The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism

[EN] Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle.The authors thank L. Corachan-Valencia for technical assistance. This work was funded by grant BIO2017¿88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-B. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain, respectively. Ministerio de Ciencia e Innovacion (PID2020-115571RB-I00), European Regional Development FundNavarro Bohigues, JA.; Sáiz-Bonilla, M.; Sanchez Navarro, JA.; Pallás Benet, V. (2021). The mitochondrial and chloroplast dual targeting of a multifunctional plant viral protein modulates chloroplast-to-nucleus communication, RNA silencing suppressor activity, encapsidation, pathogenesis and tissue tropism. The Plant Journal. 108(1):197-218. https://doi.org/10.1111/tpj.15435S197218108

Highly efficient construction of infectious viroid-derived clones

[EN] Background Viroid research generally relies on infectious cDNA clones that consist of dimers of the entire viroid sequence. At present, those dimers are generated by self-ligation of monomeric cDNA, a strategy that presents several disadvantages: (i) low efficiency, (ii) it is a non-oriented reaction requiring tedious screenings and (iii) additional steps are required for cloning into a binary vector for agroinfiltration or for in vitro RNA production. Results We have developed a novel strategy for simultaneous construction of a viroid dimeric cDNA and cloning into a multipurpose binary vector ready for agroinfiltration or in vitro transcription. The assembly is based on IIs restriction enzymes and positive selection and supposes a universal procedure for obtaining infectious clones of a viroid independently of its sequence, with a high efficiency. Thus, infectious clones of one viroid of each family were obtained and its infectivity was analyzed by molecular hybridization. Conclusion This is a zero-background strategy for direct cloning into a binary vector, optimized for the generation of infectious viroids. As a result, this methodology constitutes a powerful tool for viroid research and exemplifies the applicability of type IIs restriction enzymes and the lethal gene ccdB to design efficient and affordable direct cloning approaches of PCR products into binary vectors.This work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) Grants BIO2017-88321-R (VP) and AGL2016-79825-R (GG). The funders had no role in the experiment design, data analysis, decision to publish, or preparation of the manuscript.Márquez-Molins, J.; Navarro Bohigues, JA.; Pallás Benet, V.; Gomez, GG. (2019). Highly efficient construction of infectious viroid-derived clones. Plant Methods. 15:1-8. https://doi.org/10.1186/s13007-019-0470-4S1815Flores R, Minoia S, Carbonell A, Gisel A, Delgado S, López-Carrasco A, et al. 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A conserved motif in three viral movement proteins from different genera is required for host factor recruitment and cell-to-cell movement

[EN] Due to their minimal genomes, plant viruses are forced to hijack specific cellular pathways to ensure host colonization, a condition that most frequently involves physical interaction between viral and host proteins. Among putative viral interactors are the movement proteins, responsible for plasmodesma gating and genome binding during viral transport. Two of them, DGBp1 and DGBp2, are required for alpha-, beta- and gammacarmovirus cell-to-cell movement, but the number of DGBp-host interactors identified at present is limited. By using two different approaches, yeast two-hybrid and bimolecular fluorescence complementation assays, we found three Arabidopsis factors, eIF3g1, RPP3A and WRKY36, interacting with DGBp1s from each genus mentioned above. eIF3g1 and RPP3A are mainly involved in protein translation initiation and elongation phases, respectively, while WRKY36 belongs to WRKY transcription factor family, important regulators of many defence responses. These host proteins are not expected to be associated with viral movement, but knocking out WRKY36 or silencing either RPP3A or eIF3g1 negatively affected Arabidopsis infection by Turnip crinkle virus. A highly conserved FNF motif at DGBp1 C-terminus was required for protein-protein interaction and cell-to-cell movement, suggesting an important biological role.We thank Dr. Anne Simon and Dr. Steve A. Lommel for providing an infectious cDNA clone of the Turnip crinkle virus strain M (TCV-M) and PZP-TCV-sGFP plasmid, respectively. This work was funded by grant BIO2017-88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain.Navarro Bohigues, JA.; Serra-Soriano, M.; Corachán Valencia, L.; Pallás Benet, V. (2020). 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A Protocol to Measure the Extent of Cell-to-cell Movement of RNA Viruses in Planta. Bio-protocol 4, e1269 (2014).Koressaar, T. et al. Primer3_masker: integrating masking of template sequence with primer design software. Bioinformatics 34, 1937–1938 (2018).Lilly, S. T., Drummond, R. S., Pearson, M. N. & MacDiarmid, R. M. Identification and validation of reference genes for normalization of transcripts from virus-infected Arabidopsis thaliana. Mol. Plant Microbe Interact. 24, 294–304 (2011)

Might exogenous circular RNAs act as protein-coding transcripts in plants?

