IDENTIFICATION OF POTATO SPINDLE TUBER VIROID SMALL RNA IN OROBANCHE RAMOSA BY MICROARRAY D. Ivanova, T. Vachev, V. Baev, I. Minkov and M. Gozmanova University of Plovdiv, Department of Plant Physiology and Molecular biology, Plovdiv, Bulgaria Correspondence to: Mariyana Gozmanova E-mail:
[email protected]
ABSTRACT Post transcriptional gene silencing (PTGS) in plants is reported as a defence mechanism against pathogens, like invading viruses and viroids, transposons and transgenes. The processing of double stranded RNAs to 21-24 nt duplex RNAs (small interfering RNAs) by RNAse III–type nuclease homologs- Dicer-like (DCL) enzymes is a key step of this process. We were interested whether the viroid RNA can trigger silencing in parasitic plant O.ramosa. Therefore we infected O.ramosa attached to tomato with Potato Spindle Tuber Viroid (PSTVd). The presence of small interfering RNAs derived from PSTVd has been detected by miRNA microarray. Keywords: PSTVd, RNA silencing, small interfering RNAs, microarray, Orobanche ramosa
properties of small interfering RNAs (3, 5, 7). VsRNAs spread systemically following the phloem solutes flow in plants (10).
Introduction
We were interested to test whether a non photosynthetic host O.ramosa can support silencing mechanism and can process the viroid sequence to vsRNAs. We infected O.ramosa attached to tomato with PSTVd. Accumulation of PSTVd derived vsRNAs have been observed in viroid infected O.ramosa by microRNA Microarray.
Viroids are the smallest pathogenic RNA molecules that infect higher plants (1, 2, 9). Their host range includes crops, ornamental plants, weeds and parasitic plants (6). Viroid RNA consists of 246-401nt that do not encode any proteins and is not encapsidated. It replicates autonomously and spread systemically in infected plants. The pathogenic nature of the viroids is encoded in their specific sequence and secondary structure. PSTVd is the type member of Pospiviroidae family. PSTVd molecule is a single stranded RNA of 341-364 nt (depending on the isolate) that folds in a rod-like secondary structure with well defined structural domains: left and right terminal domains, pathogenic, central conservative and variable domain. PSTVd replicate by cellular DNA dependant RNA polymerase II in the nucleus following the asymmetric mode of rolling circle mechanism. PSTVd is a target and inducer of RNA silencing mechanism, operating as a defense against pathogens in plants. The viroid sequence can be processed into short (2224nt) viroid small RNAs (vsRNAs) with characteristic
Materials and Methods Bioassay methodology A viroid infection of O. ramosa was used to artificially provoke the siRNA generation. The viroid infection was done with longer-than-unit-length PSTVd (+) RNA of isolate KF440-2, obtained by in vitro transcription (8). The infectivity assay was done by mechanical inoculation of 600ng viroid RNA with carborundum onto the stem of O. ramosa plants. Molecular biology methods Total RNA probes were collected from stems of healthy and PSTVd infected O.ramosa plants by using mirVana miRNA isolation kit (Ambion). It was added 10 volumes of Lysis/Binding Buffer to 0.1 g frozen plant material. Then
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1/10 volume of miRNA Homogenate Additive was mixed well with the lysate and the mixture was store on ice. After that we added Acid Phenol: Chloroform in equal volume to the amount of lysate and centrifuged for 5 min at 10 000 x g, at room temperature. The upper phase was taken and mixed with 1.25 volumes of 100% ethanol. The lysate/ethanol mixture was applied on the Filter Cartridge and centrifuged for 15 sec at 10 000 x g. Afterwards the Filter Cartridge was washed with Washing Solution 1 and 2. Finally 100µl of nuclease-free water was added to the Filter Cartridge and the eluate was collected.
antigenomic strand of PSTVd RNA, the length of which was initially extended with 30nt derived from the left terminal domain of PSTVd. By using a PERL script, PSTVd sequence has been cut in 22nt. The fragments from antigenomic PSTVd strand are depicted as KF440r. A local database (http://bioinfo.uni-plovdiv.bg/~vebaev/ kf440/ si22KF440.fa) has been generated to store all PSTVd sequences with 22nt length (Fig. 2).
