Beneficial and pathogenic Arabidopsis root-interacting fungi differently affect auxin levels and responsive genes during early infection

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within three hours after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within one day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content

Studies towards the dereplication of antibiotic constituents in Alternanthera brasiliana and associated microorganisms

Alternanthera brasiliana is widely used by Brazilian people as a folk medicine for the treatment of numerous diseases. However, little is known about its chemical composition and the microorganisms that live in association with it. This work proposed the use of dereplication strategies to establish a correlation between chemical compounds present in the plant and the antibiotic activity associated with it. These strategies were also used for study endophytic micro-organisms associated with this plant, since the metabolites present in plant tissues are often result of interaction between plants and different factors present in their habitat, such as micro-organisms. 50 strains of endophytic micro-organisms associated to Alternanthera brasiliana were isolated. High-Throughput Screening strategies, which envolved analytical techniques as LC-PDA-MS, LC-PDA-MS/MS, LC-SPE-NMR and antibiosis assays allowed identification of antibiotic compounds present in the A. brasiliana stems. These compounds are member of oxylipins group, among which we highlight the 13-Z,E-KODE, 13-E,E-KODE, 9-E,Z-KODE and 9-E,EKODE. Furthermore, were identified some depsipeptides compounds produced by a strain of Fusarium fungus, isolated as endophyte of A. brasiliana. Beauvericin, which is one of these compounds , is known for its antibiotic and insecticide activities. This compound was also found in the plant extracts, suggesting that endophyte microorganism could play a role in producing antibiotic compounds inside plant tissues.Universidade Federal de Minas GeraisA planta Alternanthera brasiliana é popularmente conhecida por suas diversas atividades biológicas. Assim, o presente trabalho pretende contribuir com o uso de estratégias de desreplicação a fim de estabelecer uma correlação entre os compostos químicos presentes nesta planta e a atividade antibiótica associada a ela. Além disso, essas estratégias de desreplicação foram utilizadas para o estudo dos micro-organismos endofiticos associados a esta planta, uma vez que os metabólitos secundários presentes nos tecidos das plantas muitas vezes são resultado da interação desta planta com diversos fatores presentes em seu habitat, e dentre eles a interação com microorganismos merece destaque. Foram isoladas 50 cepas de micro-organismos endofiticos associados à planta Alternanthera brasiliana. O uso de abordagens de High Throughput Screening aliadas a técnicas analíticas como LC-PDA-MS, LC-PDA-MS/MS, LC-SPE-NMR e ensaios de antibiose permitiram a identificação dos compostos antibióticos presentes no caule de A. brasiliana. Esses compostos pertencem a classe das oxilipinas, dentre os quais se destacam o 13-Z,E-KODE, 13-E,E-KODE, 9-E,Z-KODE e 9-E,E-KODE. Tambem foram identificados compostos da classe dos depsipeptídeos nos extratos de uma cepa do gênero Fusarium, isolada como endofítico de A. brasiliana. Dentre os depsipeptídeos encontrados, a beauvericina estava presente em grande quantidade no extrato deste micro-organismo. Este composto, conhecido por suas atividades antibiótica e inseticida, foi encontrado também no extrato da planta, sugerindo que o micro-organismo endofítico poderia estar produzindo compostos desta classe no interior dos tecidos da planta, onde ele está alojado

Bioatividade, diversidade química e microbiana associadas à Alternanthera brasiliana : a influência de micro-organismos endofíticos na produção de compostos antibióticos

Alternanthera brasiliana is a plant widely used by Brazilian people as a folk medicine for the treatment of numerous diseases, especially due to its antibiotic properties. Using dereplication strategies, we identified antibiotic compounds as modified fatty acids, which are closely related to oxylipins. The biosynthesis of these compounds could be related with the presence of endophytic bacteria. Thus, we established axenic cultures of A. brasiliana and developed methods for detection and quantification of bacterial cells in plant tissues. We also performed initial re-inoculation experiments with endophytic bacteria isolated from A. brasiliana. In addition, method for quantification of phytohormones and oxylipins in plant tissues were developed. The results reinforced the correlation between both the amount of bacteria and oxylipins in A. brasiliana tissues.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Alternanthera brasiliana é uma planta amplamente utilizada na medicina popular brasileira para o tratamento de diversas doenças, principalmente devido a suas propriedades antibióticas. Durante este trabalho, utilizou-se estratégias de desreplicação a fim de se identificar os compostos antibióticos presentes nesta planta. Esses compostos foram identificados como ácidos graxos oxidados pertencentes a classe das oxilipinas. A biossíntese destes compostos esta comumente relacionada a situações de estresse biótico e abiótico em plantas. Dessa forma, nos estabelecemos métodos para a detecção e quantificação de células bacterianas em tecidos vegetais. Também foram realizados experimentos iniciais de re-inoculação de bactérias endofíticas isoladas de A. brasiliana. Além disso, foram desenvolvidos métodos para a quantificação de fitohormônios e oxilipinas em tecidos vegetais. Os resultados obtidos até o momento ressaltam a possibilidade de bactérias endofíticas influenciarem a produção de oxilipinas (compostos antibióticos) na planta A. brasiliana..FAPESP: 2007/03771-7FAPESP: 2008/07494-0FAPESP: 2010/17925-9FAPESP: 2012/20932-

Hyperspectral imaging to characterize plant–plant communication in response to insect herbivory

Abstract Background In studies of plant stress signaling, a major challenge is the lack of non-invasive methods to detect physiological plant responses and to characterize plant–plant communication over time and space. Results We acquired time series of phytocompound and hyperspectral imaging data from maize plants from the following treatments: (1) individual non-infested plants, (2) individual plants experimentally subjected to herbivory by green belly stink bug (no visible symptoms of insect herbivory), (3) one plant subjected to insect herbivory and one control plant in a separate pot but inside the same cage, and (4) one plant subjected to insect herbivory and one control plant together in the same pot. Individual phytocompounds (except indole-3acetic acid) or spectral bands were not reliable indicators of neither insect herbivory nor plant–plant communication. However, using a linear discrimination classification method based on combinations of either phytocompounds or spectral bands, we found clear evidence of maize plant responses. Conclusions We have provided initial evidence of how hyperspectral imaging may be considered a powerful non-invasive method to increase our current understanding of both direct plant responses to biotic stressors but also to the multiple ways plant communities are able to communicate. We are unaware of any published studies, in which comprehensive phytocompound data have been shown to correlate with leaf reflectance. In addition, we are unaware of published studies, in which plant–plant communication was studied based on leaf reflectance