Knockout of SlMAPK3 reduced disease resistance to Botrytis cinerea

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Agricultural and Environmental Chemistry

Knockout of SlMAPK3 reduced disease resistance to Botrytis cinerea in tomato plants Shujuan Zhang, Liu Wang, Ruirui Zhao, Wenqing Yu, Rui Li, Yujing Li, Jiping Sheng, and Lin Shen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02191 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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Journal of Agricultural and Food Chemistry

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Knockout of SlMAPK3 reduced disease resistance to

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Botrytis cinerea in tomato plants

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Shujuan Zhang†, Liu Wang†, Ruirui Zhao†, Wenqing Yu†, Rui Li†, Yujing Li†, Jiping

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Sheng‡, Lin Shen†,*

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† College of Food Science and Nutritional Engineering, China Agricultural University,

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Beijing 100083, China

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‡ School of Agricultural Economics and Rural Development, Renmin University of

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China, Beijing 100872, China

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Corresponding Author

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*E-mail: [email protected]; Phone: +86-10-62737620; Fax: +86-10-62737620.

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ACS Paragon Plus Environment


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ABSTRACT

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Mitogen-activated protein kinases (MAPKs) play an important role in defense

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responses to biotic and abiotic stresses. In order to investigate the role of SlMAPK3 in

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tomato plant resistance to Botrytis cinerea, two lines of slmapk3 mutants and wild-type

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(WT) tomato plants were used. The results showed that slmapk3 mutants were more

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susceptible to B. cinerea and that knockout of SlMAPK3 reduced the activities of

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defense enzymes, enhanced the accumulation of reactive oxygen species (ROS).

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Furthermore, we detected the expressions of salicylic acid (SA) and jasmonic acid (JA)

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signaling-related genes and found that knockout of SlMAPK3 enhanced the expressions

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of SlPR1, SlPAD4 and SlEDS1, whereas reduced the expressions of SlLoxC, SlPI I and

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SlPI II, and enhanced the expressions of SlJAZ1 and SlMYC2. We postulate that

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SlMAPK3 plays a positive role in tomato plant resistance to B. cinerea through

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regulating ROS accumulation, SA and JA defense signaling pathways.

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Keywords: tomato plant, disease resistance, Botrytis cinerea, SlMAPK3, salicylic acid,

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jasmonic acid

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INTRODUCTION

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Throughout plants life cycles, they are constantly threatened by pathogens, like

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bacteria, fungi and viruses, which causes a severe threat to food security and huge

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losses in crops production1. Pathogens were generally classified as biotrophs and

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necrotrophs according to their lifestyles. Biotrophs grow on living plant tissue, whereas

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necrotrophs kill host cells and acquire nutrition from dead or dying material.2 Botrytis

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cinerea (B. cinerea) is a kind of necrotrophic pathogens and is the pathogen agent of

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grey mold disease in more than 200 crop species.3 B. cinerea exist under most

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environment and has wild range of infection sites, including flowers, leaves, fruits and

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could infect plant at the whole process of plant development.4, 5

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To defense against pathogens invasion, plants have evolved complex immune

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systems to recognize pathogens and activate defense response. The first tier of plant

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immunity is termed pathogen-associated molecular pattern (PAMP)-triggered

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immunity (PTI), which is triggered by pathogen associated-molecular patterns

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(PAMPs/MAMPs) and activates downstream mitogen-activated protein (MAP) kinase

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cascades, reactive oxygen species (ROS) and defense genes.6 The second tier of plant

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immunity is termed effector-triggered immunity (ETI), which is driven by disease

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resistance proteins and can recognize specific pathogen effectors.7 Furthermore, PTI

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and ETI activate systemic acquired resistance (SAR), which is regulated by

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phytohormones.8 Among the phytohormones, salicylic acid (SA) and jasmonic acid (JA)

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are the best known.9 There is abundant evidence suggesting that SA is a crucial

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component of systemic acquired resistance (SAR) and is the regulator of 3



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pathogenesis-related (PR) genes.10, 11 JA promotes the expression of almost all major

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defense-related secondary metabolites and proteins, including terpenoids, alkaloids,

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amino

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proteins.12 And it is generally believed that SA is a key hormonal signaling against

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biotrophic pathogens, whereas JA is a major hormonal signaling against necrotrophic

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pathogens in plants.13, 14

acid,

anti-nutritional

proteins,

and

some

pathogenesis-related

(PR)

