Exogenous auxin affects the oxidative burst in barley roots colonized by Piriformospora indica

Abstract

Beside a cardinal role in coordination of many developmental processes in the plant, the phytohormone auxin has been recognized as a regulator of plant defense. The molecular mechanisms involved are still largely unknown. Using a sensitive chemiluminescence assay, which measures the oxidation of luminol in the presence of H₂O₂ by horseradish peroxidase (HRP), we report here on the ability of exogenously added indole-3-acetic acid (IAA) to enhance the suppressive effect of the root endophyte Piriformospora indica on the chitin-elicited oxidative burst in barley roots. Thus, the potential of P. indica to produce free IAA during the early colonization phase in barley might provide the symbiont with a means to interfere with the microbe-associated molecular patterns (MAMP)-triggered immunity.

Introduction

The beneficial root endophyte Piriformospora indica induces growth in many different hosts and is able to produce auxin.^1,2 In order to clarify the role played by fungal-derived auxin in the mutualistic interaction with barley, we recently analyzed the P. indica biochemical pathways involved in IAA production.¹ We showed elevated levels of free IAA in barley roots during the early biotrophic colonization and that exogenous IAA induced susceptibility to P. indica in this host. Silencing of the piTam1 gene, encoding a tryptophan aminotransferase, via an RNA interference (RNAi) approach resulted in P. indica strains compromised in IAA production and in reduced colonization of barley roots in the biotrophic phase, further supporting the role of IAA in the establishment of P. indica-barley symbiosis. Attenuated IAA production by the P. indica RNAi strains did not hamper growth promotion. The implication of IAA in biotrophic colonization but not in the elicitation of growth promotion prompted us to test the role of IAA in the generation of reactive oxygen species (ROS) and plant defense. ROS are important signaling molecules that are rapidly generated in response to abiotic and biotic stimuli and which regulate diverse physiological processes such as stomatal aperture, root gravitropism and cell death.^3-6 Plant peroxidases (POXs) are thought to be one of the mediator of ROS production in plants and beside their important role in plant defense, POXs bound to the cell wall were shown to be involved in cell wall loosening via the production of hydroxyl radicals (OH^.) together with NAD(P)H-oxidases which catalyze the formation of superoxide radicals (O₂^.-) at the plasma membrane.^7-11 The ability to produce hydroxyl radicals by exogenous IAA was shown for several plant systems, including the cell suspension culture of Chenopodium rubrum and maize coleoptiles.^6,12,13 There is growing evidence that POXs are the mediators of the IAA-induced cell wall loosening.^{8,10,12,14,15} However, the potential of IAA-elicited ROS production in root defense is unclear. Plants recognize microbial invaders (pathogens or mutualistic symbionts) by detecting conserved microbial structures, so-called microbe-associated molecular patterns (MAMPs) or pathogen-associated molecular patterns (PAMPs), such as fungal chitin. The elicited defense reaction is defined as MAMP/PAMP-triggered immunity (MTI or PTI, respectively). Perception of MAMPs induces an extracellular oxidative burst with production of ROS, which requires the function of the membrane bound NADPH oxidase in Arabidopsis.¹⁶ In this report we provide first evidence that IAA potentiates the suppressive effect of P. indica on the oxidative burst leading to the speculation that exogenous application of auxin, e.g., produced by microbes, increases susceptibility via manipulation of the root defense.

Results and Discussion

Combined treatment of barley roots with IAA and P. indica suppresses the chitin-elicited oxidative burst

ROS can be easily measured by chemiluminescence with a luminol assay in the presence of a catalyst, such as a peroxidase (e.g., HRP from the roots of horseradish, Fig. 1A). The horseradish peroxidase uses H₂O₂ in a first reaction step which converts the enzyme into the two-electron oxidized species known as compound I. Compound I reacts with a substrate, e.g., with luminol anion to form a luminol radical and the half-reduced compound II which returns to the native state by oxidizing a second molecule of luminol.^10,17

Figure 1. Analyses of chitin-elicited oxidative burst using the luminol-horseradish peroxidase chemiluminescence reaction. (A) Preparation of barley root segments. (B) Effect of P. indica colonization on the chitin-elicited oxidative burst. Error bars represent SE of the mean from 12 biological repetitions. Significant differences in chitin-elicited oxidative burst between P. indica-colonized and mock treated roots are visible (t-test_t(0–20) p < 0.05). Three independent replicate experiments were performed with similar results. (C) Effect of IAA on the chitin-elicited oxidative burst in P. indica colonized roots. ROS production was measured after 10 min pre-incubation with or without 1 µM IAA and subsequent elicitation with a chitin solution or with water (control). Error bars represent SE of the mean from 12 biological repetitions. Significant suppression of chitin-elicited oxidative burst in P. indica-colonized roots pretreated with 1 µM IAA is visible (t-test_t(0–20) p < 0.01). Two independent replicate experiments were performed with similar results. (D) Effect of IAA on the chitin-elicited oxidative burst in mock treated barley roots. ROS production was measured after 10 min pre-incubation with or without 1 µM IAA and subsequent elicitation with a chitin solution or with water (control). Error bars represent SE of the mean from 12 biological repetitions. Significant enhancement of chitin-elicited oxidative burst in roots pretreated with 1 µM IAA is visible (t-test_t(0–20) p < 0.05). Two independent replicate experiments were performed with similar results.

