Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions

Accepted Manuscript Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions Michał Dziurka, Anna Janeczko, Csilla Juhász, Gábor Gullner, Jana Oklestková, Ondrej Novák, Diana Saja, Andrzej Skoczowski, István Tóbiás, Balázs Barna PII:

S0981-9428(16)30402-8

DOI:

10.1016/j.plaphy.2016.10.013

Reference:

PLAPHY 4695

To appear in:

Plant Physiology and Biochemistry

Received Date: 12 July 2016 Revised Date:

13 October 2016

Accepted Date: 13 October 2016

Please cite this article as: M. Dziurka, A. Janeczko, C. Juhász, G. Gullner, J. Oklestková, O. Novák, D. Saja, A. Skoczowski, I. Tóbiás, B. Barna, Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions, Plant Physiology et Biochemistry (2016), doi: 10.1016/j.plaphy.2016.10.013. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Local and systemic hormonal responses in pepper leaves during compatible and incompatible pepper-tobamovirus interactions

RI PT

Michał Dziurka a, Anna Janeczko a, Csilla Juhász b, Gábor Gullner b, Jana Oklestková c,

Polish Academy of Sciences, The Franciszek Górski Institute of Plant Physiology,

M AN U

a

SC

Ondrej Novák c, Diana Saja a, Andrzej Skoczowski a, István Tóbiás b, Balázs Barna b,*

Niezapominajek 21, 30-239 Krakow, Poland b

Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of

Sciences, Herman Ottó út 15, 1022 Budapest, Hungary c

Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and

TE D

Agricultural Research, Institute of Experimental Botany ASCR & Palacky University,

EP

Šlechtitelů 27, 783 71 Olomouc, Czech Republic

*Corresponding author:

AC C

Tel.: +36 1 48 77 534; Fax: +36 1 48 77 555;

E-mail address: [email protected]

2

ACCEPTED MANUSCRIPT

Keywords: brassinosteroids, ethylene, hormone, pepper, phenylalanine ammonia lyase, progesterone,

RI PT

salicylic acid, tobamovirus

Abbreviations: ABA,

abscisic

acid;

ACO,

1-aminocyclopropane-1-carboxylate

oxidase;

ACS,

1-

SC

aminocyclopropane-1-carboxylate synthase; AOC, allene oxide cyclase; AOS, allene oxide synthase; BR, brassinosteroid; c-Z, cis-zeatin; c-ZR, cis-zeatin-9-riboside; dpi, days post

M AN U

inoculation; 24-epi-BR, 24-epi-brassinolide; GA1, gibberellin A1; GA3, gibberellic acid; GA4, gibberellin A4; GA6, gibberellin A6; hpi, hours post-inoculation; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; ICS, isochorismate synthase; IPA, N6-isopentenyl-adenine; ISTD, internal standard; JA, jasmonic acid; K, kinetin; KR, kinetin-9-riboside; NCED, 9-cis-

TE D

epoxycarotenoide-dioxygenase; LOX, lipoxygenase; ObPV, Obuda pepper virus; PAL, phenylalanine ammonia-lyase; PMMoV, Pepper mild mottle virus; SA, salicylic acid; SAR, systemic induced resistance; TMV, Tobacco mosaic virus; TSA, transcriptome shotgun

AC C

chromatography

EP

assembly; t-Z, trans-zeatin; t-ZR, trans-zeatin-9-riboside; UHPLC, ultra performance liquid

3

ACCEPTED MANUSCRIPT

Highlights

RI PT

1. Phytohormones were quantified in virus-infected pepper leaves by UHPLC-MS/MS.

2. ObPV infection elevated ABA, auxin, brassinosteroid, cytokinin, JA and SA levels.

SC

3. PMMoV infection increased the contents of gibberellic acid and salicylic acid.

M AN U

4. Both ObPV and PMMoV infections increased the progesterone level in pepper leaves.

AC C

EP

TE D

5. ObPV infection markedly up-regulated three PAL and one ACO genes in pepper leaves.

4

ACCEPTED MANUSCRIPT

Abstract

Phytohormone levels and the expression of genes encoding key enzymes participating in

RI PT

hormone biosynthetic pathways were investigated in pepper leaves inoculated with two different tobamoviruses. Obuda pepper virus (ObPV) inoculation led to the development of hypersensitive reaction (incompatible interaction), while Pepper mild mottle virus (PMMoV)

SC

inoculation resulted in a systemic, compatible interaction. ObPV-inoculation markedly increased not only the levels of salicylic acid (SA) (73-fold) and jasmonic acid (8-fold) but

M AN U

also those of abscisic acid, indole-3-acetic acid, indole-3-butyric acid, cis-zeatin, cis-zeatin-9riboside and trans-zeatin-9-riboside in the inoculated pepper leaves 3 days post inoculation. PMMoV infection increased only the contents of gibberellic acid and SA. Hormone contents did not change significantly after ObPV or PMMoV infection in non-infected upper leaves 20

TE D

days post inoculation. Concentrations of some brassinosteroids (BRs) and progesterone increased both in ObPV- and PMMoV inoculated leaves. ObPV inoculation markedly induced the expression of three phenylalanine ammonia-lyase (PAL) and a 1-aminocyclopropane-1-

EP

carboxylate oxidase (ACO) genes, while that of an isochorismate synthase (ICS) gene was not modified. PMMoV inoculation did not alter the expression of PAL and ICS genes but induced

AC C

the transcript abundance of ACO although later than ObPV. Pre-treatment of pepper leaves with exogenous 24-epi-brassinolide (24-epi-BR) prior to ObPV-inoculation strongly mitigated the visible symptoms caused by ObPV. In addition, 24-epi-BR pre-treatment markedly altered the level of several hormones in pepper leaves following ObPV-inoculation. These data indicate that ObPV- and PMMoV-inoculations lead to intricate but well harmonized hormonal responses that are largely determined by the incompatible or compatible nature of plant-virus interactions.

5

ACCEPTED MANUSCRIPT

1. Introduction

The resistance of plants against phytopathogenic viruses is determined by the timely

RI PT

recognition of the virus and the rapid deployment of efficient antiviral defense reactions. Viruses are generally perceived by intracellular resistance proteins (R-proteins) of host cells. Upon recognition, signals are transmitted to the nucleus leading to the rapid and extensive

SC

reprogramming of host gene expression patterns and ultimately to the development of effector-triggered immunity (ETI) in resistant plant genotypes. ETI is often associated with

M AN U

programmed cell death at sites of infection (hypersensitive response, HR) (Künstler et al., 2016). The reprogramming of the transcriptome is regulated by a complex, multilayered regulatory network, in which various transcription factors (Gatz, 2013) and defense-related plant hormones (Alazem and Lin, 2015) play critical roles.

TE D

It is generally accepted that the plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene play a key role in plant immunity (Alazem and Lin, 2015). A substantial rise of endogenous SA content was revealed in leaves of resistant tobacco plants following Tobacco

EP

mosaic virus (TMV) inoculation (Enyedi et al., 1992). SA was shown to induce virus resistance by inhibiting both virus replication and virus movement (Singh et al., 2004). In

AC C

addition, SA is essential to the development of systemic induced resistance (SAR) (Gaffney et al., 1993; Fodor et al. 1997). Exogenously applied SA can effectively induce the expression of numerous defense genes including a suite of pathogenesis-related (PR) genes (Vlot et al., 2009). The biosynthesis of SA can occur by two possible pathways. One pathway is initiated by the transformation of phenylalanine to trans-cinnamic acid catalyzed by phenylalanine ammonia-lyase (PAL, E.C. 4.3.1.5) isoenzymes (Kim and Hwang, 2014). Alternatively, SA can be synthesized from chorismate via the rate-limiting enzyme isochorismate synthase (ICS,

6

ACCEPTED MANUSCRIPT E.C. 5.4.4.2) enzyme (Catinot et al., 2008). However, little information is available about which biosynthetic pathway is predominant during the accumulation of SA in virus-infected leaves. In contrast to SA, JA was shown to negatively regulate the TMV-resistance in tobacco

RI PT

(Oka et al., 2013). The first reaction in the multi-step pathway of jasmonic acid biosynthesis is catalyzed by 13-lipoxygenase (13-LOX, E.C. 1.13.11.12) isoenzymes, which perform the peroxidation of α-linolenic acid to 13-hydroperoxy-linolenic acid (Feussner and Wasternack,

SC

2002). This metabolite is further converted by allene oxide synthase (AOS, E.C. 4.2.1.92) and allene oxide cyclase (AOC, E.C. 5.3.99.6) enzymes to (9S,13S)-12-oxo-phytodienoic acid

(Feussner and Wasternack, 2002).