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Mitogen-activated protein kinase (MAPK) cascades are signaling modules which

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could transport extracellular stimuli into intracellular.15 MAPK cascades pathway

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consists of MAPKK kinase (MAPKKK), MAPK kinase (MAPKK) and MAP kinase

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(MAPK), which transmit signals through phosphorylation successively in proper

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order.16 MAPK signaling pathway plays an important role in plant physiology,

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including cell division, hormone signaling and stress response.17

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Particularly, MAPKs participate in plant defense against pathogens. In Arabidopsis

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thaliana, MAPK1, MAPK3, MAPK4 and MAPK6 have been considered as

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defense-related MAPKs.18 Firstly, MAPKs play an important role in PTI and ETI.

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When plants recognize PAMPs, one of the earliest events is to activate of MAPKs.

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MEKK1-MKK4/MKK5-MPK3/MPK6 and MEKK1-MKK1/2-MPK4 were reported to

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involve in ETI.15 AtMAPK3 were identified as a negative role in regulating the

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transcription of defense gene, flg22-induced salicylic acid accumulation and disease

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resistance to Pseudomonas syringae.6 And it was demonstrated that AtMAPK3

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regulated camalexin [3-thiazol-2-yl-indole (23)] synthesis by regulating the

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transcription of camalexin biosynthetic genes after fungus infection.19 Moreover, PR1 4


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and most SA-response genes could be regulated by sustainability activating

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AtMAPK3.20 There are increasing evidence indicating that MAPKs play a vital role in

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plant disease resistance, especially AtMAPK3. However, the regulatory mechanism of

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MAPKs-mediated disease resistance is still unclear.

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SlMAPK3 is a tomato ortholog of Arabidopsis thaliana MAPK3 gene. There was

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increasing evidence indicating that SlMAPK3 played an important role in response to

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abiotic stress. For example, H2O2 induced chilling tolerance by activating SlMAPK3.21

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And SlMAPK3 was reported to participate in drought response by preventing cell

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membranes from oxidative damage.22 Though there was study indicating that

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overexpression of SlMAPK3 enhanced tolerance to tomato yellow leaf curl virus

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(TYLCV) and positively regulated SA signaling after the virus infection23, little is

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known about the role that SlMAPK3 plays in resistance to necrotrophic pathogens. And

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how SlMAPK3 acts in the regulation of SA/JA defense signaling pathways after

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necrotrophic pathogens infection is unknown.

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To study the function of SlMAPK3 in tomato plants disease resistance to necrotrophic

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pathogen, two independent T2 transgenic lines of slmapk3 mutants and wild-type

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tomato plants were inoculated with B. cinerea. The activities of defense enzymes,

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accumulation of ROS and expressions of SA/JA-related genes were studied to find out

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the probable regulatory mechanism of SlMAPK3 in disease resistance. This study

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provided insights on the SlMAPK3-mediated disease resistance and SA/JA-defensive

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signaling regulation in tomato plants.

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MATERIALS AND METHODS 5



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Plant Materials and Growth Conditions. Two independent T2 transgenic lines

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(L13, L8) of slmapk3 mutants and their wild-type (WT: Solanum lycopersicum cv. Ailsa

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Craig) were used in this study. The two mutant lines were from our lab and described

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previously.22 Plastic pots (7 cm diameter) containing seeding substrate, soil, and

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vermiculite (v/v/v = 2:1:1) were used for seeding, and plants were grown in the

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greenhouse at 25 ± 2°C, 60-65% relative humidity (RH), under 16 h light/8 h dark

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photoperiod. Then, six-week-old tomato plants were used for further experiment. Six

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tomato plants were used in each group (L13, L8 and WT) to observe the phenotype and

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measure lesion diameter. Twenty four tomato plants in each group were used for further

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experiment. Leaf samples were collected at 0, 6, 12, 24 h, 3 and 5 d after inoculation,

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frozen in liquid nitrogen and stored at -80°C. In this experiment, four biological

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replicates were carried out.

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Pathogen Material and Inoculation. B. cinerea (ACCC 36028) was cultured on

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potato dextrose agar medium at 28°C under darkness for two weeks. Spore suspensions

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(2 × 106 conidia mL−1) were collected by brushing the surface of the medium and

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suspending them in sterile distilled water. Then 10 µL B. cinerea spore suspension was

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spotted on the plants leaves surface, and inoculated plants were cultured at 25 ± 2°C

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and 90-95% relative humidity (RH) for phenotype observation and further experiment.

