научная статья по теме Induction of systemic acquired resistance of plants by exometabolites of causal agent of potato ring rot Биология

Текст научной статьи на тему «Induction of systemic acquired resistance of plants by exometabolites of causal agent of potato ring rot»

DOI: 10.12731/wsd-2014-10-12 UDC 571.27


Omelichkina Yu.V., Boyarkina S.V., Shafikova T.N.

Development of plant defense to the influence of the pathogen is determined by the rapid recognition. It depends on the presence of specific receptors in plants. Recognition of molecular patterns of microbial cell surface by plant receptors, which are localized in the plant cell membrane, triggers basic non-specific immunity; recognition of pathogen effectors by plant receptors, which are localized inside the plant cell, triggers specific immunity. Specific immunity involves the development of hypersensitive response, systemic acquired resistance and immune memory of the plant. The study ofpatterns and effectors as inductors of plant defenses responses is current issue to the search of biogenic activators long-term resistance of plants to a wide range of pathogens.

Clavibacter michiganensis subsp. sepedonicus (Cms) is the phytopatho-genic bacterium, that causes the plant systemic disease - "ring rot of potatoes", which is one of the most common and harmful bacterial diseases. Previously it was shown that the causal agent of potato ring rot induces development of hypersensitivity reaction and systemic acquired resistance in non-host tobacco plants. In the present study it is established that the exometabolites of this pathogen (cell free culture filtrate and heat-treated bacterial suspension) also cause the development of hypersensitivity and system sustainability. The obtained results allow to assume that thermostable effector molecules, that have the ability to induce local and systemic protective response plants, are present in the composition of exometabolites of this phytopatogene.

Keywords: systemic acquired resistance, innate immunity of plants, clavibacter michiganensis ssp. sepedonicus, effectors, microbial molecular patterns, hypersensitivity reaction.


Омеличкина Ю.В., Бояркина С.В., Шафикова Т.Н.

Развитие защитных реакций растения на воздействие патогена определяется быстрым и адекватным его распознаванием, которое зависит от наличия у растения специфических рецепторов. При распознавании мембранными рецепторами растения консервативных микробных молекулярных паттернов активируется базовый неспецифический иммунитет; при распознавании цитоплазматическими рецепторами эффекторных молекул патогена запускается специфический иммунитет. Последний включает развитие реакции сверхчувствительности, системной устойчивости и иммунной памяти растения. Изучение паттернов и эффекторов в качестве индукторов защитных ответов растении актуально для поиска биогенных активаторов долговременной устойчивости растений к широкому кругу патогенов.

Clavibacter michiga^m^^' subsp. sepedoпicus (Cms) - бактериальный фитопатоген, который вызывает у растения-хозяина системное заболевание - «кольцевую гниль картофеля», являющееся одним из наиболее распространенных и вредоносных бактериозов. Ранее было выявлено, что бактерии Cms индуцируют развитие реакции сверхчувствительности и системной приобретенной устойчивости у растения-нехозяина табака. В настоящей работе установлено, что экзометаболиты патогена - фильтрат, лишенный бактериальных клеток и термически инактивированная суспензия бактерий также вызывают развитие реакции сверхчувствительности и системной устойчивости. Полученные результаты позволяют полагать, что в составе экзометаболитов данного фитопатогена присутствуют термостабильные эффекторные молекулы, которые обладают спо-

собностью индуцировать локальный и системный защитный ответ растения.

Ключевые слова: системная приобретенная устойчивость; врожденный иммунитет растений; clavibacter michiganensis ssp. sepedonicus; эффекторы; микробные молекулярные паттерны; реакция сверхчувствительности.

To survive in natural habitats under conditions of constant aggressive phytopathogenes action, a plant has innate immune system, it is ability to recognize a pathogen in due time and activate relevant protective mechanisms. Detection of pathogen is provided by membrane plant receptors, which recognize specific molecular ligands of microbe cell surface. These molecules were called microbial-associated molecular patterns (MAMP). Microbial patterns or MAMP are conservative molecular structures, which are characteristic of practically all microorganisms' classes, regardless of their pathogenicity [1]. Detection of MAMP is performed by receptors called pattern recognition receptors (PRRs), which trigger immune signaling following activation by specific ligands [2]. This process results in activation of a number of protective responses leading to prevention of infection development. This immunity mechanism was called pattern-triggered immunity (РТС) [5] and presents the first non-specific level of plant innate immunity [3, 4]. During long-term joint evolution of plant and pathogen for suppression of the immunity of host plant pathogens developed the ability to secrete specific protein molecules - effectors (Avr-genes products), transporting them immediately to the cell through a universal type III secretion system. Effectors are delivered into the cytoplasm, bypassing the cell wall and plasmalemma and suppressed the development of PTI [5]. In the course of further joint evolution the necessity to survive resulted in emergence in plants of effector detection system. Emergence in plants of intracellular nucleotide-binding leucine-rich repeat (NB-LRR) proteins (R-genes products), which are able to recognize the effector and activate protective reactions leads to arising of the second specific level of immunity [6]. Following the NB-LRR receptor activation reactions

