ISSN 20790597, Russian Journal of Genetics: Applied Research, 2014, Vol. 4, No. 1, pp. 74–81. © Pleiades Publishing, Ltd., 2014. Original Russian Text © I.V. Zhirnov, E.A. Trifonova, A.V. Kochetov, 2013, published in Vavilovskii Zhurnal Genetiki i Selektsii, 2013, Vol. 17, No. 3, pp. 558–567.
Role of Auto and Heterologous Ribonuclease III Family Enzymes in the Resistance to Pathogensa Regulation of Gene Expression in Higher Plants I. V. Zhirnova, b, E. A. Trifonovaa, and A. V. Kochetova, b a
Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia bNovosibirsk National Research State University, Novosibirsk, Russia email:
[email protected] Received August 27, 2013; in final form, September 2, 2013
Abstract—The current view of the biological role of ribonuclease III family plant enzymes (Dicerlike dsRNAses) is considered. Emphasis is placed on their role in molecular mechanisms conferring resistance to pathogens. Keywords: genetic engineering, ribonuclease III family enzymes, plants, pathogens, gene silencing DOI: 10.1134/S2079059714010122
INTRODUCTION
siRNAs (tasiRNAs), and repeatassociated siRNAs (rasiRNAs). The enzymes associated with biogenesis of these small RNAs in TGS/PTGS processes are Dicerlike dsRNAses (DCLs), typical of higher plants, homologs of mammalian protein Dicer (Dunoyer et al., 2005; Mlotshwa et al., 2008; Bozorov et al., 2012; Udriste et al., 2012). The participation of different DCLs ribonucleases in processes are related to the protection of cell genomes from transposons, suppression of amplifica tion of viruses/viroids, regulation of the expression of genes, and repression of transgenes (Bozorov et al., 2012; Mukherjee et al., 2012). Some experimental data allow suggesting the possibility of using trans genic plants for the production of heterologous ribo nuclease III family for increasing resistance to phyto pathogenic viruses/viroids. Thus, the expression of genes of heterologous pro or eukaryotic RNases that are involved in the degradation of doublestranded RNA forms in transgenic plants enhanced the stability of the obtained transformants to a number of these pathogens (Watanabe et al., 1995; Zhang et al., 2001). At the same time, it was shown (Kreuze et al., 2005), that some viral ribonucleases that belong to the RNase III family can act as suppressors of RNA interference (RNAi). In general, with regard to the role of the anti viral immunity of plants, the role of ribonucleases depolymerizing dsRNA is understudied, indicating the need for a more detailed study of their functions in the molecular mechanisms of resistance to phyto pathogenic viruses. The goal of this study is to summarize the currently available information regarding the role of autologous and heterologous ribonuclease III family in protective systems of higher plants.
The RNase III family includes the endoribonu clease group, which catalyzes the hydrolysis of 3',5'phosphodiester bonds of doublestranded RNA (dsRNA). Ribonucleases of this family are present in a wide spectrum of organisms, including viruses, bacte ria, fungi, plants, and animals (MacRae and Doudna, 2007; Mukherjee et al., 2012). According to Xray analysis, protein molecules of the RNase III family are formed by a single polypep tide chain which has from ~200 to ~2000 amino acid residues and is characterized by the presence of two globular binding domains: dsRNA binding motifs (dsRNAbinding domain, dsRBD) and RNase III catalytic domain (RNase III). In accordance with the presence of catalytic activity, different members of the RNase III family are involved in the processing of pre cursors of ribosomal RNA and small nuclear and nucleolar RNA and also play a key role in genetic silencing, which is a process of inactivation (repres sion, silencing) of gene expression and/or of trans genes, typical of many eukaryotes (MacRae and Doudna, 2007; Bozorov et al., 2012; Mukherjee et al., 2012). The inactivation of expression of transgenes/genes can be performed both at the transcriptional level (transcriptional gene silencing, TGS) and at the post transcriptional level (posttranscriptional gene silenc ing, PTGS). In all cases, except for some variants of TGS, the process of genetic silencing is mediated by the formation of various types of small RNAs that differ in biogenesis. In higher plants, there are the following main classes of small RNAs: microRNAs (miRNAs), small interfering RNAs (siRNAs), transacting 74
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500 DExD/H
DUF283
1000
1500 RNase IIIa
PAZ
75
2000 RNase IIIb dsRBD
DCL domain structure. The plotting scale is calibrated in units of amino acid residues.
