Genetic and Functional Diversification of Small RNA Pathways in Plants

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense

Update of ASRP: the Arabidopsis Small RNA Project database

Development of the Arabidopsis Small RNA Project (ASRP) Database, which provides information and tools for the analysis of microRNA, endogenous siRNA and other small RNA-related features, has been driven by the introduction of high-throughput sequencing technology. To accommodate the demands of increased data, numerous improvements and updates have been made to ASRP, including new ways to access data, more efficient algorithms for handling data, and increased integration with community-wide resources. New search and visualization tools have also been developed to improve access to small RNA classes and their targets. ASRP is publicly available through a web interface at http://asrp.cgrb.oregonstate.edu/db

Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro

[EN] Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlappingThis work was supported by grants BFU2012-30805 (SFE) and BFU2012-36346 (MAF) from the Spanish Direccio´n General de Investigacio´n Cientı´fica y Te´cnica and by an EMBO Short-Term Fellowship and the Mentoring Program from the Foundation for Polish Science (BHJ).Hasiów-Jaroszewska, B.; Fares Riaño, MA.; Elena Fito, SF. (2014). Molecular evolution of viral multifunctional proteins: the case of Potyvirus HC-Pro. Journal of Molecular Evolution. 78(1):75-86. https://doi.org/10.1007/s00239-013-9601-0S7586781Adams MJ, Antoniw JF, Beaudoin F (2005) Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol Plant Pathol 6:471–487Atreya CD, Atryea P, Thornbury DW, Pirone TP (1992) Site-directed mutations in the potyvirus HC-Pro gene affect helper component activity, virus accumulation and symptoms expression in infected tobacco plants. Virology 191:106–111Blanc S, López-Moya JJ, Wang R, García-Lampasona S, Thornbury DW, Pirone TP (1997) A specific interaction between coat protein and helper component correlates with aphid transmission of a potyvirus. Virology 231:141–147Blanc S, Ammar ED, García-Lampasona S, Dolja VV, Llave C, Baker J, Pirone TP (1998) Mutations in the potyvirus helper component protein: effects on interactions with virions and aphid stylets. J Gen Virol 79:3119–3122Cantó T, López-Moya JJ, Serra-Yodi MT, Díaz-Ruiz JR, López-Abella D (1995) Different helper component mutations associated with lack of aphid transmissibility in two isolates of potato virus. Phytopathology 85:1519–1524Carrington JC, Freed DD, Sanders TC (1989) Autocatalytic processing of the potyvirus helper component proteinase in Escherichia coli and in vitro. J Virol 63:4459–4463Chung BY, Miller WA, Atkins JF, Firth AE (2008) An overlapping essential gene in the Potyviridae. Proc Natl Acad Sci USA 105:5897–5902Cronin S, Verchot J, Haldeman-Cahill R, Schaad MC, Carrington JC (1995) Long distance movement factor: a transport function of the potyvirus helper component-proteinase. Plant Cell 7:549–559Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797Elena SF, Rodrigo G (2012) Towards and integrated molecular model of plant-virus interactions. Curr Opin Virol 2:713–718Fares MA (2004) SWAPSC: sliding-window analysis procedure to detect selective constraints. Bioinformatics 20:2867–2868Fares MA, McNally D (2006) CAPS: coevolution analysis using protein sequences. Bioinformatics 22:2821–2822Fares MA, Travers AA (2006) A novel method for detecting intramolecular coevolution: adding a further dimension to selective constrains analyses. Genetics 173:9–23Fares MA, Elena SF, Ortiz J, Moya A, Barrio E (2002) A sliding window-based method to detect selective constraints in protein-coding genes and its application to RNA viruses. J Mol Evol 55:509–521Gibbs A, Ohshima K (2010) Potyviruses and the digital revolution. Annu Rev Phytopathol 48:205–223Guo D, Mertis A, Saarma M (1999) Self-association and mapping of interaction domains of helper component of Potato virus A potyvirus. J Gen Virol 80:1127–1131Guo B, Lin J, Ye K (2011) Structure of the autocatalytic cysteine protease domain of potyvirus helper-component proteinase. J Biol Chem 286:21937–21943Haikonen T, Rajamäki ML, Tian YP, Valkonen JPT (2013) Mutation of a short variable region in HC-Pro protein of Potato virus A affects interactions with microtubule-associated protein and induces necrotic responses in tobacco. Mol Plant Microbe Interact 26:721–733Hall TA (1999) BIOEDIT: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41:95–98Hughes AL (2009) Small effective population sizes and rare nonsynonymous variants in potyviruses. Virology 393:127–134Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292:195–202Kasschau KD, Carrington JC (1995) Requirement for HC-Pro processing during genome amplification of Tobacco etch potyvirus. Virology 209:268–273Kasschau KD, Carrington JC (2001) Long-distance movement and replication maintenance functions correlate with silencing suppression activity of potyviral HC-Pro. Virology 285:71–81Kasschau KD, Cronin S, Carrington JC (1997) Genome amplification and long-distance movement functions associated with the central domain of Tobacco etch potyvirus helper component-proteinase. Virology 