Unveiling the mechanisms for the plant volatile organic compound

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Agricultural and Environmental Chemistry

Unveiling the mechanisms for the plant volatile organic compound linalool to control gray mold on strawberry fruits Yanqun Xu, Zhichao Tong, Xing Zhang, Youyong Wang, Weiguo Fang, Li Li, and Zisheng Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03103 • Publication Date (Web): 30 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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Journal of Agricultural and Food Chemistry

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Unveiling the mechanisms for the plant volatile organic compound linalool to

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control gray mold on strawberry fruits

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Yanqun Xu1, 3, Zhichao Tong1, Xing Zhang2, Youyong Wang1, Weiguo Fang2, Li Li1,

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Zisheng Luo1, 3*

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1. Zhejiang University, College of Biosystems Engineering and Food Science, Key

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Laboratory of Agro-Products Postharvest Handling of Ministry of Agriculture and

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Rural Affairs, Zhejiang Key Laboratory for Agri-Food Processing, Hangzhou,

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310058, People’s Republic of China

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2. Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Hangzhou, 310058, People’s Republic of China 3. Ningbo Research Institute, Zhejiang University, Ningbo 315100, People’s Republic of China

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Corresponding author

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Zisheng Luo

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College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou,

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310058, People’s Republic of China. E-mail: [email protected]. Phone: +86-571-

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88982175

ACS Paragon Plus Environment


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Graphic for table of contents


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Highlights

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1. Linalool content of strawberry increased after Botrytis cinerea infection.

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2. Linalool fumigation inhibited the fungi infection on strawberry.

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3. Linalool caused damage to the fungi cell and mitochondrial membranes.

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4. Linalool induced ROSs accumulation and caused oxidative stress on B. cinerea.

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5. Linalool slowed down the rates of transcription and translation of B. cinerea.



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Abstract

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Fungal infections significantly alter the emissions of volatile organic compounds

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(VOCs) by plants, but the mechanisms for VOCs affect fungal infections of plants

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remain largely unknown. Here, we found that infection by Botrytis cinerea upregulated

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linalool production by strawberries, and fumigation with linalool was able to inhibit the

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infection of fruits by the fungus. Linalool treatment downregulated the expression of

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rate-limiting enzymes in the ergosterol biosynthesis pathway, and this reduced

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ergosterol content in the fungi cell membrane and impaired membrane integrity.

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Linalool treatment also caused damage to mitochondrial membranes by collapsing

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mitochondrial membrane potential, and it also downregulated genes involved in ATP

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production, resulting in a significant decrease in ATP content. Linalool treatment

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increased the levels of ROSs, in response to which the treated fungal cells produced

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more of the ROS scavenger pyruvate. RNA-Seq and proteomic analysis data showed

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that linalool treatment slowed down the rates of transcription and translation.

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Keywords: volatiles, linalool, Botrytis cinerea, strawberry, ATP production,

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mitochondrial membranes, oxidative stress


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1. Introduction

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Plants vigorously attempt to prevent pathogen invasion and outgrowth by

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activating multiple defensive pathways, including the production of antibacterial

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metabolites and pathogenesis-related (PR) proteins 1. Many previous studies have

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revealed the functions of plant VOCs as an essential and invisible language that is

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involved in communication between plants, insect pests, and natural enemies

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Microbial infections can significantly alter the emission of volatile organic compounds

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(VOCs) by plants 4 and the pathogen induced volatiles alternation had been used as a

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neotype of signal to evaluate the disease incidence and development in crops at

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greenhouse scale 5. For examples, wheat plants infected by Fusarium spp. emitted

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volatile chemicals that differ both quantitatively and qualitatively from undamaged

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plants, and among them linalool, linalool oxide and β‐farnesene were the major

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compounds changed 6. Microbe-induced plant volatiles could directly inhibit pathogen

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growth and indirectly promote resistance/susceptibility to subsequent plant pathogen

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attack 7. Modification of the biosynthesis of green leaf volatiles in plants via genetically

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engineer was proved to be an effective approach for improving plant resistance against

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both herbivores and pathogens in of Arabidopsis 8. Additionally, numerous studies

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reported VOC-mediated priming of resistance to pathogens in plants 9. Plant-derived

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volatile nonanal significantly enhanced PR genes expression in bean, resulting in

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reduced disease symptom appearance caused by Pseudomonas syringae pv syringae 10.