of effector-activated immunity including activation of WRKY transcription factors [7] and further molecular events, such as change in ion streams, reactive oxygen species (ROS) and nitrogen oxide accumulation, activation of PR-genes transcription for biosynthesis of salicylic and jasmonic acids, as well as ethylene [8, 9], result in triggering hypersensitivity response (HR), a form of programmed cell death (PCD) localized at the site of attempted pathogen invasion [10, 11] and accompanied by the development of non-specific systemic acquired resistance (SAR) plants to subsequent infection [12, 13]. Plant organism may also realize mechanisms of trans-generational immune memory, that is stress influence on one generation may bring about effective adaptation of the next generation to the same stress [14, 15].


The contemporary understanding of molecular-genetic mechanisms of plant-microbe interactions and relationships of different levels of innate immunity of plants has theoretical importance and practical value, since it leads to a new immunological approach to create preventive measures against plant diseases, consisting in the induction of its own immune forces of plant. It is allow giving up currently existing means of combating plant diseases, which often have a detrimental effect on both soil microbiocenoses and human health.

In this context, the aim of this study was to study the ability exometabo-lites of causal agent of potato ring rot Clavibacter michiganensis ssp. sepedonicus (Cms) induce SAR in tobacco plants Nicotiana tabacum (L.).

Materials and methods of research

Bacterial strains and culture media. Clavibacter michiganensis ssp. sepedonicus (Cms) strain Ac1405 was obtained from the All-Russian Collection of Microorganisms (Pushino). Strain was routinely grown at 26°С in C medium, which contains per liter 5 g of glucose ("Reachim", Russia), 10 g of peptone ("AppliChem", Germany), 5 g of NaCl ("Reachim", Russia) (рН 7,2) [16].

For preparation of the bacteria to be used in infiltration assays, cultures were cultivated to the logarithmic growth phase in a liquid medium at 26°C in

the dark on a rotary shaker (130 rpm). To investigate the activity of Cms ex-ometabolites, cell free culture filtrate (CF Cms) was prepared. The culture was filtered through a 0.2-^m membrane filter. This preparation was immediately used either for infiltration assays on tobacco plant leaves or for investigation of reactive oxygen species accumulation. Also in this assays we used heat-treated bacterial suspension (dead culture, DC Cms). Bacterial culture contained in Eppendorf tubes were heated to 100°C in boiling water bath for 15 min.

To determine whether Cms induces of SAR development in tobacco plants we used Escherichia coli. E. coli strain XL-1Blue was routinely grown on meat-peptone agar (MPA) ("Obolensk", Russia) at 26°C in the dark. For infiltration assays, the culture was grown stationary in meat-peptone broth (MPB) at 26°C in the dark.

Plant Materials, Growth condition and inoculation procedures. Tobacco plants (Nicotiana tabacum L.) were grown in vitro in Murashige and Skoog (MS) medium [17] under greenhouse condition with light16-h / 8-h dark pho-toperiod at 24-25°C at day and 19-20°C at night, a light intensity of 5-6 kLx.

Tobacco cultured cells were grown in the dark in MS medium. Subcultures were made weekly by tenfold dilution with fresh medium. For elicitation experiments 5-day cultures grown at 26°C on a rotary shaker at 130 rpm were used. This stage is corresponding to the second half of the logarithmic growth phase, which is characterized by the highest physiological activity of cells.

To study the response of tobacco plants on the infection with the culture of Cms and its exometabolites we used infiltration assay. Tobacco plants were grown to three weeks. Plant leaves were infiltrated with 3 ^l Cms (1*107 CFU/ml), CF Cms or DC Cms with sterile syringe. Also, infection of plants was carried out through the root system by introducing a bacterial suspension with a syringe into the culture medium. Medium C was used for infiltration assays as control. I

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