M AN U

(12-OPDA), which is transformed by several further enzymatic steps ultimately to JA

Ethylene also plays an important role as a highly inducible signal compound in the defense reactions of virus-infected plants (Alazem and Lin, 2015). The massive accumulation

TE D

of ethylene was observed in various incompatible plant-virus interactions (Barna et al., 2012b) including ObPV-infected pepper (Tóbiás et al., 1989). The last two key steps in the ethylene biosynthetic pathway are catalyzed by the 1-aminocyclopropane-1-carboxylate

EP

synthase (ACS, E.C. 4.4.1.14) and the 1-aminocyclopropane-1-carboxylate oxidase (ACO, E.C. 1.14.17.4) enzymes, which are encoded by small gene families in plants (Kim et al.,

AC C

2003).

These hormones can have synergistic and antagonistic interactions as well. A well

known example is the antagonism of SA and JA/ethylene pathways (Koorneef and Pieterse, 2008; Spoel and Dong, 2008). In addition, numerous experimental data have recently demonstrated that abscisic acid (ABA), auxins, brassinosteroids (BRs), cytokinins and gibberellins also play important roles in plant defense responses (Jameson and Clarke, 2002; Denance et al., 2013; Alazem and Lin, 2015). TMV-infection increased the concentration of

7

ACCEPTED MANUSCRIPT both free and bound forms of ABA (Whenham et al., 1986). ABA is also a negative regulator of TMV-resistance (Balázs et al., 1973), although long-term treatments with low ABA concentrations promoted virus resistance (Fraser, 1982). In the multi-step biosynthesis of ABA the 9-cis-epoxycarotenoide-dioxygenase (NCED) isoenzymes play a critical role

RI PT

(Taylor et al., 2000).

BRs are known not only to regulate plant growth and development (Bajguz, 2007), but they have been shown to play substantial roles also in plant disease resistance (Nakashita et

SC

al., 2003; Alazem and Lin, 2015). According to our previous results the pre-treatment of oilseed rape cotyledons with a BR substantially decreased the damage caused by

M AN U

Pseudomonas syringae inoculation (Skoczowski et al., 2011). Application of BR also lowered the susceptibility of rice to fungal and bacterial diseases, and induced resistance of tobacco to Tobacco mosaic virus (TMV), P. syringae, and to the powdery mildew fungus Oidium sp. (Nakashita et al., 2003). Zhang et al. (2015) proved that high BR concentrations in

TE D

Arabidopsis thaliana are positively correlated with tolerance against Cucumber mosaic virus (CMV) infection by manipulating the endogenous BR levels. BR treatment protected the photosystems, increased the activity of antioxidative enzymes and induced the expression of

EP

defense-associated genes in CMV infected plants. Progesterone is one of the mammalian hormones but its presence has already been

AC C

confirmed in some plants as well (Janeczko, 2012). Our knowledge about the roles of progesterone in plants is very limited. Progesterone has been shown to participate in plant development and abiotic stress responses (Janeczko, 2012; Janeczko et al., 2013a). Contrary to brassinosteroids, progesterone has no hormone status in plants, so every study devoted for the evaluation of its physiological functions in plants will be helpful for the evaluation of its hormonal nature.

8

ACCEPTED MANUSCRIPT To obtain a deeper knowledge about the role of plant hormones in mechanisms of plant-virus interactions, we have investigated the levels of several plant hormones in tobamovirus-infected pepper (Capsicum annuum L.) plants. Two different viruses were used. Inoculation of pepper leaves with Obuda pepper virus (ObPV) led to the appearance of

RI PT

hypersensitive necrotic lesions (incompatible interaction), while Pepper mild mottle virus (PMMoV) caused only very mild chlorotic symptoms (compatible interaction) (Tóbiás et al., 1989; Rys et al., 2014). The massive up-regulation of various 9-LOX genes and a divinyl ether

SC

synthase gene was revealed earlier in ObPV-inoculated pepper leaves (Gullner et al., 2010; Juhász et al., 2015). In this work we have studied local and systemic changes in hormone

M AN U

levels of pepper during compatible and incompatible pepper-tobamovirus interactions, and analyzed the expression of various genes participating in principal hormone biosynthetic pathways. In addition, the potential protective effect of exogenous BR pre-treatment has been investigated on symptoms of virus infections and on the endogenous levels of plant hormones

TE D

as well.

EP

2. Materials and methods

AC C

2.1. Plant culture and treatments

Seeds of the pepper (Capsicum annuum L.) cultivar TL 1791 harboring the L3 resistance gene were planted into soil and grown under greenhouse conditions (25 oC; photoperiod 16 h, radiation 160 µmol m-2 s-1; relative humidity: 75-80%). Two-month-old plants were divided into five groups (10 plants per group) to make the following treatments: (1) inoculation with ObPV (an ObPV strain isolated in Hungary, formerly used synonym: Ob strain of Tomato mosaic virus) (Tóbiás et al., 1989). (2) inoculation with an L3-resistance-breaking strain of

9

ACCEPTED MANUSCRIPT PMMoV (isolated in the USA, formerly used synonym: Samsun latent strain of Tobacco mosaic virus) (Tóbiás et al., 1989; Rys et al., 2014). (3) mock inoculation - treatment without any virus to test the effect of the slight mechanical injury caused during virus inoculation. (4) inoculation with ObPV preceded by spraying the plants with 100 nM aqueous solution of 24-

RI PT

epi-brassinolide (24-epi-BR). A 4 mM stock solution of 24-epi-BR was prepared in ethanol. (5) inoculation with PMMoV preceded by spraying the plants with a 100 nM 24-epi-BR. (6) mock inoculation - treatment without any virus preceded by spraying the plants with 100 nM

SC

aqueous solution of 24-epi-BR. Inoculations were performed on the fifth and sixth true leaves, and all the leaf surface was treated uniformly with a virus suspension as described earlier (Rys

M AN U

et al., 2014). Spraying with 24-epi-BR was carried out two days before virus inoculation. During the experiments all plants were kept in a growth chamber at 25 oC with a 16-h photoperiod. Samples for hormone analyses were taken from the virus-infected leaves 3 days post inoculation (dpi) together with samples taken from corresponding mock-inoculated

TE D

control leaves. Samples were collected also from systemic, non-infected upper leaves of virus-inoculated and control plants 20 dpi. Leaf samples for gene transcript accumulation measurements were taken 4, 8, 12, 24 and 48 hours after inoculation from the virus-infected

EP

and from corresponding control leaves.

AC C

2.2. Extraction procedure for ABA, auxins, cytokinins, gibberellins, JA and SA

Hormones were extracted from the leaf samples according to the method of Dobrev and Kaminek (2002) with some modifications. Leaf samples (1g fresh weight) were homogenized in liquid nitrogen in a mortar and suspended in 7 ml of a methanol:water:formic acid mixture (15:4:1 v/v). A mixture of internal isotopic standards was added to each sample (750 µl, 50100 ng of each standard per sample, dissolved in methanol) consisted of deuterated salicylic

10

ACCEPTED MANUSCRIPT acid, deuterated indole-acetic acid, deuterated ABA and kinetin labeled with nitrogen

15

N

(Olchemim, Czech Republic). Samples were filled up to a final volume of 10 ml with the methanol:water:formic acid mixture (15:4:1 v/v), shaken for 30 min and then centrifuged (20 min, 2 000 g, 4 °C). An aliquot (5 ml) of each supernatant was evaporated to dryness under

RI PT

nitrogen stream (TurboVap LV, Capiler Ltd., MA, USA) at 50 oC. Residues were redissolved in 1 ml of 1 M aqueous solution of formic acid, sonicated, centrifuged (10 min, 20 000 g, 4 °C) and purified on SPE cartridges (Oasis MCX, 30 mg, 1 mm, Waters, Ireland). Cartridges

SC

were activated with 1 ml of methanol followed by 1 ml of 1 M formic acid, then the samples were applied, slowly aspirated, and finally the cartridges were washed with 1 M formic acid.

