INCREASING SMALL REGULATORY RNAs WITH

IMMUNOMODULATING PROPERTIES UNDER GROWTH

REGULATORS’ ACTION IN PLANT CELLS

V. А. Tsygankova1, Ya. B. Blume2, T. R. Stefanovska3, S. P. Ponomarenko4

1Institute of Bioorganic Chemistry and Petrochemistry, NAS of Ukraine, Kyiv2 Institute of food biotechnology and genomics, NAAS of Ukraine, Kyiv3National University of Life and Environmental Science of Ukraine, Kyiv4National Enterprise Interdepartmental Science & Technology Center "Agrobiotech" of NAS and MES of Ukraine, Kyiv

Abstracts

Our laboratory experiments demonstrated that application of the PGR enhances sugar beet and rape plant resistance against nematode infection. This resistance is gained through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the nematode. As a result, translation of mRNA of the nematode is blocked and leads to the death of parasite larvae.

Over the past 15 years, much attention has been paid to isolate the small

regulatory RNAs from eukaryotic cells and to determine their biological role in

RNA interference (RNAi), a term referring to post-transcriptional gene silencing

(PTGS) phenomena found in all eukaryotes (including plants, animals and fungi)

[1, 2, 21]. Gene silencing was first demonstrated for the free-living

nematode, Caenorhabditis elegans, and the underlying

mechanism of RNAi has subsequently been studied in depth for

this nematode [3]. The impact of this work was recognized in

2006 by the award of the Nobel Prize in Physiology or Medicine to

Andrew Fire and Craig Mello [4]. Gene silencing mediated by either

degradation or translation arrest of target RNA has roles in adaptive protection

against viruses, genome defense against mobile DNA elements and developmental

regulation of gene expression. These silencing systems involve processing of two

type small regulatory RNAs: microRNA (miRNA) and short interfering RNA

(siRNA) [6, 23].

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The miRNA is generated from miRNA precursor by two rounds of

endoribonuclease cleavage by RNase III-like enzymes: pre-miRNA of ~70

nucleotides (nt) is initially processed by RNase III endoribonuclease named

Drosha and is “hairpin” primary transcript from one strand of distinct genomic loci

[10]. Then pre-miRNA is exported to the cytoplasm, where mature single-stranded

~21-22-nt miRNA is generated after processing of pre-miRNA by RNase III

endoribonuclease named Dicer and incorporated into micro-ribonucleoproteins

(miRNPs) [10, 13]. Another type of siRNA of ~22-24-nt is generated from longer

double-stranded RNA (dsRNA) molecules through their cleavage by RNase III

endoribonuclease named Dicer into short single-stranded (ss) siRNA [6]. Then one

part of (ss) siRNA is used for silencing of target mRNA, but other part (ss) siRNA

acts as a primer on complementary mRNA leading to the production of new

dsRNA with the help of RNA-dependent RNA polymerase (RdRP). It is proposed

that siRNA originates from longer transcripts derived from repetitive sequences

such as transposons and transgenes.

The si/miRNA and the anti-sense complementary structure to mRNA

function in a dual role [1, 6, 23]. The roles are: 1) together with site-specific multi-

subunit RNase, referred to as RNA-induced silencing complex (RISC), si/miRNA

determines an age period of each mRNA molecule by destroying aberrant and

defective mRNA structures that can appear faultily in the cells by specifically

mRNA degradation (cleavage) or their translation arrest (silencing) and 2)

si/miRNA provides protection against pathogens and parasites. In both cases, these

biological effects are achieved through specifically binding si/miRNA to

complementary sequences to target different classes of own plant cell mRNA

(which exhibited increased expression level at specialized feeding cells in infected

roots and involves plant developmental processes) [7, 16] or to pathogen mRNA

[8, 9, 14] or parasite mRNA [1, 23] with high degree of homology. In animal and

plant cells, si/miRNA act in different ways: si/miRNA of animals binds to the 3'-

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untranslated regions (3'-UTR) or the open reading frame (ORF) of target mRNA,

whereas si/miRNA of plants binds to the coding regions of target mRNA [15, 22].

With respect to protection against pathogens and parasites, the number of

si/miRNA molecules produced in plant cells against these antagonists in response

to a mass infection is not sufficient to provide effective protection. There are two

approaches to increase the number of si/miRNA in response to pathogen or

parasite attacks [1, 5, 14]. These are either to put a number of additional si/miRNA

genes in cells through genetic transformation or to activate the expression of

si/miRNA synthesis cellular genes themselves by certain specific inductors.