DCL RIBONUCLEASES In plants, genes encoding DCLs are represented by families with a different number of genes in different species. For example, the Arabidopsis thaliana genome contains four different DCL genes; Populus tri chocarpa, five; Oryza sativa, six; and Solanum lycoper sicum, seven genes (Margis et al., 2006). The functions of all four DCLs in A. thaliana were characterized. DCL1 participates in the biogenesis of various miRNAs, which play a key role in the suppression of gene expression (via the cleavage of transcripts of these genes or by blocking the translation of mRNA), and participates in controlling the level of accumulation of cytoplasmic mRNA of both cellular and viral origin. DCL2 is involved in virusinduced RNAsilencing (homolog of DCL2 protein of A. thaliana that was identified in Nicotiana tabacum). DCL3 is involved in RNAdirected methylation of DNA and histones. The function of DCL4 is associated with the formation of transacting siRNAs (tasiRNAs), which have func tional similarities with miRNAs and direct degrada tion in RISC (RNAinduced silencing complex) of mRNA target sequences, derived from a locus differ ent than that of tasiRNAs. Genes DCL1, DCL2, DCL3, and DCL4 are represented in the genome by a single copy. Mutations in the DCL1 gene were lethal to the plant—the death of embryos already appeared at the stage of the globule, while the inactivation of the DCL2–DCL4 genes did not have a significant impact on the viability of A. thaliana (Henderson et al., 2006; Margis et al., 2006; Liu et al., 2009; Bozorov et al., 2012). Typically, the DCL ribonuclease includes multiple functional domains (figure): Nterminal of the DE × D/H domain (presumably has an inhibitory function, reducing the rate of hydrolysis of 3',5'phos phodiester bonds of dsRNA), the DUF283 domain with an unknown function, the PAZ domain (func tions described below), two copies of the RNase III domain, and a Cterminal domain containing a dsRBD motif of dsRNA binding (Schwarz et al., 2003; Mlotshwa et al., 2008). The degradation of the doublestranded RNA forms has several stages. The initial binding of the enzyme with dsRNA is performed via the dsRBD motif of DCL. The PAZ domain provides specific contact with two unpaired 3'terminal nucleotides of dsRNA, formed as a result of the first act of DCL interaction with the target (it was shown that the pres ence of the 3'terminal singlestranded knob, which
contains 2–3 nucleotides, increases enzyme activity toward the substrate). During the final stage, two RNase III domains interact with each other, form the “internal dimer,” and catalyze the hydrolysis of 3',5'phosphodiester bonds in each strand of dsRNA at two sites at a distance of 2 nucleotides. As a result of the described targeted degradation process, there occurs the formation of short doublestranded frag ments, called small RNAs (miRNAs/siRNAs), with a length of 21–25 nucleotides, with 5'terminal phos phate group and a singlestranded overhang with the length of 2–3 nucleotides on the 3'end. The length of the nonconservative region between the PAZ and RNase III domains, which form a spiral structure, is the main factor which determines the size of the resulting small dsRNAs (Goldbach et al., 2003; Schwarz et al., 2003; Voinnet, 2005; Mlotshwa et al., 2008). DCL1 RNA silencing, also known as posttranscriptional gene silencing, is a process that plays a key role in the regulation of gene expression of many eukaryotes. In higher plants, PTGS is an important component of the miRNAmediated regulation of gene activity, per formed with the obligatory participation of DCL1 ribonuclease (Voinnet, 2009). The miRNA class includes endogenous noncoding RNAs that play a fundamental role in the inactivation of gene expression via either degradation of transcripts of these genes or via a direct block of mRNA transla tion (Llave et al., 2002; Palatnik et al., 2003). Genes that control metabolism, ion transport, signal trans duction processes, and stress response are targets for