228:251–262Kosakovsky Pond SL, Frost SDW (2005a) DATAMONKEY: rapid detection of selective pressure on individual sites of codon alignments. Bioinformatics 21:2531–2533Kosakovsky Pond SL, Frost SDW (2005b) Not so different after all: a comparison of methods for detecting amino acid sites under selection. Mol Biol Evol 22:1208–1222Kosakovsky Pond SL, Posada D, Gravenor MB, Woelk CH, Frost SDW (2006) Automated phylogenetic detection of recombination using a genetic algorithm. Mol Biol Evol 23:1891–1901Lakatos L, Csorba T, Pantaleo V, Chapman EJ, Carrington JC, Liu YP, Dojla VV, Calvino LF, López-Moya JJ, Burgyan J (2006) Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25:2768–2780Lalić J, Elena SF (2012) Magnitude and sign epistasis among deleterious mutations in a positive-sense plant RNA virus. Heredity 109:71–77Leigh JW, Susko E, Baumgartner M, Roger AJ (2008) Testing congruence in phylogenomic analysis. Syst Biol 57:104–115Li WH (1993) Unbiased estimation of the rates of synonymous and nonsynonymous substitution. J Mol Evol 36:96–99Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci USA 97:13401–13406Maia S, Haenni AL, Bernardi F (1996) Potyviral HC-Pro: a multifunctional protein. J Gen Virol 77:1335–1341Martin DP, Lemey P, Lott M, Moulton V, Posada D, Lefeuvre P (2010) RDP3: a flexible and fast computer program for analyzing recombination. Bioinformatics 26:2462–2463Moroni E, Morra G, Colombo G (2012) Molecular dynamics simulations of Hsp90 with an eye to inhibitor design. Pharmaceuticals 5:944–962Peng YH, Kadoury D, Gaol-On A, Huet H, Wang Y, Raccah B (1998) Mutations in HC-Pro gene of Zucchini yellow mosaic potyvirus: effects on aphid transmission and binding to purified virions. J Gen Virol 79:897–904Plisson C, Drucker M, Blanc S, German-Retana S, Le Gall O, Thomas D, Bron P (2003) Structural characterization of HC-Pro a plant virus multifunctional protein. J Biol Chem 278:23753–23761Posada D, Crandall KA (1998) MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818Revers F, Le Gall O, Candresse T, Maule J (1999) New advances in understanding the molecular biology of plant/potyvirus interaction. Mol Plant Microbe Interact 12:367–376Riechmann JL, Lain S, García JA (1992) Highlights and prospects of potyvirus molecular biology. J Gen Virol 73:1–16Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738Ruiz-Ferrer V, Boskovic J, Alfonso C, Rivas G, Llorca O, López-Abella D, López-Moya JJ (2005) Structural analysis of Tobacco etch potyvirus HC-pro oligomers involved in aphid transmission. J Virol 79:3758–3765Shiboleth YM, Haronsky E, Leibman D, Arazi T, Wassenegger M, Whitham SA, Gaba V, Gal-On A (2007) The conserved FRNK box in HC-Pro, a plant viral suppressor of gene silencing, is required for small RNA binding and mediates symptom development. J Virol 81:13135–13148Smoot M, Ono K, Ruschelnski J, Wang PL, Ideker T (2011) CYTOSCAPE 2.8: new features for data integration and network visualization. Bioinformatics 27:431–432Syller J (2006) The roles and mechanisms of helper component proteins encoded by potyviruses and caulimoviruses. Physiol Mol Plant Pathol 67:119–130Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739Torres-Barceló C, Martín S, Daròs JA, Elena SF (2008) From hypo- to hypersuppression: effect of amino acid substitutions on the RNA-silencing suppressor activity of Tobacco etch potyvirus HC-Pro. Genetics 180:1039–1049Torres-Barceló C, Daròs JA, Elena SF (2010a) Compensatory molecular evolution of HC-Pro, an RNA-silencing suppressor from a plant RNA virus. Mol Biol Evol 27:543–551Torres-Barceló C, Daròs JA, Elena SF (2010b) HC-Pro hypo- and hypersuppressor mutants: differences in viral siRNA accumulation in vivo and siRNA binding activity in vitro. Arch Virol 155:251–254Urcuqui-Inchima S, Walter J, Drugeon G, German-Retans S, Haeni AL, Candresse T, Bernardi F, Le Gall O (1999) Potyvirus HC-Pro self-interaction in the yeast two hybrid system and delineation of the interaction domain involved. Virology 258:95–99Urcuqui-Inchima S, Maia IG, Arruda P, Haenni AL, Bernardi F (2000) Deletion mapping of the potyviral helper component-proteinase reveals two regions involved in RNA binding. Virology 268:104–111Urcuqui-Inchima S, Haenni AL, Bernardi F (2001) Potyvirus proteins: a wealth of functions. Virus Res 74:157–175Varrelmann M, Maiss E, Pilot R, Palkovics L (2007) Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the Plum pox virus helper component proteinase suppressor of gene silencing. J Gen Virol 88:10051015Ward CW, Shukla DD (1991) Taxonomy of potyviruses: current problems and some solutions. Intervirology 32:269–296Wu S, Zhang Y (2007) LOMETS: a local meta-threading-server for protein structure prediction. Nucl Acids Res 35:3375–3382Yang Z, Bielawski JP (2000) Statistical methods for detecting molecular adaptation. Trends Ecol Evol 15:496–503Yap YK, Duangjit J, Panyim S (2009) N-terminal of Papaya ringspot virus type-W (PRSV-W) helper component proteinase (HC-Pro) is essential for PRSV systemic infection in zucchini. Virus Genes 38:461–467Zheng H, Yan F, Lu Y, Sun L, Lin L, Cai L, Hou M, Chen J (2010) Mapping the self-interaction domains of TuMV HC-pro and the subcellular localization of the protein. Virus Genes 42:110–11