M AN U

The fraction of neutral and acidic hormones (ABA, auxins, gibberellins and SA) was eluted with 1 ml of methanol:acetonitrile (1:1 v/v), then the basic compounds (cytokinins) were eluted with 1 ml of 5 % ammonia in methanol:acetonitrile (1:1 v/v). All samples were evaporated to dryness under nitrogen, redissolved in 100 µl of methanol, filtered (on a 0.22

TE D

µm nylon membrane) and used for UHPLC analyses.

EP

2.3. UHPLC-MS/MS analysis of ABA, auxins, cytokinins, gibberellins, JA and SA

Our method was a modification of that published by Żur et al. (2015). For phytohormone

AC C

analyses an Ultra High Performance Liquid Chromatography (UHPLC) apparatus was applied (Agilent Infinity 1260, Agilent, Germany) coupled to a triple quadruple mass spectrometer (6410 Triple Quad LC/MS, Agilent, USA) equipped with electrospray ionization (ESI). Separation was achieved on an Ascentis Express RP-Amide analytical column (2.7 µm, 2.1 mm × 75 mm; Supelco, Bellefonte, PA, USA). A linear gradient elution was applied with 0.01 % of formic acid solution in water (solvent A) and acetonitrile with 0.01% of formic acid (solvent B). The gradient steps were 3.5-15 % B (0-5 min), 15-60 % B (5-13 min), 60-100 %

11

ACCEPTED MANUSCRIPT B (13-13.5 min) then 100-3.5 % B (14-14.5 min) at flow rate of 0.5 ml/min. The column temperature was 40 °C and the injection volume was 0.5 µl. The following conditions were found to be optimal for analysis: capillary voltage 4 kV, gas temperature 350 °C, gas flow 12 l/min and nebulizer pressure 35 psi. Samples were analyzed by multiple reactions monitoring

RI PT

(MRM) in positive polarity. Precursor ions, product ions, and MS/MS parameters are reported in Table 1. The MassHunter software was used to control the LC–MS/MS system and for data analysis. For MRM parameters optimization the MassHunter Optimizer was used. The

SC

monitored ions according to their consecutive elutions were trans-zeatin (t-Z), cis-zeatin (cZ), kinetin (K), [15N4]kinetin (K-N15, used as internal standard - ISTD), trans-zeatin-9-

M AN U

riboside (t-ZR), cis-zeatin-9-riboside (c-ZR), N6-isopentenyl-adenine (IPA), kinetin-9riboside (KR), gibberellin A1 (GA1), gibberellic acid (GA3), gibberellin A6 (GA6), [2H5]indole-3-acetic acid (IAA-D5, used as ISTD), indole-3-acetic acid (IAA), [2H4] salicylic acid (SA-D4, used as ISTD), SA, [2H6]cis,trans-abscisic acid (ABA-D6, used as ISTD),

TE D

cis,trans-abscisic acid (ABA), indole-3-butyric acid (IBA), JA and gibberellin A4 (GA4). For all ions two transitions were monitored: a quantifier was used for quantitation purposes and an other qualifier for the additional confirmation of identity. Quantitations were based on

EP

calibration curves obtained with pure standards of all compounds taking account of the recovery rates of ISTDs. All phytohormone standards were purchased from Olchemim

AC C

(Olomouc, Czech Republic) at the highest available purity, whereas all solvents were of HPLC grade and purchased from Sigma-Aldrich (Poznan, Poland).

2.4. Determination of BRs and progesterone

The isolation and identification of BRs was performed according to a protocol described by Tarkowská et al. (2016). Briefly, the leaf material (2 g fresh weight) was homogenized in 20

12

ACCEPTED MANUSCRIPT ml ice-cold 80 % methanol. After centrifugation (20 min, 2000 g, 4 °C), the supernatants were collected, an ISTD solution was added to all samples that contained deuterium labeled BRs, and the resulting solutions were passed through Discovery columns (Supelco, Bellefonte, PA USA). The extracts were evaporated under vacuum to 3 ml on Rotavapor R-215 (BUCHI,

RI PT

Switzerland) and then to dryness under N2 at 40 oC by a TurboVap LV apparatus (Capiler Ltd., MA, USA). Samples were resuspended in 1 ml of 7.5 % MeOH in phosphate-buffered saline (50 mM NaH2PO4, 15 mM NaCl, pH 7.2), and passed through an immunoaffinity

SC

column (Laboratory of Growth Regulation, Olomouc, Czech Republic). After evaporation the samples were redissolved in methanol and the BRs were determined by UHPLC with tandem

M AN U

mass spectrometry (UHPLC-MS/MS) by using an ACQUITY UPLC® I-Class System (Waters, Milford, MA, USA) and a triple quadrupole mass spectrometer XevoTM TQ-S MS (Waters MS Technologies, Manchester, UK). The extraction and purification of progesterone as well as analysis by UHPLC-MS/MS were performed as described by Simerský et al.

TE D

(2009).

EP

2.5. RNA extraction and gene expression analysis by RT-PCR

To analyze the expression of pepper genes participating in hormone biosynthesis pathways a

AC C

reverse transcription - polymerase chain reaction (RT-PCR) procedure was applied. Total RNA was extracted from 0.1 g ObPV- , PMMoV- and mock-inoculated pepper leaves ground under liquid nitrogen with a Total RNA Miniprep kit (Viogene, Sunnyvale, CA, USA). Reverse transcription (RT) of 2.5 µg total RNA was carried out with a RevertAid H Minus First Strand cDNA Synthesis kit (MBI Fermentas, Vilnius, Lithuania) using an oligo(dT) primer. Semiquantitative PCRs for assaying gene expression levels were conducted with a PTC 200 DNA Engine extended with an ALS-1296 sample holder (Bio-Rad, Hercules, CA,

13

ACCEPTED MANUSCRIPT USA). The PCR reaction mixtures contained 4 pmol of each primer, 0.5 U of Taq DNA polymerase (MBI Fermentas, Vilnius, Lithuania), 0.2 mM of each dNTP, 2 mM MgCl2 and 1 µl of template cDNA in a total volume of 20 µl. For primer design the pepper ACO, actin, AOC, ICS, 13-LOX, NCED and PAL sequences were obtained from the GenBank database of

RI PT

the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov). All oligonucleotide primer pairs used in our studies are shown in Table 2. The PCR amplifications started with 2 mins denaturation at 94 °C, followed by 24-28 cycles of 30 s at

SC

94 °C, 30 s at specific annealing temperatures (see Table 2 for each primer pair), 30 s at 72 °C and terminated by 10 mins extension at 72 °C. Relatively low cycle numbers were used to

M AN U

maintain initial differences in target transcript amounts (semiquantitative conditions). Expression of a pepper actin gene served as a constitutive control. The amplified PCR products were separated by gel electrophoresis in 1% agarose gels and visualized by GelRed

2.6. Data analysis

TE D

nucleic acid gel stain (Biotium Inc., Hayward, CA, USA).

EP

Data presented are means of three independent parallel experiments. The significant

AC C

difference between mean values obtained in virus-inoculated and mock-inoculated control leaves were evaluated by Student's t-test. Differences were considered to be significant at P < 5 %.

3. Results

3.1 Changes in the concentrations of ABA, auxins, cytokinins, gibberellins, JA and SA

14

ACCEPTED MANUSCRIPT During our first experiments sixteen different plants hormones or their ribosyl derivatives were detected by our UHPLC-MS/MS method in pepper leaf extracts. In the case of four hormones (GA4, GA6, K, KR) the obtained concentrations were too close to the detection limits or the standard deviations were too high, therefore these data were not evaluated

RI PT

further. However, twelve phytohormones were successfully quantified in the extracts obtained from both virus-inoculated and control pepper leaves. The massive accumulation of ethylene was already detected in ObPV-infected pepper leaves (Tóbiás et al., 1989) therefore it was not

SC

investigated in the present study.

