Accepted Manuscript Title: Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria Author: W. Yim K. Kim Y.W. Lee S.P. Sundaram Y. Lee T. Sa PII: DOI: Reference:

S0176-1617(14)00079-0 http://dx.doi.org/doi:10.1016/j.jplph.2014.03.009 JPLPH 51915

To appear in: Received date: Revised date: Accepted date:

5-2-2014 28-3-2014 29-3-2014

Please cite this article as: Yim W, Kim K, Lee YW, Sundaram SP, Lee Y, Sa T, Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria, Journal of Plant Physiology (2014), http://dx.doi.org/10.1016/j.jplph.2014.03.009 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.

*Manuscript

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Real time expression of ACC oxidase and PR-protein genes mediated by

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Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv.

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vesicatoria

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W. Yima, K. Kima, Y.W. Leea, SP. Sundarama, Y. Leeb, T. Saa,*

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a

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Cheongju, Chungbuk, 361-763, South Korea

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b

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Cheongju, Chungbuk, 361-763, South Korea

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Department of Environmental and Biological Chemistry, Chungbuk National University,

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Department of Industrial Plant Science and Technology, Chungbuk National University,

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Prof. Tongmin Sa,

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E-mail address: [email protected]

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Department of Environmental and Biological Chemistry, College of Agriculture, Life and

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Environmental Sciences, Chungbuk National University, Cheongju, Chungbuk, 361-763,

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South Korea

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Telephone: (O) 82-43-261-2561; (F) 82-43-271-5921.

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SUMMARY

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Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene

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inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to

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reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas

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campestris pv. vesicatoria by modulating stress ethylene with 1-aminocyclopropane-1-

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carboxylate (ACC) deaminase activity of Methylobacterium strains.

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condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had

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low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC

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oxidase (ACO) activity with ACO related gene expression increased in Xanthomonas

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campestris pv. vesicatoria infected tomato plants over Methylobacterium strains inoculated

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plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene

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expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression

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(3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase

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in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial

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pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and

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PAL enzyme activities in pathogen challenged tomato plants. The respective increase in β-

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1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains

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were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene

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expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged

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tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that

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ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with

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ACC deaminase activity can reduce the disease severity with ultimate PR protein increase in

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tomato.

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Key words: Bacterial spot; Ethylene biosynthetic gene expression; Methylobacterium; PR-

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proteins; RT-PCR; Xanthomonas campestris pv. vesicatoria

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Under greenhouse

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Abbreviations

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ACC, 1-aminocyclopropane-1-carboxylic acid

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ACS, 1-aminocyclopropane-1-carboxylic acid synthase

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ACO, 1-aminocyclopropane-1-carboxylic acid oxidase

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AMS, ammonium mineral salts medium

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BHT, butylated hydroxytoluene as antioxidant

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CLSM, confocal laser scanning microscopy

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DAT, days after transplanting

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DI, disease index

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DNS, dinitrosalicylic acid

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FW, fresh weight

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GC, gas chromatography

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ISR, induced systemic resistance

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LSD, least significant difference

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MgSO4, magnesium sulfate

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NaOCl, sodium hypochlorite

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NaOH, sodium hydroxide

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RT-PCR, real-time reverse transcription polymerase chain reaction

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PAL, phenylalanine ammonia-lyase

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PR proteins, pathogenesis-related proteins

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SAM, S-adenosylmethionine

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S.E., standard error

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Tris HCl, trishydroxymethyl aminomethane hydrochloric acid

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XCV, Xanthomonas campestris pv. vesicatoria

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Introduction Plant pathogenic microorganisms causing various diseases are a major and serious

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threat to food production. Bacterial spot disease caused by Xanthomonas campestris pv.

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vesicatoria is a prominent disease, which can affect the foliage, fruit, blossoms, and stems of

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tomato plant (Lycopersicon esculentum). This disease occurs in many countries and is of

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great economic importance in regions with a warm and humid climate (Jones et al., 1998). It

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can severely devitalize plants by defoliation, reduced yield and affected fruit quality. The

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primary symptom is necrotic lesions that occur on leaves, stems, fruits, and flower parts (Stall,

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1995). Conventional applications of copper bactericides are not only ineffective but also pose

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severe ecological hazards (Byrne et al., 2005). An environmentally safe strategy to control

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plant diseases is the use of biocontrol agents (Domenech et al., 2006; Ji et al., 2006). The

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widely recognized mechanisms for biocontrol includes competition for an ecological niche or

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a substrate, production of inhibitory allelochemicals, and induction of systemic resistance in

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host plants to a broad spectrum of pathogens (Bloemberg and Lugtenberg, 2001; Compant et

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al., 2005; Wang et al., 2001). Mostly, plant ethylene synthesis is enhanced with severity of

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pathogenic infection.

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Though gaseous plant hormone ethylene has been recognized as being involved in a

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wide range of plant responses and developmental steps including seed germination, tissue

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differentiation, formation of root and shoot primordia, lateral bud development, flower

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initiation, fruit ripening, leaf and fruit abscission (Abeles et al., 1992; Mattoo and Suttle,

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1991; Spaink, 1997), high levels of ethylene have the ability to trigger exaggerated disease

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symptoms or exacerbate an environmental pressure. After a severe infection of pathogens,

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the mechanism of damage inflicted in plant is due to autocatalytic ethylene synthesis and not

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from direct pathogenic action. At this point, the inhibitors of ethylene synthesis or ethylene

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action can significantly decrease the severity of a fungal or bacterial infection (Glick et al.,

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2007). Bacterial ACC deaminase can reduce the stress ethylene level generated by pathogen

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invasion by converting ACC, an immediate precursor of ethylene in plants, to α- ketobutyrate

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and ammonia and thereby lowering the level of ethylene in stressed plant (Glick, 1995; 1998;

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2005; Jacobson et al., 1994). It is very likely that ACC deaminase bacteria, apart from

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directly antagonizing pathogens, augment the resistance of the plants against pathogen attack

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by way of increasing PR proteins.