[EN] Circular RNAs (circRNAs) are regulatory molecules involved in the modulation of gene expression. Although originally assumed as non-coding RNAs, recent studies have evidenced that animal circRNAs can act as translatable transcripts. The study of plant-circRNAs is incipient, and no autonomous coding plant-circRNA has been described yet. Viroids are the smallest plant-pathogenic circRNAs known to date. Since their discovery 50 years ago, viroids have been considered valuable systems for the study of the structure-function relationships in RNA, essentially because they have not been shown to have coding capacity. We used two pathogenic circRNAs (Hop stunt viroid and Eggplant latent viroid) as experimental tools to explore the coding potential of plant-circRNAs. Our work supports that the analysed viroids contain putative ORFs able to encode peptides carrying subcellular localization signals coincident with the corresponding replication-specific organelle. Bioassays in well-established hosts revealed that mutations in these ORFs diminish their biological efficiency. Interestingly, circular forms of HSVd and ELVd were found to co-sediment with polysomes, revealing their physical interaction with the translational machinery of the plant cell. Based on this evidence we hypothesize about the possibility that plant circRNAs in general, and viroids in particular, can act, under certain cellular conditions, as non-canonical translatable transcripts.This work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) PID2020-115571RB-I00VP) and AGL2016-79825-R (GG), and by the Spanish Ministry of Science and Innovation (co-supported by FEDER) Grant PID2019104126RB-I00 (GG). JMM was the recipient of a predoctoral contract supported by the Conselleria d ' Educacio, Investigacio, Cultura i Esport Generalitat Valenciana -ACIF Programme (ACIF-2017-114). The funders had no role in the experiment design, data analysis, decision to publish, or preparation of the manuscript;Conselleria d'Educacio, Investigacio, Cultura i Esport [ACIF-2017-114];Ministerio de Economia, Industria y Competitividad, Gobierno de Espana [AGL201679825-R];Ministerio de Econom?a, Industria y Competitividad, Gobierno de Espana [PID2020-115571RB-I00];Ministerio de Ciencia, Innovacion y Universidades [PID2019-104126RB-I00].Márquez-Molins, J.; Navarro Bohigues, JA.; Cervera Seco, L.; Pallás Benet, V.; Gomez, GG. (2021). Might exogenous circular RNAs act as protein-coding transcripts in plants?. RNA Biology. 18:98-107. https://doi.org/10.1080/15476286.2021.1962670S981071

Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro

[EN] Cucurbit chlorotic yellows virus (CCYV) is a new member of the genus Crinivirus (family Closteroviridae) with a bi-partite genome. CCYV RNA 1-encoded p22 has recently been reported to be a weak local suppressor of RNA silencing for which an interaction with cucumber SKP1LB1 through an F-box-like motif was demonstrated to be essential. Using a bacterially expressed maltose-binding protein (MBP) fusion of CCYV p22 in electrophoretic mobility shift assays (EMSA), we have examined in vitro its ability to bind different RNA templates. Our experiments showed that CCYV p22 is able to bind to ss and ds long RNAs, in addition to ss and ds small interfering (si) RNA molecules. CCYV p22 deletion mutants (MBP_CCYV DEL1-4) were produced that covered the entire protein, with MBP_CCYV DEL2 corresponding to the F-box motif and its flanking sequences. None of these deletions abolished the capacity of CCYV p22 to bind ss- and dsRNA molecules. However, deletions affecting the C-terminal half of the protein resulted in decreased binding efficiency for either ss- or dsRNA molecules indicating that essential elements for these interactions are located in this region. Taken together, our data add to current knowledge of the mode of action of suppressors of RNA silencing encoded by genes sited at the 3'-terminus of crinivirus genomic RNA 1, and shed light on the involvement of CCYV p22 in the suppression of RNA silencing and/or in another role in the virus life cycle via RNA binding.Salavert, F.; Navarro Bohigues, JA.; Owen, CA.; Khechmar, S.; Pallás Benet, V.; Livieratos, IC. (2020). Cucurbit chlorotic yellows virus p22 suppressor of RNA silencing binds single-, double-stranded long and short interfering RNA molecules in vitro. Virus Research. 279:1-8. https://doi.org/10.1016/j.virusres.2020.197887S18279Abrahamian, P. E., Seblani, R., Sobh, H., & Abou-Jawdah, Y. (2013). Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR. Journal of Virological Methods, 193(2), 320-326. doi:10.1016/j.jviromet.2013.06.004Bananej, K., Menzel, W., Kianfar, N., Vahdat, A., & Winter, S. (2013). First Report of Cucurbit chlorotic yellows virus Infecting Cucumber, Melon, and Squash in Iran. Plant Disease, 97(7), 1005-1005. doi:10.1094/pdis-01-13-0125-pdnBrantley, J. D., & Hunt, A. G. (1993). The N-terminal protein of the polyprotein encoded by the potyvirus tobacco vein mottling virus is an RNA-binding protein. Journal of General Virology, 74(6), 1157-1162. doi:10.1099/0022-1317-74-6-1157BUCK, K. W., & KEMPSON-JONES, G. F. (1970). Three Types of Virus Particle in Penicillium stoloniferum. Nature, 225(5236), 945-946. doi:10.1038/225945a0Cañizares, M. C., Navas-Castillo, J., & Moriones, E. (2008). Multiple suppressors of RNA silencing encoded by both genomic RNAs of the crinivirus, Tomato chlorosis virus. Virology, 379(1), 168-174. doi:10.1016/j.virol.2008.06.020Chen, S., Sun, X., Shi, Y., Wei, Y., Han, X., Li, H., … Shi, Y. (2019). Cucurbit Chlorotic Yellows Virus p22 Protein Interacts with Cucumber SKP1LB1 and Its F-Box-Like Motif Is Crucial for Silencing Suppressor Activity. Viruses, 11(9), 818. doi:10.3390/v11090818Cuellar, W. J., Tairo, F., Kreuze, J. F., & Valkonen, J. P. T. (2008). 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. Journal of General Virology, 89(2), 573-582. doi:10.1099/vir.0.83471-0Cuellar, W. J., Kreuze, J. F., Rajamaki, M.-L., Cruzado, K. R., Untiveros, M., & Valkonen, J. P. T. (2009). Elimination of antiviral defense by viral RNase III. Proceedings of the National Academy of Sciences, 106(25), 10354-10358. doi:10.1073/pnas.0806042106GYOUTOKU, Y., OKAZAKI, S., FURUTA, A., ETOH, T., MIZOBE, M., KUNO, K., … OKUDA, M. (2009). Chlorotic yellows disease of melon caused by Cucurbit chlorotic yellows virus, a new crinivirus. Japanese Journal of Phytopathology, 75(2), 109-111. doi:10.3186/jjphytopath.75.109Herranz, M. C., & Pallás, V. (2004). RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus. Journal of General Virology, 85(3), 761-768. doi:10.1099/vir.0.19534-0Huang, L.-H., Tseng, H.-H., Li, J.-T., & Chen, T.-C. (2010). First Report of Cucurbit chlorotic yellows virus Infecting Cucurbits in Taiwan. Plant Disease, 94(9), 1168-1168. doi:10.1094/pdis-94-9-1168bKataya, A. R. A., Suliman, M. N. S., Kalantidis, K., & Livieratos, I. C. (2009). Cucurbit yellow stunting disorder virus p25 is a suppressor of post-transcriptional gene silencing. Virus Research, 145(1), 48-53. doi:10.1016/j.virusres.2009.06.010Klaassen, V. A., Mayhew, D., Fisher, D., & Falk, B. W. (1996). In VitroTranscripts from Cloned cDNAs of the Lettuce Infectious Yellows Closterovirus Bipartite Genomic RNAs Are Competent for Replication inNicotiana benthamianaProtoplasts. Virology, 222(1), 169-175. doi:10.1006/viro.1996.0407Kreuze, J. F., Savenkov, E. I., Cuellar, W., Li, X., & Valkonen, J. P. T. (2005). Viral Class 1 RNase III Involved in Suppression of RNA Silencing. Journal of Virology, 79(11), 7227-7238. doi:10.1128/jvi.79.11.7227-7238.2005Kubota, K., & Ng, J. C. K. (2016). Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures. Phytopathology®, 106(6), 653-662. doi:10.1094/phyto-09-15-0219-rLandeo-Ríos, Y., Navas-Castillo, J., Moriones, E., & Cañizares, M. C. (2016). The p22 RNA silencing suppressor of the crinivirus Tomato chlorosis virus preferentially binds long dsRNAs preventing them from cleavage. Virology, 488, 129-136. doi:10.1016/j.virol.2015.11.008Marcos, J. F., Vilar, M., Pérez-Payá, E., & Pallás, V. (1999). In VivoDetection, RNA-Binding Properties and Characterization of the RNA-Binding Domain of the p7 Putative Movement Protein from Carnation Mottle Carmovirus (CarMV). Virology, 255(2), 354-365. doi:10.1006/viro.1998.9596Mashiko, T., Wang, W.-Q., Hartono, S., Suastica, G., Neriya, Y., Nishigawa, H., & Natsuaki, T. (2019). The p27 open reading frame of tomato infectious chlorosis virus encodes a suppressor of RNA silencing. Journal of General Plant Pathology, 85(4), 301-305. doi:10.1007/s10327-019-00850-0Navarro, J. A., Genovés, A., Climent, J., Saurí, A., Martínez-Gil, L., Mingarro, I., & Pallás, V. (2006). RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology, 356(1-2), 57-67. doi:10.1016/j.virol.2006.07.040Okuda, M., Okazaki, S., Yamasaki, S., Okuda, S., & Sugiyama, M. (2010). Host Range and Complete Genome Sequence of Cucurbit chlorotic yellows virus, a New Member of the Genus Crinivirus. Phytopathology®, 100(6), 560-566. doi:10.1094/phyto-100-6-0560Orfanidou, C., Maliogka, V. I., & Katis, N. I. (2014). First Report of Cucurbit chlorotic yellows virus in Cucumber, Melon, and Watermelon in Greece. Plant Disease, 98(10), 1446-1446. doi:10.1094/pdis-03-14-0311-pdnOrfanidou, C. G., Mathioudakis, M. M., Katsarou, K., Livieratos, I., Katis, N., & Maliogka, V. I. (2019). Cucurbit chlorotic yellows virus p22 is a suppressor of local RNA silencing. Archives of Virology, 164(11), 2747-2759. doi:10.1007/s00705-019-04391-xOrílio, A. F., Fortes, I. M., & Navas-Castillo, J. (2014). Infectious cDNA clones of the crinivirus Tomato chlorosis virus are competent for systemic plant infection and whitefly-transmission. Virology, 464-465, 365-374. doi:10.1016/j.virol.2014.07.032Pumplin, N., & Voinnet, O. (2013). RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nature Reviews Microbiology, 11(11), 745-760. doi:10.1038/nrmicro3120Richmond, K. E., Chenault, K., Sherwood, J. L., & German, T. L. (1998). Characterization of the Nucleic Acid Binding Properties of Tomato Spotted Wilt Virus Nucleocapsid Protein. Virology, 248(1), 6-11. doi:10.1006/viro.1998.9223Salem, N. M., Chen, A. Y. S., Tzanetakis, I. E., Mongkolsiriwattana, C., & Ng, J. C. K. (2009). Further complexity of the genus Crinivirus revealed by the complete genome sequence of Lettuce chlorosis virus (LCV) and the similar temporal accumulation of LCV genomic RNAs 1 and 2. Virology, 390(1), 45-55. doi:10.1016/j.virol.2009.04.025Serra-Soriano, M., Antonio Navarro, J., & Pallás, V. (2016). Dissecting the multifunctional role of the N-terminal domain of theMelon necrotic spot viruscoat protein in RNA packaging, viral movement and interference with antiviral plant defence. 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In memoriam of Ricardo Flores: The career, achievements, and legacy of an inspirational plant virologist