The most striking effects were observed in the infected leaves during the incompatible

M AN U

pepper-ObPV interaction, where the levels of eight hormones or hormone- ribosyl derivatives were markedly elevated. ObPV inoculation strongly increased the contents of ABA (9.6-fold), of two auxins (IAA and IBA, 4.2- and 4.4-fold, respectively), of three cytokinins (c-Z, t-ZR, c-ZR, 4-, 7- and over 100-fold, respectively) as well as of SA (73-fold) and JA (8-fold) in the

TE D

infected pepper leaves 3 dpi as compared to mock-inoculated leaves (Fig. 1). Interestingly, the elevation of hormone levels was highly significant (P < 0.1%) not only in the case of the typical stress hormones as ABA, JA and SA, but also in the case of c-Z, c-ZR and t-ZR (Fig.

EP

1). In addition, the amounts of two auxin-type hormones, IAA and IBA rose significantly (P < 1%) as well in the ObPV-inoculated leaves. On the other hand, PMMoV inoculations led to

AC C

the significant elevation in the contents of only two phytohormones. A substantial increase of gibberellic acid (GA3) level as well as a small, but significant rise of SA level was observed in the PMMoV-inoculated pepper leaves 3 dpi (Fig. 1). No significant changes were observed in the above hormone levels in the systemic, upper leaves of virus-inoculated plants 20 dpi, neither after ObPV nor after PMMoV inoculation (Fig. 1).

3.2. Changes in concentrations of BRs and progesterone

15

ACCEPTED MANUSCRIPT

A particularly interesting observation was the detection of the steroid regulator progesterone in uninfected and virus-inoculated pepper leaves. A massive, 11-fold increase of progesterone content was found in ObPV-inoculated leaves 3 dpi as compared to mock-inoculated leaves.

RI PT

PMMoV inoculation also induced a significant elevation of progesterone level 3 dpi (Fig. 2). The amount of progesterone in the upper leaves was under the limit of detection, therefore changes after virus infection could not be evaluated (Fig. 2). As regards the other steroid

SC

regulators we could detect only few BRs in pepper leaves. In addition, the amount of 24-epiBR could be detected only in the systemic, upper leaves of ObPV infected pepper plants (Fig.

M AN U

2). However, castasterone and 28-homocastasterone levels were determined both in infected lower and in systemic, upper leaves. Interestingly, the amount of castasterone increased significantly only in the PMMoV inoculated lower leaves, while the 28-homocastasterone content decreased in upper leaves of PMMoV infected pepper plants (Fig. 2). Only these two

TE D

changes in the amount of BRs were significant after virus infection.

3.3. Transcript levels of genes involved in hormone biosynthesis in ObPV and PMMoV

EP

infected pepper leaves

AC C

To explore further the role of plant hormones in virus-infected plants we examined the expression of eleven pepper genes in ObPV- and PMMoV-inoculated leaves that encode key enzymes in hormone biosynthetic pathways. Three PAL genes and an ICS gene were studied due to their potential roles in SA biosynthesis. Two 13-LOX genes (13-LOX2 and 13-LOX6) and an AOC gene were examined, which can participate in JA biosynthesis. The two selected pepper 13-LOX genes showed high sequence homologies to the Nicotiana attenuata 13-LOX3 gene that was proved to participate in JA biosynthesis (Allmann et al., 2010). Furthermore an

16

ACCEPTED MANUSCRIPT ACO and three NCED genes were also investigated for their determining roles in ethylene and ABA biosynthetic pathways, respectively. The expression of an actin gene was studied as constitutive control. Specific PCR primers were designed and the virus-induced changes in the expression of all genes were studied by semi-quantitative RT-PCR.

RI PT

ObPV-inoculation strongly induced the expression of all three PAL genes in the inoculated leaves. The transcript abundance of PAL1 was markedly induced already 24 hours post-inoculation (hpi), while those of PAL2 and PAL3 increased only 48 hpi, particularly

SC

strongly in the case of PAL2 (Fig. 3). The up-regulation of all PAL genes preceded the appearance of the hypersensitive lesions. In contrast to PAL genes, the expression of ICS1

M AN U

was not altered significantly by ObPV-inoculation (Fig. 3). The expression of PAL1, PAL2, PAL3 and ICS1 genes did not change in the compatible pepper-PMMoV interaction (Fig. 3). The transcript abundance of the ACO gene was strongly elevated by both ObPV- and PMMoV-inoculations. However, the substantial up-regulation of ACO gene was already

TE D

observed 1 dpi in the ObPV-inoculated leaves, whereas in the compatible pepper-PMMoV interaction this effect was observed only one day later (Fig. 3). The expression of AOC, 13LOX2, 13-LOX6, NCED1, NCED2, NCED3 and actin genes was influenced neither by ObPV-

AC C

studied genes.

EP

nor by PMMoV-inoculation (Fig. 3). Mock inoculations did not modify the expression of any

3.4. Effect of BR pre-treatment on the visual symptoms of ObPV inoculation

To obtain more information about the effects of BRs on pepper-virus interactions we investigated the effect of a BR pre-treatment on the visual symptoms of ObPV inoculation. Previous studies showed that the infection of pepper leaves with ObPV resulted in the appearance of visible necrotic lesions 72 hpi, while PMMoV inoculation caused no visible

17

ACCEPTED MANUSCRIPT symptoms on the infected leaves (Tóbiás et al., 1989; Rys et al., 2014). In the present experiments, pre-treatment (spraying) of pepper leaves with 100 nM 24-epi-BR two days before ObPV inoculation strongly suppressed the development of necrotic lesions caused by ObPV (Fig. 4A). The number of ObPV-elicited hypersensitive necrotic lesions per leaf

was not modified significantly by 24-epi-BR pre-treatment.

SC

3.5. Effect of BR pre-treatment on hormone contents

RI PT

decreased by 39% as a consequence of 24-epi-BR pre-treatment (Fig. 4B). The lesion size

M AN U

To gain an insight into the interaction of BRs with other plant hormones during virus infections, pepper plants were sprayed with 100 nM 24-epi-BR and two days later the lower leaves were inoculated with ObPV or PMMoV. Mock inoculations were also carried out as controls. The endogenous levels of twelve plant hormones were analyzed by UHPLC-MS/MS

TE D

in the infected lower leaves as well as in the uninfected, systemic upper leaves. Pre-treatment of leaves with by 100 nM 24-epi-BR two days prior to mock inoculation did not influence significantly the level of any hormones as compared to mock inoculation alone in the mock-

EP

inoculated lower leaves (Fig. 5A). Interestingly however, pre-treatment with 100 nM 24-epiBR two days prior to ObPV inoculation drastically increased the GA3 and ABA contents in

AC C

the inoculated leaves as compared to samples taken from ObPV-inoculated leaves without 24epi-BR pre-treatment (Fig. 5B). On the other hand, 24-epi-BR pre-treatment significantly decreased the contents of c-ZR and t-ZR in the ObPV-inoculated lower leaves (Fig. 5B). The rest of the investigated hormones were unaffected by 24-epi-BR pre-treatment. The effect of 24-epi-BR pre-treatment was also investigated in the upper, systemic leaves following ObPVinoculation. Pre-treatment with 24-epi-BR markedly decreased (by 55%) the ABA content in upper, systemic leaves following ObPV inoculation of lower leaves (Fig. 5C). However, the

18

ACCEPTED MANUSCRIPT 24-epi-BR pre-treatment exerted no significant influence on the level of other hormones in the upper leaves (Fig. 5C). PMMoV inoculations preceded with 24-epi-BR pre-2treatment did not modify the content of any of the twelve plant hormones in the upper, non-infected leaves 20

RI PT

dpi (Fig. 5D).