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Species of genus Methylobacterium symbiotically associate and ubiquitously colonize

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the terrestrial and aquatic plants (Corpe and Rheem, 1989). Methylobacterium species using

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plant methanol as the source of carbon and energy (Trotsenko et al., 2001), in turn produce

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phytohormones (cytokinins and auxins), which are known to stimulate plant growth (Ivanova

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et al., 2001), lower the stress ethylene (Madhaiyan et al., 2006a) and activation of induced

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systemic resistance (ISR) in plants against pathogens (Madhaiyan et al., 2004). Though there

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are studies demonstrating stress ethylene reduction and ISR induction by Methylobacterium

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inoculation in pathogen infected plants, their respective related genes transcriptional studies

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are meager. Hence the present study was conducted to understand Methylobacterium strains

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mediated ACO and PR proteins related gene expressions in bacterial pathogen, Xanthomonas

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campestris pv. vesicatoria infected tomato plants.

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Materials and methods

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Strains and culture conditions

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The Methylobacterium strains: Methylobacterium fujisawaense CBMB12 (EF126740), M.

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fujisawaense CBMB15 (EF126745), M. oryzae CBMB20 (AY683045),

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phyllosphaerae CBMB27 (EF126746) already isolated in this lab (Madhaiyan et al., 2006a;

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Yim et al., 2012) were grown in ammonium mineral salts (AMS) medium (Whittenbury et al.,

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1970) supplemented with 0.5% sodium succinate. The bacterial pathogen Xanthomonas

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campestris pv. vesicatoria KACC11157, obtained from Korean Agricultural Culture

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Collection (KACC) in Republic of Korea, was maintained in Nutrient broth (NB) medium.

128 Greenhouse bioassay

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Bacterial cells of Methylobacterium strains from 72 h old cultures were harvested by

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centrifugation at 10,000 g for 10 min at 4°C, washed twice and suspended in 0.03 M MgSO4.

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Homogenous bacterial suspensions were adjusted to 1.0 OD at 600 nm. The surface

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disinfected (first with 70 % ethanol for 1 min followed by 2 % sodium hypochlorite for 1

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min) tomato seeds were subjected to the following treatments for 4 h: (i) 0.03 M MgSO4

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(control), (ii) M. fujisawaense CBMB12, (iii) M. fujisawaense CBMB15, (iv) M. oryzae

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CBMB20, (v) M. phyllosphaerae CBMB27.

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The treated seeds were sown in seedling trays (50 hole tray-1 and one seed hole-1)

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containing 150 g of nutrient-free bed soil and 150 g of Biosangto-Mix bed soil [Heung Nong

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Co., Ltd, Incheon, Gyeonggi-do, Republic of Korea; it contains 65-70 % coco peat, 15-20 %

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peat moss, 8-10 % perlite and macronutrient (mg L-1) NH4-N, 80-100; NO3-N, 150-200;

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available P2O5, 230-330; K2O, 80-120; pH 5.5 to 6.5; moisture content 50-60 % and water

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holding capacity 35-40 %] and incubated in a growth chamber (DS 54 GLP, DASOL

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Scientific Co., Ltd., Korea) at 25 °C with 14/10 h day/night photoperiod. After germination,

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seedlings were transferred and allowed to grow in the greenhouse with a temperature regime

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of 28 °C/22 °C (day/night) under natural illumination. Five mL methylobacterial suspension

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adjusted to 1.0 OD at 600 nm was applied to the soil at each plant, every week after

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germination.

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Twenty six days old tomato seedlings were transplanted into 400 mL plastic pots

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containing bed soil fertilized with 10 mL of Hoagland’s nutrient solution. After establishment,

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the plants were subjected to the following treatments: (i) Control without any inoculation (ii)

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Pathogen X.campestris pv. vesicatoria (XCV)

alone, (iii) Bacterial pathogen XCV +

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CBMB12, (iv) Bacterial pathogen XCV + CBMB15, (v) Bacterial pathogen XCV +

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CBMB20, (vi) Bacterial pathogen XCV + CBMB27 and (vii) Bacterial pathogen XCV +

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germicide. Eight replications were maintained per treatment, each pot with a single plant and

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arranged in a completely randomized design. X. campestris pv. vesicatoria (108 cfu mL-1)

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inoculation was performed with a hand-held pneumatic sprayer at 3 and 17 d after

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transplanting (DAT). The Coside® (copper hydroxide) was used as germicide following

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guidelines on the safe use of pesticides.

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The effect of Methylobacterium spp. inoculation on the growth of pathogen

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challenged tomato was recorded as increment in plant height every week after transplanting

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and plant biomass production was determined as dry matter content of plants on 35 DAT.

