RNA-seq analysis of the response of plant-pathogenic oomycete Phytophthora parasitica to the fungicide dimethomorph / Análisis RNA-seq de la respuesta del oomiceto fitopatógeno Phytophthora parasitica al fungicida dimetomorf

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

Inhibición del crecimiento y modificación genética de Phytophthora capsici usando quitosano de bajo grado de polimerización / Growth inhibition and genetic modification of Phytophthora capsici using chitosan with low degree of polymerization

Phytophtora capsici es un patógeno que incide sobre cultivos de la familia de las solanáceas causando pérdidas económicas en cultivos de pimientos, tomates, berenjenas y cur-cubitáceas. En este trabajo evaluamos el efecto del quitosano de bajo grado de polimerización (QBP) sobre el crecimiento de P. capsici y sobre la regulación génica de este fitopatógeno a nivel transcripcional. A una concentración de 0,4mg/l de QBP se obtuvo un 88% de inhibición en el crecimiento; concentraciones superiores a 1,6 mg/l inhibieron el crecimiento en un 100%. Mediante ensayos de cambio en la movilidad electroforética de ácidos nucleicos se comprobó que el quitosano interactúa con el ADN y el ARN del hongo frente a concentraciones entre 2 y 4 mg/l de ADN y entre 0,5 y 3 mg/l de ARN. Además, se efectuó un análisis de despliegue diferencial de los productos de amplificación por RT-PCR de los ARN mensajeros de P. capsici obtenidos en presencia o ausencia de QBP; este mostró cambios en el perfil de expresión inducidos por el tratamiento con quitosano. El análisis bioinformático de las secuencias de los transcritos expresados diferencialmente sugiere que el QBP afectó la regulación génica de elementos involucrados en la síntesis de quitina y de proteínas de unión a hidratos de carbono.

Phytophthora blight of peppers, caused by oomycete Phytophthora capsici, currently causes economic losses in crops such as peppers, tomatoes, eggplant and cucurbits. In this work, we evaluated the effect of chitosan with low degree of polymerization (LDP) on growth and gene expression of P. capsici cultures. LDP chitosan inhibited 88% of P. capsici mycelial growth at concentrations up to 0,4 mg/l, whereas at concentrations higher than 1,6 mg/l it completely inhibit growth. Gel mobility shift assays demonstrated that chitosan interacts with DNA and RNA of the fungus at concentrations ranging from 2 to 4 mg/l for DNA and 0,5 to 3 mg/l for RNA. The differential display analysis of RT-PCR-amplification products of P. capsici messenger RNA revealed changes in gene expression profiles after the chitosan treatment. Bioinformatic analysis of sequences from selected differentially-expressed bands showed the gene regulation of elements involved in chitin synthesis and carbohydrate-binding proteins.

Cloning, characterization and expression of a novel laccase gene Pclac2 from Phytophthora capsici

Laccases are blue copper oxidases (E.C. 1.10.3.2) that catalyze the one-electron oxidation of phenolics, aromatic amines, and other electron-rich substrates with the concomitant reduction of O2 to H2O. A novel laccase gene pclac2 and its corresponding full-length cDNA were cloned and characterized from Phytophthora capsici for the first time. The 1683 bp full-length cDNA of pclac2 encoded a mature laccase protein containing 560 amino acids preceded by a signal peptide of 23 amino acids. The deduced protein sequence of PCLAC2 showed high similarity with other known fungal laccases and contained four copper-binding conserved domains of typical laccase protein. In order to achieve a high level secretion and full activity expression of PCLAC2, expression vector pPIC9K with the Pichia pastoris expression system was used. The recombinant PCLAC2 protein was purified and showed on SDS-PAGE as a single band with an apparent molecular weight ca. 68 kDa. The high activity of purified PCLAC2, 84 U/mL, at the seventh day induced with methanol, was observed with 2,2'-azino-di-(3-ethylbenzothialozin-6-sulfonic acid) (ABTS) as substrate. The optimum pH and temperature for ABTS were 4.0 and 30 ºC, respectively . The reported data add a new piece to the knowledge about P. Capsici laccase multigene family and shed light on potential function about biotechnological and industrial applications of the individual laccase isoforms in oomycetes.

Genetic diversity and differentiation of Indian isolates of Phytophthora infestans as revealed by RAPD analysis.

Sixty-seven isolates of Phytophthora infestans collected from Himalayan hill regions and subtropical planes of India were characterized by RAPD markers to assess diversity and differentiation based on location of origin. Ten random decamer primers generated 161 polymorphic fragments. Association of P. infestans isolates on the dendrogram and PCO plot revealed two clear grouping based on geographical location of origin-hill isolates and plane isolates. Quantification of diversity by Shannon index of diversity analysis demonstrated that most of the diversity was present with a particular population (hill or plane) of P. infestans isolates, with 85% variation being within and 15% being between hill and plane isolates. Subtropical plane isolates of P. infestans exhibited higher variability compared to hill isolates and they were more dispersed on the PCO plot. No clear differentiation of isolates based on mating type was reflected on the dendrogram and PCO plot.