4. Discussion

SC

It has been known for a long time that plant hormones play pivotal roles in the regulation of defense signaling networks in both antagonistic and synergistic manners

M AN U

(Alazem and Lin, 2015). However, not only plants can use these small molecules to improve their resistance, but pathogens can also manipulate the plant defense signaling networks for their own benefit (Pieterse et al., 2009). It is noteworthy that changes in hormone quantity and composition greatly depend on the lifestyle of the pathogen. Generally in compatible

TE D

interactions biotrophic pathogens prefer and induce the accumulation of juvenility/antisenescence hormones (auxins, cytokinins, and gibberellins), while the immune response of plants to biotrophs upregulates the salicylic acid (SA) pathway and induce the accumulation

EP

of SA. To the contrary, necrotrophic pathogens induce the accumulation of stress or senescence hormones like ABA, BRs, ethylene or JA (Barna et al., 2012a). Necrotic lesion

AC C

formation by virus infections has similarities to necrotroph symptoms; it induces a strong accumulation of ethylene (Tóbiás et al., 1989; Barna et al., 2012b). Currently it is already widely accepted that not only three hormones (ethylene, JA and

SA) regulate plant defense to pathogens, but ABA, auxins, BRs, cytokinins and gibberellins are also claimed as important players in disease resistance (Denance et al., 2013; Alazem and Lin, 2015). This hypothesis was supported by our data as well. In our experiments ObPVinoculation substantially increased not only the levels of the typical stress hormones (JA and

19

ACCEPTED MANUSCRIPT SA), but also those of ABA, IAA, IBA, c-Z, c-ZR and t-ZR in the inoculated leaves (Fig. 1). Further studies are necessary to clarify the synergistic and antagonistic nature of interactions between these hormones. A well-known example is the antagonism of SA and JA/ethylene pathways (Pieterse et al., 2009). However, in TMV-infected Nicotiana benthamiana the

RI PT

simultaneous accumulation of SA and JA was also observed (Zhu et al., 2014). Interestingly, treatment of N. benthamiana plants with JA and subsequently with SA enhanced their systemic resistance against TMV (Zhu et al., 2014). It seems that plants respond with the

SC

production of a specific hormone pattern to different pathogen attacks and thus intricate 'hormone signatures' can be detected also in virus-inoculated leaves. In our experiments

M AN U

ObPV infection significantly increased not only the SA, JA and ethylene levels but also those of ABA, auxins, cytokinins and BRs, thus a complex hormone signature could be observed (Figs. 1 and 2). It is important to note that the virus-elicited hormone signatures markedly differed between the compatible and incompatible pepper-virus interactions. In the compatible

TE D

pepper-PMMoV interaction only the levels of two hormones (GA3 and SA) increased significantly in the inoculated leaves (Fig. 1). These results showed that in the symptomless, compatible pepper-PMMoV interaction the hormonal changes are substantially weaker than in

EP

the incompatible pepper-ObPV interaction.

A particularly interesting finding of the present work was the detection of

AC C

progesterone in pepper leaves, and to our knowledge this is the first report of progesterone from pepper plants. In addition, progesterone content was strongly elevated in ObPVinoculated, and also significantly increased in PMMoV-inoculated leaves (Fig. 2). The possible role of progesterone in disease resistance is a new and open question, but we found earlier that similarly to BRs, pre-treatment of leaves with progesterone diminished the necrotic symptoms, the electrolyte leakage and improved the efficiency of photosystem II in Arabidopsis thaliana infected with Pseudomonas syringae (Janeczko et al., 2013b).

20

ACCEPTED MANUSCRIPT We could detect relatively few BRs in pepper leaves, and interestingly the amount of 24-epi-BR was detected only in the upper leaves of ObPV-inoculated pepper plants. Whether this systemic accumulation of a BR is in connection with the induction of systemic acquired resistance of pepper is an open question. On the contrary, castasterone and 28-

RI PT

homocastasterone were detected in both lower and upper leaves. The amount of castasterone markedly increased in the ObPV-inoculated lower leaves 3 dpi, while the 28homocastasterone content decreased in the systemic, upper leaves of PMMoV-inoculated

SC

plants (Fig. 2). BRs are known to protect plants against various abiotic and biotic stresses (Skoczowski et al., 2011; Zhang et al. 2015). Now we have proved that the exogenous

M AN U

application of 24-epi-BR can mitigate the formation of hypersensitive necrotic lesions in pepper leaves following ObPV inoculation (Fig. 4). Probably the increase in steroid accumulation is a part of stress resistance of plants that helps to avoid cell and tissue damage by 'strengthening' plant membranes, but this hypothesis requires further and more detailed

TE D

studies. Another possibility is that BR themselves possess antiviral effect (Wachsman et al. 2000). Pre-treatment with 24-epi-BR prior to ObPV-inoculation markedly changed the level of some hormones presumably due to modifying their biosynthesis (Fig. 5), but again these

EP

observations need further studies. As shown in experiments on BR-deficient mutants, these steroids are important players in plant hormonal networks and a decrease in endogenous BR

AC C

contents results in disturbances in the biosynthesis of other hormones including ABA (Janeczko et al. 2016).

Virus inoculations strongly influenced the expression of several pepper genes that

encode key enzymes in hormone biosynthetic pathways. As in the case of hormone levels, marked differences were observed again by comparing gene expression levels between the compatible and incompatible pepper-virus interactions (Fig. 3). In connection with SA biosynthesis we compared the expression of PAL and ICS genes, which are the critical

21

ACCEPTED MANUSCRIPT enzymes of two possible SA biosynthetic pathways (Vlot et al., 2009). ObPV-inoculation significantly up-regulated the expression of three PAL genes while that of the ICS1 gene was not modified (Fig. 3). In contrast to ObPV, PMMoV-inoculation did not modify the expression of PAL or ICS genes (Fig. 3). These results suggest that the phenylpropanoid

RI PT

pathway of SA biosynthesis (Kim and Hwang, 2014) is responsible for the SA accumulation observed during the incompatible pepper-ObPV interaction, whereas the effect of the isochorismate pathway (Catinot et al., 2008) seems to be less important. These results support

SC

earlier findings of Pallas et al. (1996), who observed a markedly decreased SA accumulation in TMV-inoculated tobacco plants, in which the PAL expression was epigenetically

M AN U

suppressed.

Our earlier study already revealed the rapid and strong accumulation of ethylene in ObPV-inoculated pepper leaves, while the ethylene level did not change in PMMoVinoculated leaves (Tóbiás et al., 1989). In the present work, we revealed the marked up-

TE D

regulation of an ACO gene both in ObPV- and in PMMoV-inoculated leaves. However, the transcript accumulation of ACO was observed 1 day earlier in the ObPV- than in the PMMoV-inoculated leaves (Fig. 3). The early up-regulation of this particular ACO gene by

EP

ObPV, which coincides with the accumulation of ethylene (Tóbiás et al., 1989), presumably markedly enhances the rate of ethylene biosynthesis. Interestingly, PMMoV-inoculation also

AC C

led to the accumulation of ACO transcript 2 dpi, although in this system Tóbiás et al. (1989) did not find any significant ethylene accumulation. Previously TMV-inoculation was already shown to up-regulate the expression of both ACO and ACS genes in resistant tobacco leaves (Kim et al., 2003). However, the massive accumulation of ethylene in virus-infected plants can also be explained by the post-transcriptional activation of the ACS protein by a kinase cascade (Kim et al., 2003). The transcript accumulations of the AOC, 13-LOX2, 13-LOX6, NCED1, NCED2, NCED3 and actin genes were not modified by any virus inoculation (Fig.

22

ACCEPTED MANUSCRIPT 3), which showed that the elevated levels of ABA and JA following ObPV-inoculation was not a consequence of the transcriptional up-regulation of these genes. Earlier we showed that the expression of a pepper 13-AOS gene was not modified neither by ObPV nor by PMMoVinoculation (Gullner et al., 2010).

RI PT

To further explore the effect of BR on the defense reactions of virus-infected pepper plants we analyzed the effects of 24-epi-BR pre-treatment on visual symptoms as well as on endogenous hormone contents in ObPV-inoculated pepper leaves. Pre-treatment with 24-epi-

SC

BR prior to ObPV-inoculation considerably attenuated the visible necrotic symptoms caused by ObPV (Fig. 4). Previously we observed a similar protective effect of 24-epi-BR pre-

M AN U

treatment in rape cotyledons following infection with an incompatible bacterium (Skoczowski et al., 2011).