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Disease scoring started after the initial development of symptoms 7 d after pathogen

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inoculation. Disease severity of X.campestris pv. vesicatoria treatments were evaluated

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visually according to Campbell and Madden (1990) on 14, 21, 28 and 35 DAT, using a

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disease index (DI) with a range of 0 - 7 (0 - no disease; 1 - less than half of a leaf symptoms;

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2 - between one half of a leaf but 75 % of the leaves with symptoms; 7 - plant dying or

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dead). Disease indices were calculated based on the following formula (Bora et al., 2004):

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∑ (rating x no. of plants rated)

DI (%) =

× 100 Total no. of plants x highest rating

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Ethylene estimation

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Tomato leaves collected on 14, 21, 28, and 35 DAT from plants grown in the greenhouse

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were placed in 120 mL vials and sealed with a rubber septum for 4 h. One mL sample of the

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headspace was injected into a Gas Chromatograph (dsCHROM 6200, Donam Instruments

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Inc., Republic of Korea) packed with Poropak-Q column at 70 °C and equipped with a flame

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ionization detector. The amount of ethylene emission was expressed as nmol of ethylene h-1 g

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dry weight-1 and compared to a standard curve generated with pure ethylene (Praxair, Praxair

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Korea Co., Ltd).

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179 ACC accumulation

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ACC concentration in plant tissue was estimated following the protocol of Madhaiyan et al.

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(2007). One gram of leaves collected on 35DAT was immediately frozen in liquid nitrogen

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and ground well. ACC from frozen ground tissue was extracted using 5 mL of 80% methanol

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containing butylated hydroxytoluene as antioxidant (BHT, 2 mg L-1) and incubated at room

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temperature for 45 min. Samples were centrifuged at 2,000 g at 20 °C for 15 min, the pellet

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was re-suspended in 4 mL methanol and again centrifuged under the same conditions. The

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combined supernatants were evaporated to dryness under vacuum in a rotary evaporator.

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ACC levels were determined by the method of Wachter et al. (1999) using the protocol

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described by Lizada and Yang (1979). Residues were re-suspended in 2 mL distilled water

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and then the upper aqueous phase (0.5 mL) was obtained by extraction with dichloromethane

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mixed with 0.1 mL HgCl2 (80 mM) in test tube and sealed with rubber septum. Then 0.2 mL

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of NaOCl solution (40 mL NaOH, 80 mL 12.5 % NaOCl solution, 30 mL distilled water) was

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injected into each tube, shaken, and incubated for 8 min. One milliliter of the gaseous portion

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was taken and assayed for ethylene with a GC. The efficiency of ACC oxidation, on average

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60 %, were estimated by analyzing replicated samples containing internal standards of ACC.

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ACC oxidase activity

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The protein extracts for measuring the in vitro ACO activities in tomato leaves were prepared

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according to Petruzzelli et al. (2000) and Madhaiyan et al. (2007). The frozen tissues were

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pulverized in liquid nitrogen and homogenized in 2 mL g-1 of extraction buffer consisting of

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100 mM Tris HCl (pH 7.2), 10 % (w/v) glycerol and 30 mM sodium ascorbate. The

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homogenate was centrifuged at 15,000 rpm for 15 min at 4 °C. The supernatants were used

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for the in vitro ACO assay (Malerba et al., 1995). The enzyme activity was assayed in 10 mL

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screw cap tubes fitted with a Teflon-coated septum containing 1.5 mL of supernatant, 50 μM

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FeSO4, 1 mM ACC, and 5 % (v/v) CO2 at 30 °C for 15 min. At the end of this time period,

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the quantity of ethylene released into the headspace was determined by GC.

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β-1,3-glucanase and phenylalanine ammonia-lyase activity

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Leaf samples (1 g) collected on 35 DAT were frozen and homogenized immediately with

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liquid nitrogen. The resulting powder was macerated for 30 s with 100 mM potassium

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phosphate buffer, pH 7.0 (1:1.25 w/v). Then the crude homogenates were centrifuged at

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20,000 g for 20 min at 4 °C. The supernatants were kept in an ice bath and used for the

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determination of PR proteins. β-1,3-glucanase activity was assayed using laminarin (from

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Laminaria digitata) (Sigma-Aldrich Co. St. Louis, Missouri, USA) as substrate (Liang et al.,

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1995). The assay mixture of 0.1 mL consisted of 50 μL enzyme extract, 50 μL laminarin (10

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mg mL-1) in 50 mM sodium acetate buffer (pH 5.0) was incubated at 37 °C for 1 h. The

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amount of reducing sugar released from laminarin was determined by termination of the

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reaction with 1.5 mL dinitrosalicylic acid (DNS) reagent and boiling the mixture in a water

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bath for 5 min. The reducing sugar equivalents were measured at 530 nm and enzyme activity

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was expressed as μg glucose min-1 mg protein-1.

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Phenylalanine ammonia-lyase activity was measured according to the procedure described by

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El-Shora (2002). The assay mixture consisted of 1.9 mL of 100 mM Tris–Cl buffer (pH 8.5),

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1 mL of 15 mM L-phenylalanine and 100 μL enzyme extract was incubated at 30 °C for 15

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min. The reaction was terminated by adding 200 μL of 6 M HCl and the absorbance of the

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solution was measured at 290 nm. One unit represents the conversion of 1 μmol L-

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phenylalanine to trans-cinnamic acid min-1. The amount of trans-cinnamic acid synthesized

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was calculated using its molar absorptivity of 9630 M-1 cm-1 and expressed as nmol trans-

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cinnamic acid min-1 mg protein-1.