[EN] Ricardo Flores (1947-2020) focused his research on the identification, replication, pathogenesis, and evolution of viroids, the minimal non-protein-coding circular RNAs (250-400 nt) able to replicate and incite diseases in plants that are remarkable for being at the lowest step of the biological scale. He and his collaborators initially identified and characterized additional group members, adding six new ones to the family Pospiviroidae, and expanding the Avsunviroidae from one to four members. They showed that members of the second family "encode" ribozymes, a property that, together with others, makes them candidates for being the most primitive replicons that emerged on our planet 3500 million years ago. He also made important contributions regarding how viroids replicate, providing relevant data on the templates, enzymes, and ribozymes that mediate this process and on the mutation rate, which turned out to be the highest reported for any biological entity. More recently, he concentrated on the role that RNA silencing could play on viroid-host interactions, describing details of this process. Ricardo also worked on citrus tristeza virus, a widely different type of subcellular pathogen, and made important contributions on the structure, localization and functions of its unique p23 protein. His research has produced 170 original articles and reviews, according to Web of Science. He encouraged the scientific careers of a large number of researchers, and collaborated with many others, some of whom have recapitulated his scientific legacy in this review and contributed with other chapters in this special issue.This work was supported by the Spanish Agencia Estatal de Investigaci ' on (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), grant number PID2020-115571RB-100. We apologize to colleagues whose work was not cited in this review due to the page limit.Pallás Benet, V.; Hernandez Fort, C.; Marcos, JF.; Daròs, J.; Ambrós, S.; Navarro, B.; Navarro Bohigues, JA.... (2022). In memoriam of Ricardo Flores: The career, achievements, and legacy of an inspirational plant virologist. Virus Research. 312(198718):1-9. https://doi.org/10.1016/j.virusres.2022.1987181931219871