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Necrotic lesions were observed every day after inoculation. On the second day after

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inoculation, photographs were taken and lesion diameters were measured. In addition,

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lesions were distributed according to their size and divided into three classes: small (LD

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< WT first quartile), medium (WT first quartile < LD < WT third quartile) and large 6


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(LD > WT third quartile). Based on the rule of distribution mentioned above, lesions

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were divided into three classes: small (LD < 3.0 mm), medium (3.0 mm < LD < 5.0 mm)

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and large (LD > 5.0 mm).

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Quantitative Real-Time PCR (qRT-PCR). EasyPure Plant RNA Kit (Beijing

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Transgen Biotech Co.Ltd., Beijing, China) was used for extracting total RNA from 0.2

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g of frozen leaf samples. First-strand cDNA was synthesized using the TransScript

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One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (Beijing Transgen

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Biotech Co. Ltd., Beijing, China).

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TransStart Top Green qPCR SuperMix (Beijing Transgen Biotech Co.Ltd., Beijing,

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China) and the Bio-Rad CFX96 real-time PCR system (Bio-Rad, USA) were used for

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performing qRT-PCR. The procedure for qRT-PCR was designed as follow: 94°C for

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30 s, followed by 40 cycles at 94°C for 5 s, 60°C for 15 s, and 72°C for 15 s. β-actin

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gene (NM_001308447) was used as control and gene specific primers for qRT-PCR

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were listed in table1.

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Assay of Defense Enzyme Activities. To determine the activities of CHI (Chitinase,

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EC 3.2.1.14), GLU (β-1,3-glucanase, EC 3.2.1.39) and PPO (polyphenol oxidase, EC

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1.10.3.2), 0.4 g of frozen leaf samples were extracted with 5 mL extraction buffer (100

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mM, pH 5.2 acetic acid buffer for CHI and GLU, 100 mM, pH 6.8 PBS for PPO). The

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activities of CHI, GLU and PPO were assayed followed the method described by Zheng

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et al24, and expressed as U·g−1 FW.

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For the assay of CHI activity, the absorbance was measure at 585 nm. And one unit

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of CHI activity was defined as the production of 10−9 mol N-acetyl-D-glucosamine per 7



143 144 145

hour. For the assay of GLU activity, the absorbance was measured at 540 nm. One unit of GLU activity was defined as the degradation of 10−9 mol laminarin per hour.

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For the assay of PPO activity, 140 µM PBS, 40 µL enzyme extract and 100 mM

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catechol were mixed. And the change in absorbance at 420 nm was recorded rapidly for

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6 min. One unit of PPO activity was defined as a change of 1 in absorbance per minute.

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Assay of Hydrogen Peroxide Content and DAB, NBT Staining. To assay

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hydrogen peroxide (H2O2) content, 0.4 g frozen leaf samples were extracted with 5 mL

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cold 100 mM PBS (pH 7.0). The homogenates were centrifuged at 12 000g for 8 min at

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4°C for twice, and the supernatant were collected for H2O2 content analysis. Hydrogen

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Peroxide Detection Kit (A064-1, Jiancheng, Nanjing, China) was used for measuring

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H2O2 content.

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To determine the in situ synthesis of H2O2 and superoxide ( O2•−), leaf samples were

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collected on 0 and 2 d after inoculation and infiltrated in 0.1% 3,3′-diaminobenzidine

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solution (DAB, containing 0.05% v/v Tween 20 and 10 mM PBS at pH 7.0) or 0.1%

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nitroblue tetrazolium solution (NBT, containing 25 mM HEPES and 10 mM PBS at pH

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7.0) at dark for 8 h at room temperature. To remove chlorophyll, leaf samples were

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dipped into 95% ethanol, boiled for 10 min and maintained in 95% ethanol.

