INDUCTION OF SYSTEMIC ACQUIRED RESISTANCE OF PLANTS

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In the World of Scientific Discoveries, 10(58), 2014
DOI: 10.12731/wsd-2014-10-12
UDC 571.27
INDUCTION OF SYSTEMIC ACQUIRED
RESISTANCE OF PLANTS BY EXOMETABOLITES
OF CAUSAL AGENT OF POTATO RING ROT
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 of patterns 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 phytopathogenic 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.
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157
ИНДУКЦИЯ СИСТЕМНОЙ
ПРИОБРЕТЕННОЙ УСТОЙЧИВОСТИ РАСТЕНИЙ
ЭКЗОМЕТАБОЛИТАМИ ВОЗБУДИТЕЛЯ
КОЛЬЦЕВОЙ ГНИЛИ КАРТОФЕЛЯ
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 michigaпeпsis 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-
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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 (РTI) [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
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159
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].
Purpose
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 exometabolites 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
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the dark on a rotary shaker (130 rpm). To investigate the activity of Cms exometabolites, cell free culture filtrate (CF Cms) was prepared. The culture was
filtered through a 0.2-µm 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°С 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 photoperiod at 24–25°С at day and 19–20°С at night, a light intensity of 5–6 кLx.
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 µl 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. Inoculated tobacco plants were moved back to the grown
chamber under the same conditions prior to inoculation and visually monitored for reactions. To determine SAR development tobacco plants, which
pretreated by Cms, CF Cms or DC Cms, were infiltrated with 3 µl E. coli
(1×108 CFU/ml) with sterile syringe after 7 days post priming.
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161
Hydrogen peroxide measurements. To study such response reactions of
the plant innate immunity as the accumulation of reactive oxygen species, the
method of co-cultivation of tobacco cell cultures and bacterial suspensions
was carried out. During co-cultivation, the formation of hydrogen peroxide
plant cells was determined using a method based on the reduction reaction of
a hydroperoxide with iron ions in the acid solution to form an iron complex
with xylenol orange indicator [18].
Research results and their interpretation
In our previous study, it was shown that inoculation of bacteria Cms into
tobacco leaves induced a HR development and accompanied by SAR. Bacterial culture was also able to induced rapid cell death of cell-suspension culture of tobacco. Observed morphology changes in infected, cultured tobacco
cells were in line with features of programmed cell death development. We
have obtained that infected cultured cells showed response defensive reactions, such as the rapid generation of reactive oxygen species [19, 20].
Here we report that along with the bacterial suspension of Cms, cell free
culture filtrate CF Cms as well as heat-treated dead culture DC Cms able to
induce HR development at the site of infection (Fig. 1.).
Fig. 1. A local reaction of tobacco plants at the site of infection Cms, CF Cms and
DC Cms
Similar results were found when cell free culture filtrate and dead culture
were infiltrated into medium around roots of tobacco plant. Necrotic lesions
HR formed on the roots of tobacco plants after infiltration bacteria, CF Cms
and DC Cms (Fig. 2).
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It should be noted that the development of SAR is accompanied by development of protection long-term plant responses to a wide range of pathogens,
including atypical pathogens for plants, for example, the causative agent of
the disease in humans and animals E.coli [19].
It was shown, that local necrosis HR was induced at the site of E. coli
infiltration on the plants pretreated by Cms, CF Cms or DC Cms (Fig. 2).
Thus, not only Cms induces SAR development, but SAR-induced activity was found in cell free culture filtrate (CF Cms) and dead culture (DC
Cms).
Fig. 2. Necrotic lesions formed on the roots of tobacco plants after infiltration bacteria Cms, CF Cms and DC Cms; local necrosis formed at the site of E. coli infiltration
on the plants pretreated by Cms, CF Cms or DC Cms
It is known that pathogen infections induce a rapid accumulation of ROS
in plant tissue, which is involved not only in limiting pathogen ingress,
but in induction of cell death and in signal transduction of many defense
processes. To investigate accumulation of ROS, co-cultivation of tobacco
cells with Cms, cell free culture filtrate Cms or heat-treated dead culture
Cms was performed. Time-course analyses using this systems showed, that
content of apoplast forms of hydrogen peroxide increased rapidly during
1h after onset co-cultivation with not only bacterial cells, but also its metabolites (CF Cms) and heat-treated metabolites (DC Cms) (Fig. 3). These
results clearly indicated that exometabolites of Cms is sufficient to activate
immune responses.
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163
Fig. 3. Time-course analyses of accumulation of hydrogen peroxide during elicitation of tobacco cultured cells by Cms, CF Cms or DC Cms (percentage)
Conclusion
The obtained results suggest that the activation local and systemic plants
resistance occurs not only with the direct contact with Cms cells [19, 20], but
also in the perception of pathogen exometabolites.
Furthermore, it should be noted, that the activity of exometabolites retained after heat treatment, suggesting it is heat stability. Further work in this
direction will lead to the creation of long-term biogenous inductors of plant
resistance to a wide range of pathogens that meet all the criteria required for
plant protection products of the new generation. It may be essential for agriculture and food safety.