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Real-Time RT-PCR analysis of ACO and PR protein expressions

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Total RNA (1 μg) was extracted from each part of internodes of tomato leaves on 35 DAT

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using a Plant RNase Mini Kit (Qiagen, Germany) and subjected to real-time reverse

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transcription polymerase chain reaction (RT-PCR). cDNA was synthesized using the

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Superscript III First Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). SYBR Green

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Master Mix (Bio-Rad, USA) was used for quantification with iQ5 optical system (Bio-Rad,

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USA). The data were analyzed using the delta-delta-Ct method (Livak and Schmittgen, 2001).

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The primers constructed using the gene sequences of Tomato ACO (accession: AB013101),

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PAL (accession: AB269917) and β-1,3-glucanase (accession: FJ151171) were used as

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internal controls (Table 1). The following protocol was used for PCR: denaturation program

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(95 °C for 3 min), amplification and quantification program repeated 40 cycles (95 °C for 10

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s; 56 °C for 10 s; 72 °C for 30 s). All samples were amplified in triplicate from the same

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RNA preparation and the mean value was considered. All data points were calculated from

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biological triplicates and technically duplicated measurements.

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Detection of gfp tagged Methylobacterium spp.

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Plasmid pFAJ1820 containing a PUT miniTn5 transposon carrying kanamycin resistance

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gene and two copies of the gfp gene controlled by nptII promoter (Xi et al., 1999) was

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transferred

to

methylobacteria

through

triparental

mating

(Unge

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1998).

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Methylobacterial strains were grown in TSA (Trptic soy broth, Difco, with agar 15 g L-1) for

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conjugation. The donor strain E. coli S17-1 (pFAJ1820) and helper strain E. coli HB101

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(pRK2013) were grown on LB broth with kanamycin (50 µg mL-1). The transconjugants in

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methyloabcteria were selected on AMS containing 0.5 % succinate supplemented with

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kanamycin 20 µg mL-1 and nalidixic acid 10 µg mL-1. Colonies that showed fluorescence

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under UV were selected and the presence of gfp gene was confirmed by PCR. Colonization

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of gfp tagged Methylobacterium spp. in tomato was determined using CLSM. Leaf samples

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were randomly collected at 30 DAT from each treatment. The leaves were cut into three parts

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(tip, blade and petiole) and fixed in glass slides under a cover slip (Poonguzhali et al., 2008).

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Microscopic observations were performed using Leica TCS SP2 confocal system (Leica

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Microsystems Heidelberg GmbH, Manheim, Germany) equipped with excitation wavelength

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of 488 nm (Ar laser). Emission light was measured in a range of 510 - 580 nm for green

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fluorescence, and 620 - 660 nm for red fluorescence. Image acquisitions were carried out

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using a ×63 oil immersion objective with a numerical aperture (NA) of 1.4, and processed

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using the standard software package with the CLSM system (version 2.5.1227a).

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Statistical analysis

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Data were subjected to analysis of variance (ANOVA) and the significance at P ≦ 0.05 was

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tested by least significant difference (LSD) using SAS package, Version 9.1 (SAS, 2009).

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Results

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Methylobacterium spp. inoculation effect on tomato growth challenged with X. campestris pv.

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vesicatoria under greenhouse condition

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The tomato plant growth in terms of plant height observed at 7, 14, 21, 28 and 35 DAT are

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presented in Fig. 1. A significant increase in plant growth at 7, 14 and 21 DAT was observed

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in CBMB12 and CBMB15 inoculated and pathogen challenged tomato over X. campestris pv.

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vesicatoria infected plants. At 35 DAT, inoculation with Methylobacterium strains CBMB15

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and CBMB20 significantly increased tomato plant growth.

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consistently increased the plant growth throughout the study. Germicide application resulted

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to a significant increase in plant growth from 21 DAT onwards over pathogen alone

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inoculated plants.

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Moreover, CBMB15 strain

The inoculation effect of Methylobacterium spp. on pathogen challenged tomato root

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growth on 35 DAT is shown in Table 2. Among the Methylobacterium spp. treatments,

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inoculation with CBMB20 strain recorded significant increase in root length (29.8 cm plant -1),

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shoot biomass (12.5 g plant-1) and root biomass (0.75 g plant-1) over X. campestris pv.

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vesicatoria alone inoculated plants. A significant increase in root length was recorded in

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germicide treated (31.6 cm plant-1) tomato plants compared to the bacterial pathogen

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treatment (19.9 cm plant-1). The shoot biomass accumulation was found to be higher in all

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Methylobacterium spp. inoculated plants compared to other treatments. Highly significant

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root biomass accumulation was observed in Methylobacterium spp. treated plants compared

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to bacterial pathogen treatment which recorded the lowest root biomass 0.39 g plant-1.

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Among the Methylobacterium spp. inoculated tomato, CBMB20 strain recorded significantly

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higher root biomass (0.75 g plant-1) over other treatments. Overall, Methylobacterium spp.

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inoculated plants consistently showed a significant increase in shoot and root lengths along

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with plant biomass production.

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Bacterial spot disease severity

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X. campestris pv. vesicatoria pathogen infected plants showed small, water-soaked lesions on

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the leaves of tomato plants while Methylobacterium strains were able to reduce disease

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incidence in the inoculated plants (Fig. 2). A similar result was also observed on the apical

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leaves of tomato treated with Methylobacterium strains on X. campestris pv. vesicatoria

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challenged plants (Fig. 3). Methylobacterium strains were effective in reducing disease

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symptoms on apical leaves. Percentage disease indices of Methylobacterium strains against X.

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campestris pv. vesicatoria was observed on 14, 21, 28 and 35 DAT (Fig. 4). The X.