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Statistical Analysis. One-way analysis of variance (ANOVA) and Duncan’s

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multiple range tests were used for statistical evaluations using the statistical analysis

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software SPSS version 16.0. Significant differences relative to WT at P < 0.01 and P
5.0 mm). And more

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proportion of large lesions and fewer small lesions were observed in the slmapk3

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mutants than in WT (Figure 2C). All of the results indicated that knockout of SlMAPK3

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reduced disease resistance to B. cinerea in tomato plants. 9



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Knockout of SlMAPK3 reduced the activities of defense enzymes

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To further verify the results mentioned above, the activities of defense enzymes CHI,

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GLU and PPO were assayed on 0, 1, 3 and 5 d after inoculation. The activity of CHI

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increased gradually in WT after inoculation, while the values of slmapk3 mutants

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showed fluctuating changes. From the first day, L13 and L8 showed significantly lower

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CHI activity than WT, and on the fifth day, CHI activity in L13 and L8 was 45.99% and

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34.30% lower than that in WT respectively (Figure 3A, P < 0.05). In addition, the

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activities of GLU and PPO in WT and slmapk3 mutants increased gradually. However,

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GLU activity in L13 and L8 was 31.68% and 34.16% lower than that in WT plants on

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the third day, respectively (Figure 3B, P < 0.01). Meanwhile, PPO activity in L13 and

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L8 was always lower than WT, especially on the fifth day after inoculation (Figure 3C,

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P < 0.01). The results indicated that knockout of SlMAPK3 reduced the activities of

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defense enzymes CHI, GLU and PPO, which further confirmed the result that knockout

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of SlMAPK3 reduced resistance to B. cinerea in tomato plant.

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Knockout of SlMAPK3 enhanced ROS accumulation

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ROS play a vital role in pathogen-plant interactions as signaling modules, especially

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in necrotroph-plant interactions.25 Thus, we detected the content of H2O2 at different

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time points (0, 1 , 3 and 5 d) after inoculated with B. cinerea and found that H2O2

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content increased gradually after B. cinerea infection in WT and slmapk3 mutants . In

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addition, L13 and L8 showed significantly higher H2O2 content than WT, and the

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values on the third day were 40.40% and 51.51% higher than that in WT, respectively

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(Figure 4A, P < 0.01). 10


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Furthermore, the accumulation of H2O2 and O2•− in the leaves was detected by DAB

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and NBT staining on the second day after inoculation, respectively. The localization of

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H2O2 was confirmed by DAB staining, which exhibited dark brown precipitates in

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leaves, and more intensely stained spots were observed in the leaves of slmapk3

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mutants than in WT (Figure 4B). The localization of O2•− was visualized in the leaves

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by the staining with NBT which would react with O2•− and produce blue stained spots.

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Compared with WT plants, L13 and L8 showed more blue spots in leaves (Figure 4C).

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All of these results indicated that knockout of SlMAPK3 enhanced the accumulation of

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ROS in leaves after inoculation with B. cinerea.

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SlMAPK3 regulated the expression of SA/JA defense-related genes

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SA and JA are two important hormones involved in plant defense response. In order

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to study the regulatory mechanism that SlMAPK3-mediated resistance to B. cinerea in

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tomato plants, relative expressions of eight SA/JA signaling-related genes were assayed.

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The expressions of SA-biosynthetic genes EDS1 (Enhanced Disease Susceptibility 1),

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PAD4 (Phytoalexin Deficient 4) and SA marker gene PR1 (pathogenesis-related protein

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1) increased in L13 and L8 at 24 h after B. cinerea infection (Figure 5). In particular,

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compared with WT, L13 and L8 showed significantly higher levels of EDS1 and PAD4

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than WT at 24 h after inoculation (Figure 5A,5B, P < 0.05). PR1 gene expressions in

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L13 and L8 were 4.45 and 5.50 times of that in WT, respectively (Figure 5C, P < 0.05).

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The results suggested that the knockout of SlMAPK3 up-regulated SA defense

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signaling.

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However, the expression of JA-biosynthetic gene LoxC (lipoxygenase) decreased at 11



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24 h after inoculation in L13, L8 and WT, the values in L13 and L8 were 58% and 39%

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lower than that in WT, respectively (Figure 5D, P < 0.01). Meanwhile, the expressions

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of two JA-dependent genes, PI I and PI II (proteinase inhibitors I and II) were

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significantly lower in L13 and L8 than WT at 0 h and 24 h after inoculation (Figure 5E,

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5F, P < 0.01). In addition, at 24 h after inoculation, L13 and L8 showed higher

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expressions of JAZ1 (JASMONATE ZIM DOMAIN 1) which is a negative regulator of

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JA signaling26, and the value was 2.83 and 3.00 times of that in WT respectively (Figure

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5G, P < 0.01). Compared with WT, L13 and L8 showed significantly higher levels of

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MYC2 which is the negative regulator of pathogen-responsive genes in JA signaling

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pathways27 at 24 h after inoculation (Figure 5H, P < 0.01). The results suggested that

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knockout of SlMAPK3 down-regulated JA signaling pathways. All of these indicated

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that SlMAPK3 played a positive role in JA defense signaling, whereas a negative role in

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SA defense signaling.

