THE ROLE OF VIRAL EFFECTOR PROTEINS IN SUPPRESSION OF PLANT ANTIVIRAL DEFENSES BASED ON RNA SILENCING AND INNATE IMMUNITY Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch‐Naturwissenschaftlichen Fakultät der Universität Basel von Golyaev Victor von Moscau, Russland Basel, 2017 Originaldokument gespeichert auf dem Dokumentenserver der Universität Basel edoc.unibas.ch Genehmigt von der Philosophisch–Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Thomas Boller, PD Dr. Mikhail Pooggin und Dr. Todd Blevins Basel, den 20 Juni 2017 Prof. Dr. Martin Spiess Dekan TABLE OF CONTENT Summary ...................................................................................................................................................... 3 1. General Introduction ............................................................................................................................ 4 1.1. Plant viruses ....................................................................................................................................... 4 1.2. Family Caulimoviridae ....................................................................................................................... 4 1.3. Rice tungro bacilliform virus ............................................................................................................. 6 1.4. Cauliflower mosaic virus ................................................................................................................. 11 1.5 RNA silencing in plants .................................................................................................................... 19 1.5.1 Introduction ................................................................................................................................ 19 1.5.2 RNA silencing pathways ............................................................................................................ 21 1.5.3 RNA silencing in rice ................................................................................................................. 25 1.5.4 Antiviral RNA silencing ............................................................................................................. 27 1.5.5 Viral suppressors of RNA silencing ........................................................................................... 30 1.6 Plant innate immunity ....................................................................................................................... 35 1.6.1 Introduction ................................................................................................................................ 35 1.6.2 Pattern-triggered immunity ........................................................................................................ 36 1.6.3 Effector-triggered immunity ...................................................................................................... 37 1.6.4 Innate immunity in rice .............................................................................................................. 39 1.6.4 Plant viruses and innate immunity ............................................................................................. 42 2. Aims ...................................................................................................................................................... 45 3. Materials and methods........................................................................................................................ 46 4. Results .................................................................................................................................................. 55 4.1 RTBV P4 can suppress cell-to-cell spread of RNA silencing ........................................................... 55 4.2 RTBV P4 and CaMV P6 suppress oxidative burst ............................................................................ 58 4.3 RTBV P4 counteracts TBSV p19-mediated suppression of cell autonomous RNA silencing.......... 60 4.4 Mutation of the P4 F-Box motif inhibits suppression of cell-to-cell spread of RNA silencing and oxidative burst ......................................................................................................................................... 62 4.5 The N-terminal RTBV P4 region is required for P4 anti-silencing activity, but dispensible for P4- mediated suppression of innate immunity ............................................................................................... 66 4.6 P6 from strain D4 failed to suppress oxidative burst, but not RNA silencing .................................. 68 4.7 RTBV circular dsDNA evades cytosine methylation in infected rice plants .................................... 70 4.8 Rice plants overexpressed OsAGO18 protein are resistant to RTBV infection ................................ 73 4.9 The P4 F-box is likely required for RTBV infectivity ...................................................................... 74 5. Discussion ............................................................................................................................................. 77 5.1 RTBV P4 is a supressor of host plant antiviral responses ................................................................. 77 5.2 The importance of RTBV P4 F-box-like and N-terminal motifs for P4-mediated supression of host plant antiviral responses .......................................................................................................................... 79 5.3 Pathogenicity and the host range of different camv strains is determined by P6-mediated suppression of innate immunity .............................................................................................................. 81 5.4 RTBV evades siRNA-directed DNA methylation in infected rice plants ......................................... 82 1 5.5 OsAgo18 transgenic rice plants are more resistant to RTBV infection ............................................ 83 6. Concluding remarks ............................................................................................................................ 85 Acknowledgments ..................................................................................................................................... 86 References .................................................................................................................................................. 87 List of abbreviations ............................................................................................................................... 119 2 SUMMARY Plant viruses are widespread and economically important pathogens. Currently, there are more than one thousand viruses that are known to be potentially capable of infecting plants and new viruses are being discovered every day. Many of them could cause important diseases of various cultivated plants that humans grow for food, fiber, feed, construction material and biofuel. Therefore understanding the biology of plant viruses is important for development and improvement of cultivated plant resistance to viral pathogens. A major role in plant resistance against viruses belongs to the process called RNA silencing, that targets both RNA and DNA viruses through the small RNA-directed RNA degradation and DNA methylation pathways. In addition, plants respond to virus infection using an innate immune system that recognizes microbe-associated molecular patterns (MAMPs) of potential pathogens and elicits both local and systemic defense responses. However, in order to be succesfull and break the host resistance, plant viruses have evolved a variety of counter-defense mechanisms such as expressing effector proteins, which are used to downregulate plant antiviral responses. Here, we performed comparative investigation of viral effector proteins from two distanly-related pararetroviruses, Cauliflower mosaic virus (CaMV) and Rice tungro bacilliform virus (RTBV), to understand their role in the suppression of plant antiviral defenses based on RNA silencing and innate immunity. The CaMV P6 protein has previously been shown to serve as a silencing suppressor, while the function of RTBV P4 protein was unknown. Through the use of a classical transient assay in leaves of the N. benthamiana transgenic line 16c we show that RTBV P4 can suppress cell-to-cell spread of transgene silencing, but enhance cell autonomous transgene silencing, which correlates with reduced accumulation of 21-nt siRNAs and increased accumulation of 22-nt siRNAs, respectively. Furthermore, we demonstrate that CaMV P6 from strain CM1841 and RTBV P4 proteins are able to suppress the early plant innate immunity responses, such as oxidative burst. In contrast, CaMV P6 from strain D4 failed to suppress innate immunity, but was capable of suppressing RNA silencing as P6 protein from strain CM1841. We also elucidated the role of P4 F-box-like motif and N-terminal domain that are required for RTBV P4 effector functions and protein stability, respectively. Finally, through the use of agroinoculation of Oryza sativa plants with RTBV infectious clone we tested if the P4 F-box motif is required for infectivity and our preliminary results show that the F-box mutant virus exhibts drastically reduced infectivity. Furthermore, we found that RTBV circular double-stranded DNA evades siRNA-directed cytosine methylation in infected rice plants and that rice plants overexpressing an OsAGO18 protein