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Likewise, the direct vitro inhibitory effects of the plant essential oils containing

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terpenes on plant pathogenic fungi growth had been studied widely in vitro by using

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mycelial growth inhibitory technique

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(R)-camphor, (R)-carvone, 1,8-cineole, cuminaldehyde, (S)-fenchone, geraniol, (S)-

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limonene, (R)-linalool, (1R,2S,5R)-menthol, myrcene and thymol showed antifungal

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activities against plant pathogenic fungi, including Rhizoctonia solani, Fusarium

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oxysporum, Penecillium digitatum and Asperigallus niger 15.

11-14.

2, 3.

Diverse monoterpenes, such as camphene,

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Botrytis cinerea (teleomorph: Botryotinia fuckeliana) is an airborne plant

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pathogenic fungus that can infect over 200 crop hosts worldwide and it ranked second

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into the world Top 10 fungal plant pathogens list based on scientific and economic



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importance 16. As a typical soft tissue fruit, strawberry (Fragaria × ananassa, Duch) is

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a host of B. cinerea, which causes gray mold as the major disease occurring in the field

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and after harvest

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maximum incidence of 89 % in the second fruit harvest season

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destined for long-term storage are generally treated with various fungicides to control

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gray mold, which has resulted in serious health concerns 19. Therefore, alternative safe

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and effective control options for gray mold are urgently needed. Several environmental

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friendly approaches have been studied recently to control postharvest fungal rotting of

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fruit and vegetables with encouraging results from the use of natural products 20, 21.

17.

The main losses occur during the postharvest phase, with a 186.

Strawberries

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A complex blend of more than 300 VOCs has been identified in strawberries,

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among which terpenes are a major volatile class. Linalool is the most abundant

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monoterpene with a low odor threshold value 22. This acyclic monoterpene has a sweet,

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floral, and citrus-like odor

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strawberries and the infection by M. fragariae can upregulate its production in

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strawberry leaves 24. The applications of plant volatile terpenes as crop disease control

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agent had been tested in the studies of plant essential oils against plant fungal pathogens

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25-27.

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stolonifera was decreased up to 70 % by the essential oils from Thymus vulgaris

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Carvacrol and thymol mixture showed good inhibition on the germination and

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mycelium growth of B. cinerea and the volatiles had been loaded with thermoplastic

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starch/clay nanocomposites as packaging for strawberry during postharvest storage 28.

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A previous study showed that the linalool monomer can inhibit the growth of B. cinerea

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on an artificial medium 29. Thus, linalool fumigation treatment presented high potential

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to be a new approach for postharvest disease control of fruits.

23.

Linalool is constitutively produced by healthy

The postharvest decay of strawberry fruits caused by B. cinerea and Rhizopus 11.

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It had been reported that the monoterpenes thymol and (S)-limonene affected the

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expression of important fungal metabolic enzymes, including pectin methyl esterase,

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cellulase and polyphenol oxidase

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Mentha piperita oil with major component of

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menthol caused intracellular ROS accumulation, mitochondrial fragmentation and

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chromatin condensation 30. The ocimum sanctum essential oil with linalool as one of

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the main ingredient showed synergistic inhibition of H+ extrusion of fungal cell


31.

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Although the antifungal effect of linalool had been revealed in some studies, the

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underlying mechanism of the impacts that linalool exerts on the fungal physiology was

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far from clear. In this study, the interactive mechanisms between the volatile linalool

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and B. cinerea were revealed. Here, we found that B. cinerea infection upregulated

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linalool production by strawberry fruits, and exogenous application (fumigation) with

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linalool was able to inhibit infection of the fruits by the fungus. To better understanding

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the role of linalool in the plant-microbe interactions, we further investigated the key

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mechanisms for linalool to inhibit the growth of gray mold at the biochemical and

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transcriptomic level.

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2. Materials and methods

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2.1 Fungal strains and growth conditions

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Botrytis cinerea Persoon (ATCC 48342) was purchased from the American Type

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Culture Collection (Rockville, MD, USA). Malt extract agar (MEA) and malt extract

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broth (MEB) were purchased from Oxoid, Basingstoke, UK.

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2.2 MFC and MIC assays

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Minimum inhibition concentration (MIC) and the minimum fungicidal

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concentration (MFC) of linalool were determined by the half dilution method as

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previously described 32. MFC was the lowest dose of linalool that completely inhibited

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spore germination in MEB at 25 °C for 9 h. MIC was the lowest dose that significantly

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inhibited the growth of mycelium cultured at 25 °C in MEB for 9 h.

