The article published in “Communications in agricultural applied biological sciences”, Belgium, Ghent University, 2012. Vol. 77/4. P. 779 – 788.

INDUCING EFFECT OF PGRs ON SMALL REGULATORY si/miRNA IN RESISTANCE TO SUGAR BEET CYST NEMATODE

V. А . TSYGANKOVA 1 , T. R. STEFANOVSKA2. A.P. GALKIN 3,S. P. PONOMARENKO4, Ya. B. BLUME3

1 Institute of Bioorganic Chemistry and Petrochemistry, Natl Acad. Of Sci. of Ukraine, Kiev 2National University of Life and Environmental Science of Ukraine, Kiev

3 Institute of Food Biotechnology and Genomics, Natl Acad. of Scie. of Ukraine, Kiev4National Enterprise Interdepartmental Science & Technology Center "Agrobiotech", Natl

Acad. of Sci. of Ukraine and MES of Ukraine, Kiev

SUMMARY

Sugar beet cyst nematode Heterodera schachtii Schmidt is an economically important plant parasite of sugar beet in Ukraine. The pest control options are limited. Sugar beet cyst nematode resistant varieties are not available on the market. Carbamate and organophosphate pesticides have been banned due to the high toxicity. The problem is aggravated by continuously increasing of oilseed rape (which is suitable host for H. schachtii ) growing area due to biofuel demands. Several studies’ results indicate that PGRs have role in management of plant parasitic nematodes but for sugar beet it is not studied well. We had an objective- studying of the role of four compositional PGRs created based of avermectin in suppression of sugar beet cyst nematode population on sugar beet and oilseed rape caused by enhancing of endogeneous si/miRNA complementary to H. schachtii mRNA. Laboratory study was conducted in 2011 with using method DOT-blot hybridization si/miRNA with mRNA and by testing inhibitory activity in cell free system protein biosynthesis. That was shown that application of the PGRs enhances sugar beet and oilseeds rape plant immune-protective properties and resistance against plant-parasitic nematode Heterodera schachtii through enhancement of synthesis of small regulatory si/miRNA related (complementary) to an mRNA structure of the parasitic organisms. As a result, translation of mRNA of the nematode is blocked and causes the mortality of plant parasite juveniles.

Key words: sugar beet cyst nematode, oilseed rape, PGRs, resistance, si/mi RNA, mRNA synthesis, enhancement.

INTRODUCTION

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 plants, animals and fungi (Filipowicz et al., 2005; Vaucheret, 2006). 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 (Fire et al.,1998) 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 ( Fuller et al.;2008). 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) (Hamilton et al.,2002; Zhang et al., 2007). 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. 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) (Lee et al., 2003; Mourelatos et al., 2002). 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 (Hamilton et al.,2002). 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 (Bakhetia et al., 2005, Hamilton et al.,2002; Zhang et al., 2007). 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) (Hewezi et al., 2008; Pate

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et al., 2010) or to pathogen mRNA (Jin, 2008; Katiyar-Agarwal, 2006; Padmanabhan et al., 2009) or

parasite mRNA (Bakhetia et al., 2005; Zhang et al., 2007) with highly degree of homology. In animal and plant cells, si/miRNA act in different ways: si/miRNA of animals binds to the 3'-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 (Park et al.,2002; Yang et al., 2007).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 (Bakhetia et al., 2005; Gheysen et al., 2006; Padmanabhan et al., 2009). 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.Plant grow regulators (PGRs) has an important role in pesticide use reduction to prevent human health hazards (Pidlisnyuk, 2009). 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 (Tsygankova et al., 2011) 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. Shmidt.

MATERIALS AND METHODS

Field experiments

Field experiments were conducted in 2010 at Uladovo-Lyulynetska experimental breeding station (Vinnitsa region) on natural background by invasive favorite small plots research, at the Department of Entomology and Phytopathology of the Institute of Bioenergy Crops and Sugar Beet NAS Ukraine, Department of Entomology, National University of Life and Environmental science. The plant growth regulators -Radostim, Radostim-super, Biogen and Biolan) were used as emulsion concentrate on target objects: sugar beets hybrid “Ukrainian World Cup 70”. Seeding rate: 130 000 seeds per hectare.The PGRs: Radostim, Rasostin-super, and Biogen were applied to sugar beet seeds (Ukrainian World Cup 70) at the rate of 250 ml/liter of water for April. The PGR, Biolan, was applied to sugar beets at the rate of 25 ml/liter of water for June. The treated sugar beets were sown on 24 April 2010 at the rate of 130,000 seeds/ha. Untreated seeds were placed at the same time and included a control and a PGR foliar treatment. For the PGR foliar treatment, when the sugar beet plants were at the 6-8 leaves stage (on 25 June), Biolan-extra was applied at the rate of 50 ml/250 liters of water per 1 ha. Date of occurrence of stairs: 25. 06. 2010. Each experiment consisted from 5 variants: the 1st – control sugar beets and 4 others – sugar beets treated by PGRs (3 replicates per treatment/Sugar beet cyst nematode population density in the soil was determined by the number of cyst per 100 cm3, larvae and eggs selected from 100 cm3 soil by Flotation-Funnel method.