High-Throughput Sequencing of Arabidopsis microRNAs: Evidence for Frequent Birth and Death of MIRNA Genes

In plants, microRNAs (miRNAs) comprise one of two classes of small RNAs that function primarily as negative regulators at the posttranscriptional level. Several MIRNA genes in the plant kingdom are ancient, with conservation extending between angiosperms and the mosses, whereas many others are more recently evolved. Here, we use deep sequencing and computational methods to identify, profile and analyze non-conserved MIRNA genes in Arabidopsis thaliana. 48 non-conserved MIRNA families, nearly all of which were represented by single genes, were identified. Sequence similarity analyses of miRNA precursor foldback arms revealed evidence for recent evolutionary origin of 16 MIRNA loci through inverted duplication events from protein-coding gene sequences. Interestingly, these recently evolved MIRNA genes have taken distinct paths. Whereas some non-conserved miRNAs interact with and regulate target transcripts from gene families that donated parental sequences, others have drifted to the point of non-interaction with parental gene family transcripts. Some young MIRNA loci clearly originated from one gene family but form miRNAs that target transcripts in another family. We suggest that MIRNA genes are undergoing relatively frequent birth and death, with only a subset being stabilized by integration into regulatory networks

Genetic and Functional Diversification of Small RNA Pathways in Plants

Multicellular eukaryotes produce small RNA molecules (approximately 21–24 nucleotides) of two general types, microRNA (miRNA) and short interfering RNA (siRNA). They collectively function as sequence-specific guides to silence or regulate genes, transposons, and viruses and to modify chromatin and genome structure. Formation or activity of small RNAs requires factors belonging to gene families that encode DICER (or DICER-LIKE [DCL]) and ARGONAUTE proteins and, in the case of some siRNAs, RNA-dependent RNA polymerase (RDR) proteins. Unlike many animals, plants encode multiple DCL and RDR proteins. Using a series of insertion mutants of Arabidopsis thaliana, unique functions for three DCL proteins in miRNA (DCL1), endogenous siRNA (DCL3), and viral siRNA (DCL2) biogenesis were identified. One RDR protein (RDR2) was required for all endogenous siRNAs analyzed. The loss of endogenous siRNA in dcl3 and rdr2 mutants was associated with loss of heterochromatic marks and increased transcript accumulation at some loci. Defects in siRNA-generation activity in response to turnip crinkle virus in dcl2 mutant plants correlated with increased virus susceptibility. We conclude that proliferation and diversification of DCL and RDR genes during evolution of plants contributed to specialization of small RNA-directed pathways for development, chromatin structure, and defense

