Mechanismus der Weitergabe und Eliminierung einer parasitären RNA in der männlichen Keimbahn anhand ökonomisch wichtiger Viroid-Spezies

• Antragsteller: Professor Dr. Gerhard Steger
• Fachliche Zuordnung: Bioinformatik und Theoretische Biologie ; Virologie ; Zell- und Entwicklungsbiologie der Pflanzen
• Förderung: Förderung von 2018 bis 2020
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 391033695

• Erstellungsjahr: 2024

Zusammenfassung der Projektergebnisse

Viroids are plant-infectious, single-stranded, circular, non-coding, autonomously replicating RNAs. Several viroid diseases are a threat to agriculture; recent examples are hop latent viroid infecting hemp and cannabis, and citrus bark cracking viroid (CBCVd) as the causal agent of a severe hop stunt disease. Viroids can be either pollen-transmissible (spread via pollen) or eliminated from developing pollen, depending on the plant-pathogen combination. We used Nicotiana tabacum (tobacco) as a pollen research model to analyze transcriptomic, degradomic and proteomic data after infection with a hop isolate of apple fruit crinkle viroid (AFCVd) for the identification of mechanism(s) suppressing viroid-propagation and leading to viroid elimination at defined check points during male germline development. These data were supplemented by analyses of Nicotiana benthamiana pollen after plant infection and/or ectopic transformation with cDNAs of AFCVd, CBCVd, and potato spindle tuber viroid (PSTVd). All viroids propagated to high levels in immature anthers similar to leaves, while their levels were drastically reduced in mature pollen of infected N. benthamiana, respectively. The decrease of viroid levels during pollen development correlated with mRNA accumulation of several RNA-degrading factors. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZF and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucine zipper family protein in apical meristem, is essential for a network responsible for some morphological changes and modifications of the plant degradome within shoot meristem regulation and secondary xylem differentiation. A low level of TFIIIA-7ZF in pollen is involved in depression of viroid replication but also enables a high level of degradome expression during pollen development, resulting in complete viroid elimination. Forcing of viroid RNA to express in higher levels in pollen by transgenosis confirmed a strong pathogenesis caused by pollen non-transmissible viroids. This suggests some adaptation of pollen-transmissible viroids to pollen metabolism.

Projektbezogene Publikationen (Auswahl)

• Predicting the Structure of a Viroid. Methods in Molecular Biology, 331-371. Springer US.
  Steger, Gerhard
  
• Elimination of apple fruit crinkle viroid (AFCVd) during anther development and its depressed propagation in pollen of Nicotiana tabacum. International Conference on Viroids and Viroid-Like RNAs. July 5–7, 2018, Valencia, Spain
  Matoušek J., Steinbachová L., Killi U.K., Mishra A.K., Sano T., Honys D. & Steger G.
  
• Viroid research and its significance for RNA technology and basic biochemistry. Nucleic Acids Research.
  Steger, Gerhard & Riesner, Detlev
  
• Viroids: at the forefront of RNA technology and RNA biology. International Conference on Viroids and Viroid-Like RNAs. July 5–7, 2018, Valencia, Spain
  Steger G.
  
• Elimination of hop viroids from pollen. In: Proceedings of the Scientific-Technical Commission; International Hop Growers Convention Bischoffsheim, Alsace, France. July 07-11, 2019, p. 104, ISSN 2512-3785.
  Matoušek J., Honys D., Killi U.K., Mishra A.K., Steinbachová L., Kocábek T., Radišek S., Sano T. & Steger G.
  
• Some changes in RNA expression caused by hop viroids in pollen. In: Proceedings of the Scientific-Technical Commission; International Hop Growers Convention Bischoffsheim, Alsace, France. July 7–11, 2019, p. 105, ISSN 2512-3785.
  Mishra A.K., Steinbachová L., Honys D., Sukumari Nath V., Mishra D., Kumar A., Steger G. & Matoušek J.
  
• Elimination of Viroids from Tobacco Pollen Involves a Decrease in Propagation Rate and an Increase of the Degradation Processes. International Journal of Molecular Sciences, 21(8), 3029.
  Matoušek, Jaroslav; Steinbachová, Lenka; Drábková, Lenka Záveská; Kocábek, Tomáš; Potěšil, David; Mishra, Ajay Kumar; Honys, David & Steger, Gerhard
  
• Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum. International Journal of Molecular Sciences, 21(22), 8700.
  Shrestha, Ankita; Mishra, Ajay Kumar; Matoušek, Jaroslav; Steinbachová, Lenka; Potěšil, David; Nath, Vishnu Sukumari; Awasthi, Praveen; Kocábek, Tomáš; Jakse, Jernej; Drábková, Lenka Záveská; Zdráhal, Zbyněk; Honys, David & Steger, Gerhard
  
• The decrease of the propagation rate of hop viroids in pollen and a potential multifunctional role of transcription factor TFIIIA. In: Proceedings of the Vth International Humulus Symposium, March 8–12, 2021; Stuttgart, Germany; International Society for Horticultural Science, pp. 36.
  Matoušek J., Kocábek T., Mishra A.K., Radisek T. & Steger G.
  
• Transformation of Seed Non-Transmissible Hop Viroids in Nicotiana benthamiana Causes Distortions in Male Gametophyte Development. Plants, 10(11), 2398.
  Steinbachová, Lenka; Matoušek, Jaroslav; Steger, Gerhard; Matoušková, Helena; Radišek, Sebastjan & Honys, David
  
• Conserved Motifs and Domains in Members of Pospiviroidae. Cells, 11(2), 230.
  Wüsthoff, Kevin-Phil & Steger, Gerhard
  
• The Splicing Variant TFIIIA-7ZF of Viroid-Modulated Transcription Factor IIIA Causes Physiological Irregularities in Transgenic Tobacco and Transient Somatic Depression of “Degradome” Characteristic for Developing Pollen. Cells, 11(5), 784.
  Matoušek, Jaroslav & Steger, Gerhard
  
• Viroids: Non-coding Circular RNAs Are Tiny Pathogens Provoking a Broad Response in Host Plants. RNA Technologies, 295-309. Springer International Publishing.
  Steger, Gerhard; Wüsthoff, Kevin P.; Matoušek, Jaroslav & Riesner, Detlev
  
• “Pathomorphogenic” Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco. International Journal of Molecular Sciences, 24(9), 7790.
  Matoušek, Jaroslav; Wüsthoff, Kevin P. & Steger, Gerhard