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering.We thank Veronica Aragones for excellent technical assistance. We also thank Ash N. Watson (Smurfit Institute of Genetics, Trinity College Dublin, Ireland) for English edition. This research was supported by grants BIO2011-26741 and BIO2014-54269-R from Ministerio de Economia y Competitividad (MINECO, Spain) to J.-A.D. E.M. was the recipient of a predoctoral fellowship (AP2012-3751) from Ministerio de Educacion, Cultura y Deporte (Spain). J.-A.N was supported by grant BIO2011-25018 (MINECO).Majer, E.; Navarro Bohigues, JA.; Daros Arnau, JA. (2015). A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein. Biotechnology Journal. 10(11):1792-1802. https://doi.org/10.1002/biot.201500042S17921802101

A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector

[EN] Plant viruses express RNA silencing suppressor (RSS) proteins to counteract plant defence mechanisms. Here, we describe a method to assess the RSS activity based on an alfalfa mosaic virus (AMV) RNA 3 expression vector and transgenic Nicotiana tabacum plants that express the P1 and P2 subunits of the AMV replicase (P12 plants). Inoculation of P12 plants with different AMV RNA 3 constructs expressing different HC-Pro mutants that differ in their RSS capabilities, revealed a perfect correlation between necrotic lesions on inoculated leaves and RSS activity. Protoplast analysis showed that the RSS activity correlated with the accumulation of the AMV RNAs. A direct comparison between three RSS activity assays and the AMV-P12 system revealed that the coat protein of carnation mottle virus displays RSS activity with the four assays and reduced the accumulation of the siRNAs.We thank L. Corachan for her excellent technical assistance. We thank Dr. S. Elena, I2SysBio (Valencia, Spain), for supplying us the different HC-Pro mutants. This work was supported by grant BIO2017-88321-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2015/010 from the Generalitat Valenciana.Martínez-Pérez, M.; Navarro Bohigues, JA.; Pallás Benet, V.; Sanchez Navarro, JA. (2019). A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector. Virus Research. 272:1-5. https://doi.org/10.1016/j.virusres.2019.197733S1527

Molecular characterization, targeting and expression analysis of chloroplast and mitochondrion protein import components in Nicotiana benthamiana

[EN] Improved bioinformatics tools for annotating gene function are becoming increasingly available, but such information must be considered theoretical until further experimental evidence proves it. In the work reported here, the genes for the main components of the translocons of the outer membrane of chloroplasts (Toc) and mitochondria (Tom), including preprotein receptors and protein-conducting channels of N. benthamiana, were identified. Sequence identity searches and phylogenetic relationships with functionally annotated sequences such as those of A. thaliana revealed that N. benthamiana orthologs mainly exist as recently duplicated loci. Only a Toc34 ortholog was found (NbToc34), while Toc159 receptor family was composed of four orthologs but somewhat different from those of A. thaliana. Except for NbToc90, the rest (NbToc120, NbToc159A and NbToc159B) had a molecular weight of about 150 kDa and an acidic domain similar in length. Only two orthologs of the Tom20 receptors, NbTom20-1 and NbTom20-2, were found. The number of the Toc and Tom receptor isoforms in N. benthamiana was comparable to that previously reported in tomato and what we found in BLAST searches in other species in the genera Nicotiana and Solanum. After cloning, the subcellular localization of N. benthamiana orthologs was studied, resulting to be identical to that of A. thaliana receptors. Phenotype analysis after silencing together with relative expression analysis in roots, stems and leaves revealed that, except for the Toc and Tom channel- forming components (NbToc75 and NbTom40) and NbToc34, functional redundancy could be observed either among Toc159 or mitochondrial receptors. Finally, heterodimer formation between NbToc34 and the NbToc159 family receptors was confirmed by two alternative techniques indicating that different Toc complexes could be assembled. Additional work needs to be addressed to know if this results in a functional specialization of each Toc complex.Funding This work was funded by grant PID2020-115571RB-I00 from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). JN and MS-B are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain, repectively.Sáiz-Bonilla, M.; Martín Merchán, A.; Pallás Benet, V.; Navarro Bohigues, JA. (2022). Molecular characterization, targeting and expression analysis of chloroplast and mitochondrion protein import components in Nicotiana benthamiana. Frontiers in Plant Science. 13:1-21. https://doi.org/10.3389/fpls.2022.10406881211