The NADPH Oxidases Nox1 and Nox2 Differentially Regulate Volatile Organic Compounds, Fungistatic Activity, Plant Growth Promotion and Nutrient Assimilation in Trichoderma atroviride.

Valter Cruz-Magalhães,John Hampton,Maria Fernanda Nieto-Jacobo,Johanna M Steyaert,Artemio Mendoza-Mendoza,Fabiola Padilla-Arizmendi,Diwakar Kandula,Janaki Kandula,Alfredo Herrera-Estrella,Alison Stewart,Leandro Lopes Loguercio,Michael Rostás,Eline Van Zijll De Jong

doi:10.3389/fmicb.2018.03271

Abstract

In eukaryotic systems, membrane-bound NADPH oxidases (Nox) generate reactive oxygen species (ROS) as a part of normal physiological functions. In the soil-borne mycoparasitic and plant facultative symbiont Trichoderma atroviride, Nox1 and the regulator NoxR are involved in differentiation induced by mechanical damage, while the role of Nox2 has not been determined. The knock-out strains Δnox1, ΔnoxR and Δnox2 were compared to the parental strain (WT) in their ability to grow and conidiate under a series of stress conditions (osmotic, oxidative, membrane, and cell-wall stresses). All three genes were differentially involved in the stress-response phenotypes. In addition, several interactive experiments with biotic factors (plant seedlings and other fungi) were performed comparing the mutant phenotypes with the WT, which was used as the reference strain. Δnox1 and ΔnoxR significantly reduced the antagonistic activity of T. atroviride against Rhizoctonia solani and Sclerotinia sclerotiorum in direct confrontation assays, but Δnox2 showed similar activity to the WT. The Δnox1, ΔnoxR, and Δnox2 mutants showed quantitative differences in the emission of several volatile organic compounds (VOCs). The effects of a blend of these volatiles on plant-growth promotion of Arabidopsis thaliana seedlings were determined in closed-chamber experiments. The increase in root and shoot biomass induced by T. atroviride VOCs was significantly lowered by ΔnoxR and Δnox1, but not by Δnox2. In terms of fungistatic activity at a distance, Δnox2 had a significant reduction in this trait against R. solani and S. sclerotiorum, while fungistasis was highly increased by ΔnoxR and Δnox1. Identification and quantification of individual VOCs in the blends emitted by the strains was performed by GC-MS and the patterns of variation observed for individual volatiles, such as 6-Pentyl-2H-pyran-2-one (6PP-1) and (E)-6-Pent-1-enylpyran-2-one (6PP-2) were consistent with their negative effects in plant-growth promotion and positive effects in fungistasis at a distance. Nox1 and NoxR appear to have a ubiquitous regulatory role of in a variety of developmental and interactive processes in T. atroviride either as positive or negative modulators. Nox2 may also have a role in regulating production of VOCs with fungistatic activity.

Highlights

Reactive Oxygen Species (ROS) are small molecules derived from oxygen, capable of interacting with proteins, lipids, carbohydrates, nucleic acids, and several other small organic molecules (Bedard and Krause, 2007; Hernandez-Oñate and Herrera-Estrella, 2015)
Both noxR and nox1 were distinct from the wildtype strain (WT) for all conditions tested, with exception of cell-wall stresses
Our dataset suggests that NoxR and Nox1 proteins are relevant for cell metabolisms that deal with growth, development, reproductive differentiation, and common biotic interactions with plants and other fungi

Summary

Introduction

Reactive Oxygen Species (ROS) are small molecules derived from oxygen, capable of interacting with proteins, lipids, carbohydrates, nucleic acids, and several other small organic molecules (Bedard and Krause, 2007; Hernandez-Oñate and Herrera-Estrella, 2015). Most ROS are produced by NADPH oxidases (Nox family), which transfer electrons from NADPH to oxygen (Bedard and Krause, 2007; Marschall and Tudzynski, 2016), and play a critical role in host defense, posttranslational modification of proteins, cell communication, regulation of gene expression, and cell differentiation (Aguirre et al, 2005; Cano-Dominguez et al, 2008; Hernandez-Oñate et al, 2012; Hernandez-Oñate and HerreraEstrella, 2015). NoxR has been shown to regulate activation of Nox1/A and Nox2/B (Takemoto et al, 2007; Scott and Eaton, 2008; Tanaka et al, 2008; Marschall and Tudzynski, 2016) as its gene deletion has resulted in phenotypes similar to those of nox and nox (CanoDominguez et al, 2008; Segmuller et al, 2008; Scott, 2015)

Methods

Results

Conclusion