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DISCUSSION

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MAPKs have been reported to play an important role in plant defense response to

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pathogen invasion in many species, such as Arabidopsis, oryza sativa, pearl millet and

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tomato.15, 28, 29 In Arabidopsis, MAPK1, MAPK3, MAPK4 and MAPK6 have been

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reported to positively or negatively regulate defense response against different types of

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pathogens.17, 30 AtMAPK3 have been reported to be a positive regulator in camalexin

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biosynthesis, and atmapk3 mutants showed compromised B. cinerea-induced

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camalexin accumulation, which was related to reduced resistance to B. cinerea.19

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SlMAPK3 is the ortholog of Arabidopsis thaliana AtMAPK3 gene in tomato. Previous 12


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study

reported

that

MAPKs

inhibitor

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[1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)] treatment increased the

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disease incidence and lesion areas in mature green tomato fruits after inoculation with B.

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cinerea.24 However, little is known about the function of SlMAPK3 in disease resistance,

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especially in resistance to necrotrophic pathogens. In this study, relative expression of

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SlMAPK3 was remarkably induced by B. cinerea, which suggested that SlMAPK3 may

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be involved in defense response to B. cinerea (Figure 1). This result was consistent with

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previous studies that MAPKs, as signaling modules, played an important role in

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defense response, and the activation of MAPKs is one of the earliest signaling events

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after plant recognizing PAMPs.31, 32 However, the regulatory mechanism of SlMAPK3

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in disease resistance to B. cinerea is still unclear. This study provided insights on

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SlMAPK3-mediated disease resistance to B. cinerea.

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Two independent lines (L13 and L8) of slmapk3 mutants mediated by CRISPR/Cas9

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system were used to further investigate the function of SlMAPK3 in disease resistance

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to B. cinerea. When plants were inoculated with B. cinerea for two days, more severe

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necrosis and higher mean diameter of lesions were shown in transgenic lines, which

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indicated that SlMAPK3 played a positive role in tomato plants resistance to B. cinerea

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(Figure 2). It has been reported that overexpression of SlMAPK3 enhanced tolerance to

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tomato yellow leaf curl virus (TYLCV).23 However, in Arabidopsis, after inoculated

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with P. syringae pv. tomato DC3000 (Pst DC3000), mapk3 mutants exhibited

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significantly lower bacterial titres and reduced susceptibility to P. syringae.6 Thus,

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MAPK3 may play different roles in resistance to different types of pathogens based on 13



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different signaling pathways.9

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CHI, GLU and PPO have been regarded as primary defense enzymes against

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fungus.24 CHI and GLU belong to the PR-3 and PR-2 family, respectively, and play a

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vital role in the degradation of fungal cell walls.33 PPO which catalyzes the oxidation of

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phenolic compounds to form quinines, is related to induced defense responses.34

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Previous studies demonstrated that enhanced disease resistance is associated with

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increased activities of CHI, GLU or PPO.35, 36 In this study, the knockout of SlMAPK3

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reduced CHI, GLU and PPO activities during most of the time after inoculation with B.

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cinerea (Figure 3). However, there was no significant difference in GLU activity

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between mutants and WT on 5 d after inoculation (Figure 3B), which may be because

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the knockout of SlMAPK3 would not prevent GLU activity from increasing after

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inoculation, it just delayed the peak time of GLU activity. These findings confirmed

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that knockout of SlMAPK3 reduced resistance to B. cinerea in tomato plants.

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ROS are the metabolic by-products and signaling components in stress responses.37

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Previous studies pointed out that one of the early defense responses against pathogen

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invasion is oxidative burst, ROS generation during oxidative burst is associated with

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the perception of MAMPs/PAMPs, and ROS were considered as antimicrobial

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molecules, cross-linkers of the plant cell wall to defense pathogen.