>KF440-0 CGGAACTAAACTCGTGGTTCCT >KF440-2 GAACTAAACTCGTGGTTCCTGT >KF440-4 ACTAAACTCGTGGTTCCTGTGG >KF440-6 TAAACTCGTGGTTCCTGTGGTT >KF440-8 AACTCGTGGTTCCTGTGGTTCA >KF440-10 CTCGTGGTTCCTGTGGTTCACA >KF440-12 CGTGGTTCCTGTGGTTCACACC >KF440-14 TGGTTCCTGTGGTTCACACCTG >KF440-16 GTTCCTGTGGTTCACACCTGAC >KF440-18 TCCTGTGGTTCACACCTGACCT >KF440-20 CTGTGGTTCACACCTGACCTCC
Bioinformatics studies The genome of PSTVd isolate (KF440-2) was truncated in 22nt sequences (the defined size of siRNAs) and saved in a local database. The unique PSTVd siRNAs were filtered and used to design a microarray chip.
Results and Discussion Two independent RNA probes were collected from O.ramosa plants infected with PSTVd isolate KF-440 2 at 14 days post inoculation (dpi) and healthy plants by using mirVana miRNA isolation kit (Ambion) (see materials and methods). The quality of RNA samples was checked on 1.5% agarose gel (Fig. 1). ORPS1 ORPS2
ORH1
ORH2
>KF440r-0 TGAACCACAGGAACCACGAGTT >KF440r-2 AACCACAGGAACCACGAGTTTA >KF440r-4 CCACAGGAACCACGAGTTTAGT >KF440r-6 ACAGGAACCACGAGTTTAGTTC >KF440r-8 AGGAACCACGAGTTTAGTTCCG >KF440r-10 GAACCACGAGTTTAGTTCCGAG >KF440r-12 ACCACGAGTTTAGTTCCGAGGA >KF440r-14 CACGAGTTTAGTTCCGAGGAAC >KF440r-16 CGAGTTTAGTTCCGAGGAACCA >KF440r-18 AGTTTAGTTCCGAGGAACCAAC >KF440r-20 TTTAGTTCCGAGGAACCAACTG
Fig 2. In silico generated small PSTVd RNAs corresponding to the part of array data stored in the local database
The results of miRNA microarray showed a presence of few viroid small RNAs in infected with PSTVd O.ramosa plants compared to the healthy plants. The results are summarized below (Fig. 3). All data were statistically validated.
Fig. 1 Total RNA isolated from PSTVd infected (ORPS1, ORPS2) and healthy O. ramosa (ORH3, ORH4) was separated on 1.5% agarose gel and visualized with EtBr staining
The quantity of probes was determined spectrophotomertrically. 5µg of total RNAs was analyzed using a custom made microRNA microarray done by LC Science, Huston USA. Microarray chip has been covered by in silico prepared 22nt long fragments derived from genomic and
Small RNAs that were detected with p-value below 0.05 originated from genomic PSTVd strand. They derive from pathogenicity domain, central conservative domain, variable and terminal left domains. A schematic view of these results is present on Fig. 4. It is poorly understood how the viroid small RNAs are produced in plants. It is assumed that small interfering RNAs of viruses derived from dsRNAs produced during replication, while the experimental evidence about biogenesis of viroid small RNAs remains to be provided. So far it is known that viroid RNA could be a substrate for DCL activities.
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Conclusions Our data are in consistence with this hypothesis additional evidence about the existence of phenomenon in parasitic plants. Identification vsRNAs with other molecular biology methods sequencing is foreseen.
providing silencing of these and their
Acknowledgment This work is supported by NSF, grant DOO2-235.
p-value < 0.05
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p-value < 0.1
Fig 3. Clustering of PSTVd small RNAs based on their expression profile. ORH1 and ORH2 correspond to healthy controls, while ORPS1 and ORPS2 to PSTVd infected O. ramosa
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Fig.4 Mapping of statistically validated PSTVd small interfering RNAs derived from infected O.ramosa plants on genomic PSTVd sequence
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