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Список литературы
1. Staal J., Dixelius C. Tracing the ancient origins of plant innate immunity //
Trends in Plant Science, 2007, vol. 12. Р. 334–342.
2. Nicaise V., Roux M., Zipfel C. Recent advances in PAMP-triggered immunity
against bacteria: pattern recognition receptors watch over and raise the alarm
// Plant Physiology, 2009, vol. 150. Р. 1638–1647.
3. Jones J.D., Dangl J.L. The plant immune system // Nature, 2006, vol. 444.
Р. 323–329.
4. Zhang J., Zhou J.M. Plant immunity triggered by microbial molecular signatures // Molecular Plant, 2010, vol. 3. Р. 783–793.
5. Subterfuge and manipulation: type III effector proteins of phytopathogenic
bacteria / Grant S.R., Fisher E.J., Chang J.H., Mole B.M., Dangl J.L. // Annual Review of Microbiology, 2006, vol. 60. Р. 425–449.
6. Eitas T.K., Dangl J.L. NB-LRR proteins: pairs, pieces, perception, partners,
and pathways // Current Opinion in Plant Biology, 2010, vol. 13. Р. 472–477.
7. Pandey S.P., Somssich I.E. The role of WRKY transcription factors in plant
immunity // Plant Physiology, 2009, vol. 150. Р. 1648–1655.
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In the World of Scientific Discoveries, 10(58), 2014
8. Moore J.W., Loake G.J., Spoel S.H. Transcription dynamics in plant immunity // Plant Cell, 2011, vol. 23. Р. 2809–2820.
9. Hormonal modulation of plant immunity / Pieterse C.M., Van der Does D.,
Zamioudis C., Leon-Reyes A., Van Wees S.C. // Annual Review of Cell and
Developmental Biology, 2012, vol. 28. Р. 489–521.
10.The hypersensitive response: the centenary is upon us but how much do we
know? / Mur L.A., Kenton P., Lloyd A.J., Ougham H., Prats E. // Journal of
Experimental Botany, 2008, vol. 59. Р. 501–520.
11.Coll N.S., Epple P., Dangl J.L. Programmed cell death in the plant immune
system // Cell Death and Differentiation, 2011, vol. 18. Р. 1247–1256.
12.Durrant W.E., Dong X. Systemic acquired resistance // Annual Review of
Phytopathology, 2004, vol. 42. Р. 185–209.
13.Vallad G.E., Goodman R.M. Systemic acquired resistance and induced systemic resistance in conventional agriculture // Crop Science, 2004, vol. 44.
Р. 1920–1934.
14.Descendants of primed Arabidopsis plants exhibit enhanced resistance to biotic stress / Slaughter A., Daniel X., Flors V., Luna E, Hohn B, Mauch-Mani
B. // Plant Physiology, 2012, vol. 158. Р. 835–843.
15.Next generation systemic acquired resistance / Luna E., Bruce T.J., Roberts
M.R., Flors V., Ton J. // Plant Physiology, 2012, vol. 158. Р. 844–853.
16.Murashige T., Skoog F. A revised medium for rapid growth and bioassays with
tobacco tissue cultures // Physiologia Plantarum, 1962, no. 15. Р. 473–497.
17.Meletzus D., Eichenlaub R. Transformation of the phytopathogenic bacterium Clavibacter michiganense subsp. michiganense by electroporation and
development of a cloning vector // Journal of Bacteriology, 1991, no. 173(1).
Р. 184–190.
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DATA ABOUT THE AUTHORS
Omelichkina Yulia Viktorovna, Postgraduate Student
Siberian Institute of Plants Physiologies and Biochemistry
132, Lermontova st., Irkutsk, 664054, Russian Federation
e-mail: omelichkina@yandex.ru
Boyarkina Svetlana Vladimirovna, Postgraduate Student
Siberian Institute of Plants Physiologies and Biochemistry
132, Lermontova st., Irkutsk, 664054, Russian Federation
Shafikova Tatyana Nikolaevna, Senior Researcher, Cand. of Biol. Sc.
Siberian Institute of Plants Physiologies and Biochemistry
132, Lermontova st., Irkutsk, 664054, Russian Federation
e-mail: t-shafikova@yandex.ru
ДАННЫЕ ОБ АВТОРАХ
Омеличкина Юлия Викторовна, аспирант
Сибирский институт физиологии и биохимии растений
ул. Лермонтова, 132, г. Иркутск, 664054, Российская Федерация
e-mail: omelichkina@yandex.ru
Бояркина Светлана Владимировна, аспирант
Сибирский институт физиологии и биохимии растений
ул. Лермонтова, 132, г. Иркутск, 664054, Российская Федерация
Шафикова Татьяна Николаевна, старший научный сотрудник, к.б.н.
Сибирский институт физиологии и биохимии растений
ул. Лермонтова, 132, г. Иркутск, 664054, Российская Федерация
e-mail: t-shafikova@yandex.ru