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2.3 Fungal inoculation and linalool fumigation

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Strawberry fruits (Fragaria × ananassa Duch. “Akihime”) with 70 % red were

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hand-harvested from greenhouses and transported to the lab within 1 h. Fruits with

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homogeneous qualities were sterilized by soaking in 1 % sodium hypochlorite for 5

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min and washing with sterile water three times. Five microliters of B. cinerea spore

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suspension (1 × 104 spores mL–1) were inoculated on the equator. Thirty fruits were

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then placed in a 266 × 205 ×166 mm (volume of 9 L) LOCK&FRESH crisper as one

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replicate. For linalool fumigation, 1.5 mL of linalool solution (125.7 mM in 0.05 % (v/v)

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Tween 80), was dispersed on filter paper stuck inside the crisper to create a final vapor

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linalool concentration of 20.95 µM. The control was Tween 80 solution. The crisper



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was sealed immediately. After incubation at 23 °C for 24 h, the lip of the crisper was

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removed, and the container with fruits inside was incubated at 23 °C with 90%

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humanity for an additional 4 days. Disease incidence and lesion diameters were

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recorded daily. The experiment was repeated two times with two replicates per repeat.

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In each replicate, 30 strawberry fruits were used.

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2.4 Profiling plant volatile organic compounds

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Strawberry fruits were homogenized with a saturated salt solution (1:1, m/v), and

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2 mL of the extract was moved into a 20 mL headspace bottle. VOCs were assayed as

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previously described by Xu et al. 33 using gas chromatography-mass spectrometry (GC-

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MS) (Agilent Technologies, Santa Clara, CA, USA) combined with automatic

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headspace solid-phase microextraction (HS-SPME) by a Multi-Purpose Sampler

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(GERSTEL, GmbH & Co. KG, Germany). After a 15-min balance, a 50/30 mm

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DVB/CAR/PDMS fiber (Supelco, Bellefonte, PA, USA) was used for volatile

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extraction at 45 °C for 30 min. VOCs were separated by an HP-5MS capillary column

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(30 m × 0.25 mm, 0.25 µm) (Agilent Technologies) and identified by mass spectrum

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searching in the NIST05 spectral library and Kovats retention indices

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quantitative results were calculated with an internal standard of (+)-sativene (Sigma

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Aldrich, Shanghai, China).

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2.5 RNA-Seq analysis

34, 35.

Semi-

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B. cinerea (1 × 106 spores) was inoculated into 100 mL MEB and cultured at 25 °C

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for 72 h with 180 rpm shaking. One milliliter of linalool solution (209.5 mM in 0.05 %

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Tween 80) was added and cultured for additional 30 min (L30) or 120 min (L120).

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Fungal cultures with only 1 ml of 0.05 % Tween 80 added were also incubated for 30

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min (C30) or 120 min (C120). The mycelia were then harvested for RNA extraction

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with TRIzol reagent (Invitrogen). Construction of libraries and sequencing with the

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Illumina HiSeq 2500 platform were performed by Tianjin Novogene Bioinformatics

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Technology Co., Ltd (Tianjin, China). The clean reads were mapped to the genome of

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B. cinerea B05.10. Differentially expressed genes (DEGs) were identified using the

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DESeq R package (1.18.0) 36 using cutoffs of an adjusted P value of 0.05 and a ≥ 2-fold

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change. Two biological replicates were established for each treatment. To identify the


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metabolic pathways populated with these DEGs, the annotated DEG sequences were

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mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database.

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GOSeq software (Gene Ontology, GO, www.geneontology.org) was used for

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functional classification of all annotated DEGs.

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2.6 ROS, GSH, pyruvate, ATP content, and ATPase activity determination

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ROS levels in fungal cells were determined using a fluorometric intracellular ROS

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Kit (Sigma Aldrich). The pyruvate content was assayed using a Pyruvate Assay Kit

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(Solarbio Life Science, China). The ATP content was determined using a Molecular

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Probes ATP Determination Kit (Invitrogen detection technologies).

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To quantify GSH and assay ATPase activity, fungal mycelia were ground into a

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fine powder in liquid nitrogen, and the total protein was extracted using a buffer [0.1

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M Tris-HCl (pH 7.4), 0.15 M NaCl, 1 mM EDTA, 1 % (v/v) Triton X-100, 0.5 % SDS].

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The total protein was quantified using the Bicinchoninic Acid Protein Quantitation

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Assay Kit (KeyGen Bio TECH, Nanjing, China). GSH was quantified using a GSH

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assay kit (Beyotime Biotechnology, Shanghai, China). ATPase activity was assayed

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using the ATPase/GTPase Activity Assay Kit (Sigma Aldrich, USA).

