Adaptation of aphid stylectomy for analyses of proteins and mRNAs in barley phloem sap

Sieve tubes are transport conduits not only for photoassimilates but also for macromolecules and other compounds that are involved in sieve tube maintenance and systemic signalling. In order to gain sufficient amounts of pure phloem exudates from barley plants for analyses of the protein and mRNA composition, a previously described stylectomy set-up was optimized. Aphids were placed in sealed cages, which, immediately after microcauterization of the stylets, were flooded with water-saturated silicon oil. The exuding phloem sap was collected with a capillary connected to a pump. Using up to 30 plants and 600 aphids (Rhopalosiphum padi) in parallel, an average of 10 μl of phloem sap could be obtained within 6 h of sampling. In first analyses of the macromolecular content, eight so far unknown phloem mRNAs were identified by cDNA-amplified fragment length polymorphism. Transcripts in barley phloem exudates are related to metabolism, signalling, and pathogen defence, for example coding for a protein kinase and a pathogen- and insect-responsive WIR1A (wheat-induced resistance 1A)-like protein. Further, one-dimensional gel electrophoresis and subsequent partial sequencing by mass spectrometry led to the identification of seven major proteins with putative functions in stress responses and transport of mRNAs, proteins, and sugars. Two of the discovered proteins probably represent isoforms of a new phloem-mobile sucrose transporter. Notably, two-dimensional electrophoresis confirmed that there are >250 phloem proteins awaiting identification in future studies

Repression of Flowering by the miR172 Target SMZ

The flowering repressors SMZ and FLM, members of the AP-2 and MADS domain transcription factor families, unexpectedly work together to regulate flowering time via their effects on expression of the FT gene

Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems

Conclusions: We compare for the first time the sRNA profile of four different tissues, including source, sink and conductive (phloem) tissues, in two plant-virus pathosystems. Our results indicate that antiviral silencing machinery in melon and cucumber acts mainly through DCL4. Upon infection, the total sRNA pattern in phloem remains unchanged in contrast to the rest of the analyzed tissues indicating a certain tissue-tropism to this polulation. Independently of the accumulation level of the vsRNAs both viruses were able to modulate the host sRNA pattern.We thank Dr A. Niehl for critical reading and helpful comments on the manuscript. This work was funded by a supporting program for the research from the Universidad Politecnica de Valencia (PAID-05-10), a grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica and the PROMETEO program 2011/003 from the Generalitat Valenciana. MCH is the recipient of a contract from JAE-DOC program of the CSIC, JAN is the recipient of a postdoctoral contract from the Ministerio de Educacion y Ciencia of Spain.Herranz Gordo, MDC.; Navarro Bohigues, JA.; Sommen, E.; Pallás Benet, V. (2015). Comparative analysis among the small RNA populations of source, sink and conductive tissues in two different plant-virus pathosystems. BMC Genomics. 16:1-15. https://doi.org/10.1186/s12864-015-1327-5S11516Pumplin N, Voinnet O. RNA silencing suppression by plant pathogens: defence, counter-defence and counter-counter-defence. Nat Rev Microbiol. 2013;11(11):745–60.Brodersen P, Voinnet O. The diversity of RNA silencing pathways in plants. Trends Genet. 2006;22(5):268–80.Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;10(2):94–108.Ciaudo C, Jay F, Okamoto I, Chen CJ, Sarazin A, Servant N, et al. RNAi-dependent and independent control of LINE1 accumulation and mobility in mouse embryonic stem cells. PLoS Genet. 2013;9(11):e1003791.Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell. 2007;130(3):413–26.Szittya G, Moxon S, Pantaleo V, Toth G, Rusholme Pilcher RL, Moulton V, et al. Structural and functional analysis of viral siRNAs. PLoS Pathog. 2010;6(4):e1000838.Donaire L, Wang Y, Gonzalez-Ibeas D, Mayer KF, Aranda MA, Llave C. Deep-sequencing of plant viral small RNAs reveals effective and widespread targeting of viral genomes. Virology. 2009;392(2):203–14.Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell. 2009;136(4):669–87.Liu Q, Feng Y, Zhu Z. Dicer-like (DCL) proteins in plants. Funct Integr Genomics. 2009;9(3):277–86.Henderson IR, Zhang X, Lu C, Johnson L, Meyers BC, Green PJ, et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat Genet. 2006;38(6):721–5.Margis R, Fusaro AF, Smith NA, Curtin SJ, Watson JM, Finnegan EJ, et al. The evolution and diversification of Dicers in plants. FEBS Lett. 2006;580(10):2442–50.Deleris A, Gallego-Bartolome J, Bao J, Kasschau KD, Carrington JC, Voinnet O. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science. 2006;313(5783):68–71.Blevins T, Rajeswaran R, Shivaprasad PV, Beknazariants D, Si-Ammour A, Park HS, et al. Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing. Nucleic Acids Res. 2006;34(21):6233–46.Bouche N, Lauressergues D, Gasciolli V, Vaucheret H. An antagonistic function for Arabidopsis DCL2 in development and a new function for DCL4 in generating viral siRNAs. EMBO J. 2006;25(14):3347–56.Moissiard G, Voinnet O. RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc Natl Acad Sci U S A. 2006;103(51):19593–8.Qu F, Ye X, Morris TJ. Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc Natl Acad Sci U S A. 2008;105(38):14732–7.Vaucheret H. Plant ARGONAUTES. Trends Plant Sci. 2008;13(7):350–8.Hutvagner G, Simard MJ. Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol. 2008;9(1):22–32.Voinnet O. Use, tolerance and avoidance of amplified RNA silencing by plants. Trends Plant Sci. 2008;13(7):317–28.Palauqui JC, Elmayan T, Pollien JM, Vaucheret H. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 1997;16(15):4738–45.Yoo BC, Kragler F, Varkonyi-Gasic E, Haywood V, Archer-Evans S, Lee YM, et al. A systemic small RNA signaling system in plants. Plant Cell. 2004;16(8):1979–2000.Buhtz A, Pieritz J, Springer F, Kehr J. Phloem small RNAs, nutrient stress responses, and systemic mobility. BMC Plant Biol. 2010;10:64.Buhtz A, Springer F, Chappell L, Baulcombe DC, Kehr J. Identification and characterization of small RNAs from the phloem of Brassica napus. Plant J. 2008;53(5):739–49.Rodriguez-Medina C, Atkins CA, Mann AJ, Jordan ME, Smith PM. Macromolecular composition of phloem exudate from white lupin (Lupinus albus L.). BMC Plant Biol. 2011;11:36.Pallas V, Gomez G. Phloem RNA-binding proteins as potential components of the long-distance RNA transport system. Frontiers in Plant Science. 2013;4:130.Tournier B, Tabler M, Kalantidis K. Phloem flow strongly influences the systemic spread of silencing in GFP Nicotiana benthamiana plants. Plant J. 2006;47(3):383–94.Hamilton A, Voinnet O, Chappell L, Baulcombe D. Two classes of short interfering RNA in RNA silencing. EMBO J. 2002;21(17):4671–9.Voinnet O. MicroRNA and autophagy--C. elegans joins the crew. EMBO Rep. 2013;14(6):485–7.Dunoyer P, Schott G, Himber C, Meyer D, Takeda A, Carrington JC, et al. Small RNA duplexes function as mobile silencing signals between plant cells. Science. 2010;328(5980):912–6.Brosnan CA, Mitter N, Christie M, Smith NA, Waterhouse PM, Carroll BJ. Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc Natl Acad Sci U S A. 2007;104(37):14741–6.Silva TF, Romanel EA, Andrade RR, Farinelli L, Osteras M, Deluen C, et al. Profile of small interfering RNAs from cotton plants infected with the polerovirus Cotton leafroll dwarf virus. BMC Mol Biol. 2011;12:40.Martinez G, Donaire L, Llave C, Pallas V, Gomez G. High-throughput sequencing of Hop stunt viroid-derived small RNAs from cucumber leaves and phloem. Mol Plant Pathol. 2010;11(3):347–59.Donaire L, Barajas D, Martinez-Garcia B, Martinez-Priego L, Pagan I, Llave C. Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol. 2008;82(11):5167–77.Qi X, Bao FS, Xie Z. Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS One. 2009;4(3):e4971.Pantaleo V, Saldarelli P, Miozzi L, Giampetruzzi A, Gisel A, Moxon S, et al. Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine. Virology. 2010;408(1):49–56.Lin KY, Cheng CP, Chang BC, Wang WC, Huang YW, Lee YS, et al. Global analyses of small interfering RNAs derived from Bamboo mosaic virus and its associated satellite RNAs in different plants. PLoS One. 2010;5(8):e11928.Navarro B, Pantaleo V, Gisel A, Moxon S, Dalmay T, Bisztray G, et al. Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction. PLoS One. 2009;4(11):e7686.Martin R, Arenas C, Daros JA, Covarrubias A, Reyes JL, Chua NH. Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants. Virology. 2007;367(1):135–46.St-Pierre P, Hassen IF, Thompson D, Perreault JP. Characterization of the siRNAs associated with peach latent mosaic viroid infection. Virology. 2009;383(2):178–82.Di Serio F, Gisel A, Navarro B, Delgado S, de Alba AE M, Donvito G, et al. Deep sequencing of the small RNAs derived from two symptomatic variants of a chloroplastic viroid: implications for their genesis and for pathogenesis. PLoS One. 2009;4(10):e7539.Li R, Gao S, Hernandez AG, Wechter WP, Fei Z, Ling KS. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS One. 2012;7(5):e37127.Hu Q, Hollunder J, Niehl A, Korner CJ, Gereige D, Windels D, et al. Specific impact of tobamovirus infection on the Arabidopsis small RNA profile. PLoS One. 2011;6(5):e19549.Hibi T, Furuki I. Melon Necrotic Spot Virus. In: CMI: AAB Descriptions of Plants Viruses N° 302. Kew, UK: Commonwealth Mycological Institute; 1985.Riviere CJ, Rochon DM. Nucleotide sequence and genomic organization of melon necrotic spot virus. J Gen Virol. 1990;71(Pt 9):1887–96.Diaz JA, Nieto C, Moriones E, Truniger V, Aranda MA. Molecular characterization of a Melon necrotic spot virus strain that overcomes the resistance in melon and nonhost plants. Mol Plant Microbe Interact. 2004;17(6):668–75.Navarro JA, Genoves A, Climent J, Sauri A, Martinez-Gil L, Mingarro I, et al. RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins. Virology. 2006;356(1–2):57–67.Genoves A, Navarro JA, Pallas V. A self-interacting carmovirus movement protein plays a role in binding of viral RNA during the cell-to-cell movement and shows an actin cytoskeleton dependent location in cell periphery. Virology. 2009;395(1):133–42.Genoves A, Navarro JA, Pallas V. The Intra- and intercellular movement of Melon necrotic spot virus (MNSV) depends on an active secretory pathway. Mol Plant Microbe Interact. 2010;23(3):263–72.Serra-Soriano M, Pallas V, Navarro JA. A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements. Plant J. 2014;77(6):863–79.Genoves A, Navarro JA, Pallas V. Functional analysis of the five melon necrotic spot virus genome-encoded proteins. J Gen Virol. 2006;87(Pt 8):2371–80.Pallas V, Aparicio F, Herranz MC, Amari K, Sanchez-Pina MA, Myrta A, et al. Ilarviruses of Prunus spp.: a continued concern for fruit trees. Phytopathology. 2012;102(12):1108–20.Pallas V, Aparicio F, Herranz MC, Sanchez-Navarro JA, Scott SW. The molecular biology of ilarviruses. Adv Virus Res. 2013;87:139–81.Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods. 2007;3:12.Blevins T, Rajeswaran R, Aregger M, Borah BK, Schepetilnikov M, Baerlocher L, et al. Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res. 2011;39(12):5003–14.Takeda A, Tsukuda M, Mizumoto H, Okamoto K, Kaido M, Mise K, et al. A plant RNA virus suppresses RNA silencing through viral RNA replication. EMBO J. 2005;24(17):3147–57.Andersson MG, Haasnoot PC, Xu N, Berenjian S, Berkhout B, Akusjarvi G. Suppression of RNA interference by adenovirus virus-associated RNA. J Virol. 2005;79(15):9556–65.Himeno M, Maejima K, Komatsu K, Ozeki J, Hashimoto M, Kagiwada S, et al. Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome. Virology. 2010;396(1):69–75.Aparicio F, Vilar M, Perez-Paya E, Pallas V. The coat protein of prunus necrotic ringspot virus specifically binds to and regulates the conformation of its genomic RNA. Virology. 2003;313(1):213–23.Ruiz-Ruiz S, Navarro B, Gisel A, Pena L, Navarro L, Moreno P, et al. Citrus tristeza virus infection induces the accumulation of viral small RNAs (21-24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile. Plant Mol Biol. 2011;75(6):607–19.Folimonova SY, Folimonov AS, Tatineni S, Dawson WO. Citrus tristeza virus: survival at the edge of the movement continuum. J Virol. 2008;82(13):6546–56.Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, et al. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology. 2009;388(1):1–7.Karyeija RF, Kreuze JF, Gibson RW, Valkonen JP. Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology. 2000;269(1):26–36.Melnyk CW, Molnar A, Bassett A, Baulcombe DC. Mobile 24 nt small RNAs direct transcriptional gene silencing in the root meristems of Arabidopsis thaliana. Curr Biol. 2011;21(19):1678–83.Gosalvez-Bernal B, Genoves A, Navarro JA, Pallas V, Sanchez-Pina MA. Distribution and pathway for phloem-dependent movement of Melon necrotic spot virus in melon plants. Mol Plant Pathol. 2008;9(4):447–61.Harper SJ, Cowell SJ, Robertson CJ, Dawson WO. Differential tropism in roots and shoots infected by Citrus tristeza virus. Virology. 2014;460–461:91–9.Andika IB, Kondo H, Tamada T. Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves. Mol Plant Microbe Interact. 2005;18(3):194–204.Mi S, Cai T, Hu Y, Chen Y, Hodges E, Ni F, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide. Cell. 2008;133(1):116–27.Takeda A, Iwasaki S, Watanabe T, Utsumi M, Watanabe Y. The mechanism selecting the guide strand from small RNA duplexes is different among argonaute proteins. Plant Cell Physiol. 2008;49(4):493–500.Wu L, Zhang Q, Zhou H, Ni F, Wu X, Qi Y. Rice MicroRNA effector complexes and targets. Plant Cell. 2009;21(11):3421–35.Xu Y, Huang L, Fu S, Wu J, Zhou X. Population diversity of rice stripe virus-derived siRNAs in three different hosts and RNAi-based antiviral immunity in Laodelphgax striatellus. PLoS One. 2012;7(9):e46238