miRNA (Dugas and Bartel, 2004; Sunkar and Zhu, 2004). A significant part of the target is mRNA encod ing transcription factors, and, as usual, one miRNA regulates the expression of several transcription factor of the same family (Kidner and Martienssen, 2005). It should be noted that the mRNAs of DCL1 and protein AGO1 were also targets of miRNAs (see below). This in turn indicates the existence of a feedback mecha nism by which miRNA determines the level of its own production (Du and Zamore, 2005). Genes encoding plant miRNAs are located in the intergenic spacing and are sufficiently removed from proteinencoding genes, which indicates their inde pendent transcription (Bartel and Bartel, 2003; Pari zotto et al., 2004). miRNA biogenesis is carried out in
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the cell nucleus in several stages. During the first stage, synthesis of primary capped and polyadenylated tran scripts (primary miRNAs, primiRNAs), which have a characteristic hairpin secondary structure, catalyzed by RNA polymerase II, takes place. During the next stage of biogenesis, there occurs sequential formation of a long premiRNA and its shorter form (short pre miRNA), which has a length of 64 to 303 nucleotides and forms the future 3'end of the mature miRNA, from a primiRNA precursor (~1 million nucleotides) under the action of a DCL1 ribonuclease. During the third and last stage of biogenesis, as the result of DCL1 catalysis, the cleavage of short premiRNA at the 5'end of the mature miRNA form with a length of 20–24 nucleotides takes places (Bonnet et al., 2006; Vazquez, 2006). Along with DCL1, the necessary fac tor for the completion of miRNA biogenesis is the activity of two proteins localized in the nucleus: dsRNA, which binds protein HYL1 (hyponastic leaves 1), and the methyltransferase HEN1 (HUA enhancer 1), which performs the methylation of the 3'terminal ribose residues at the 2'hydroxyl group (Yu et al., 2005). It is interesting that the most effective viral sup pressors, RNAi (see below), are capable of blocking the methylation by the formation of a complex with small RNAs (Yu et al., 2006). Transport of mature miRNAs in plants from nucleus to cytoplasm is controlled by the HASTY pro tein (HST), which is a homolog of animal exportin5 (Bollman et al., 2003). In the cytoplasm, miRNa interacts with the AGO1 protein (Argonaute family), which is a noncatalytic element of the multiprotein complex RISC and exhibits RNase H activity. The final result of the described interaction is the degrada tion of the mRNA target sequences (Vaucheret, 2008). It should be noted that for plants the only case of translational repression that is known is the interac tion of miRHA of A. thaliana miR172 and mRNA of APETALA2 gene (Chen, 2004). DCL2 Genomes of most pathogenic viruses are repre sented by singlestranded RNA; however, during the life cycle of these pathogens, doublestranded RNA is formed in the replicative intermediates. In addition, the formation of long dsRNA can also be caused by transcription expressed by the transgene of viral or plant RNAdependent RNA polymerase (RdRP). The natural molecular component of tolerance responsible for the selective recognition of long dsRNAs (in partic ular, of viral and/or viroid origin) and their subsequent degradation in higher plants is the RNAi system. RNA silencing can be induced by viruses and thus can be considered as operating on the nucleic acid level and is a highly specific part of the immune system (Vazquez, 2006; Mlotshwa et al., 2008). To date, there is a commonly accepted model according to which the RNA silencing mechanism can