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necrotrophs can also regulate the accumulation of ROS to achieve full pathogenicity,

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and fungal NADPH oxidases (Nox) are necessary for pathogenic development.39 In our

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study, after inoculated with B. cinerea, L13, L8, and WT showed increased

296

accumulation of ROS, and slmapk3 mutants showed a higher level of ROS content and 14


38

However,

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reduced resistance to B. cinerea than WT (Figure 4), which were consistent with

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previous studies mentioned above38, 39. In addition, in Arabidopsis, exogenous H2O2

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activated the expression of AtMAPK3, AtMAPK4 and AtMAPK6.40 AtMAPK3 had a

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negative effect on ROS production, and the accumulation of ROS was increased in

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atmapk3 mutant than control upon flg22 and elf18 activation.41, 42 Meanwhile, slmapk3

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mutants showed significantly higher H2O2 content than WT, associated with lower CAT

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and SOD activities in response to drought stress.22 Overexpression of SlMAPK3 in

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tobacco plants reduced H2O2 content under low temperature stress.43 These results were

305

consistent with ours that knockout of SlMAPK3 up-regulated the accumulation of ROS

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after inoculated with B. cinerea, and indicated that SlMAPK3 was involved in the

307

regulation of H2O2 accumulation in response to abiotic and biotic stress. Taken together,

308

we postulated that knockout of SlMAPK3 resulted in the accumulation of ROS after

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infected with B. cinerea, which could enhance the susceptibility to B. cinerea in tomato

310

plants.

311

To further explore the regulatory mechanisms of SlMAPK3-mediated disease

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resistance to B. cinerea, relative transcript levels of SA marker gene SlPR1,

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SA-biosynthetic genes SlEDS1 and SlPAD4, JA-biosynthetic gene SlLoxC,

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JA-dependent genes, SlPI I and SlPI II, and JA signaling negative regulators SlJAZ1

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and SlMYC2 were analyzed. The expressions of SlEDS1, SlPAD4 and SlPR1 were

316

increased in slmapk3 mutants, which suggested that knockout of SlMAPK3

317

up-regulated SA signaling (Figure 5A, 5B and 5C). This was consistent with that

318

AtMAPK3 played a negative role in flg22-induced salicylic acid accumulation.6 15



319

However, the relative expressions of SlLoxC, SlPI I and SlPI II decreased, and the

320

relative expressions of SlJAZ1 and SlMYC2 increased in slmapk3 mutants, which

321

indicated that knockout of SlMAPK3 down-regulated JA signaling (Figure 5D, 5E and

322

5F). This was consistent with the previous study that silencing of SlMAPK3 reduced the

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relative expressions of JA signaling marker genes SlLapA, SlPI I and SlPI II after

324

tomato yellow leaf curl virus infection23. Previous studies demonstrated that plants

325

display resistance to necrotrophic pathogens mainly through JA signaling, whereas SA

326

played a negative role in responding to necrotrophic pathogens.14 For example, in

327

Arabidopsis and tobacco, deficiency of SA signaling enhanced resistance to B. cinerea,

328

whereas deficiency of JA signaling enhanced susceptibility to B. cinerea.44 These may

329

explain why knockout of SlMAPK3 reduced the resistance to B. cinerea. In addition,

330

there was an interesting phenomenon that the knockout of SlMAPK3 did not reduce

331

expression of the SA/JA-related genes except SlPI I and SlPI II at 0 h, which indicated

332

that SlMAPK3 played a vital role in the regulation of SlPI I and SlPI II expressions. The

333

reason why knockout of SlMAPK3 reduced the disease resistance to B. cinerea may be

334

related to the interaction between SlMAPK3 and SlPI I, SlPI II. The exact regulatory

335

mechanism should be further studied.

336

One the other hand, it has been reported that exopolysaccharide produced by B.

337

cinerea activated SA pathway, while SA signaling pathway antagonized the JA

338

signaling pathway to promote necrotrophs development in tomato.45 Meanwhile,

339

SA had a negative effect on the transcriptional activator ORA59, which leads to

340

suppression of JA signaling.46 In addition, previous studies pointed out that SA induced 16


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the accumulation of H2O2 by directly inactivating catalases (CATs) and ascorbate

342

peroxidases (APXs), and H2O2 could promote the accumulation of SA.47,48 Moreover,

343

JA-deficient tomato mutant defenseless-1 showed ROS-associated injury phenotypes

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with lower activity of non-enzymatic antioxidants and enzymatic antioxidants.49 To

345

sum up, SA and H2O2 positively regulates each other, while SA signaling pathway

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antagonizes JA signaling pathway and JA plays an important role in ROS scavenge,

347

which was consistent with our results that slmapk3 mutant showed more susceptible

348

phenotypes associated with ROS accumulation, up-regulation of SA-related genes, and

349

down-regulation of JA-related genens. Thus, we postulated that the knockout

350

of SlMAPK3 up-regulated SA signaling, down-regulated JA signaling, which resulted

351

in reduced resistance to B. cinerea in tomato plants.