HRP + H₂O₂ → HRP-I + H₂O

HRP-I + LH^- → HRP-II + L^.- + H₂O

HRP-II + LH^- → HRP + L^.-

Where LH^- is the substrate, e.g., luminol, and HRP-I and HRP-II are the intermediates formed from HRP by electron donation from the substrate to form the luminol radical L^.-.

It was reported that P. indica can suppress the MAMP-elicited oxidative burst reaction in Arabidopsis and in barley.^18,19 In this study we confirm this data and additionally we show that exogenously added IAA enhances P. indica’s ability to suppress the chitin-induced oxidative burst (Fig. 1B and C). IAA alone did not suppress the chitin-induced oxidative burst and pretreatment of the non-inoculated roots with 1 µM IAA in the presence of HRP resulted in a stronger chemiluminescence upon addition of chitin compared with non-IAA treated roots, showing a potentiating effect of the IAA on the luminol-roots-horseradish peroxidase reaction (Fig. 1D). It was reported that IAA can be oxidized by HRP in the presence of O₂ yielding IAA cation radicals and O₂^.- superoxide radicals that rapidly undergoes dismutation in aqueous solution to H₂O_2.^10,14

HRP^Fe3+ + IAA ↔ [HRP-IAA]

[HRP-IAA] + O₂ ↔ [HRP-IAA-O₂]

[HRP-IAA-O₂] → HRP + IAA^.+ + O₂^.-

In our assay addition of IAA at the concentration of 1 and 10 µM did not induce an oxidative burst reaction in the roots of barley neither these concentrations of IAA produced chemiluminescence in the luminol-HRP system in the absence of barley roots (Fig. 2A and data not shown). Addition of 100 and 1,000 µM IAA induced the production of a detectable amount of H₂O₂ in the presence and absence of barley roots, confirming that IAA can act as a substrate for the HRP in an O₂-dependent oxidative reaction (Fig. 2A and data not shown). HRP can oxidize IAA via a second known mechanism that requires H₂O₂ but does not display strict substrate specificity:^10,14

Figure 2. Effect of exogenously applied IAA on luminol-horseradish peroxidase chemiluminescence with and without barley roots. (A) Auxin-dependent induction of H₂O₂ production in barley roots. The values are given as the average of the chemiluminescence measured over 10 min after application of different concentrations of IAA. Error bars represent SE of the mean from 12 biological repetitions. Asterisks represent significant differences (ANOVA *p < 0.01). Two independent replicate experiments were performed with similar results. (B) Effect of P. indica and IAA on HRP activity after H₂O₂ treatment. Working solutions with and without P. indica germinated chlamydospores were pre-incubated with or without 1 µM IAA for 10 min. Subsequently, 1 µM H₂O₂ was applied in order to start the HRP-dependent luminol oxidation reaction. Working solutions without HRP were used as controls. Error bars represent SE of the mean from 12 biological repetitions. Two independent replicate experiments were performed with similar results.

HRP + H₂O₂ → HRP-I + H₂O

HRP-I + IAA → HRP-II + P

HRP-II + IAA + H⁺ → HRP + H₂O + P

Where P is the product of one-electron oxidation of IAA. Additionally, it was shown that compounds with radicals with a greater or lower reduction potential than luminol can act as enhancers or inhibitors of chemiluminescence in the luminol-H₂O₂-HRP system. In particular, phenol derivatives can function as enhancers or inhibitors of the luminol-H₂O₂-HRP chemiluminescence in a conventional H₂O₂-dependent HRP cycle:¹⁷