Recently, in laboratory and field studies, we found that the plant growth

regulators (PGRs) significantly increased plant resistance to viral pathogens,

nematodes and insect herbivores [19, 20]. Thus, we further explored the possibility

of enhancement by PGRs of sugar beet plant immune-protective properties through

increasing synthesis of small regulatory si/miRNA and, as a result, to achieve

increased plant resistance to the plant-parasitic nematode, Heterodera schachtii.

The materials and methods

In our experiments the sugar beet and rape plants infected by the cyst sugar

beet nematode, Heterodera shcachtii Shmidt were used. Experimental plants were

treated by new plant growth regulators (PGRs) Biolan, Biogen and Radostim-

super, created in the Institute of Bioorganic Chemistry and Petrochemistry, NAS of

Ukraine, jointly with National Enterprise Interdepartmental Science & Technology

Center "Agrobiotech" of NAS and MES of Ukraine. These polycomponent PGRs

contain complex biologically active substances (mixture of amino acids,

carbohydrates, fat acids, polysaccharides, plant hormones and microelements),

produced in vitro culture by symbiotic micromycete isolated out of ginseng root

system and aversectines – complex antiparasitic antibiotics, that are metabolites

produced by soil micromycete Streptomyces avermitilis [19].

Experimental parameters: Field experiments were conducted in 2010 at

Uladovo-Lyulynetska experimental breeding station (Vinnitsa region). The

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laboratory experiments are fulfilled in the Department of Entomology and

Phytopathology of the Institute of Bioenergy Crops and Sugar Beet NAS Ukraine

and have a natural background infestation of the sugar beet nematode.

The 15 experimental plots of 13.5 m2 were established in a complete

randomized block design. The soil composition of experimental plots was deep

black earth. Structure of profile is typical for northwest agro-soil region. The depth

of humus horizon is 60 cm with dust-lump structure. Granulometric composition of

arable layer of soil: total sand content - 61.19 %; silt content – 25.08 %; clay

content – 11.24 %; humus content – 4,8 %; salt pH – 5,8 – 6,2.

The soil was tilled to a depth of 30-32 cm and leveled before setting up the

plots. Prior to sowing, fertilizer was applied at the following rates per hectare:

manure at 40 tons, N at 110 tons, P at 100 kg, and K at 100 kg. Normal farming

procedures were carried out in the plots including intercrop tillage and application

of a leaf fungicide, Alto Super 400, at 0.5 kg/ha.

Treatment and planting of sugar beets: Sugar beet seeds (Ukrainian World

Cup 70) were soaked and carefully shaken for 10 min. in water solution of PGRs,

Radostim, Rasostim-super, or Biogen, at the rate of 250 ml/liter, and Biolan - at the

rate of 25 ml/liter. After this procedure, the seeds were dried for 2 days at room

temperature. Untreated seeds were placed in water and handled the same way as

the treated seeds. The treated and untreated sugar beet seeds were sown at the rate

of 130,000 seeds/ha (planting date: 24 April 2010, date of occurrence of sprouts:

02 May 2010, meteorological data at planting: air temperature: 10-14ºC, air

moisture: 80-85 %, wind speed: 5-7 m/sec, extreme weather condition: dry and hot

weather on July and August). For the PGRs foliar treatment, when the sugar beet

plants were at the 6-8 leaves stage (on 25 June, 2010), Biolan-extra was applied at

the rate of 50 ml/250 liters of water per 1 ha. Each experiment was repeated 3

times.

Sampling for nematodes: Soil samples were taken to assess the number of

sugar beet nematodes per cm3 of soil. Soil samples were taken (1) before sowing

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the sugar beets seeds on 22 April, (2) after the development of the first generation

of nematodes on 1 July, and (3) after harvesting of sugar beet crop on 20

September following the procedure ISO 6057:2008 outlined in the State Standard

of Ukraine 6057:2008 “Sugar beets. Test methods of beet nematode harmfulness”.

Studies carried out on an isolated plot with total area of 2 ha. Discount land - 13,5

m2, placing at randomise areas. Reversibility of the experiment - three times.

Sowing was conducted 22/04/2010 with a hybrid seed Ukrainian World Cup 70.