352

In conclusion, knockout of SlMAPK3 reduced resistance to B. cinerea. The activities

353

of defense enzymes CHI, GLU and CHI were reduced in slmapk3 mutants, which

354

further confirmed the result mentioned above. Knockout of SlMAPK3 enhanced the

355

content of ROS which played an important role in pathogen-plant interactions,

356

indicating that SlMAPK3 played a negative role in ROS accumulation. On the other

357

hand, knockout of SlMAPK3 up-regulated SA defense signaling and down-regulated JA

358

defense signaling after inoculation. In summary, we postulated that SlMAPK3

359

participated in tomato disease resistance to B. cinerea by regulating ROS accumulation,

360

SA and JA defense signaling.

361 362

ABBREVIATIONS USED 17



363

MAPK, mitogen-activated protein kinase; CRISPR, clustered regularly interspaced

364

short palindromic repeats; B. cinerea, Botrytis cinerea; WT, wild type; H2O2, hydrogen

365

peroxide; ROS, reactive oxygen species; SA, salicylic acid; JA, jasmonic acid; CHI,

366

chitinase; GLU, β-1,3-glucanase; O2•−, superoxide; DAB, 3,3′-diaminobenzidine; NBT,

367

nitroblue tetrazolium

368

AUTHOR INFORMATION

369

Corresponding Author

370

*E-mail: [email protected]; Phone: +86-10-62737620; Fax: +86-10-62737620.

371

Funding

372

The research was supported by the National Natural Science Foundation of China (NO.

373

31272215, 31371847 and 31571893) and the National Basic Research Program of

374

China (2013CB127102 and 2013CB127106).

375

Notes

376

The authors declare that they have no competing interests.

18


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532

FIGURE CAPTIONS

533

Figure 1. The expression of SlMAPK3 in wild-type plant leaves at different time points

534

(0, 6, 12, 24 h) after inoculation with B. cinerea. Values followed by different letters

535

were significantly different according to Duncan’s multiple range test at P < 0.05. The

536

data (mean ± SD) were calculated on the basis of four biological replicates.

537

Figure 2. Effects of SlMAPK3 mutants on disease resistance to B. cinerea. (A) Disease

538

phenotype in wild-type (WT), L13 and L8 plants on 2 d after infected with B.

539

cinerea. Leaves of six-week-old plants have been used for inoculation tests, and six

540

plants per genotype were infected with B. cinerea. (B) Lesion diameters were measured

541

on 2 d after inoculation. (C) Lesions were distributed according to their size and divided

542

into three classes: small (LD < 3.0 mm), medium (3.0 mm < LD < 5.0 mm) and large

543

(LD > 5.0 mm).

544

Figure 3. Effects of slmapk3 mutants on the activities of defense enzymes: (A) CHI, (B)

545

GLU, (C) PPO after inoculation of B. cinerea. Significant differences between the

546

slmapk3 mutant lines and WT were compared by Duncan’s multiple range test.

547

Significant differences at P < 0.01 and P < 0.05 were marked as double (**) and single

548

(*), respectively.

549

Figure 4. Effects of slmapk3 mutants on the accumulation of ROS: (A) H2O2 content,

550

(B) DAB staining, (C) NBT staining. Localization of H2O2 and O2•− was visualized in

551

the leaves by DAB and NBT staining on second day after inoculation. Significant

552

differences between the slmapk3 mutant lines and WT were compared by Duncan’s

553

multiple range test. Significant differences at P < 0.01 and P < 0.05 were marked as 26


Page 26 of 34

Page 27 of 34


554

double (**) and single (*), respectively.

555

Figure 5. Effects of slmapk3 mutants on SA/JA signaling-related gene expressions: (A)

556

SlEDS1, (B) SlPAD4, (C) SlPR1, (D) SlLoxC, (E) SlSPI I, (F) SlPI II, (G) SlJAZ1, (H)

557

SlMYC2. Significant differences between the slmapk3 mutant lines and the WT were

558

compared by Duncan’s multiple range test. Significant differences at P < 0.01 and P

Knockout of SlMAPK3 reduced disease resistance to Botrytis cinerea

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