HRP + H₂O₂ → HRP-I + H₂O

HRP-I + EH → HRP-II + E· + H₂O

HRP-II + EH → HRP + E·

LH^- + E· ↔ L^.- + EH

Where EH represent the chemiluminescence enhancer and E· is the enhancer radical that convert luminol into the luminol radicals favoring the step of light emission. This leads to the speculation that the indolic compound IAA might also accelerate or retard the formation of luminol radicals. In our study IAA was not able to enhance or suppress the luminol-H₂O₂-HRP chemiluminescence in the absence of barley roots (Fig. 2B). This suggests that the higher elicited levels of H₂O₂ in the IAA-pretreated roots following addition of chitin is not due to the H₂O₂-dependent oxidation of IAA by HRP neither to the production of IAA radicals with higher reduction potential than luminol, but it may represent an auxin priming effect on the roots. The presence of the endophyte in this system overturned the situation and led to the suppression of the chitin-elicited burst in the presence of IAA. In the absence of host roots, P. indica was not able to suppress the luminol-H₂O₂-HRP chemiluminescence upon pre-incubation with IAA, showing that the IAA-P. indica induced suppression of the oxidative burst reaction in barley roots is not a direct effect of P. indica on IAA, HRP or H₂O₂ but rather a combined effect of P. indica and barley roots (Fig. 2B). Further analyses are required to clarify if the suppression of the chitin-elicited oxidative reaction by IAA in P. indica colonized roots is a consequence of an increased H₂O₂ scavenging activity or it resides upstream of H₂O₂ production in the root. A possible explanation for this might be that upon colonization of barley roots by P. indica a considerable level of fungal-derived peroxidases that can oxidize IAA in the presence of H₂O₂ but not in the presence of O₂¹⁴ accumulate in the extracellular milieu. These enzymes possibly function as scavenger and prevent the accumulation of the signaling molecule H₂O₂ upon chitin-elicitation. Under this condition low amount of exogenous IAA would serve as antioxidant and hence provide the symbiont with a means to interfere with barley MAMP-triggered immunity.

Conclusions

Detection of H₂O₂ via luminol-horseradish peroxidase chemiluminescence in the supernatant of plant leaves and roots is often used to study the effect of microbes and elicitors/effectors on the MAMP-induced oxidative burst reaction. The sensitivity and simplicity of this assay make it attractive for a wide range of biological questions, but it should not be forgotten that in this detection system there are several players, such as HRP, luminol, H₂O₂, plant cells and the microbes and therefore the effects detected might be multilayered making the interpretation of the data difficult.

Figure 3 summarizes the results of the luminol-HRP assay in the presence of barley roots colonized and non-colonized by P. indica. Although the mechanism of ROS suppression by exogenous IAA on P. indica colonized roots of barley remains unclear, our results indicate a potential role of IAA in chitin-elicited oxidative burst. The success of microbes to invade plants reflects their ability to evade and/or to manipulate the immune response and reprogram host metabolism. Many microbes are known to produce auxin during colonization of the host plant were it interferes with plant developmental processes and defense. In this report we provided some hints on the ability of free IAA to possibly interfere with MAMP-triggered immunity. The characterization of the IAA effects on POXs and on roots will be an important step in the understanding of the mechanism by which microbial-derived IAA influence defense in host plants.

Figure 3. Schematic overview of the influence of IAA and P. indica on the chitin-elicited ROS production in barley roots. Chitin-elicited oxidative burst is suppressed in the presence of P. indica. This effect is potentiated by exogenous application of 1 µM IAA.

Materials and Methods

Oxidative burst assay

Three-day-old barley germlings (Hordeum vulgare L. cv Golden Promise) were inoculated with P. indica (5 × 10⁵ chlamydospores/ml) or mock treated and cultivated on 1/10 PNM medium in sterile jars for 3 d in a growth chamber (Persival AR-95XL) with a day/night cycle of 16/8 h (light intensity: approx. 390 μmol m²/s) and temperature of 22/18°C. For determination of the oxidative burst, the part of the root corresponding to the elongation zone (first 4 cm below the seed) was subjected to a luminol-based assay. The 0.5-cm-long root segments were incubated overnight in H₂O and then transferred to a 96-well plate (2 pieces per well, corresponding to approximately 10 mg fresh weight) with 195 µl of working solution [3.4 mg/l Luminol (Sigma); 0.4 mM NaOH and 10 µg/ml horseradish peroxidase (Sigma) in sterile ddH₂O] and 5 µl of 0.2 M sodium phosphate buffer pH 8. Luminescence was measured over 40 min after elicitation with 50 µl of chitin solution (approximately 10 mg/ml, Sigma) with Tecan Infinite^® PRO 200 microplate multimode reader (Tecan). Effect of exogenous auxin on oxidation of luminol in the presence of barley roots was assessed by application of 1, 10, 100 or 1,000 µM IAA in the working solution. Additionally, influence of 1 µM IAA and/or P. indica germinated chlamydospores on luminol oxidation was examined in the absence of roots. 200 µl of working solution with or without HRP buffered with sodium phosphate was mixed with approximately 3 × 10⁵ P. indica chlamydospores/ml or 50 µl sterile water and pre-incubated 10 min with or without 1 µM IAA. Oxidation of luminol was started by application of 1 µM H₂O₂. For each assay 12 biological replicates and two to three independent replicate experiments were performed.

Glossary

Abbreviations:

HRP

horseradish peroxidase

IAA

indole-3-acetic acid

MAMP

microbe-associated molecular patterns

POXs

plant peroxidases

OH^.

hydroxyl radicals

O₂^.-

superoxide radicals

Hilbert M, Voll LM, Ding Y, Hofmann J, Sharma M, Zuccaro A. Indole derivative production by the root endophyte Piriformospora indica is not required for growth promotion but for biotrophic colonization of barley roots. New Phytol. 2012;196:520–34. doi: 10.1111/j.1469-8137.2012.04275.x.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.