Changes in cellular infrastructure after induced endoplasmic reticulum stress in Moniliophthora perniciosa

Abstract

Moniliophthora perniciosa is a basidiomycete fungus that causes witches’ broom disease in Theobroma cacao. We analyzed the morphology and survival of fungal hyphae and endoplasmic reticulum (ER) remodeling in either glucose- or glycerol-grown M. perniciosa after treatment with ER stress-inducing chemicals dithiothreitol (DTT) or tunicamycin (TM). Changes in intracellular redox potential can cause endoplasmic reticulum (ER) stress due to diminished efficiency in protein folding that could in turn reduce cell survival. Such stress diminishes protein-folding efficiency that could in turn reduce cell survival. Light microscopy revealed morphological changes in hyphae after TM but not after DTT treatment, regardless of the media carbon source. Decrease in fungal survival, after both TM and DTT treatments, was dose-dependent and glycerol-grown cells showed a higher resistance to both chemicals compared to glucose-grown cells. Electron microscopy showed TM and DDT-induced ER stress in M. perniciosa as evidenced by structural alterations of the organelle. The volume of ER structures increased as a typical consequence of unfolded protein stress, and the number of autophagosomes was higher. In glycerol-grown fungus DTT treatment slightly induced expression of molecular chaperone BiP. The TM exposure-induced expression of gene MpIRE1, involved in signaling of the unfolded protein response, was higher in glycerol than glucose-grown cells. Such difference was not observable with expression of gene MpATG8, encoding a key protein in autosome formation, that was induced 1.4-fold and 1.2-fold in glucose or glycerol-grown cells, respectively. DHE-based fluorescence assay showed M. perniciosa oxidative stress induced by H₂O₂, and treated cells had a higher level of oxidative stress compared to control. A comprehensive study of remodeling of ER is important in understanding M. perniciosa fungus resistance to oxidative stress and its ability to implement a successful infection in T. cacao.

• autophagosomes
• endoplasmic reticulum expansion
• ER stress
• witches’ broom disease

Acknowledgments

Evelyn Vita-Santos, Tatiana Setenta Basso and Cristina Pungartnik held CNPq ( Conselho Nacional de Desenvolvimento Científico e Tecnológico) fellowships. MB held a FAPESB fellowship (Fundação de Apoio a Pesquisa no estado da Bahia). CNPq and FAPESB also provided financial support. We thank Dr Fátima C. Alvim for providing BiP antibody and the staff of the Centro de Microscopia at UESC for valuable contributions, especially Dr Eduardo Gross.

Notes

Authors’ contributions

Tatiana Setenta Basso and Evelyn Vita-Santos contributed equally to this work. Gabriele Marisco performed the fluorescent experiments. Martin Brendel and Cristina Pungartnik devised experimental procedures and contributed in writing and editing the manuscript.