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campestris pv. vesicatoria treated plants showed increase in disease index during greenhouse

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bioassay. A significant decrease in disease incidence was found in Methylobacterium spp.

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inoculated plants compared to only pathogen X. campestris pv. vesicatoria treatment. On 35

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DAT, CBMB27 strain had higher inhibitory effect on bacterial disease incidence, followed by

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CBMB20 strain in tomato plants when challenged with X. campestris pv. vesicatoria .

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308 Ethylene emission

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Stress ethylene levels were recorded in tomato plants at 14, 21, 28 and 35 DAT. Under

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greenhouse conditions, ethylene emission in uninoculated plants remained low compared to

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pathogen infected plants. A significant increase in ethylene levels in pathogen infected plants

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was observed 7 d after challenge inoculation and reached maximum at 14 DAT (Fig. 5).

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Methylobacterium strains treated tomato plants showed reduced ethylene levels. CBMB27

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strain was most efficient in reducing stress ethylene, followed by CBMB12, CBMB20 and

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CBMB15 at 14 DAT, whereas ethylene emission was significantly higher in X. campestris pv.

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vesicatoria treated plants. A week later, though ethylene levels greatly decreased in all

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treatments, Methylobacterium strains treated plants recorded significant reduction over other

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treatments. Re-inoculation of X. campestris pv. vesicatoria at 21 DAT caused ethylene levels

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again to rise on 27 DAT in pathogen alone and in germicide treated plants. On the other hand,

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Methylobacterium strains inoculated plants did not significantly differ. At 35 DAT, the

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ethylene level variation in all treatments was not significant, except in bacterial pathogen

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inoculated plants. When relationship between stress ethylene level and disease index was

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studied, an excellent correlation was observed (data not shown) with R2 value of 0.90.

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325 ACC accumulation and ACO activity

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ACC accumulation in the tissue extracts of tomato plants was 100.7 nmol g-1 FW, when

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grown under controlled conditions. But increased to 373.6 nmol g-1 FW, when infected with

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X. campestris pv. vesicatoria (Fig. 6). Inoculation of tomato plants by ACC deaminase

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producing methylobacterial strains CBMB12, CBMB15, CBMB27 and CBMB20 reduced

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ACC accumulation by 67.5, 66.9, 58.2 and 43.2 %, respectively, over Xanthomonas

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campestris pv. vesicatoria treatment. Germicide treatment also reduced ACC accumulation

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compared to Xanthomonas campestris pv. vesicatoria inoculation. Excellent correlation

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coefficient (R2 = 0.89) between ACC accumulation and stress ethylene emission was

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observed.

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The activity of the ACO enzyme, which converts ACC into ethylene, was measured by GC

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by quantifying the amount of ethylene produced. Similar to ethylene production and ACC

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concentration, ACO activity also increased in tomato treated with X. campestris pv.

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vesicatoria (Fig. 6). The ACO activity of tissue extracts of tomato was 28.9 pmol ethylene g-1

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FW h-1, when grown under controlled conditions. However, it increased to 52.4 pmol

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ethylene g-1 FW h-1 in leaves treated with X. campestris pv. vesicatoria. ACO activity in

342

tomato plants treated with methylobacterial starins CBMB20, CBMB12, CBMB15 and

343

CBMB27 and challenged with pathogen, decreased by 47.5, 39.9, 37.2 and 33.3 %,

344

respectively, when compared with X. campestris pv. vesicatoria treatment. The germicide

345

treatment also recorded reduced ACO activity over bacterial pathogen treatment. In this study,

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346

ACO activity was significantly correlated to ACC accumulation (R2 = 0.84) and ethylene

347

level (R2 = 0.90).

348 β-1, 3-glucanase and PAL activities

350

The β-1,3-glucanase and PAL activities were extremely high in treatment with

351

Methylobacterium spp. compared to X. campestris pv. vesicatoria treated tomato plants (Fig.

352

7). Methylobacterium spp. treated and X. campestris pv. vesicatoria challenged plants

353

increased the total β-1,3-glucanase activity with a range from 20.7 and 28.3 μg glucose min-1

354

mg-1 protein. Tomato inoculated with Methylobacterium spp. showed comparatively high

355

PAL activity over X. campestris pv. vesicatoria treatment. Among the treatments, lowest

356

PAL activity was found in control (2.39 μmol trans-cinnamic acid min-1 mg-1 protein) while

357

highest was found in CBMB12 inoculated tomato plants (4.28 μmol trans-cinnamic acid min-

358

1

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mg-1 protein).

d

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ACO, β-1,3-glucanase and PAL-related genes expression

361

As seen in the ACC accumulation and ACO activity, ACO related gene expression also

362

increased in tomato infected with X. campestris pv. vesicatoria compared to all other

363

treatments. The plant treated with pathogen alone showed significant increase in ACO gene

364

expression (7.09 Normalized Fold Expression). ACC deaminase producing methylobacterial

365

strains treated plants have reduced ACO related gene expression compared to X. campestris

366

pv. vesicatoria treatment. Among the Methylobacterium spp. treatments, CBMB12

367

inoculation recorded lowest ACO related gene expression (1.46 Normalized Fold Expression)

368

and CBMB20 inoculation recorded high (3.42 Normalized Fold Expression) in tomato plants.

369

In general, Methylobacterium spp. treatments induced a significant decrease in ACO related

370

gene expression in tomato when challenged with X. campestris pv. vesicatoria (Fig. 8). β-

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Page 15 of 41

1,3-glucanase related gene expression increased in tomato treated with X. campestris pv.