Laboratory experiments

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, Natl Acad. of Sci. of Ukraine, jointly with National Enterprise Interdepartmental Science & Technology Center "Agrobiotech" of Natl Acad. of Sci. of Ukraine 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 (Tsygankova et al., 2011).

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 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 Maniatis et al.; 1982). Isolation of total RNA from plant cells and separation of high-purity si/miRNA preparations using our modified methods we published early (Tsygankova et al.,

2010; Tsygankova& Blume, 2011). For research on plant si/miRNA hybridization with own plant mRNA

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and pathogen mRNA, before receiving si/miRNA, it was intensely marked in vivo with 33P using Na2HP33O4 (Tsygankova et al., 2010) (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 ( Marcus et al.,1974). 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 DISCUSSIONS

Results showed the effectiveness of PGRs applied for treatment of sugar beet seeds and spraying in vegetation period against beet nematode varied and ranged 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.

Table1. Effect of PGRs on sugar beet nematode in soil, Uladovo- Lulynetska research station, Vinnitza region, 2010

Experiment variants Applicationrate

(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

Radostim 250 3671±112 2487±96* (-)1.5

Radostim-super 250 4375±134 1131±34* (-) 3.9Biogen 250 4625±142 2074±63* (-) 2.2

Biolan 25 4336±116 3367±107 (-)1.3

* - availability of differences between groups, p<0.05, n=3 **- (-) decreasing; (+) - increasing

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 2. Plant growth regulators’ influence on sugar been yield and sugar content, Uladovo- Lulynetska research station,Vinnitza region, 2010

Experiment variants Application rate ml/ton

Yield and sugar content

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*

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

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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) ( Hewezi et al., 2008 ; Patel et al.,2010 ) well as to

pathogen mRNA ( Jin, 2008; Katiyar-Agarwal, 2006; Padmanabhan et al., 2009) or parasite mRNA (with highly degree of homology) (Bakhetia et al., 2005; Zhang et al., 2007)

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.

The results of polyacrylamide-gel (PAGE) electrophoresis are presented in Fig. 1 demonstrating that we obtained si/miRNA preparations of high purity; their size is 20 – 25 nucleotides that are in

accordance to classic parameters of these RNA types.

Figure 1. 1. PAGE-electrophoresis of si/miRNA isolated from rape germs: the marker polynucleotides (the 1st gel path) and si/miRNA preparations (the 2nd gel path) were saturated by ethidium bromide; radioautograph of 33P-labelled si/miRNA out of gel (the 3rd gel path)

In vivo experiments (Fig.2) showed that 5-days sugar beet sprouts that were obtained from seeds treated by PGR Radostim-super and grown on infectious background were more viable and resistant to nematode H. schachtii as compared to control sprouts.

Figure. 2. 5-days sprouts of sugar beet infested with H. schachtii Left: treated by PGR Radostim-superRight: without PGR (control)

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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 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 highly degree of homology) is increased in plants, infected by nematodes and treated with Radostim-super.

Table 3 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 ± 98.0

2. 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 larvae 3760 ± 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 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 on hybridization of 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 %

3. Hybrids of mRNA from control plants with Р33 si/miRNAfrom plants incubated withnematode larvae

5583 ± 164 (64%)** 46 % 36 %

4. Hybrids 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

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

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1. Control (sprouts of plants incubated on aqueous medium) 4760 ± 146

2. 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 larvae 5380 ± 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 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.

CONCLUSIONS

Field studies showed that seed processing of sugar beets by the plant growth regulators (PGRs), Radostim, Radostim-super, Biogen, and Biolan, and spraying by Biolan-extra on sugar beet plants at the 6-8 leaf stage reduced by 22.4-74.2% the number of sugar beet nematodes in the soil.Laboratory experiments demonstrated that application of the PGR enhances sugar beet and rape plant resistance against nematode infection.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.

AKNOWLEDGMENT

This research have been carried out within the STCU project P490

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