In addition, 24-epi-BR pre-treatment markedly increased the endogenous ABA and GA3 contents, and significantly decreased the c-ZR and t-ZR levels in ObPV inoculated,

TE D

lower leaves (Fig. 5B). In the upper, systemic leaves 24-epi-BR pre-treatment influenced only the ABA content (Fig. 5C). PMMoV inoculations preceded by 24-epi-BR pre-treatment did not influence the hormone levels in the systemic, upper leaves (Fig. 5D). BRs are poorly (or

EP

not) transported in plants when applied via spraying (Janeczko and Swaczynova, 2010), which may be a reason for the inefficiency of BR in the upper leaves. Whether these changes

AC C

in endogenous hormone contents are in direct or indirect connections with changes of biotic stress resistance of plants pre-treated with BRs is not known yet. In conclusion, in our experiments not only the accumulation of SA and JA, but also

those of ABA, auxins, cytokinins, gibberellins, steroid regulators were detected in the hypersensitively reacting ObPV-inoculated pepper leaves. On the other hand, there were also significant, although much weaker changes in some hormone levels in the compatible pepperPMMoV interaction. Our data indicated the appearance of complex hormonal changes as a

23

ACCEPTED MANUSCRIPT result of virus inoculations. We revealed also the marked up-regulation of an ACO and three PAL genes that encodes key enzymes of ethylene and SA biosynthetic pathways, respectively. In addition, to our knowledge this is the first report about the determination of BRs and progesterone in pepper leaves as well as about the virus-elicited changes in their foliar

RI PT

contents. Moreover, our data about the alleviation of foliar symptoms of ObPV-inoculation by the exogenous application of 24-epi-BR in this economically important plant species can be

SC

valuable from a practical point of view.

M AN U

Acknowledgments

The experiments were conducted within a bilateral cooperation project between the Polish and Hungarian Academy of Sciences during 2014-2016. We thank Dr. Lajos Zatykó (Research Institute of Vegetable Crops, Budatétény, Hungary) for kindly providing the pepper seeds.

TE D

The study was partly financed by the Grant Agency of the Czech Republic (GA14-34792S). The financial support of the Hungarian Scientific Research Fund (OTKA K83615) is

AC C

References

EP

gratefully acknowledged.

Alazem, M., Lin, N.S., 2015. Roles of plant hormones in the regulation of host-virus interactions. Mol. Plant Pathol. 16, 529-540.

Allmann, S., Halitschke, R., Schuurink R.C., Baldwin, I.T., 2010. Oxylipin channelling in Nicotiana attenuata: lipoxygenase 2 supplies substrates for green leaf volatile production. Plant Cell Environ. 33, 2028-2040.

24

ACCEPTED MANUSCRIPT

Bajguz, A., 2007. Metabolism of brassinosteroids in plants. Plant Physiol. Biochem. 45, 95107.

RI PT

Balázs, E., Király, Z., Gáborjányi, R., 1973. Leaf senescence and increased virus susceptibility in tobacco. The effect of abscisic acid. Physiol. Plant Pathol. 3, 341-346.

SC

Barna, B., Fodor, J., Harrach, B.D., Pogány, M., Király, Z., 2012a. The Janus face of reactive oxygen species in resistance and susceptibility of plants to necrotrophic and biotrophic

M AN U

pathogens. Plant Physiol. Biochem. 59, 37-43.

Barna, B., Pogány, M., Koehl, J., Heiser, I., Elstner, E.F., 2012b. Induction of ethylene synthesis and lipid peroxidation in damaged or TMV infected tobacco leaf tissues by

TE D

light. Acta Physiol. Plant. 34, 1905-1914.

Catinot, J., Buchala, A., Abou-Mansour, E., Métraux, J.P., 2008. Salicylic acid production in

EP

response to biotic and abiotic stress depends on isochorismate in Nicotiana

AC C

benthamiana. FEBS Lett. 582, 473-478.

Denance, N., Sanches-Vallet, A., Goffner, D., Molina, A., 2013. Disease resistance or growth: the role of plant hormones in balancing immune responses and fitness costs. Front. Plant Sci. 4, 155.

25

ACCEPTED MANUSCRIPT Dobrev, P.I., Kaminek, M., 2002. Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. J. Chromatography A 950, 21-29.

RI PT

Enyedi, A.J., Yalpani, N., Silverman, P., Raskin, I., 1992. Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco

SC

mosaic virus. Proc. Natl. Acad. Sci. USA 89, 2480-2484.

Feussner, I., Wasternack, C., 2002. The lipoxygenase pathway. Annu. Rev. Plant Biol. 53,

M AN U

275-297.

Fodor, J., Gullner, G., Ádám, A. L., Barna, B., Kőmíves, T., Király, Z., 1997. Local and systemic responses of antioxidants to tobacco mosaic virus infection and to salicylic

TE D

acid in tobacco: Role in systemic acquired resistance. Plant Physiol. 114, 1443-1451.

Fraser, R.S.S., 1982. Are pathogenesis-related proteins involved in acquired systemic

EP

resistance of tobacco plants to tobacco mosaic virus? J. Gen. Virol. 58, 305–313.

AC C

Gaffney, T., Friedrich, L., Vernooij, B., Negrotto, D., Nye, G., Uknes, S., Ward, E., Kessmann, H., Ryals, J., 1993. Requirement of salicylic acid for the induction of systemic acquired resistance. Science. 261, 754-756.

Gatz, C., 2013. From pioneers to team players: TGA transcription factors provide a molecular link between different stress pathways. Mol. Plant-Microbe Interact. 26, 151-159.

26

ACCEPTED MANUSCRIPT Gullner, G., Künstler, A., Király, L., Pogány, M., Tóbiás, I., 2010. Up-regulated expression of lipoxygenase and divinyl ether synthase genes in pepper leaves inoculated with Tobamoviruses. Physiol. Mol. Plant Pathol. 74, 387-393.

RI PT

Jameson, P.E., Clarke, S.F., 2002. Hormone-virus interactions in plant. Crit. Rev. Plant Sci. 21, 205-228.

M AN U

epibrassinolide. Biol. Plant. 54, 477-482.

SC

Janeczko, A., Swaczynová, J., 2010. Endogenous brassinosteroids in wheat treated with 24-

Janeczko, A., 2012. The presence and activity of progesterone in the plant kingdom. Steroids. 77, 169-173.

TE D

Janeczko, A., Oklestková, J., Siwek, A., Dziurka, M., Pociecha, E., Kocurek, M., Novák, O., 2013a. Endogenous progesterone and its cellular binding sites in wheat exposed to

EP

drought stress. J. Steroid Biochem. Mol. Biol. 138, 384–394.

Janeczko, A., Tóbiás, I., Skoczowski, A., Dubert, F., Gullner, G., Barna, B., 2013b.

AC C

Progesterone moderates damage in Arabidopsis thaliana caused by infection with Pseudomonas syringae or P. fluorescens. Biol. Plant. 57, 169-173.

Janeczko, A. Gruszka, D., Pociecha, E., Dziurka, M., Filek, M., Jurczyk, B., Kalaji, H.M., Kocurek, M., Waligórski, P., 2016. Physiological and biochemical characterisation of watered and drought-stressed barley mutants in the HvDWARF gene encoding C6oxidase involved in brassinosteroid biosynthesis. Plant Physiol. Biochem. 99, 126-141.

27

ACCEPTED MANUSCRIPT

Juhász, C., Tóbiás, I., Ádám, A.L., Kátay, G., Gullner, G., 2015. Pepper 9- and 13lipoxygenase genes are differentially activated by two tobamoviruses and by hormone

RI PT

treatments. Physiol. Mol. Plant Pathol. 92, 59-69.

Kim, C.Y., Liu, Y., Thorne, E.T., Yang, H., Fukushige, H., Gassmann, W., Hildebrand, D., Sharp, R.E., Zhang, S., 2003. Activation of a stress-responsive mitogen-activated

SC

protein kinase cascade induces the biosynthesis of ethylene in plants. Plant Cell. 15,

M AN U

2707-2718.

Kim, D.S., Hwang, B.K., 2014. An important role of the pepper phenylalanine ammonia-lyase gene (PAL1) in salicylic acid-dependent signalling of the defence response to microbial

TE D

pathogens. J. Exp. Bot. 65, 2295-2306.

Koorneef, A., Pieterse, C.M.J., 2008. Cross talk in defence signaling. Plant Physiol. 146, 839-

EP

844.

Künstler, A., Bacsó, R., Gullner, G., Hafez, Y.M., Király, L., 2016. Staying alive - is cell

AC C

death dispensable for plant disease resistance during the hypersensitive response? Physiol. Mol. Plant Pathol. 93, 75-84.