372

vesicatoria compared to control. However, β-1,3-glucanase related gene expression in tomato

373

plants treated with ACC deaminase producing Methylobacterium strains CBMB12, CBMB15,

374

and CBMB20 was 66.3, 25.5 and 10.4 % higher than X. campestris pv. vesicatoria treatment.

375

Interestingly, tomato plants treated with CBMB27 strain showed lower β-1,3-glucanase

376

related gene expression compared to X. campestris pv. vesicatoria treatment. PAL related

377

gene expression was 0.11 Normalized Fold Expression in control tomato plants and it was

378

doubled with a Normalized Fold Expression of 0.22 in bacterial pathogen treated plants.

379

Respective high gene expression of PAL was recorded with 0.67 and 0.30 Normalized Fold

380

Expression in CBMB12 and CBMB15 inoculated plants and challenged with bacterial

381

pathogen (Fig. 8).

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Detection of gfp tagged Methylobacterium spp. in tomato leaves

384

Tomato leaves were scanned using CLSM to confirm the localization of the gfp tagged

385

Methylobacterium, strains. When the leaves of plants treated with pathogen alone and control

386

did not show any fluorescent bacterial cells, the Methylobacterium strains treated plants

387

showed fluorescence. The methylobacterial fluorescence could be easily differentiated with

388

chlorophyll autofluorescence. Most of the fluorescent bacterial cells were found to be

389

localized nearer to the stomata and also distributed throughout the leaf blade. The GFP

390

emission from the bacterial cells was found to be dominant in intercellular spaces (Fig. 9).

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Discussion

393

The gaseous plant hormone ethylene is known to be enhanced in plants due to pathogenic

394

stress (Ma et al., 2003) and accumulation of ACC, the immediate precursor of ethylene

395

biosynthesis and an important mediator of stress response in plants (Abeles et al., 1992).

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Page 16 of 41

Chen et al. (2003) suggested biotrophic and necrotrophic phases with two distinct ethylene

397

peaks during pathogenesis in plants. While the ethylene in the biotrophic phase is stimulating

398

the defence responses by regulating a wide range of defence related genes, in the

399

necrotrophic phase it is involved in a negative effect of advancement in pathogenesis. The

400

divergent role of ethylene in pathogenic infection warrants the ethylene release after the

401

induction of defense proteins should be inhibited. Plant growth promoting bacteria possessing

402

ACC deaminase activity were found to modulate ethylene level in plants by hydrolytic

403

cleavage of ACC (Glick and Penrose, 1998). We have tested the hypothesis that lowering

404

plant ethylene concentrations by ACC deaminase activity of Methylobacterium strains

405

can decrease the degree of plant disease susceptibility by increasing defence response in

406

tomato plants.

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Under greenhouse condition, the Methylobacterium spp. inoculated plants, when

408

challenged with X. campestris pv. vesicatoria had higher plant growth with less disease

409

incidence compared to solitary pathogen inoculation. Also, tomato plants treated with

410

Methylobacterium strains and challenged with X. campestris pv. vesicatoria significantly

411

reduced disease severity with low ethylene emission (Fig.1, Fig.2). Wang et al. (2000)

412

contradicted the role of ACC deaminase activity in Pseudomonas fluorescens CHA0 having

413

introduced ACC deaminase gene, capable of increasing resistance against Pythium damping-

414

off and susceptibility to Fusarium crown and root rot in tomato. The methylobacterial strains

415

used in this study have been isolated from rice plants and screened for plant growth

416

promotion activities in rice, red pepper and tomato (Lee et al., 2006; Madhaiyan et al., 2007;

417

Yim et al., 2012). Further the positive canola seedlings root growth promotion effect of

418

Methylobacterium in plant growth chamber (Madhaiyan et al., 2006a) and their negative

419

effect by RNAi silencing of the ACC deaminase gene in Trichoderma asperellum (Viterbo et

420

al., 2010) confirms the additional plant growth promotion effect due to ACC deaminase

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Page 17 of 41

enzyme activity. The classic work of Hao et al. (2007) on inhibition of crown gall formation

422

in tomato and castor bean by transferring ACC deaminase gene from Pseudomonas putida

423

UW4 into crown gall pathogen Agrobacterium tumefaciens C58 is a further proof for

424

ethylene induced pathogenesis and imply that bacterial ACC deaminase could play a potential

425

role in inducing disease tolerance in plants. The results obtained in our study support the

426

hypothesis that ethylene plays an important role in disease severity and that ACC deaminase

427

possessing Methylobacterium modulates the ethylene level in plants.

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Biosynthesis of ethylene begins with the compound S-adenosylmethionine

429

(SAM) that is also required as the precursor in many other pathways. The committed step in

430

the biosynthesis of ethylene from SAM is the conversion of ACC by the enzyme ACC

431

synthase (ACS). ACC is converted to ethylene by ACO (Stearns and Glick, 2003). In this

432

study, ACC and ACO accumulation were significantly reduced in the tomato leaves

433

due to Methylobacterium strains inoculation compared to pathogen inoculated control.