be presented as follows. During the first stage, degra dation of dsRNA occurs by induction of activity of ribonuclease DCL2/dsRNA formed by any of the mechanisms described above. Hydrolytic cleavage of the extended doublestranded RNA catalyzed by DCL2 ultimately leads to the formation of short dou blestranded fragments, called siRNA, having the length of 21–26 nucleotides, with the 5'terminal phosphate group and overhang of 2–3 nucleotides at the 3'end (Goldbach et al., 2003; Mlotshwa et al., 2008). In the subsequent RNAi step, owing to the activity of ATPdependent RNA helicase, the separation of two strands of siRNAs that were previously formed occurs. One of the strands (characterized by lower thermodynamic stability of 5'end) remains in the mature RISC complex, where it binds to mRNA target sequences (Schwarz et al., 2003; Mlotshwa et al., 2008). AGO1 protein, which is the catalyst component of the RISC complex, exhibits endonuclease activity against mRNA, complementary to the bound frag ment of siRNA (Vaucheret, 2008; Wang et al., 2011). Singlestranded fragments of siRNA, complementary to the site of mRNAtarget sequences, can be used as primers for plant RdRP, which synthesizes the second strand, using the RNAtarget sequences as a template (the effect is limited to the distance ~300–500 nucle otides in the 5'direction from the initial cleavage site). It was shown that only siRNA with the length of 22 nucleotides can act as triggers of RNAi recycle, while the more typical siRNA with the length of 21 nucleotides do not have capability for such induc tion. During the ongoing degradation of newly synthe sized dsRNA, performed by DCL2 ribonuclease, the formation of new siRNA, called secondary siRNA, occurs. In this way, the signal is amplified (Lipardi et al., 2001; Mlotshwa et al., 2008). Afterwards, sec ondary siRNA can not only participate in a direct deg radation of mRNA target sequences as a part of the RISC, but they can also be transported between cells by plasmodesmata as signaling molecules. Such sym plastic transport is proteinmediated (in particular, a protein which selectively transports siRNA with a length of 25 nucleotides was identified) (Yoo et al., 2004). DCL3 As already mentioned, inactivation of expression of transgenes/genes may be performed also at the tran scriptional level. There are three main mechanisms of TGS: RNAdependent DNA methylation (RdDM), DNA methylation not associated with participation of small RNAs, and chromatin modification. RdDM, the characteristic feature of which is the methylation of the promoter of the target gene in the region of homology with rasiRNA, is the most wellstudied mechanism of repression of DNA transcription (Marenkova and Deyneko, 2010).
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In this case, one of the sources of long dsRNA can be transcripts read by an RNA polymerase II from inverted repeats and forming a doublestranded struc ture at the complementary sites. Another source of long dsRNA may be higher or lower repetitious meth ylated sequences (including transposable elements), which are transcribed by DNAdependent RNA poly merase IV or DNAdependent RNA polymerase V and then converted into double a stranded form as a result of the catalysis by RNAdependent RNA poly merase RdRP2 or RdRP6, unique to plants (Baul combe, 2004; Vazquez, 2006; Marenkova and Dey neko, 2010). Formation of rasiRNA having the size of 24 nucleotides is accomplished under the action of DCL3RNase. In the cytoplasm, rasiRNA interacts with the RISC complex, which contains AGO4 or AGO6 proteins. During the final stage, this effector complex directs specific methylation of DNA target sequences and chromatin compaction (Qi et al., 2006; Zheng et al., 2007). DCL4 Another class of small RNAs, called transacting siPHK, was identified in mosses and angiosperms. The tasiRNA class includes endogenous noncoding RNAs that have functional similarities to miRNAs and direct degradation in RISC of mRNA target sequences derived from a locus different from that of tasiRNA (Allen et al., 2005; TalmorNeiman et al., 2006). Capped and polyadenylated tasiRNA precursors (pretasiRNA) are formed as a result of the transcrip tion of five genes: TAS1a, TAS1b, TAS1c, TAS2, and TAS3 (Yoshikawa et al., 2005). PretasiRNAs TAS1a, TAS1b, TAS1c, and TAS2 are not conservative (found only in A. thaliana). In contrast, TAS3 RNA was