RESUMEN
Bacterial secondary metabolites exhibit diverse remarkable bioactivities and are thus the subject of study for different applications. Recently, the individual effectiveness of tripyrrolic prodiginines and rhamnolipids against the plant-parasitic nematode Heterodera schachtii, which causes tremendous losses in crop plants, was described. Notably, rhamnolipid production in engineered Pseudomonas putida strains has already reached industrial implementation. However, the non-natural hydroxyl-decorated prodiginines, which are of particular interest in this study due to a previously described particularly good plant compatibility and low toxicity, are not as readily accessible. In the present study, a new effective hybrid synthetic route was established. This included the engineering of a novel P. putida strain to provide enhanced levels of a bipyrrole precursor and an optimization of mutasynthesis, i.e., the conversion of chemically synthesized and supplemented monopyrroles to tripyrrolic compounds. Subsequent semisynthesis provided the hydroxylated prodiginine. The prodiginines caused reduced infectiousness of H. schachtii for Arabidopsis thaliana plants resulting from impaired motility and stylet thrusting, providing the first insights on the mode of action in this context. Furthermore, the combined application with rhamnolipids was assessed for the first time and found to be more effective against nematode parasitism than the individual compounds. To obtain, for instance, 50% nematode control, it was sufficient to apply 7.8 µM hydroxylated prodiginine together with 0.7 µg/ml (~ 1.1 µM) di-rhamnolipids, which corresponded to ca. » of the individual EC50 values. In summary, a hybrid synthetic route toward a hydroxylated prodiginine was established and its effects and combinatorial activity with rhamnolipids on plant-parasitic nematode H. schachtii are presented, demonstrating potential application as antinematodal agents. Graphical Abstract.
RESUMEN
Serendipita indica is an axenically cultivable fungus, which colonizes a broad range of plant species including the model plant Arabidopsis thaliana. Root colonization by this endophyte leads to enhanced plant fitness and performance and promotes resistance against different biotic and abiotic stresses. The involvement of MPK6 in this mutualistic interaction had been previously shown with an mpk6 A. thaliana mutant, which failed to respond to S. indica colonization. Here, we demonstrate that mpk6 roots are significantly less colonized by S. indica compared to wild-type roots and the foliar application of plant hormones, ethylene, or jasmonic acid, restores the colonization rate at least to the wild-type level. Further, hormone-treated mpk6 plants show typical S. indica-induced growth promotion effects. Moreover, expression levels of several genes related to plant defense and hormone signaling are significantly changed at different colonization phases. Our results demonstrate that the successful root colonization by S. indica depends on efficient suppression of plant immune responses. In A. thaliana, this process relies on intact hormone signaling in which MPK6 seems to play a pivotal role.
RESUMEN
Pectin in the primary plant cell wall is thought to be responsible for its porosity, charge density, and microfibril spacing and is the main component of the middle lamella. Plant-parasitic nematodes secrete cell wall-degrading enzymes that macerate the plant tissue, facilitating the penetration and migration within the roots. In sedentary endoparasitic nematodes, these enzymes are released only during the migration of infective juveniles through the root. Later, nematodes manipulate the expression of host plant genes, including various cell wall enzymes, in order to induce specific feeding sites. In this study, we investigated expression of two Arabidopsis pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780), together with pectic epitopes with different degrees of methylesterification in both syncytia induced by the cyst nematode Heterodera schachtii and giant cells induced by the root-knot nematode Meloidogyne incognita. We confirmed upregulation of PLL18 and PLL19 in both types of feeding sites with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Furthermore, the functional analysis of mutants demonstrated the important role of both PLL genes in the development and maintenance of syncytia but not giant cells. Our results show that both enzymes play distinct roles in different infected root tissues as well as during parasitism of different nematodes.