Transcriptional activity of transposable elements in maize

<p>Abstract</p> <p>Background</p> <p>Mobile genetic elements represent a high proportion of the Eukaryote genomes. In maize, 85% of genome is composed by transposable elements of several families. First step in transposable element life cycle is the synthesis of an RNA, but few is known about the regulation of transcription for most of the maize transposable element families. Maize is the plant from which more ESTs have been sequenced (more than two million) and the third species in total only after human and mice. This allowed us to analyze the transcriptional activity of the maize transposable elements based on EST databases.</p> <p>Results</p> <p>We have investigated the transcriptional activity of 56 families of transposable elements in different maize organs based on the systematic search of more than two million expressed sequence tags. At least 1.5% maize ESTs show sequence similarity with transposable elements. According to these data, the patterns of expression of each transposable element family is variable, even within the same class of elements. In general, transcriptional activity of the <it>gypsy</it>-like retrotransposons is higher compared to other classes. Transcriptional activity of several transposable elements is specially high in shoot apical meristem and sperm cells. Sequence comparisons between genomic and transcribed sequences suggest that only a few copies are transcriptionally active.</p> <p>Conclusions</p> <p>The use of powerful high-throughput sequencing methodologies allowed us to elucidate the extent and character of repetitive element transcription in maize cells. The finding that some families of transposable elements have a considerable transcriptional activity in some tissues suggests that, either transposition is more frequent than previously expected, or cells can control transposition at a post-transcriptional level.</p

Identification of Pns6, a putative movement protein of RRSV, as a silencing suppressor

RNA silencing is a potent antiviral response in plants. As a counterdefense, most plant and some animal viruses encode RNA silencing suppressors. In this study, we showed that Pns6, a putative movement protein of Rice ragged stunt virus (RRSV), exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns6 of RRSV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Deletion of a region involved in RNA binding abolished the silencing suppressor activity of Pns6. Further, expression of Pns6 enhanced Potato virus × pathogenicity in N. benthamiana. Collectively, these results suggested that RRSV Pns6 functions as a virus suppressor of RNA silencing that targets an upstream step of the dsRNA formation in the RNA silencing pathway. This is the first silencing suppressor to be identified from the genus Oryzavirus

Simultaneous Mutations in Multi-Viral Proteins Are Required for Soybean mosaic virus to Gain Virulence on Soybean Genotypes Carrying Different R Genes

BACKGROUND: Genetic resistance is the most effective and sustainable approach to the control of plant pathogens that are a major constraint to agriculture worldwide. In soybean, three dominant R genes, i.e., Rsv1, Rsv3 and Rsv4, have been identified and deployed against Soybean mosaic virus (SMV) with strain-specificities. Molecular identification of virulent determinants of SMV on these resistance genes will provide essential information for the proper utilization of these resistance genes to protect soybean against SMV, and advance knowledge of virus-host interactions in general. METHODOLOGY/PRINCIPAL FINDINGS: To study the gain and loss of SMV virulence on all the three resistance loci, SMV strains G7 and two G2 isolates L and LRB were used as parental viruses. SMV chimeras and mutants were created by partial genome swapping and point mutagenesis and then assessed for virulence on soybean cultivars PI96983 (Rsv1), L-29 (Rsv3), V94-5152 (Rsv4) and Williams 82 (rsv). It was found that P3 played an essential role in virulence determination on all three resistance loci and CI was required for virulence on Rsv1- and Rsv3-genotype soybeans. In addition, essential mutations in HC-Pro were also required for the gain of virulence on Rsv1-genotype soybean. To our best knowledge, this is the first report that CI and P3 are involved in virulence on Rsv1- and Rsv3-mediated resistance, respectively. CONCLUSIONS/SIGNIFICANCE: Multiple viral proteins, i.e., HC-Pro, P3 and CI, are involved in virulence on the three resistance loci and simultaneous mutations at essential positions of different viral proteins are required for an avirulent SMV strain to gain virulence on all three resistance loci. The likelihood of such mutations occurring naturally and concurrently on multiple viral proteins is low. Thus, incorporation of all three resistance genes in a soybean cultivar through gene pyramiding may provide durable resistance to SMV