Care of crops - generally for this forest-steppe zone of Ukraine. Soil samples were

taken using shovel from a depth of 10 – 20 cm along two diagonals or in a zigzag

fashion. Ten subsamples were taken combined into one sample per plot. Each

subsample contained 50 cm3 of soil and therefore each plot had a total of 500 cm3.

Soil samples were placed in polyethylene bags, labelled to indicate the

sampling date and plot number, and transferred to the Department of

Phytopathology and Entomology, which conducted the analysis. In the laboratory

soil samples were thoroughly stirred, sieved through a sieve with a diameter of 2

mm and air-dried.

Beet nematode population density in the soil was determined by the number

of cyst, larvae and eggs in 100 cm3 soil by the Flotation-Funnel method. A 100 cm3

sample was placed into 1-liter cup and 750 ml of water was added. Soil was mixed

with a glass rod for 2-3 min and left to settle for 5 min. The supernatant containing

the nematode cysts that had floated to the surface and organic particles were

processed through a sieve with pore size diameter of 0.1-0.2 mm. This procedure

was repeated three times. The cysts and particles on the sieve were washed through

a funnel containing a piece of filter paper. The filter paper was examined for the

nematode cysts with a dissecting microscope MBS-9 (at magnification 8 X). Cysts

found on the filter were transferred into water and then counted to get the total

number per sub sample. The eggs and juveniles of sugar beet nematode in the soil

were determined by counting the number of larvae and eggs per 100 cm3 soil. The

effectiveness of PGRs against the sugar beet nematode in soil was determined by

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the percentage difference between the number of sugar beet nematodes in soil

before sowing and population density after development of the first generation on

July (01/07/2010).

To further assess the effectiveness of the PGRs on increasing of sugar been

yield and sugar content, the sugar beet was harvested and weighed from each plot

to calculate the yield per hectare. In addition, the root crop sugar content was

determined on the processing line "Venema" by cold digestion.

The objects and methods used in the laboratory experiments: Beet and rape

seeds were sprouted in Petri dishes (9.5 cm in diameter) in nematode-free aqueous

medium (control) or with a suspension of nematode eggs (at the correlation of 20-50

nematode eggs/ 20 beet and rape seeds). The seeds were incubated at 23 0C and the

nematode larvae hatched in 5-7 days later in average. Each experiment performed

in three replicates and consisted of 4 variants: 1) seeds incubated on aqueous

medium (control), 2) seeds incubated on aqueous medium with Radostim-super, 3)

seeds incubated on aqueous medium with a suspension of nematode eggs, 4) seeds

incubated on aqueous medium with Radostim-super and a suspension of nematode

eggs. In variant 3,4 dose of nematodes for treatment was 20-50 eggs/20 beet and rape

seeds. In all experiments Radostim-super was applied at the rate of 1 mg of acting

substance/1ml of water (in 10-9 dilution).

In the molecular-genetic experiments we determined percent of homology

between cytoplasmic mRNA population and small regulatory si/miRNA of control

and experimental plants using DOT-blot hybridization method [11]. Isolation of

total RNA from plant cells and separation of high-purity si/miRNA preparations

using our modified methods we published early [17-20]. For research on plant

si/miRNA hybridization with own plant mRNA and pathogen mRNA, before

receiving si/miRNA, it was intensely marked in vivo with 33P using Na2HP33O4 [17,

18]. Testing of functional (silence) activity si/miRNA, isolated from treated by

regulator Radostim-super plants, inhibiting translation of own plant mRNA and

nematode mRNA conducted in wheat germ cell-free protein synthesis system [12].

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Determination of inhibition of protein synthesis in cell-free system was carried out

according to index of decreasing level of incorporation [35S] methionine into

proteins and was accounted on (count per min/1mg of protein). For testing its

inhibitory activity in cell-free protein synthesis systems, we used unmarked

si/miRNA. The dispersion statistical analysis of the data was carried out according

to Student.

Results and Discussion

The results of soil samples revealed that the number of sugar beet nematodes

before sowing averaged 4,647 eggs and larvae/100 cm3 of soil which is 23 times

higher than the economic nematode threshold of 200 eggs + larvae/ 100 cm3 of

soil) (Table 1).