Asunto(s)
Arabidopsis/enzimología , Interacciones Huésped-Parásitos , Enfermedades de las Plantas/parasitología , Polisacárido Liasas/metabolismo , Tylenchida/fisiología , Tylenchoidea/fisiología , Animales , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/parasitología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pared Celular/metabolismo , Femenino , Regulación de la Expresión Génica de las Plantas , Genes Reporteros , Células Gigantes/citología , Masculino , Mutagénesis Insercional , Fenotipo , Raíces de Plantas/citología , Raíces de Plantas/enzimología , Raíces de Plantas/genética , Raíces de Plantas/parasitología , Brotes de la Planta/citología , Brotes de la Planta/enzimología , Brotes de la Planta/genética , Brotes de la Planta/parasitología , Polisacárido Liasas/genética , Plantones/citología , Plantones/enzimología , Plantones/genética , Plantones/parasitología , Especificidad de la Especie , Tylenchida/citología , Tylenchoidea/citología , Regulación hacia ArribaRESUMEN
The beneficial endophytic fungus Piriformospora indica colonizes the roots of many plant species, including the model plant Arabidopsis thaliana. Its colonization promotes plant growth, development, and seed production as well as resistance to various biotic and abiotic stresses. In the present work, P. indica was tested as potential antagonist of the sedentary plant-parasitic nematode Heterodera schachtii. This biotrophic cyst-forming nematode induces severe host plant damage by changing the morphogenesis and physiology of infected roots. Here it is shown that P. indica colonization, as well as the application of fungal exudates and cell-wall extracts, significantly affects the vitality, infectivity, development, and reproduction of H. schachtii.
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Arabidopsis/microbiología , Arabidopsis/parasitología , Basidiomycota/fisiología , Tylenchoidea/microbiología , Tylenchoidea/fisiología , Animales , Agentes de Control Biológico , Quimiotaxis , Microscopía Electrónica de Transmisión , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/prevención & control , Raíces de Plantas/microbiología , Raíces de Plantas/parasitología , Reacción en Cadena de la Polimerasa , Tylenchoidea/crecimiento & desarrolloRESUMEN
Pyk10 is a root and hypocotyl specific myrosinase from Arabidopsis thaliana. Northern analysis revealed the root specific expression of pyk10. In order to study the pyk10 promoter and the genomic structure of the gene, a genomic clone was isolated and sequenced. The clone contained the complete pyk10 gene and a promoter region of 3569 bp. The gene spans 2963 bp and consists of 12 exons and 11 introns, a structure that reflects the common gene organization of myrosinases. Within the promoter sequence, different development specific, organ specific, elicitor and plant hormone responsive regulatory elements could be identified, which also occur in other promoters. To determine the pattern of expression, four different 5'-promoter deletion fragments were linked to a ss-glucuronidase (gus) reporter gene and transformed into A. thaliana. The results demonstrated that the pyk10 promoter mediates a developmental gene activity with a strong emphasis in the root. Cis-acting sequences regulating root specific expression were identified to reside in the two promoter fragments B and C.