found in many flowering plants, although the similar ity of proposed homologs was only within the frag ment, giving rise to (see below) the mature form of ta siRNA (Vazquez et al., 2004; Allen et al., 2005; Zhang et al., 2012). All TAS RNAs serve as targets for miRNA, which direct the next stage of the biogenesis of tasiRNAs: TAS1a, TAS1b, TAS1c, and TAS2 RNAs interact with miRNA miR173, and TAS3 RNA interacts with miR390. The result of this interaction is the hydrolytic cleavage of TAS RNA, which leads to a shorter form of tasiRNA precursor, which is on the 5'end (in the case of TAS3) or 3'terminal (for TAS1a, TAS1b, TAS1c, and TAS2) fragments of the primary tran script. Singlestranded fragments are formed during this step and then converted into double strands by RNAdependent RNA polymerase RdRP6. The cleavage of these dsRNA fragments with the formation of the mature 21nucleotide tasiRNA occurs during the last step of biogenesis, as a result of DCL4 catalysis (Allen et al., 2005; Yoshikawa et al., 2005; Zhang et al., 2012).
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mRNAs of transcription factors ARF2, ARF3, and ARF4 (auxin response factors) are the target for prod ucts of TAS3 RNA processing. The targets of other ta siRNAs are several genes with unidentified functions to date (Vazquez et al., 2004; Allen et al., 2005; Zhang et al., 2012). SUPPRESSOR OF RNA SILENCING The most effective strategy developed by phyto pathogenic viruses in the course of evolution that allow them to overcome the effect of RNAi is a block of genetic silencing by a specific suppressor protein. A majority of viral proteins known to date as suppressors of RNA silencing were originally identified as factors of pathogenicity, since their expression is largely deter mined by the formation and symptoms of viral infec tion (deformation and leaf mosaic, necrosis, dwarf ism, slow growth rate, etc.). In most cases, the expres sion of this protein group is not an obligatory factor for genome replication of an infectious agent; however, viral RNAi suppressors are essential for efficient accu mulation of virions in plant tissues and their subse quent dissemination (Scholthof, 2005; Li and Ding, 2006; Scholthof, 2007). One of the most wellknown suppressors of RNA silencing is HCPro protease (helper component pro teinase), found in members of the Potyviridae family, in particular, in potato virus Y (PVY). HCPro pro tease is a classic example of a viral multifunctional protein involved in many processes: replication of genomic RNA, proteolytic processing of the transla tion product of polycistronic viral RNA, and extracel lular transport of virions; but participation in the sup pression of RNAi is the most important function of HCPro (Ebhardt et al., 2005; Yu et al., 2006; Scholthof, 2007). The mechanism of action of HC Pro is not fully understood; however, using A. thaliana as a model, it was shown that expression of this protein leads to defects in the plant growth and differentiation, which presumably can be associated with inhibition of miRNAassociated hydrolysis of mRNA transcription factors. Furthermore, the suppression of RNA silenc ing by HCPro protease can be due to binding of formed siRNAs and decrease in their stability, since HCPro prevents functional methylation of siRNA and miRNA (Lakatos et al., 2006; Yu et al., 2006). The fact of the interaction of HCPro protease with rgsCaM protein, which is an endogenous suppressor of RNAi in plants, is also interesting (Shiboleth et al., 2007). Protein P19 is an important factor in the pathoge nicity in representatives of the Tombusviridae family; it has a maximum affinity for siRNA and 21nucle otide miRNA regardless of the presence of 3'terminal overhangs (Scholthof, 2006; Hsieh et al., 2009). The efficiency of binding siRNA/miRNA by P19 protein decreases significantly when the length of these small RNAs increases, even by one nucleotide. As in the