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2.7 Assay of mitochondrial membrane potential (ΔΨm) of B. cinerea

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ΔΨm was determined using the JC-1 Staining Kit (Beyotime Biotechnology,

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Shanghai, China). After JC-1 staining, the fluorescence intensities at λex = 490/λem =

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530 nm (green) and at λex = 525 /λem =590 nm (red) were measured by confocal

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microscopy (Leica TCS SP8, Wetzlar, Germany). The extent to which the ΔΨm

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collapsed was determined by the ratio of green/red fluorescence intensity. The

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experiment was repeated four times with three replicates per repeat.

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2.8 Specimen processing for SEM

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The fruit tissues infected by fungi were fixed in 2.5 % glutaraldehyde in 0.1 M

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phosphate buffer solution (pH 7.0) at 4 °C overnight. The fruit tissues were then

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prepared as previously described by Zhang et al.

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electron microscope (Hitachi SU-8010, Tokyo, Japan).

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2.9 Determination of ergosterol content

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for SEM assays on a scanning

Lyophilized hyphae (0.1 g) were ground into a fine powder in liquid nitrogen and



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then mixed with 10 mL dichloromethane. Then 100 μL of 1 mg/L 7-dehydrocholesterol

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was added to the solution as an internal standard. The solution was then treated with

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ultrasonication for 60 min at 35 °C (120 W, DS-2510DT, SHENGXI, Shanghai)

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followed by centrifugation at 10,000 g for 20 min at 4 °C. Three milliliters of the

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supernatant were transferred to a tube and evaporated with nitrogen at 40°C. The

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residue was then dissolved in 300 μl of N,O- Bis(trimethylsilyl)trifluoroacetamide

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(BSTFA) by incubation at 80 °C for 45 min. The solution was filtered by a 0.45-μm

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filter, and the filtrate was then placed into a 2.0 mL sample bottle for GC-MS analysis

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(Agilent Technologies). The injected volume was 2 μL, and the injector was set at

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260 °C. The compounds were separated by an HP-5MS capillary column (30 m × 0.25

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mm, 0.25 µm) (Agilent Technologies). The oven program for GC was set as follows:

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100°C for 1 min and then increased to 280 °C by 30 °C min-1, then maintained for 24

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min. The MS detector was run in SIM mode with a mass scanning range of 50~550 m/z

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with a solvent delay of 10 min. Ergosterol was quantified by an external standard curve.

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2.10 PI-staining of linalool-treated hyphae

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Spores of B. cinerea on an object slide were cultured in MEB for 20 h and then

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treated with 2095 µM linalool for 30 min, 60 min, or 120 min. Untreated hyphae were

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used as controls. Immediately after the treatments, the hyphae were washed twice with

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50 mM Tris-HCl (pH 7.4) and stained with 500 nM PI solution for 5 min. The stained

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hyphae were then washed three times with 50 mM Tris-HCl (pH 7.4). The fluorescence

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signal was observed at λex = 535/λem = 617 nm by a fluorescence microscope (Ni-U,

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Nikon, Japan). The fluorescence intensity was measured by Image-Pro Plus 6.0.

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2.11 Real-time quantitative PCR

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Total RNA was extracted as described above. First-strand cDNA was synthesized

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in a total volume of 20 µL using a PrimeScriptTM RT reagent Kit with gDNA eraser

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(Takara, Japan). The cDNA was diluted with DEPC-H2O by 10-fold and used as a

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template for RT-qPCR analysis. RT-qPCR was performed using a CFX96 Touch™

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Real-time PCR detection system (Bio-Rad, Shanghai, Chian) with Thunderbird SYBR

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qPCR mix (no ROX reference dye; Toyobo, Japan). The relative normalized transcript

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level of each gene was computed using the 2−ΔΔCt method 38. BctubA was selected as a


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housekeeping gene based on the BestKeeper analysis (Table S4). Primers used in RT-

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qPCR are listed in Table S5. All of the qRT-PCR analyses were repeated three times

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with three replicates per repeat.

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2.12 TMT-labeling quantitative proteomic analysis

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Tandem mass tag (TMT)-labelling quantitative proteomic assays were conducted

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by Tianjin Novogene Bioinformatics Technology Co., Ltd (Tianjin, China). Hyphal

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protein was extracted with lysis buffer (50 mM Tris-HCl (pH 8), 8 M urea, and 0.2%

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SDS), and subsequent protein extraction was conducted as previously described by Han

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et al. 39. Protein concentration was determined with a Bradford assay (Thermo Fisher).