434

It was expected a higher rate of ethylene production with a corresponding increase in

435

ACC concentration along with ACO activity in pathogen-stressed tomato. In contrast, the

436

ACC deaminase producing Methylobacterium, decreased ACC accumulation and ACO

437

activity in the inoculated canola plants (Madhaiyan et al., 2006a). The present study

438

evidencing, the low expression of ACO-related gene in tomato plants inoculated with

439

Methylobacterium strains provides proof at molecular level transcription for reduced ethylene

440

synthesis (Fig. 8). This may be because of less content of ACC in the plants due to

441

ACC deaminase activity of Methylobacterium as suggested earlier by Penrose and Glick

442

(2001) reporting ACC deaminase possessing bacteria

443

leading to low concentrations of ACC in root exudates. Additionally, suppressing the genes

444

responsible for ethylene production and inducing the genes involved in plant growth by ACC

445

deaminase possessing Paenibacillus polymyxa in roots of Arabidopsis thaliana was also

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18

sequester and hydrolyze ACC,

Page 18 of 41

446

demonstrated by differential display PCR (Timmusk and Wagner, 1999). Furthermore, we found that Methylobacterium strains inoculated tomato plants

448

when challenged with X. campestris pv. vesicatoria, could enhance significant protection

449

against bacterial spot disease with significant increase in β-1,3-glucanase and PAL defence

450

enzymes and their related genes expression. The increased activity of defence proteins

451

parallel with low level of ethylene reflects the role of ACC deaminase producing

452

Methylobacterium strains in the induction of defence responses. Similar result was observed

453

in tomato and groundnut with higher accumulation of PR proteins when inoculated with

454

Methylobacterium and challenged with Pseudomonas syringae pv. tomato and Aspergillus

455

niger/Sclerotium rolfsii respectively (Indiragandhi et al., 2008; Madhaiyan et al., 2006b).

456

Our Methylobacterium strains could considerably lower the ethylene level with simultaneous

457

increase in defence enzymes in pathogen challenged tomato plants. The quantitative real time

458

RT-PCR studies also revealed the simultaneous decrease in the expressions of ethylene

459

biosynthetic ACO and increase in β-1,3-glucanase

460

expressions. Using the Arabidopsis mutants jar1, etr1, and npr1, Pieterse et al. (1998)

461

showed the regulatory protein NPR1 playing a crucial role in Pseudomanas fluorescens

462

WCS417r–mediated ISR against Pseudomonas syringae pv. tomato in Arabidopsis following

463

a novel jasmonate and ethylene induced signaling pathway, similar to salicylic acid mediated

464

system acquired resistance (SAR). They further provided evidence that the processes

465

downstream of NPR1 in the ISR pathway are divergent from those in the SAR pathway,

466

indicating that NPR1 differentially regulates defense responses. The biocontrol spectrum of

467

plant diseases using Pseudomonas fluorescens is further expanding after the invention of an

468

plant ubiquitous colonizing Methylobacterium .While the downstream signaling events in the

469

rhizobacteria-mediated ISR pathway is clearly defined, it is yet to be understood in

470

Methylobacterium mediated plant defence mechanism.

and

PAL enzymes related genes

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Page 19 of 41

The ubiquitous occurrence of Methylobacterium in the plants has been earlier

472

documented with epiphytic and endophytic colonization of roots and leaves in various plants

473

(Benediktyova and Nedbal, 2009; Lee et al., 2011; Poonguzhali et al., 2008). We confirmed

474

the colonization of gfp tagged Methylobacterium strains throughout the leaf blade and

475

particularly concentrating the stomata, by their fluorescence under CLSM (Fig. 9). The

476

methanol release of the plants through stomata facilitates the primary colonization of

477

methylotrophic bacteria nearer to the stomata thereby facilitating their endophytic entry as

478

well (Omer et al., 2004; Sy et al., 2005). The absence of fluorescent cells in control and

479

pathogen alone treated tomato leaves confirmed the absence of methylobacterial strains used

480

in the study in those plants. The autofluorescence of chlorophyll was easily differentiated

481

from bacterial fluorescence, since green photons are absorbed less strongly than red light and

482

penetrate deep into the plant tissue (Vogelmann and Evans, 2002) causing chlorophyll

483

autofluorescence. But fluorescence from Methylobacterium originated mostly in stomata and

484

mesophyll chloroplast and a combination with red light made it easily distinguishable

485

(Benediktyova and Nedbal, 2009). The colonization of the seed treated gfp tagged

486

methylobacterial strains in the mesophyll and stomata indicates their ability for endophytic

487

movement reaching the phylloplane. Our confirmation on

488

Methylobacterium strains in tomato with gfp tagged methylobacterial strains supports their

489

nonspecific induction of ISR in tomato against

490

vascular pathogen (Yim et al., 2013) and X. campestris pv. vesicatoria, another epiphytic

491

bacterial spot pathogen as evidenced in the current study. Earlier studies indicate the leaf

492

application of the rhizobacteria could be effective in inducing resistance in plants to leaf

493

pathogens as well as root pathogens (Leeman et al., 1995; Liu et al., 1995). Our findings

494

documents methylobacteria mediated ISR induction against multiple pathogens in tomato, as

495

done earlier in Pseudomonas fluorescens induced ISR in radish against two fungal pathogens

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the ubiquitous nature of the

Ralstonia solanacearum, a soil borne

Page 20 of 41

496

(Hoffland et al., 1996). The reduction in bacterial spot disease severity in X. campestris pv. vesicatoria

498

challenged tomato plants evidenced in the current study could be attributed to the reduced

499

stress ethylene level due to ACC deaminase activity of inoculated Methylobacterium

500

strains. Furthermore, the increased activity of defence enzymes and their relative genes

501

expressions as a result of reduced ethylene level substantiates the potential role of

502

Methylobacterium strains in the induction of defense responses in plants. In addition to the

503

known plant growth promotion effects, the Methylobacterium strains may prevent the

504

pathogenesis in the challenged plants. We therefore suggest the possible using of

505

Methylobacterium as a potential biocontrol means, for controlling bacterial diseases in tomato

506

plants after field evaluation and further understanding on the molecular mechanism of

507

methylobacterial mediated defence system in plants.