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case of HCPro, it was shown that P19 prevents func tional methylation of siRNA and miRNA, thus reduc ing their stability (Omarov et al., 2007; Pantaleo et al., 2007). Unlike most known viral RNAi suppressors, trans port protein 2b (found in members of the Cucumovir idae family) directly interacts in vitro and in vivo with AGO1, which is the RISC catalytic center and exhibits slicer activity (Zhang et al., 2006). It was shown that the result of such interaction between 2b and AGO1 is the specific inhibition of the enzymatic hydrolysis of the mRNA target sequence. In addition, the signifi cant reduction of pools of all siRNA types associated with the expression of 2b was shown for A. thaliana (DiazPendon et al., 2007; Goto et al., 2007). An interesting feature of the mechanism of sup pression of RNA silencing was shown for the P38 capsid protein of turnip crinkle virus (TVC), Car moviridae family. Unlike the abovedescribed viral suppressors HCPro, P19 or 2b, P38 TVC protein is capable of binding doublestranded RNAs, regardless of molecule size (Qu et al., 2003). A consequence of this feature of P38 may be the reduced availability of dsRNA substrate for DCL ribonucleases, which in turn would inhibit the production of siRNA. Interest ingly, even though this relationship was demonstrated for DCL4 of A. thaliana, P38 TVC protein does not inhibit DCL2 activity (Thomas et al., 2003; Merai et al., 2006). RNAi suppressors were identified in representa tives from at least 15 families of DNA and RNAcon taining plant viruses. The detection of viral RNAi sup pressors is consistent with the idea that RNA silencing is a natural antiviral defense mechanism of plants rather than the only way to control the expression of plant genes. It was shown that suppression of RNA silencing is performed by different mechanisms; how ever, the majority of studied viral RNAi suppressors take effect by binding to the doublestranded RNA forms. It was suggested that the binding of dsRNA may be a general strategy used by phytopathogenic viruses for suppression of RNA silencing. Thus, it was shown that the genome of sweet potato chlorotic stunt virus (SPCSV) contains the RNase III gene, which is a syn ergist of RNA silencing suppressor protein P22 SPCSV. Homologs of RNase III, as well as examples of use of two cooperatively acting proteins to suppress RNAi, were not found in other RNA viruses (also within Closteroviridae family, where SPCSV belongs). Interestingly, RNase III SPCSV homologs were found in A. thaliana and O. sativa, but their functions in plants remain unknown (Kreuze et al., 2005; Cuellar et al., 2008; Lin et al., 2012).
TRANSGENIC PLANTS AS A MODEL FOR THE STUDY OF RIBONUCLEASE III FAMILY The genomes of most pathogenic viruses are ssRNAs and the formation of dsRNA occurs in the course of their life cycle, namely, during the replica tion stage. It was assumed that the understanding of the mechanism of expression of genes of heterologous RNAses capable of hydrolysis of such dsRNA replica tive intermediates could form the basis for developing promising strategies for the production of virustoler ant plants (Watanabe et al., 1995; Zhang et al., 2001). It was been shown that transgenic N. tabacum plants, expressing the PAC1 gene of Schizosaccharo myces pombe (encodes intracellular ribonuclease, cat alyzing the hydrolysis of doublestranded RNA forms), showed resistance to several infectious agents: PVY, cucumber mosaic virus (CMV), and tomato mosaic virus (ToMV). In all these cases, the resistance of the transformants was the delay of symptoms of viral infection (Watanabe et al., 1995). In further studies, it was demonstrated that the production of RNase PAC1 by transgenic plants also induces resistance to a num ber of infections of viroid etiology. It has been shown that Solanum tuberosum transformants, expressing PAC1 gene, in the case of infection by potato spindle tuber viroid (PSTVd) were characterized by the sup pression of infection and reduced accumulation of infectious RNA in tissues (Sano et al., 