Nakashita, H.,Yasuda, M., Nitta, T., Asami, T., Fujioka, S., Arai, Y., 2003. Brassinosteroid functions in a broad range of disease resistance in tobacco and rice. Plant J. 33, 887898.

28

ACCEPTED MANUSCRIPT Oka, K., Kobayashi, M., Mitsuhara, I., Seo, S., 2013. Jasmonic acid negatively regulates resistance to Tobacco mosaic virus in tobacco. Plant Cell Physiol. 54, 1999-2010.

Pallas, J.A., Paiva, N.L., Lamb, C., Dixon, R.A., 1996. Tobacco plants epigenetically

RI PT

suppressed in phenylalanine ammonia lyase expression do not develop systemic acquired resistance in response to infection by tobacco mosaic virus. Plant J. 10, 281-

SC

293.

Pieterse, C.M.J., Leon-Reyes, A., Van der Ent, S., Van Wees, S.C.M., 2009. Networking by

M AN U

small-molecule hormones in plant immunity. Nat. Chem. Biol. 5, 308-316.

Rys, M., Juhász, C., Surówka, E., Janeczko, A., Saja, D., Tóbiás, I., Skoczowski, A., Barna, B., Gullner, G., 2014. Comparison of a compatible and an incompatible pepper-

TE D

tobamovirus interaction by biochemical and non-invasive techniques: chlorophyll a fluorescence, isothermal calorimetry and FT-Raman spectroscopy. Plant Physiol.

EP

Biochem. 83, 267-278.

Simerský, R., Novák, O., Morris, D.A., Pouzar, V., Strnad, M., 2009. Identification and

AC C

quantification of several mammalian steroid hormones in plants by UPLC-MS/MS. J. Plant Growth Regul. 28, 125-136.

Singh, D.P., Moore, C.A., Gilliland, A., Carr, J.P., 2004. Activation of multiple antiviral defence mechanisms by salicylic acid. Mol. Plant Pathol. 5, 57-63.

29

ACCEPTED MANUSCRIPT Skoczowski, A., Janeczko, A., Gullner, G., Tóbiás, I., Kornas, A., Barna, B., 2011. Response of brassinosteroid-treated oilseed rape cotyledons to infection with the wild type and HR-mutant of Pseudomonas syringae or with P. fluorescens. J. Thermal Anal. Calorim.

RI PT

104, 131-139.

Spoel, S.H., Dong, X., 2008. Making sense of hormone crosstalk during plant immune

SC

responses. Cell Host Microbe 3, 348-351.

Tarkowská, D., Novák, O., Oklestková, J., Strnad, M., 2016. The determination of 22 natural

Bioanal. Chem. 408, 6799-6812.

M AN U

brassinosteroids in a minute sample of plant tissue by UHPLC–ESI–MS/MS. Anal.

Taylor, I.B., Burbidge, A., Thompson, A.J., 2000. Control of abscisic acid synthesis. J. Exp.

TE D

Bot. 51, 1563-1574.

Tóbiás, I., Fraser, R.S.S., Gerwitz, A., 1989. The gene-for-gene relationship between

EP

Capsicum annuum L. and tobacco mosaic virus: effects on virus multiplication, ethylene synthesis and accumulation of pathogenesis-related proteins. Physiol. Mol. Plant Pathol.

AC C

35, 271-286.

Vlot, A.C., Dempsey, D.A., Klessig, D.F., 2009. Salicylic acid, a multifaceted hormone to combat disease. Annu. Rev. Phytopathol. 47, 177-206.

30

ACCEPTED MANUSCRIPT Wachsman, M.B., López, E.M., Ramirez, J.A., Galagovsky, L.R., Coto, C.E., 2000. Antiviral effect of brassinosteroids against herpes virus and arenaviruses. Antivir. Chem. Chemother. 11, 71-77.

RI PT

Whenham, R.J., Fraser, R.S., Brown, L.P., Payne, J.A., 1986. Tobacco-mosaic-virus-induced increase in abscisic-acid concentration in tobacco leaves: Intracellular location in light

SC

and dark-green areas, and relationship to symptom development. Planta 168, 592-598.

Zhang, D.W., Deng, X.G., Fu, F.Q., Lin, H.H., 2015. Induction of plant virus defense

M AN U

response by brassinosteroids and brassinosteroid signaling in Arabidopsis thaliana. Planta 241, 875-885.

Zhu, F., Xi, D.H., Yuan, S., Xu, F., Zhang, D.W., Lin, H.H., 2014. Salicylic acid and

TE D

jasmonic acid are essential for systemic resistance against tobacco mosaic virus in Nicotiana benthamiana. Mol. Plant-Microbe Interact. 27, 567-577.

EP

Żur, I., Dubas, E., Krzewska, M., Janowiak, F., 2015. Current insights into hormonal

AC C

regulation of microspore embryogenesis. Front. Plant Sci. 6, 424.

31

ACCEPTED MANUSCRIPT

Table 1. Optimized mass spectrometry parameters for ABA, auxins, cytokinins, gibberellins, JA and SA.

[M+H]+

K-N15

[M+H]+

K

[M+H]+

t-ZR

[M+H]+

c-ZR

[M+H]+

IPA

[M+H]+

KR

[M+H]+

GA1

[M-H2O+H]+

GA3

[M-H2O+H]+

GA6

[M-H2O+H]+

IAA-D5

[M+H]+

IAA

[M+H]+

SD-D4

[M+H]+

SA

[M+H]+

ABA-D6

[M-H2O+H]+

ABA

[M-H2O+H]+

JA

[M+H]+

GA4

AC C

IBA

[M+H]+

[M-H2O+H]+

Fragment Collision Dwell time electric energy (V) voltage (V) 350 85 9 350 85 33 350 85 9 350 85 33 200 90 9 200 90 9 200 90 9 200 90 9 200 120 9 200 120 29 200 120 9 200 120 29 200 90 9 200 90 9 350 116 9 350 116 21 200 100 14 200 100 14 200 100 14 200 100 14 80 104 14 80 104 98 200 38 14 80 38 50 80 51 9 80 51 53 80 80 14 80 80 46 80 80 14 80 80 46 200 80 14 200 80 14 200 80 14 200 80 14 250 80 14 250 80 14 250 69 9 250 69 25 400 100 14 400 100 14

MRM Start Time (min)

RI PT

c-Z

Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier Quantifier Qualifier

TE D

[M+H]+

EP

t-Z

Precursor/product ions 220.2/136.3 220.2/119.2 220,2/136,3 220.2/119.2 220,1/192,3 220,1/152,3 216,1/188,3 216,1/148,3 352,2/220,3 352,2/136,3 352,2/220,3 352,2/136,3 204,1/148,3 204,1/119,2 348,2/216,3 348,2/148,3 331,3/285,3 331,3/257,3 329,3/311,3 329,3/237,3 329,3/283,3 329,3/115,1 181,1/135,1 181,1/82,1 176,1/130,3 176,1/130,3 143,2/125,2 143,2/125,2 139,2/121,2 139,2/39,1 253,4/191,3 253,4/178,3 247,4/187,2 247,4/173,3 211,3/151,2 211,3/133,2 204,1/186,4 204,1/130,3 315,3/269,3 315,3/241,3

M AN U

Type of ion

SC

Transition Analyte

1 1 1 1 2,7 2,7 2,7 2,7 4,4 4,4 4,4 4,4 4,4 4,4 5,9 5,9 6,6 6,6 6,6 6,6 7,15 7,15 7,15 7,15 7,15 7,15 7,15 7,15 7,15 7,15 8,8 8,8 8,8 8,8 10 10 10 10 11,3 11,3

32

ACCEPTED MANUSCRIPT Table 2. Primers used for PCR assays in 5' to 3' direction.