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Acknowledgement: This research was supported by Basic Science Research Program

510

through the National Research Foundation of Korea (NRF) funded by the Ministry of

511

Education, Science and Technology (2012R1A2A1A01005294).

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d

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te

681

684 685

Ac

683

ce p

682

28

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686

Figures legend

687 Figure 1 Shoot length of tomato treated with Methylobacterium spp. and challenged with

689

Xanthomonas campestris pv. vesicatoria (XCV). Each value represents the mean ± S.E (n=8).

690

Asterisk shows significant difference in values from the control, per group by LSD test (P ≦

691

0.05). * P ≦ 0.05, ** P ≦ 0.001

ip t

688

cr

692

Figure 2 Efficacy of Methylobacterium spp. inoculation in reducing the severity of bacterial

694

spot caused by Xanthomonas campestris pv. vesicatoria (XCV) on tomato leaves. (a) XCV

695

alone; (b) XCV + CBMB12; (c) XCV + CBMB15; (d) XCV + CBMB20; (e) XCV +

696

CBMB27; (f) XCV + Germicide.

an

us

693

M

697

Figure 3 Efficacy of Methylobacterium spp. inoculation in reducing the severity of bacterial

699

spot caused by Xanthomonas campestris pv. vesicatoria (XCV) on tomato apical leaves. (a)

700

XCV alone; (b) XCV + CBMB12; (c) XCV + CBMB15; (d) XCV + CBMB20; (e) XCV +

701

CBMB27; (f) XCV + Germicide.

ce p

te

d

698

702

Figure 4 Percent disease index of bacterial spot caused by Xanthomonas campestris pv.

704

vesicatoria (XCV) on tomato treated with Methylobacterium spp. Each value represents the

705

mean ± S.E (n=8).

Ac

703

706 707

Figure 5 Ethylene emission of tomato treated with Methylobacterium spp. and challenged

708

with Xanthomonas campestris pv. vesicatoria (XCV). Each value represents the mean ± S.E

709

(n=3).

710

29

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711

Figure 6 ACC and ACO accumulation of tomato treated with Methylobacterium spp. and

712

challenged with Xanthomonas campestris pv. vesicatoria (XCV). Each value represents the

713

mean ± S.E (n=3).

714 Figure 7 β-1,3-glucanase and PAL activity in tomato treated with Methylobacterium spp. and

716

challenged with Xanthomonas campestris pv. vesicatoria (XCV). Each value represents the

717

mean ± S.E (n=3).

cr

ip t

715

us

718

Figure 8 Quantitative reverse-transcriptase-PCR analysis of ACC oxidase, β-1,3-glucanase

720

and PAL related gene expression of tomato treated with Methylobacterium spp. and

721

challenged with Xanthomonas campestris pv. vesicatoria (XCV). Each value represents the

722

mean ± S.E (n=3).

M

an

719

723

Figure 9 CLSM images showing colonization of gfp tagged Methylobacterium spp. strains in

725

tomato leaves. The green fluorescence within the squares indicates the colonization of gfp

726

tagged methylobacteria.

te

ce p Ac

727

d

724

30

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ip t

Table 1

Table 1 Primer sets used for the real-time quantitative reverse-transcriptase-PCR Primer Host plants

Primer sequence (5'→3')

Accession no.

cr

Gene name

us

name Lycopersicon

YL991 AAGATGGCACTAGGATGTCAATAG

ACCesculentum

AB013101

an

oxidase

YL992 TCCTCTTCTGTCTTCTCAATCAAC Mill. Lycopersicon esculentum

AB269917

M

PAL

YL993 CGCTATGCTCTCCGAACATCTC

glucanase

YL995 GCGGTGTTCAGCCTGGATG

FJ151171

Ac ce p

esculentum

te

Lycopersicon β-1,3-

d

YL994 ATTCACCGAGTTAATCTCCCTCTC

Mill.

YL996 AGCATGAGCAAGAAGTATGTTGTG

Mill.

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Table 2

Table 2 The inoculation effect of Methylobacterium spp. on pathogen challenged tomato root growth on 35th day after transplanting Shoot dry weight

Root dry weight

(cm plant-1)

(g plant-1)

Control

24.0 ± 1.5b

10.0 ± 0.3b

XCV alone

19.9 ± 1.7c

8.2 ± 1.0c

XCV + CBMB12

29.5 ± 1.6a

12.3 ± 0.7a

0.58 ± 0.06bc

XCV + CBMB15

28.2 ± 1.1a

12.3 ± 0.2a

0.62 ± 0.02b

XCV + CBMB20

29.8 ± 1.0a

12.5 ± 0.3a

0.75 ± 0.05a

XCV + CBMB27

22.3 ± 0.8bc

XCV + Bactericide

31.6 ± 2.0a

ip t

Root length

(g plant-1)

M

an

0.45 ± 0.01d

cr

us

Treatment

0.39 ± 0.04d

0.48 ± 0.03cd

9.7 ± 0.4bc

0.43 ± 0.01d

d

10.5 ± 0.3b

Ac ce p

te

Values (mean ± S.E. n=8) with the same letters do not differ significantly at 0.05% (LSD).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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Figure 9

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