1997). Produc tion of PAC1 by transgenic Dendranthema grandiflo rum plants contributed to the development of increased resistance to chrysanthemum stunt viroid (CSVd) and tomato spotted wilt virus (TSWV) (Ogawa et al., 2005). It should be noted that the expression of genes encoding heterologous ribonuclease genes in trans genic plants may be associated with a number of phe notypic abnormalities caused by the expressed cyto toxic action of this group of enzymes. Thus, transgenic N. tabacum plants, expressing a mutated form of the rnc gene of Escherichia coli, encoding intracellular RNase III, were resistant to pepper ringspot virus (PepRSV), impatiens necrotic spot virus (INSV), tobacco etch virus (TEV), alfalfa mosaic virus (AMV), tobacco mosaic virus (TMV), and TSWV. Triticum aestivum transformants, expressing the same mutant form of rnc, showed resistance to infection caused by barley stripe mosaic virus (BSMV). The product of a mutant form of the gene had replacement Glu39 → Lys39 and did not exhibit the appropriate catalytic activity while maintaining the ability to bind dsRNA. The experiment demonstrated the same efficiency when using both normal and mutant forms of rnc, but N. tabacum and T. aestivum transforms, expressing a wild type transgene, were characterized by a delay in the development. In addition, it was noted that, in the case where construction with the normal form of the transgene was used, there was a significant decrease in
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the efficiency of Agrobacteriummediated plant trans formation (Langenberg et al., 1997; Zhang et al., 2001). Thus, to date, participation of different ribonu cleases of the ribonuclease III family in induction (DCL RNases) and suppression (RNAse III SPCSV) of RNA silencing was demonstrated. This fact, as well as the availability of data on increased resistance of transgenic plants expressing heterologous proteins of this family to viruses, demonstrates the need for a more detailed study of the role of autologous and het erologous ribonucleases of ribonuclease III in the pro tection systems of higher plants. ACKNOWLEDGMENTS This work was supported by the Russian Founda tion for Basic Research (grant no. 120401478a), Integration Project of the Siberian and Far East Branches of the Russian Academy of Sciences, and program of the Russian Academy of Sciences “Wild life: Current Status and Problems of Development.” REFERENCES Allen, E., Xie, Z., Gustafson, A.M., and Carrington, J.C., MicroRNAdirected phasing during transacting siRNA biogenesis in plants, Cell, 2005, vol. 121, pp. 207– 221. Bartel, B. and Bartel, D., MicroRNAs: at the root of plant development?, Plant Physiol., 2003, vol. 132, pp. 709– 717. Baulcombe, D., RNA silencing in plants, Nature, 2004, vol. 431, pp. 356–363. Bollman, K.M., Aukerman, M.J., Park, M.Y., et al., HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis, Develop ment, 2003, vol. 130, pp. 1493–1504. Bonnet, E., van de Peer, Y., and Rouze, P., The small RNA world of plants, New Phytol., 2006, vol. 171, pp. 451–468. Bozorov, T.A., Pandey, S.P., Dinh, S.T., et al., DICERlike proteins and their role in plant–herbivore interactions in Nicotiana attenuata, J. Integ. Plant Biol., 2012, vol. 54, pp. 189–206. Chen, X., A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development, Science, 2004, vol. 303, pp. 2022–2025. Cuellar, W.J., Tairo, F., Kreuze, J.F., and Valkonen, J.P.T., Analysis of gene content in sweet potato chlorotic stunt virus RNA1 reveals the presence of the p22 RNA silencing suppressor in only a few isolates: implications for viral evolution and synergism, J. Gen. Virol., 2008, vol. 89, pp. 573–582. DiazPendon, J.A., Li, F., Li, W.X., and Ding, S.W., Sup pression of antiviral silencing by cucumber mosaic virus 2b protein in Arabidopsis is associated with drastically reduced accumulation of three classes of viral small interfering RNAs, Plant Cell, 2007, vol. 19, pp. 2053– 2063.
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