Rapid suppression of defence enzymes and compounds by sheath blight (Rhizoctonia solani) and sheath rot (Sarocladium oryzae) toxins in rice cell suspension cultures

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To understand the suppression mechanisms against disease resistance in rice, we took advantage of the fact that suspension cultured cells exhibit many of the defence responses that are characteristic of intact tissues. In this study we constitutively
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   PLEASE SCROLL DOWN FOR ARTICLE This article was downloaded by: [Kandan, A.]  On: 17 September 2010  Access details: Access Details: [subscription number 927030697]  Publisher Taylor & Francis  Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Archives Of Phytopathology And Plant Protection Publication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t713454295 Rapid suppression of defence enzymes and compounds by sheath blight  hizoctonia solani ) and sheath rot Sarocladium oryzae ) toxins in rice cellsuspension cultures A. Kandan a ; R. Radja Commare b ; R. Nandakumar c ; G. Amutha d ; A. Vijayasamundeeswari d ; A.Ramanathan d ; T. Raguchander d ; P. Balasubramanian d ; R. Samiyappan da  Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan, R.O.C. b Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taiwan, R.O.C., Taipei 115 c Department of Plant Pathology and Crop Physiology, Louisiana State University, Louisiana, USA d Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu AgriculturalUniversity, Coimbatore, IndiaOnline publication date: 16 September 2010 To cite this Article  Kandan, A. , Commare, R. Radja , Nandakumar, R. , Amutha, G. , Vijayasamundeeswari, A. ,Ramanathan, A. , Raguchander, T. , Balasubramanian, P. and Samiyappan, R.(2010) 'Rapid suppression of defenceenzymes and compounds by sheath blight ( Rhizoctonia solani  ) and sheath rot ( Sarocladium oryzae  ) toxins in rice cellsuspension cultures', Archives Of Phytopathology And Plant Protection, 43: 14, 1407 — 1422 To link to this Article: DOI: 10.1080/03235400802476658 URL: http://dx.doi.org/10.1080/03235400802476658 Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdfThis article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.  Rapid suppression of defence enzymes and compounds by sheath blight( Rhizoctonia solani  ) and sheath rot ( Sarocladium oryzae ) toxins in rice cellsuspension cultures A. Kandan a *, R. Radja Commare b , R. Nandakumar c , G. Amutha d ,A. Vijayasamundeeswari d , A. Ramanathan d , T. Raguchander d , P. Balasubramanian d andR. Samiyappan d a Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan,R.O.C.;  b Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei 115, Taiwan,R.O.C.;  c Department of Plant Pathology and Crop Physiology, Louisiana State University, Louisiana,USA;  d  Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641 003, India ( Received 5 September 2008; final version received 8 September 2008 )To understand the suppression mechanisms against disease resistance in rice, we tookadvantage of the fact that suspension cultured cells exhibit many of the defence responsesthat are characteristic of intact tissues. In this study we constitutively measured the Rhizoctonia solani   and  Sarocladium oryzae  toxins, induced and suppressed levels of phenylalanineammonialyase,peroxidase,superoxidedismutase,phenols,catalase, b -1,3-glucanase and chitinase in rice suspension cultured cells. The addition of   Rhizoctoniasolani   and  Sarocladium oryzae  toxins separately in suspension cultured cells shows thesuppression of defence enzymes and compounds at 24 h and 48 h respectively exceptSOD. The rice cultivar IR50 delays the disease suppression effect when compared to theother cultivars viz., Pusa Basmati and Co 43. The PR proteins (namely  b -1,3-glucanaseand chitinase) activities in rice suspension cultured cells were reduced during 48 h and72 h after the addition of   Rhizoctonia solani   toxin, whereas the activities were suppressedonly after 72 h when inoculated with  Sarocladium oryzae  toxin. Selective suppression of these defence enzymes and compounds by  Rhizoctonia solani   and  Sarocladium oryzae toxin shows that toxins play a major role during pathogenesis in rice cells. Keywords:  defence enzymes; rice; sheath blight; sheath rot; suspension cultures; toxins Introduction Plants have developed an impressive array of defence mechanisms and the genes inducedas part of the general defence response can be divided into several functional groups(Dixon et al. 1994). Generally,  in vitro  selection for disease resistance and the defencemechanisms of the cell are attractive approaches in host–pathogen systems wherepathotoxins are involved in pathogenesis. The use of suspension cultured cells can beexpected to have some advantage over intact plant tissues in order to study the mode of action of toxins because the suspension cultured cells are grown synchronously and can beuniformly treated with toxins. Moreover, the cultured cell systems have been utilised forconfirmation of defence mechanisms and also for selection of toxin tolerant cells *Corresponding author. Email: kandan@gate.sinica.edu.tw Archives of Phytopathology and Plant Protection Vol. 43, No. 14, 20 September 2010, 1407–1422 ISSN 0323-5408 print/ISSN 1477-2906 online   2010 Taylor & FrancisDOI: 10.1080/03235400802476658http://www.informaworld.com  D o w nl o ad ed  B y : [ K a nd a n ,  A .]  