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Protein (0.1 mg) was digested with Trypsin Gold (Promega, Beijing, China) at 37°C

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for 16 h. After trypsin digestion, the peptides were desalted with a C18 cartridge to

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remove the high urea and were dried by vacuum centrifugation (Vacufuge plus,

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Eppendorf,). Desalted peptides were labeled with TMT6/10-plex reagents

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(TMT6/10plex™ Isobaric Label Reagent Set, Thermo Fisher). The TMT-labeled

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peptide mix was fractionated using a C18 column (Waters BEH C18 4.6 × 250 mm, 5

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μm) on a Rigol L3000 HPLC. Eluent was collected every 1 min and then merged into

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15 fractions. The samples were dried under a vacuum and reconstituted in 0.1% (v/v)

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formic acid (FA) in water for subsequent analyses. Shotgun proteomics analyses were

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performed using an EASY-nLCTM 1200 UHPLC system (Thermo Fisher) coupled to

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an Orbitrap Q Exactive HF-X mass spectrometer (Thermo Fisher) operating in the data-

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dependent acquisition (DDA) mode.

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The mass spectrometry proteomics data has been deposited to the 40

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ProteomeXchange Consortium through the PRIDE repository

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number PXD013498. The mass spectrum raw data were matched to the protein database

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of B. cinerea B05.10 (https://www.ncbi.nlm.nih.gov/genome/494) by Proteome

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Discoverer 2.2 with Max Missed Cleavage Sites set at 2, Precursor Mass Tolerance at

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10 ppm, and Fragment Mass Tolerance at 0.02 Da. A unique peptide was used for

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protein quantification. The fold changes (FC) of each protein of the treated groups were

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normalized to the control groups. Proteins with a value of FC > 1.0, p-value < 0.05 via

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t test were regarded as upregulated, while FC < 1.0, p-value < 0.05 was downregulated.


under accession


protein

sequences

were

mapped

with

Gene

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Identified

Ontology

Terms

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(http://geneontology.org/) to determine their functional and biological properties.

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Cluster of Orthologous Groups of Proteins System (ftp://ftp.ncbi.nih.gov/pub/

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COG/KOG) was employed for the functional annotation of genes from new genomes

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and for research into genome evolution. A hypergeometric test was employed to

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perform GO enrichment and KEGG pathway enrichment.

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2.13 Statistical analysis

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The statistics were performed using SAS software (SAS Institute Inc., Cary, NC,

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USA), and bar graphs were generated by GraphPad Prism 6.01 (GraphPad Software,

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Inc., La Jolla, CA, USA). Data were transformed, if necessary, to meet the requirements

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of homogeneity of variance and back-transformed for data presentation. Where

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appropriate, the significance level was determined by one-way analysis of variance

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(ANOVA), and means were compared by the least significant difference (LSD) t test at

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P < 0.05.

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3. Results

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3.1 B. cinerea infection increased linalool production by strawberry fruits

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To investigate the effect of B. cinerea infection on the production of VOCs by

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strawberry fruits, VOCs of inoculated fruits were compared with uninoculated fruits.

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Forty-seven VOCs were identified in the uninoculated fruits, including 17 esters, 11

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terpenes, 7 aldehydes, 5 alcohols, 1 furanone, and 6 others (Table S1). Linalool is the

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dominant terpene and accounts for around 14 % of the total VOCs in the fruits at harvest

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(Table S1). Within four days post-inoculation, fungal infection did not significantly

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alter the composition of VOCs produced by the fruit but changed their contents. At 2

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days post-inoculation, the concentrations of 14 VOCs in the infected fruits were

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significantly higher than in the uninoculated fruits (Fig. 1A), among which 8 were

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terpenes. Principal component analysis (PCA) also showed that VOCs at day 2 post-

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inoculation were separated from others in the 3D diagram based on three PCs that

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accounted for over 80 % of the whole profile (Fig. 1B). Particularly, at two days post-

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fungal inoculation, the concentration of linalool increased by 10.45-fold (Fig. 1C) as

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compared to a 13.58–58.9 % increase of total VOCs (Fig. 1D). At three days post-


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inoculation, the linalool concentration was still significantly (P < 0.05) higher than in

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the uninoculated control fruits (Fig. 1D).