Forward primer

Reverse primer

Product Annealing length temperature (bp) (°C) __________________________________________________________________________________________ ACO (XM_016698789)

ttggagaagttggctgaggagtta

aagttggcgcgggatagattac

433

56

actin (AY572427)

agcaactgggacgatatggagaaga

aagagacaacaccgcctgaatagca

198

55

AOC (XM_016699708)

aggggtttccggtcaagta

gaccccaacaaaagtcgtga

236

60

ICS1 (AY743431)

tcatcggtgcacccaactc

ccaagacccttttcaaccaa

179

58

13-LOX2 (JQ219046)

ggaccggcgatgcagaga

atgtacttgcatcttaa

234

50

13-LOX6 (XM_016692059)

acttggcagcatgcaatttgt

cgcgtatcttcttcgaaagac

164

59

NCED1 (XM_016697078)

gaaaaggaatggaaatcgga

cggggacgtatattctaaac

231

55

NCED2 (XM_016691094)

agtaccaccaaaaattatagg

tctgagctttccgaatc

208

57

NCED3 (XM_016719296)

aaagagtgccatatggtt

tcaagaagggtattcaataga

200

54

PAL1 (KF279696)

ggaaggaacttggaactga

gcacttgacaagcactaaca

204

58

ggcaagtcatccattcctaa

ggggacgagatgaaccat

214

52

gaatctgaaaaatggcacta

188

52

PAL3 (XM_016687267)

SC

M AN U

TE D

EP

AC C

PAL2 (XM_016710142)

aggtgaaagagtccgatca

RI PT

Target gene (GenBank accession)

__________________________________________________________________________________________

33

ACCEPTED MANUSCRIPT

Figure legends

Figure 1

RI PT

Effects of ObPV- and PMMoV-inoculations on the level of twelve phytohormones in the inoculated, lower leaves as well as in the systemic, uninfected upper leaves of pepper plants. Local and systemic effects of virus inoculations were investigated 3 and 20 days post

SC

inoculation (dpi), respectively. Means of three independent experiments ± SD are shown. The symbols * , ** and *** show significant differences between virus-inoculated and mock

M AN U

inoculated (control) leaves at P < 5%, P < 1% and P < 0.1%, respectively.

Figure 2

Effects of ObPV- and PMMoV-inoculations on the level of brassinosteroids and progesterone

TE D

in the inoculated, lower leaves as well as in the systemic, uninfected upper leaves of pepper plants. Local and systemic effects of virus inoculations were investigated 3 and 20 days post inoculation (dpi), respectively. Means of three independent experiments ± SD are shown. For

EP

the explanation of symbols see the caption of Fig. 1.

AC C

Figure 3

Effects of ObPV- and PMMoV-inoculations on the expression of eleven pepper genes encoding enzymes participating in hormone biosynthetic pathways. Abbreviations: ACO, 1aminocyclopropane-1-carboxylic acid oxidase; AOC, allene oxide cyclase; ICS, isochorismate synthase;

LOX,

lipoxygenase;

NCED,

9-cis-epoxycarotenoid

dioxygenase;

PAL,

phenylalanine ammonia-lyase. The expression of actin was examined as control,

34

ACCEPTED MANUSCRIPT housekeeping gene. GenBank accession numbers of all genes are shown in Table 2. Representative results of three independent parallel experiments are shown.

Figure 4

RI PT

Visible disease symptoms and lesion numbers on pepper leaves 4 days after mockinoculation, after ObPV-inoculation as well as after ObPV-inoculation preceded by a spraying of leaves with 100 nM 24-epi-brassinolide (24-epi-BR) 2 days before ObPV inoculation. A:

SC

disease symptoms observed on the 5th leaf positions, B: lesion numbers on the 5th leaf positions (n = 20). The symbol * shows significant differences between 24-epi-BR pre-

M AN U

treated and not pre-treated plants at P < 5%.

Figure 5

Effect of spraying with 100 nM 24-epi-brassinolide (24-epi-BR) 2 days prior to mock, ObPV

TE D

or PMMoV inoculations on the endogenous level of twelve plant hormones in pepper leaves. A: mock inoculation, inoculated leaves (3 dpi), B: ObPV inoculation, inoculated leaves (3 dpi), C: ObPV inoculation, systemic upper leaves (20 dpi), D: PMMoV inoculation, systemic

EP

upper leaves (20 dpi). Means of three independent experiments ± SD are shown. The symbols * , ** and *** show significant differences between 24-epi-BR pre-treated and not pre-treated

AC C

plants at P < 5%, P < 1% and P < 0.1%, respectively. Values of mock and ObPV infected plants are taken as 100%.

35

ACCEPTED MANUSCRIPT Figure 1

200

600 400

ng g FW

-1

100 80 60 40

0.8 0.6 0.4

0

AC C

gibberellin A1 (GA1)

6 4

trans-zeatin 9-riboside (t-ZR)

***

abscisic acid (ABA)

1

0

-1

ng g FW

500 400 300 200 100 0 30

-1

-1

EP

2

***

2

600

ng g FW

N -isopentenyladenine (IPA)

3

8

0.50

3

6

0

0.75

-1

cis-zeatin9-riboside (c-ZR)

10

1

1.00

0.00

***

15

4

trans-zeatin (t-Z)

0.25

TE D

-1

ng g FW

ng g FW

1.0

5

1.25

M AN U

cis-zeatin (c-Z)

ng g FW

-1

ng g FW

***

-1

0

5

-1

20

1.50

0.0

ng g FW

30

1.4

0.2

ng g FW

40

0

20

indole-3-butyric acid (IBA)

50

10

20

1.2

**

75

0

indole-3-acetic acid (IAA)

**

100

50 -1

120

jasmonic acid (JA)

25

*

0

***

125

SC

200

150

RI PT

800

ng g FW

ng g FW

1000

175 -1

salicylic acid (SA)

-1

1200

***

ng g FW

1400

25 20

gibberellic acid (GA3) **

15 10

2

5

0

mock ObPV PMMoV

0

mock ObPV PMMoV

infected leaf

upper leaf

3 dpi

20 dpi

mock ObPV PMMoV

mock ObPV PMMoV

infected leaf

upper leaf

3 dpi

20 dpi

36

ACCEPTED MANUSCRIPT Figure 2

RI PT

0.3

24-epi-brassinolide 0.2

0.0

n.d. n.d. n.d.

n.d.

M AN U

*

0.4 0.2 0.0

28-homocastasterone

10

TE D

-1

n.d.

castasterone

0.6

pmol g FW

SC

0.1

5

**

0

EP

0.06

***

progesterone

AC C

0.04 0.02

**

n.d. n.d.

0.00 mock ObPV PMMoV

mock ObPV PMMoV

infected leaf

upper leaf

3 dpi

20 dpi

37

ACCEPTED MANUSCRIPT Figure 3

ObPV-inoculated

PMMoV-inoculated

RI PT

mock-inoculated ICS1 PAL1 PAL2

SC

PAL3 13-LOX2

M AN U

13-LOX6 AOC NCED1 NCED2 NCED3 actin 0

TE D

ACO

4 8 12 24 48

0

4 8 12 24 48

0 4

AC C

EP

Hours post-inoculation (hpi)

8 12 24 48

38

ACCEPTED MANUSCRIPT Figure 4

B 70

lesion number

60 50 40 30 20

24-epi-BR + ObPV

*

TE D

10

ObPV

M AN U

mock

SC

RI PT

A

0

AC C

EP

ObPV

24-epi-BR + ObPV

39

ACCEPTED MANUSCRIPT Figure 5

300

A

200

mock inoculation

mock 24-epi-BR + mock

RI PT

250

150 100 50

SA JA ABA IAA IBA c-Z t-Z c-ZR t-ZR IPA GA1 GA3 ObPV 24-epi-BR + ObPV

B

400

inoculated leaf

300

**

200 100

*

0

***

M AN U

500

*

SA JA ABA IAA IBA c-Z t-Z c-ZR t-ZR IPA GA1 GA3

300 250

C

200

upper leaf

150 100

0

SA JA ABA IAA IBA c-Z t-Z c-ZR t-ZR IPA GA1 GA3

D

AC C

250 200

ObPV 24-epi-BR + ObPV

*

EP

50

TE D

relative hormone content

600

SC

0

PMMoV 24-epi-BR + PMMoV

upper leaf

150 100 50

0 SA JA ABA IAA IBA c-Z t-Z c-ZR t-ZR IPA GA1 GA3

leaf hormone content

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

AS, BB, GG and IT conceived the experiments, BB, GG, IT and AJ made the sample preperations, CJ and GG carried out the RT-PCR experiments, MD, AJ and DS carried out the hormone analysis, JO and ON made the steroid hormone analysis, AS, GG, AJ, DS and BB prepared the manuscript, IT and CJ carried out the virus inoculations