A t : 01 :47 17  S e p t e mb e r 2010  (Kodama et al. 1995). Recognition of pathogens triggers a large range of inducible defenceresponses. The induced mechanisms associated with the HR include strengthening of theplant cell wall by alterations in the synthesis of cell wall structural proteins (Lamb et al.1989). First the phenylpropanoid products play diverse roles in the response of plants todifferent biotic and abiotic stimuli. Second, the synthesis of peroxidases is induced. Thesedefence gene products may also contribute to the formation of a structural barrier topathogen invasion. Third, chitinase and  b -1,3-glucanse enzyme synthesis occurs. Theseenzymes may either promote degradation of the cell walls of invading fungi which furtherstimulates the defence responses (Dietrich et al. 1990).Rice ( Oryza sativa  L.) is one of the most important cereal crops feeding three-quartersof the world’s population. One of the major limiting factors in rice production is damagethrough diseases caused by fungi, bacteria, viruses and other abiotic stresses. Among thefungal diseases, sheath blight caused by  Rhizoctonia solani   Kuhn and sheath rot caused by Sarocladium oryzae  W. Gams and D. Hawksw are the most serious diseases in all rice-growing countries (Sawada 1922; Chen and Chien 1964; Jimenez and Parizos 1975;Shajahan et al. 1977; Roy 1993; Mariappan 1996). Several fungal pathogens are able toproduce phytotoxins (Cutler 1993), varying in molecule size, chemical type and hostspecificity, but all modify the physiology of host cells. Suspension culture should alsoprove valuable for the identification and purification of individual defence enzymes, whichmay serve as indicators of disease resistance in plants (Popp et al. 1997). Considering theimportance of phenylpropanoid metabolism in plant defence mechanisms, it seemspossible that some phytotoxins might be capable of specifically suppressing the activationof this pathway in the host tissues. To investigate this possibility, we took advantage of thefact that rice suspension cultured cells that are characteristic of intact tissues where theinfluence of fungal toxins viz.,  Rhizoctonia solani   toxin (RS toxin) and  Sarocladium oryzae toxin (SO toxin) on the suppression of defence mechanisms such as phenylalanineammonia-lyase (PAL), peroxidase, phenol, catalase, superoxide dismutase (Vidhyasekaranet al. 1992; Mohan et al. 1993; Mona 1994; Baker and Orlandi 1995; Shiraishi et al. 1995; Vurro and Ellis 1997), chitinase and  b -1,3-glucanse (Graham and Graham 1991; Poppet al. 1997) were evaluated. Materials and methods Preparation of toxin RS toxin RS toxin from  R. solani   was purified as described earlier (Vidhyasekaran et al. 1997).  R.solani   isolates were grown on Richards’s broth (Fahmy 1923). The one-litre culture filtratewas collected after 21 days of incubation at room temperature. The filtrate was condensedto one tenth of its srcinal volume by flash evaporation  in vacuo  at 50 8 C. An equal volumeof methanol was added to the condensed culture filtrate and incubated overnight at 5 8 C.The precipitate was removed by filtration and methanol was evaporated at 50 8 C using avacuum flash evaporator. The water fraction was partitioned with equal volumes of ethylacetate, hexane and chloroform in sequence using a separating funnel. The partitionedwater fraction showing toxic action was condensed to half of its volume and subjected tocolumn chromatography (Pharmacia, USA) using Sephadex G-75 with water as eluant.Five mililitre fractions were collected, toxic fractions were pooled, evaporated to dryness in vacuo  at 40 8 C, weighed and re-dissolved in 5 ml of water. This partially purified toxinwas used in all further studies.1408  A. Kandan  et al.  D o w nl o ad ed  B y : [ K a nd a n ,  A .]  A t : 01 :47 17  S e p t e mb e r 2010  SO toxin SOtoxinfrom S.oryzae waspurifiedasdescribedearlier(Hemalathaetal.1999;Samiyappanet al.2003). Richard’s broth (Fahmy 1923) was used for culturingthepathogen. Flasks withculture were incubated at 25 8 C under stationary conditions for 20 days, then the culturefiltrates were harvested. After filtration, the culture fluid was concentrated to 10% of itssrcinal volume at 50 8 C using a vacuum flask. The condensed culture fluid was treated withan equal volume of MeOH and kept overnight at 4 8 C. Precipitates developed were removedby filtering throughWhatmanNo.1 filter paper. Methanolwasevaporated  in vacuo  and thewater fraction was partitioned with equal volumes of chloroform, ethylacetate, carbontetrachloride, hexane and ether in sequence using a separating funnel. The water fractionshowing the toxic activity was condensed to half of its volume, applied to Sephadex G-75column (2.5  6  60 cm Pharmacia, USA) and eluted with distilled