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3.2 Linalool fumigation inhibited infection of strawberry fruits by B. cinerea

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As described above, linalool production is highly upregulated by B. cinerea

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infection, and we thus suspected that exogenous application of linalool (fumigation)

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could inhibit the infection of strawberry fruits by B. cinerea. We first determined the

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concentration of linalool that could be used for fumigation. First, we found that the MIC

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and the MFC of linalool upon B. cinerea in MEB medium were 20.95 and 2,095 μM,

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respectively (Table 1). Scanning electron microscopy (SEM) assays showed that

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untreated hyphae are tubular and linearly shaped, which produced spherical or nearly

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spherical spores (Fig. 2A, B). Linalool treatment at the MFC severely changed hyphal

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morphology. After 30 min of fumigation, hyphae were distorted and partly collapsed

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(Fig. 2C), and spores were slightly depressed (Fig 2D). Elongated linalool fumigation

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(120 min) caused more serious damage to the hyphae and spores. Hyphae were broken

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and shrunken (Fig. 2E), and some spores were also broken (Fig. 2F).

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We then assayed the effect of linalool fumigation on strawberry fruits. Fumigation

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at a concentration of 209.5 µM caused visual damage to the fruits while 20.95 µM did

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not (Fig. S1). In addition, 20.95 µM of linalool is much lower than the minimum

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toxicity dose (4.87 mM) of linalool on human skin cells 41, so this concentration could

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be safe to human beings. The linalool rapidly diffused after fumigation, and by one day

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post-fumigation, the linalool concentration in the treated fruits was not significantly

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different from that of untreated control fruits (Fig. S2A, B). Linalool fumigation also

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changed the VOC emissions, with 15 VOCs upregulated in the treated fruits at the end

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of 24 h post-fumigation, but by 3 days post-fumigation, no significant differences in

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the VOC profile were seen between the treated and untreated fruits (S. Fig. 2C, D).

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Therefore, a concentration of 20.95 µM was used to assay the effects of linalool

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fumigation on gray mold development on strawberry fruits. Strawberry fruits were

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fumigated for 24 h and were then incubated at 23 °C to allow B. cinerea infection. Two

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and three days after fumigation, the disease incidence (Fig. 3A) and lesion development

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(Fig. 3B) in the fumigated fruits were significantly (P < 0.05) less than in the control



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(unfumigated fruits with B. cinerea inoculated). At four days post-fumigation, the

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disease incidence (100 %) in the fumigated fruits was the same as in the control, but

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the disease lesion size in the fumigated fruits was still smaller (P < 0.05) than in the

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control (Fig. 3B).

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Consistent with the disease incidence and lesion development in fumigated fruits

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and the control, at two days post-fumigation, visible fungal colonies were seen at the

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inoculation sites on the control fruits, while almost no colonies were seen on the

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fumigated fruits (Fig. 3C-a, b). At three days post-fumigation, the hyphae in the control

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fruits invaded the tissue adjacent to the inoculation sites, but this was not found in the

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fumigated fruits (Fig. 3C-c, d). Tissue colonization by the fungus in the control fruits

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was more intense than in the fumigated fruits. Furthermore, the fungal biomass and

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spore yield on the control fruits appeared to be greater than on the treated ones (Fig.

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3C-c, d).

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3.3 Overview of RNA-Seq analysis of the B. cinerea response to linalool treatment

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To investigate the mechanisms by which linalool inhibits the infection of

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strawberry fruits by B. cinerea, the transcriptome of B. cinerea grown in MEB medium

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(control) was compared using RNA-Seq to that grown in MEB medium supplemented

330

with linalool for 30 min (designated as L30) and 120 min (designated as L120). The

331

controls that were grown for 30 min and 120 min are designated as C30 and C120,

332

respectively. Overall, 93.24–95.5 % of clean reads were mapped to the genome (B.

333

cinerea B05.10, NCBI), and a total of 12,695 protein-coding genes were detected in the

334

samples, indicating the high abundance and excellent quality of the sequencing data

335

(GenBank accession number PRJNA521298). A high correlation of the qualitative

336

agreement of expression levels was detected between the replicates by Pearson

337

correlation coefficients (Fig. S3).

338

Only a few differentially expressed genes (DEGs) were found between C30 and

339

C120. There were 166 DEGs between C30 and L30 with 96 upregulated and 70

340

downregulated by linalool treatment (Fig. S4). Linalool treatment for a longer time (120

341

min) caused a greater difference in gene expression, and 931 DEGs existed between

342

C120 and L120, with 491 upregulated and 440 downregulated (Fig. S4). Only 94 DEGs


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343

were shared by 30 min and 120 min linalool treatment (Fig. S4). One hundred genes

344

were differentially expressed between L120 and L30, with 82 downregulated and 18

345

upregulated in L120.