Einsatz von Telepräsenzsystemen in der Lehre - Transfer eines interprofessionellen Lehrmoduls zur digital gestützten Kommunikation in das Blockpraktikum "Innere Medizin" während der Covid-19-Pandemie

Objective: The contact restrictions caused by the Covid-19 pandemic fundamentally limit patient-centered teaching. To realize a patient-oriented education in the block training "Internal Medicine" at the University Hospital Halle (Saale) despite the challenges, the already established teaching module "Interprofessional Teleconsultation" was adapted. The short article outlines the interprofessional teaching module including first evaluation results and describes the adapted block training.Method: In the "Internal Medicine" block training, students in a lecture hall navigated a telepresence system, which was accompanied by a physician across the ward and conducted an anamnesis via video and audio transmission without actual patient contact. Results: Students, physicians, and patients were open-minded about this form of communication during the Covid-19 pandemic and quickly got accustomed to the use of the telepresence system. To be able to react to technical challenges (e.g. unstable connection between the communication partners), a careful preparation of the lecturers is necessary. Conclusion: In using a telepresence system, patient-oriented teaching of students in the block training "Internal Medicine" can be ensured with low-threshold technical effort during the Covid-19 pandemic. The telepresence system allows for the involvement of patients into teaching while adhering to the necessary hygiene measures. Despite technical challenges, the teaching format based on telepresence is suitable as an alternative to face-to-face teaching if actual patient contact is not possible.Zielsetzung: Die Kontakteinschränkungen durch die Covid-19-Pandemie beeinträchtigen die patientennahe Lehre grundlegend. Um trotz der besonderen Herausforderungen eine patientennahe Ausbildung im Blockpraktikum "Innere Medizin" am Universitätsklinikum Halle (Saale) zu realisieren, wurde das bereits etablierte Lehrmodul "Interprofessionelles Telekonsil" adaptiert. Der Kurzbeitrag skizziert das interprofessionelle Lehrmodul mit ersten Evaluationsergebnissen und beschreibt das adaptierte Blockpraktikum.Methodik: Im Blockpraktikum "Innere Medizin" navigieren die Studierenden von einem Hörsaal aus ein von einem*r Ärzt*in begleitetes Telepräsenzsystem über die Station und führen so via Video- und Audio-Übertragung ein Anamnesegespräch ohne unmittelbaren Patientenkontakt. Ergebnisse: Sowohl die Studierenden und Ärzt*innen als auch die Patient*innen standen dieser Form der Kommunikation während der Covid-19-Pandemie aufgeschlossen gegenüber und gewöhnten sich schnell an den Umgang mit dem Telepräsenzsystem. Um gegebenenfalls auf technische Herausforderungen (z.B. instabile Verbindung zwischen den Kommunikationspartner*innen) reagieren zu können, ist eine sorgfältige Vorbereitung der Dozent*innen notwendig. Schlussfolgerung: Durch den Einsatz des Telepräsenzsystems kann die patientennahe Ausbildung der Studierenden im Blockpraktikum "Innere Medizin" während der Covid-19-Pandemie mit niederschwelligem technischen Aufwand gewährleistet werden. Das Telepräsenzsystem ermöglicht dabei die Einbindung von Patient*innen in die studentische Lehre bei gleichzeitiger Einhaltung der erforderlichen Hygienemaßnahmen. Trotz technischer Herausforderungen ist das Lehrformat mit Telepräsenz als Alternative zur Präsenzlehre geeignet, wenn ein unmittelbarer Patientenkontakt nicht möglich ist