Table 1

Effect of plant growth regulators’ (PGRs) influence on sugar beet nematode number in soil

TreatmentsApplication

rate (ml/tons)

Sugar beet nematode number, eggs + larvae/ 100 cm3 of soil ± SE

Changingnematode population

density (times) **

before sowingseeds

(April)

after the 1st generation

(July)

Seeds incubated on water medium

- 4420±125 5384±176 (+) 1.2

Seeds treated by PGRs:Radostim 250 3671±112 2487±96* (-)1.5Radostim-super 250 4375±134 1131±34* (-) 3.9Biogen 250 4625±142 2074±63* (-) 2.2Biolan 25 4336±116 3367±107 (-)1.3

* - availability of differences between groups, p<0.05, n=3 **- (-) decreasing; (+) - increasing Results showed the effectiveness of PGRs applied for treatment of sugar beet

seeds and spraying in vegetation period against beet nematode varied and ranged

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from 22.4 to 74.2% (Table 1). High effectiveness in reducing the number of beet

nematode was shown with the application of Radostim-super. The number of

nematode in the soil showed a decrease of 74.2%. Seed treatment by Biogen

provided somewhat lower anti-nematode action with a reduction of the pest

population density by 55.2%. Application of Biolan and Radostim provided

decline of beet nematode numbers in soil by 22.4% and 32.2%. On the contrary, in

control experiments (seeds treated by water) the beet nematode numbers in soil

increased by 22 %.

In addition to the reduction in nematode numbers, the application of PGRs

increased the sugar beet yield and sugar content which were significantly higher

than in the control by 1.7-6.5 and 0.6-1.4 tons/ha, respectively (Table 2). The

highest indexes of sugar beet yield and sugar content were obtained after treatment

of sugar beet seeds by Biogen and Radostim-super, 40.3 and 40.0 t/ha and 6.1 and

6.2 t/ha respectively.

Treatment of sugar beet seeds by Radostim and Biolan provided increase of

root crop yield by 2.7 and 1.7 t/ha, respectively compared to control sugar beet

seeds treated with water.

Table 2Plant growth regulators’ influence on sugar been yield and sugar

content at the end of September

Experiment variantsNorm, ml/ton

Yield data*

sugarbeet

yield, tons/ha ± SE

Sugar content,%± SE

sugar yield, tons/ha± SE

Control (seed treated with water) - 33.6±1.2 14.2±0.02 4.8±0.01

Seeds treated by PGRs:Radostim

250 36.3±1.6 15.2±0.03* 5.5±0.02*

Radostim-super 250 40.0±1.8* 15.6±0.04* 6.2±0.03*

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Biogen 250 40.1±1.7* 15.3±0.02* 6.1±0.03*

Biolan 25 35.3±1.1 15.2±0.01* 5.4±0.02*

* - availability of differences from control, p<0.05, n=3

Our molecular-genetic experiments were based on the assumption that in

organisms infected with different types of pathogens or parasites is induced

synthesis of si/miRNA pools specific to own plant cell mRNA (which exhibited

increased expression level at specialized feeding cells in infected roots and

involves plant developmental processes) [7, 16] or to pathogen mRNA [8, 9, 14] or

parasite mRNA [1, 23] with high degree of homology. We further assumed that the

PGRs stimulated synthesis of si/miRNA which improved plant immunity through

the specified mechanism of si/miRNA action. We need to be sure that our

assumptions are correct so that a new generation of PGRs with the properties of

selective activation of synthesis of si/miRNA specific to own plant cell mRNA or

to mRNA of pathogen or parasite (with highly degree of homology) can be created.

Table 3 provides the data on the level of si/miRNA synthesis in control

plants, plants treated with Radostim-super, plants incubated with nematodes or

plants infected with nematodes treated with Radostim-super. Our results show that

Radostim-super dramatically increased cell si/miRNA synthesis, and on the

contrary plant infection with nematodes reduced sharply synthesis of this class of

RNA. Radostim-super compensated somewhat si/miRNA synthesis but this level,

though higher than in the control, did not reach the level of synthesis induced by

the plant growth regulator without nematodes.