346

3.4 Linalool treatment impaired cell membrane integrity by reducing ergosterol

347

production

348

Linalool treatment (30 min) downregulated two ergosterol biosynthesis genes

349

(Bcerg1 and Bcerg3), and 120 min linalool treatment reduced the expression of an

350

additional ergosterol biosynthesis gene (Bccyp51) (Fig. 4A). Downregulation of these

351

three genes by linalool was validated by quantitative qRT-PCR (Table S2). Consistent

352

with the gene expression changes, the membrane ergosterol content (2141.64 mg/kg,

353

dry weight) in the mycelium fumigated with 120 min linalool was 42.6 % lower (P
0.05) different from that of the untreated group (Fig. 4B).

357

Propidium iodide (PI) stains cells that have lost their membrane permeability

358

barrier, a common indirect proxy for the loss of membrane integrity and cell viability.

359

The fluorescence intensity in the mycelium treated with linalool for 30 min was stronger

360

than that in the untreated group. The mycelium treated with linalool for 120 min had

361

the strongest fluorescence intensity (P < 0.05) (Fig. 4C). The PI-stained hypha

362

proportion also increased with the length of time of linalool treatment (Fig. 4D).

363

3.5 Linalool treatment caused mitochondrial dysfunction

364

The mitochondrial gene Bccox11, which encodes a respiratory oxidase for ATP

365

production, was significantly (P < 0.05) downregulated by both 30 and 120 min linalool

366

treatments (Fig. 5A). Consistent with this reduction, the ATP content in the 30 min-

367

treated hyphae was 29.6 % lower (P > 0.05) than in the untreated, while 60 min and

368

120 min linalool treatments decreased ATP contents by 54.9 % (P < 0.05) and 37.2 %

369

(P < 0.05), respectively (Fig. 5B). In contrast, the ATPase activity was increased after

370

120 min treatment, which was 3.21-fold higher (P < 0.05) than in the control (Fig. 5C).

371

Based on the discrepancy between the changes in ATP content and ATPase activity, we

372

suspected that linalool treatment caused other damage to mitochondria. Indeed, we



373

found that linalool treatment (30 min) significantly collapsed the mitochondrial

374

membrane potential (ΔΨm), and prolonged linalool treatment caused damage to the

375

mitochondrial membrane to a greater extent (Fig. 5D, E).

376

3.6 Linalool-treated hyphae were under oxidative stress

377

Linalool treatment (120 min) upregulated glutathione (GSH) biosynthesis genes

378

including Bcoxp1, a putative glutathione hydrolase gene, Bcglr1, and a putative

379

phosphogluconate dehydrogenase gene, while the expression of the glutathione

380

reductase gene Bcglr1 was increased both in L30 and L120 (Fig. 6A). Meanwhile, the

381

genes Bcgst8 and Bcgst9, encoding glutathione transferase that transforms GSH into

382

oxidized glutathione (GSSG), were downregulated (Fig. 6A). In contrast to the

383

difference in gene transcription, the GSH titer in the linalool-treated hyphae was less

384

than 50 % (P < 0.05) of that of the untreated control (Fig. 6B).

385

Linalool treatment also changed the expression of pyruvate metabolism genes.

386

Four genes encoding rate-limiting enzymes in the glycolysis/gluconeogenesis pathway

387

for the production of pyruvate, pyruvate kinase (Bcpic7), phosphoenolpyruvate

388

carboxykinase (Bcpck1), phosphoglycerate kinase (Bcpgk1), and phosphoglycerate

389

mutase , were upregulated by the linalool treatment (120 min) (Fig. 6C). In contrast,

390

pyruvate consumption genes in the glyoxylate and dicarboxylate pathways were

391

significantly downregulated by 30 min and 120 min linalool treatment. Consistent with

392

the gene expression, the pyruvate concentration was elevated in the mycelium treated

393

with linalool for 120 min, which was 40.1 % higher (P < 0.05) than in the untreated

394

control (Fig. 6D). No significant difference in pyruvate concentration was found

395

between the control and the 30 min and 60 min linalool-treated hyphae (Fig. 6D).

396

Besides pyruvate, no other known reactive oxygen species (ROS) scavengers, such

397

as catalases, and SODs were upregulated by linalool treatment; instead, a catalase gene

398

(Bccat6) was downregulated by 4.71-fold compared to the untreated control (Fig. 6D).

399

We further assayed the effect of linalool treatment on the levels of ROS. Compared

400

to the untreated control, 30, 60, and 120 min linalool treatment increased the ROS levels

401

significantly (P < 0.05) by 60.6 %, 98.2 %, and 175 %, respectively (Fig. 6F).