water. Five mililitrefractions were collected and the toxic fractions were pooled. Callus induction The induction of callus and the initiation of cell suspension cultures were followed fromthe slightly modified protocols of Zhang et al. (1996). Rice cv. Pusa Basmati, IR 50 and Co43 were used for callus induction and further studies in all the experiments. The seeds werecollected from Paddy Breeding Station, Tamil Nadu Agricultural University, Coimbatore,India. Rice calli were produced from dehusked mature seeds. Dehusked seeds were surfacesterilised in 70% ethanol for 1 min, followed by 0.1% mercuric chloride for 8 min andrinsed with several changes of sterile distilled water. The seeds were plated on Petri dishescontaining MS (Murashige and Skoog 1962) medium supplemented with 2,4-D (2.0 mg/l)and incubated at 25  +  2 8 C in darkness for four weeks. Then subclutured on fresh MSmedium supplemented with 2,4-D (2.5 mg/l), proline (500 mg/l), casein (300 mg/l) andkept under dark at 25  +  2 8 C for 3–4 weeks. Phytagel 0.4% (w/v) was used as solidifyingagent instead of agar in all solid media used in this study. Preparation of suspension cultured cells Suspension-cultured cells were prepared by transferring 3 g of small, compact,embryogenic calli to 250 ml Erlenmeyer flask containing 50 ml of modified R 2  medium(Ohira et al. 1973) supplemented with maltose (30 g/l) and 2,4-D (2 mg/l ). Flasks werekept on a rotary shaker at 125 rpm at 25  +  2 8 C. Suspension-cultured cells weretransferred to fresh medium at weekly intervals. Treatment of suspension-cultured cells One mililitre of toxin (50  m g/ml glucose equivalents) was added to the suspension-culturedcells and incubated on a rotary shaker with 125 rpm at 25  +  2 8 C. Suspension-culturedcells treated with 1 ml of sterile distilled water served as control. Cells were harvested atperiodic intervals and used for various analyses. Assay of phenylalanine ammonia-lyase (EC. 4.5.1.5) The PAL assay was carried out as per the method described by Brueske (1980). Suspension-cultured cells (200 mg) were extracted in 1.0 ml of 0.1 M sodium borate buffer (pH 7.0) and Archives of Phytopathology and Plant Protection  1409  D o w nl o ad ed  B y : [ K a nd a n ,  A .]  A t : 01 :47 17  S e p t e mb e r 2010  centrifuged at 10,000  g  for 10 min at 4 8 C, the supernatant served as the enzyme source. Theenzyme activity was expressed as  m mol/min/g fresh weight of cultured cells. Assay of peroxidase (EC.1.11.1.7) Peroxidase activity was assayed using guaiacol as a hydrogen donor as previously reportedby Rathmell and Sequiera (1974). Suspension-cultured cells (200 mg) were extracted in1.0 ml of 0.1 M sodium phosphate buffer (pH 6.5) and centrifuged at 10,000  g  for 10 minat 4 8 C, the supernatant served as the enzyme source. The peroxidase activity was expressedas change in absorbance min/g of fresh weight of cultured cells. Assay of phenols The phenolic content was estimated as per the method of Zieslin and Benzaken (1993).Suspension-cultured cells (10 mg) were homogenised in 1 ml of 80% methanol and thismethanolic extract was utilised for phenol estimation. The phenol content was expressed inmg/g fresh weight of cultured cells. Assay of catalase (EC. 1.11.1.6) Catalase activity was assayed based on the decrease in H 2 O 2  concentration was measuredas the decline in A 240  during the first 10 min after initiation of the reaction (BeleidEl-Moshaty et al. 1993). Enzyme activity was expressed as change in absorbance min/gfresh weight of cultured cells. Assay of superoxide dismutase (SOD) (EC. 1. 15. 1. 1) Suspension-culturedcells(100 mg)werehomogenisedinachilledpestleandmortarin0.2 Mcitratephosphatebuffer(pH6.5)containing0.5%TritonX-100(Sigma,USA),in1:10(w/v)ratio, centrifuged in the homogenate for 15 min at 20,000  g  at 4 8 C the supernatant wasused as the enzyme source. The assay mixture (3 ml) was prepared with 50 mM sodiumphosphate buffer (pH 7.8), 13 mM methionine, 75  m M Nitroblue tetrazolium (NBT),0.1 mM EDTA, 100  m l of the enzyme extract and 2  m M riboflavin. The assay was carriedout according to the protocol described earlier (Beleid El-Moshaty et al. 1993). The SODactivity was expressed as percentage inhibition of NBT photoreduction. Assay of   b -1,3-glucanase (EC.3.2.1.39) The enzyme activity was colorimetrically assayed (Pan et al. 1991). Five hundredmilligrams of cultured cells were extracted with 0.05 M sodium citrate buffer (pH 5.0) at4C. The homogenate was centrifuged at 4C for 20 min at 10,000 rpm. The supernatantwas used as crude enzyme extract. The enzyme activity was expressed as g equivalents of glucose/min/g fresh weight of cultured cells. Assay of chitinase (EC.3.2.1.14) The colorimetric assay of chitinase was carried out as per Boller and Mauch (1988). Fivehundred milligrams of cultured cells were extracted with 1 ml of 0.1 M sodium citratebuffer (pH 5.0) at 4C. The homogenate was centrifuged at 4C for 20 min at 10,000 rpm.1410  A. Kandan  et al.  D o w nl o ad ed  B y : [ K a nd a n ,  A .]  A t : 01 :47 17  S e p t e mb e r 2010
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