of fever onset who do not exhibit signs of severe disease are eligible for the study. A broad range of clinical and laboratory parameters are assessed daily for up to 6 days during the acute illness, and also at a follow up visit 1 week later. Discussion: Data from this large cohort of patients, enrolled early with undifferentiated fever, will be used to develop a practical diagnostic algorithm and a robust clinical case definition for dengue. Additionally, among patients with confirmed dengue we aim to identify simple clinical and laboratory parameters associated with progression to a more severe disease course. We will also investigate early virological and serological correlates of severe disease, and examine genetic associations in this large heterogeneous cohort. In addition the results will be used to assess the new World Health Organization classification scheme for dengue in practice, and to update the guidelines for “Integrated Management of Childhood Illness” used in dengue-endemic countries. Trial registration: NCT01550016. Registration Date: March 7, 201

Recent advances in Pseudomonas biocontrol

Fluorescent Pseudomonads have been intensively studied as biocontrol agents. These organisms exhibit an enormous metabolic versatility and especially isolates from the P. fluorescens group produce a remarkable spectrum of secondary metabolites. Multilocus sequence analysis and phylogenomics have revealed the presence of at least five distinct subgroups in the P. fluorescens group with biocontrol isolates previously classified as P. fluorescens interspersed with strains classified in other species. Antibiotics such as phenazines, diacetylphloroglucinol, and hydrogen cyanide are produced by certain taxonomic groups within the genus Pseudomonas and appear to be ancestral. These compounds often have a physiological role for the producing strain, independent from their antibiotic activity. Other secondary metabolites are only found in certain Pseudomonas isolates and are apparently obtained by horizontal gene transfer. The recent boost in genome sequencing has revealed many orphan biosynthetic gene clusters in the genomes of Pseudomonas biocontrol strains. Genome mining has led to the discovery of many new antimicrobial compounds with a role in biocontrol of plant pathogenic fungi, oomycetes and bacteria. Moreover, some Pseudomonas biocontrol strains produce potent insect toxins. The versatility of Pseudomonas biocontrol agents continues to surprise

Mutations introduced in susceptibility genes through CRISPR/Cas9 genome editing confer increased late blight resistance in potatoes

The use of pathogen-resistant cultivars is expected to increase yield and decrease fungicide use in agriculture. However, in potato breeding, increased resistance obtained via resistance genes (R-genes) is hampered because R-gene(s) are often specific for a pathogen race and can be quickly overcome by the evolution of the pathogen. In parallel, susceptibility genes (S-genes) are important for pathogenesis, and loss of S-gene function confers increased resistance in several plants, such as rice, wheat, citrus and tomatoes. In this article, we present the mutation and screening of seven putative S-genes in potatoes, including two DMR6 potato homologues. Using a CRISPR/Cas9 system, which conferred co-expression of two guide RNAs, tetra-allelic deletion mutants were generated and resistance against late blight was assayed in the plants. Functional knockouts of StDND1, StCHL1, and DMG400000582 (StDMR6-1) generated potatoes with increased resistance against late blight. Plants mutated in StDND1 showed pleiotropic effects, whereas StDMR6-1 and StCHL1 mutated plants did not exhibit any growth phenotype, making them good candidates for further agricultural studies. Additionally, we showed that DMG401026923 (here denoted StDMR6-2) knockout mutants did not demonstrate any increased late blight resistance, but exhibited a growth phenotype, indicating that StDMR6-1 and StDMR6-2 have different functions. To the best of our knowledge, this is the first report on the mutation and screening of putative S-genes in potatoes, including two DMR6 potato homologues

Interplay between orfamides, sessilins and phenazines in the control of Rhizoctonia diseases by Pseudomonas sp CMR12a

We investigated the role of phenazines and cyclic lipopeptides (CLPs) (orfamides and sessilins), antagonistic metabolites produced by Pseudomonas sp. CMR12a, in the biological control of damping-off disease on Chinese cabbage (Brassica chinensis) caused by Rhizoctonia solaniAG 2-1 and root rot disease on bean (Phaseolus vulgaris L.) caused by R. solaniAG 4-HGI. A Pseudomonas mutant that only produced phenazines suppressed damping-off disease on Chinese cabbage to the same extent as CMR12a, while its efficacy to reduce root rot on bean was strongly impaired. In both pathosystems, the phenazine mutant that produced both CLPs was equally effective, but mutants that produced only one CLP lost biocontrol activity. In vitro microscopic assays revealed that mutants that only produced sessilins or orfamides inhibited mycelial growth of R. solani when applied together, while they were ineffective on their own. Phenazine-1-carboxamide suppressed mycelial growth of R. solaniAG 2-1 but had no effect on AG 4-HGI. Orfamide B suppressed mycelial growth of both R. solani anastomosis groups in a dose-dependent way. Our results point to an additive interaction between both CLPs. Moreover, phenazines alone are sufficient to suppress Rhizoctonia disease on Chinese cabbage, while they need to work in tandem with the CLPs on bean