402

3.7 Analysis of the protein-protein interaction network identified connections of


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403

the pyruvate metabolism pathway with the GSH-GSSG system and P450s with

404

ergosterol biosynthesis

405

The major pathways with DEG enrichment that were affected by linalool treatment

406

were found by KEGG analysis (Fig. S5). These included ergosterol biosynthesis,

407

pyruvate

408

glycolysis/gluconeogenesis pathway, GSH metabolism, and aminoacyl-tRNA

409

biosynthesis. Additionally, five putative genes encoding cytochrome P450 enzymes

410

(P450s) were significantly upregulated in the 120 min linalool treated sample, with two

411

of them also having increased expression in 30 min-treated hyphae (Fig. S6A). The set

412

of genes in aminoacyl-tRNA biosynthesis for different amino acid transport in protein

413

biosynthesis were upregulated in linalool-treated hyphae (Fig. S6B).

metabolism,

glyoxylate

and

dicarboxylate

metabolisms,

the

414

Fifty-six DEGs in these pathways were selected and subjected to PPI network

415

visualization analyses. Forty-three genes, including 15 genes of pyruvate biosynthesis,

416

9 in the glycolysis/gluconeogenesis pathway, 5 in the glyoxylate and dicarboxylate

417

metabolism pathway, 5 of GSH metabolism, 16 of aminoacyl-tRNA biosynthesis, 4 in

418

steroid metabolism, and 4 P450 genes, were mapped to neighboring nodes and arranged

419

according to their interactions with a string score over 700 in the String database (Fig.

420

7). The genes in the pyruvate metabolism were connected with each other, among which

421

Bcpic7 was an important node connected with the glycolysis/gluconeogenesis pathway

422

and with the glutathione reductase gene, Bcglr1 (Fig. 7). An intimate connection

423

between the GSH metabolism and glyoxylate/dicarboxylate metabolism was also found,

424

with Bcoxp1 and Bcglr1 as connecting nodes (Fig. 7).

425

A close connection was also found between the rate-limiting gene Bcerg3

426

(encoding the enzyme catalyzing squalene to (S)-squalene-2, 3-epoxide) in the

427

ergosterol biosynthesis pathway and the 4 putative genes encoding P450 enzymes that

428

could be involved in linalool oxidation (Fig. 7). In addition, 15 genes in aminoacyl-

429

tRNA biosynthesis were connected with each other, one of which was connected with

430

the ergosterol biosynthesis gene Bcerg1 (Fig. 7).

431

3.8 Linalool treatment changed the protein abundance in nutrition transport and

432

metabolism



433

Proteomic analysis detected 465,750 mass spectra form two biological samples,

434

resulting in the identification of 5638 proteins at a corresponding protein false

435

discovery rate of 5 %. Compared to the control, linalool-treated fungi samples showed

436

159 different abundant proteins (DAPs) in hyphae treated for 120 min (L120), and 129

437

DAPs in the 30 min group (L30) (Fig. S7A). Although only 21 were common between

438

30 min and 120 min treatments, their classification into clusters of orthologous genes

439

(COG) showed similar profiles of identified comparable sets of DAPs (over 50% of

440

proteins had COG annotation) (Fig. S7B). Translation, ribosomal structure, and

441

biogenesis; carbohydrate transport and metabolism; amino acid transport and

442

metabolism; posttranslational modification, protein turnover, and chaperones; and

443

coenzyme transport and metabolism were the 5 major COG classes of DAPs in both

444

groups (Fig. S7B). In addition, energy production and conversion was also a notable

445

functional class of DAPs, especially after treatment with linalool for 30 min (Fig. S7B).

446

The details of the DAPs and their COG analysis are shown in Table S3, with a fold

447

change over 1 being upregulated and that less than 1 being downregulated.

448

Among the DAPs involved in energy production (Fig. 8A), NAD(P)H-dependent

449

FMN reductase, lactate dehydrogenase, or the related 2-hydroxyacid dehydrogenase

450

accumulated in linalool-treated hyphae, while one ATPase subunit was decreased in

451

protein amount, especially in L120. Compared to 120 min, 30 min linalool treatment

452

more heavily altered the abundance of proteins involved in the energy production. The

453

set of proteins of B. cinerea involved in amino acid transport and metabolism had

454

decreased translation, especially those in glycine and histidinol metabolism, as well as

455

of argininosuccinate lyase, and DAHP synthase, especially after 120 min of linalool

456

treatment (Fig. 8B). This indicated a decrease in amino acid transport and metabolism.

457

Isopentenyl diphosphate isomerase, which is attributed to isoprenoid biosynthesis in

458

microorganisms

459

carbohydrate metabolism and transport (Fig. 8D), most of the 4 proteins were

460

significantly (P < 0.05) downregulated in L30, while 8 DAPs were significantly (P

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