RESUMEN
Using a promoter tagging approach, a gene upregulated in nematode feeding structures (NFS) of Heterodera schachtii was identified in Arabidopsis thaliana plants. Sequence analysis of the transgenic line bearing gus reporter gene and the wild-type plant revealed that the T-DNA had been inserted into the promoter of the gene, however, with transcription start points at different sites for the gus reporter gene and for the endogenous gene. This tagged gene, designated pyk20, encodes a transcript of 2.6 kb. Southern blot analysis revealed a single gene copy for pyk20 in the Arabidopsis C-24 genome. Other cruciferous plants were shown to possess pyk20 or homologous genes. The predicted amino acid sequence of the PYK20 protein contains 695 residues with a molecular mass of 78 kDa and includes a glutamine-rich domain in the C-terminal region. IAA and kinetin treatment increased the level of the pyk20 transcript in the plant, whereas ABA treatment and temperature stress reduced the pyk20 transcript level. In-situ hybridisation and Northern blot analysis revealed that the gene is expressed in NFS. Based on homologies of the glutamine-rich domain, the biological role of the pyk20 gene product as a transcription factor is assumed.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Genes de Plantas/genética , Nematodos/crecimiento & desarrollo , Secuencia de Aminoácidos , Animales , Arabidopsis/parasitología , Secuencia de Bases , Brassicaceae/genética , ADN de Plantas/genética , ADN de Plantas/aislamiento & purificación , Dosificación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Glutamina/genética , Intrones , Datos de Secuencia Molecular , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Reguladores del Crecimiento de las Plantas/farmacología , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , ARN de Planta/efectos de los fármacos , ARN de Planta/genética , ARN de Planta/metabolismo , Temperatura , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The Hs1(pro-1) locus confers resistance to the beet cyst nematode (Heterodera schachtii Schmidt), a major pest in the cultivation of sugar beet (Beta vulgaris L.). The Hs1(pro-1) gene was cloned with the use of genome-specific satellite markers and chromosomal break-point analysis. Expression of the corresponding complementary DNA in a susceptible sugar beet conferred resistance to infection with the beet cyst nematode. The native Hs1(pro-1) gene, expressed in roots, encodes a 282-amino acid protein with imperfect leucine-rich repeats and a putative membrane-spanning segment, features similar to those of disease resistance genes previously cloned from higher plants.
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Clonación Molecular , Genes de Plantas , Proteínas de la Membrana/genética , Nematodos/patogenicidad , Enfermedades de las Plantas/genética , Proteínas de Plantas , Verduras/genética , Verduras/parasitología , Secuencia de Aminoácidos , Animales , Membrana Celular/química , ADN Complementario/genética , Prueba de Complementación Genética , Leucina/química , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia Molecular , Enfermedades de las Plantas/parasitología , Raíces de Plantas/genética , Raíces de Plantas/parasitología , Transformación GenéticaRESUMEN
In the quest for plant regulatory sequences capable of driving nematode-triggered effector gene expression in feeding structures, we show that promoter tagging is a valuable tool. A large collection of transgenic Arabidopsis plants was generated. They were transformed with a beta-glucuronidase gene functioning as a promoter tag. Three T-DNA constructs, pGV1047, p delta gusBin19, and pMOG553, were used. Early responses to nematode invasion were of primary interest. Six lines exhibiting beta-glucuronidase activity in syncytia induced by the beet cyst nematode were studied. Reporter gene activation was also identified in galls induced by root knot and ectoparasitic nematodes. Time-course studies revealed that all six tags were differentially activated during the development of the feeding structure. T-DNA-flanking regions responsible for the observed responses after nematode infection were isolated and characterized for promoter activity.
Asunto(s)
Arabidopsis/genética , Arabidopsis/parasitología , Genes de Plantas , Nematodos/patogenicidad , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Genes Reguladores , Genes Reporteros , Vectores Genéticos , Glucuronidasa/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Activación TranscripcionalRESUMEN
Phloem unloading of both the fluorescent probe carboxyfluorescein (CF) and 14C-labeled solutes was induced in Arabidopsis thaliana L. roots by the parasitic nematode Heterodera schachtii Schmidt. Confocal laser scanning microscopy demonstrated that anomalous unloading of CF from the sieve element companion cell complexes occurred specifically into the syncytium, the nematode-induced feeding structure located within the stele of the root. From this syncytial complex of modified root cells, both fluorescent and radioactive labels were withdrawn by feeding nematodes. Movement of CF was unidirectional from the phloem to the syncytium. A range of low-molecular-weight fluorescent probes (including CF) microinjected into the syncytium stayed in this structure, demonstrating that it is symplastically isolated from the surrounding root tissue. The mechanism of unloading in this host-pathogen relationship therefore appears to be apoplastic. Our results provide unequivocal evidence that sedentary cyst-forming nematodes have direct access to phloem-derived solutes.