Table 3

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Impact of Radostim-super plant growth regulator on incorporation of Na2HP33O4 in si/miRNA in cells of 5-day beet sprouts incubated and non-

incubated with nematode

No. Experiment variants Count per min/mg of si/miRNA± SE

1. Control (sprouts of plants incubated on aqueous medium) 2680 ± 98.02. Sprouts got on aqueous medium with Radostim-super 4309 ± 121*3. Sprouts got on aqueous medium with nematode larvae 1970 ± 83*4. Sprouts got on aqueous medium with Radostim-super and

nematode larvae3760 ± 112*

Note: five-day sprouts of the plants were incubated with Na2HP33O4 during one hour in Petri dishes. Regulatory si/miRNA being anti-sense, complementary to mRNA were separated by our procedure of obtaining highly purified native preparations of si/miRNA. Aliquots of radioactive si/miRNA were placed on nitrocellulose substrate with subsequent counting of radioactivity.* - availability of differences from control, p<0.05, n=3

Table 4 shows the data revealing the essence of the results listed in the Table

3. As it can be seen, this is connected to the fact that nematode infection reduces

synthesis of plant si/miRNA, which increases significantly at plants non-infected

by nematodes and treated with Radostim-super, but this increased level is

suppressed by larvae. At the same time, synthesis of protective anti-nematode

si/miRNA (targeted to own plant mRNA, synthesized in specialized feeding cells

in infected roots and involves plant developmental processes, as well as nematode

mRNA with high degree of homology) is increased in plants, infected by

nematodes and treated with Radostim-super.

Table 4

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Increase of anti-nematode properties of si/miRNA of 5-day sugar beet sprouts under impact of Radostim-super and sugar beet nematode juveniles

№

Experiment variants

Level of homology of hybridization mRNAwith Р33

si/miRNA (count per min/20µg ± SE of mRNA)

Indicator of inhibition of protein synthesis in cell-free system(count per min/1mg of protein)*mRNA from plants + Р33 si/miRNA from plants

mRNA from larvae+ Р33 si/miRNA from plants

1

Hybrids of mRNA with Р33 si/miRNA of control plants

8724 ± 146 (100%)

100 % 10 %

2

Hybrids of mRNA from control plants with Р33

si/miRNAfrom plants treated with Radostim-super

6850 ± 224 (83%)** 82 % 15 %

3Hybrids of mRNA from control plants with Р33

si/miRNAfrom plants incubated withnematode larvae

5583 ± 164 (64%)** 46 % 36 %

4Hybrids of mRNA from control plants with Р33 si/miRNA from plants incubated with Radostim-super and nematode larvae

6358 ± 182 (73%)**

65 % 58 %

Note: free-cell system of protein synthesis from wheat sprouts was used; S35

methionine amino acid was taken as a marked predecessor. * the same experiment variants as in the investigations on hybridization were used in the cell-free protein synthesis system.** - availability of differences from control, p<0.05, n=3

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Similar results were obtained in our experiments and with the rape plants

infecting by nematode larvae. Data presented at Table 5 allows to see that

conformity with a law in the changes of si/miRNA synthesis at rape as it is

observed at beet plants: the considerable increasing level of si/miRNA synthesis

under action of growth regulator Radostim-super; decreasing level of own cellular

si/miRNA synthesis because plants are infected by nematode larvae; decreasing of

nematode infection under growth regulator action due to increasing level of

synthesis si/miRNA with antinematode activity.

Table 5

Impact of Radostim-super plant growth regulator on incorporation of Na2HP33O4 in si/miRNA in cells of 5-day rape sprouts incubated and non-

incubated with nematode

No. Experiment variants Count per min/mg of si/miRNA± SE

1 Control (sprouts of plants incubated on aqueous medium) 4760 ± 1462 Sprouts got on aqueous medium with Radostim-super 6420 ± 208*3 Sprouts got on aqueous medium with nematode larvae 2910 ± 117*4 Sprouts got on aqueous medium with Radostim-super and

nematode larvae5380 ± 185*

Note: five-day sprouts of the plants were incubated with Na2HP33O4 during one hour in Petri dishes. Regulatory si/miRNA being anti-sense, complementary to mRNA were separated by our procedure of obtaining highly purified native preparations of si/miRNA. Aliquots of radioactive si/miRNA were placed on nitrocellulose substrate with subsequent counting of radioactivity.* - availability of differences from control, p<0.05, n=3

Conclusions

Thus, the results of molecular-genetic experiments indicate considerable

increase in plant cells of si/miRNA synthesis (i. e. plant immunity), that leads to

inhibition of reproduction nematode larvae, formation of cyst in sugar beet cells

and according to decreasing accumulation of nematode in soil (the cycle or

exclusive circle of nematode reproduction are destroyed), therefore plant

productivity - crop capacity per unit of sown area increases.

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