Inhibition of siRNA methylation is one of the widely accepted mechanisms by which HCPro suppresses antiviral RNA silencing (Jamous et al., 2011). However, the mechanism underlying deactivation of HEN1-mediated siRNA methylation was unknown. In this study we show that HCPro interacts with SAMS and SAHH in planta and downregulates SAMS activity. This act of disrupting methionine cycle could be a strategy adopted by HCPro to inhibit HEN1-mediated siRNA methylation. Accordingly, downregulation of SAMS, SAHH and HEN1 was shown to rescue PVADHCPro expression, further consolidating relevance of this strategy in context of PVA infection. Interestingly, disruption of the methionine cycle was also found to be one of the instrumental factors underlying induction of synergism during a mixed PVX-PVA infection. In addition to siRNA destabilization, we also observed synergism-specific downregulation of GSH biosynthesis. Since, this phenomenon was observed only when methionine cycle disruption / HCPro overexpression was carried out in conjunction to PVX infection, we proposed involvement of a yet to be identified PVX component in this process. Moreover, the mode of action of this PVX-component remains to be studied. A recent finding on potex-potyviral synergism indicated that PVX pathogenicity determinant P25 along with HCPro, induces ER stress and UPR ultimately leading to PCD (Aguilar et al., 2018). Multiple lines of evidence suggesting correlation between oxidative stress, ER stress and UPR (reviewed in Malhotra and Kaufman, 2007;
Eletto et al., 2014; Cao and Kaufman 2014; Amodio et al., 2018) makes it reasonable to hypothesize that, these two independent observations during synergism might somehow be interconnected. If so, P25 could well be the missing PVX-factor responsible for synergism specific GSH depletion, we proposed in this study. Therefore, implication of P25-HCPro interaction in induction of oxidative stress, could be a potential point to be studied in future.
Molecular cues involved in perturbation of these pathways and possible involvement of other PVX proteins therein could also be investigated. In another line of study, tissue / cell overlapping of PVXGFP and PVARFP could be studied. This would be convenient to address as both the viruses are already tagged with fluorescent markers. However, it could reveal a lot s of information on PVX-PVA mixed infection like the temporal and spatial distribution of the individual viruses and the extent of their overlap during the period of infection.
In this study presence of a core complex composed of several host and viral components has been envisioned. This complex has been proposed to guide vRNAs from replication to encapsidation. During different stages of infection, the complex is predicted to dynamically release or recruit host and viral factors to generate specialized bodies like PGs and translation associated HMW complexes. HCPro and VCS are currently thought to be two essential components of this core complex. In the future, attempt could be made to identify other factors required to form this complex. Also, in planta association of HCPro and VCS is demonstrated in this work, however, whether they interact directly or not is not known yet.
In future, development of a method to elucidate their nature of interaction, might be worth
54
exploring. This study also proposes the presence of a 5’UTR associated HMW complex promoting PVA translation and encapsidation, and provided evidence for increased RLUC/RNA ratio as a possible sign of translational enhancement. One of the questions unresolved in this study is the mechanism behind HCPro-VCS-VPg co-ordinated active translation of PVA RNA from lower amount of total RNA. This could well be a case of release in translational repression, however, with methods available at the moment it is difficult to predict the actual reason. During active infection in the cells the total vRNA pool can be divided into many fractions: within VRCs, PGs, polysome bound and stored in virions. Since we do not know the actual amount of vRNA in active translation, any estimation of the relief of translational repression could be biased due to misinterpretation of the polysome-bound vRNA amount. In the future, a method to segregate and quantify polysome-bound vRNAs contributing to active translation could be developed. With this information the questions about the regulatory roles of 5’UTR associated HMW complexes in PVA translation could be easier to address. Role of another host factor, which we did not address in this study, is AGO1. Evidences exist to suggest that it could have both anti- and pro-viral roles (Iwakawa and Tomari 2013; Garcia-Ruiz et al., 2015; Hafren et al., 2015).
We found AGO1 to be present in HCPro-derived ribosome-associated HMW complexes. It could be possible that AGO1, though an innately anti-viral host protein, is hijacked to serve in a pro-viral purpose. An in depth study of HCPro-AGO1 interaction is proposed as a prospective future work.
55 6. ACKNOWLEDGEMENTS
First of all, I would like to express my heartfelt gratitude to my supervisor Dr. Kristiina Mäkinen for giving me the opportunity to work in her group. When I joined this group I had no prior experience of working with viruses or plant systems. I thank her profusely for placing her trust on me. She always encouraged me to think independently and made time for discussions, even from her busiest schedules. She happily took the pains of critically going through all my writings, and refining them with necessary corrections and constructive inputs. She has taken care of even the smallest details of my wellbeing both in and out of the lab. No words are enough to express my gratitude towards her. It is the fruits of her excellent supervision and meticulous planning that I have reached up to the point of defending my thesis.
My next earnest gratitude is reserved for Dr. Konstantin Ivanov. I thank him for training me during my initial days. He helped me familiarize in this lab and taught me most of the techniques, which I used throughout the following years on a daily basis. Despite having shared a really short journey with him, he left a profound impact on my overall thought process. I was deeply inspired by him, and still now while designing an experiment or analysing a result, I feel his influence in the inner recesses of my mind.
I sincerely thank Professor Uwe Sonnewald for accepting the invitation for being my opponent. I am also grateful to the pre-examiners Dr. Saijaliisa Kangasjärvi and Professor Miguel Aranda for painstakingly going through my thesis. Their constructive suggestions and critical comments were instrumental in helping me improve my thesis.
This journey wouldn’t have been complete without regular evaluation and really apt inputs time-to-time from my thesis committee members: Dr. Katri Eskelin and Dr. Markku Varjosalo. I thank them for their close monitoring of my progress and ensuring that everything sticks to the timeline. I thank Professor Jari Valkonen, for always being there as the co-ordinator of BRAVE program and the professor-in-charge for my thesis, and dealing with so much of official things throughout this period. I thank him for arranging learning journal sessions with my fellow BRAVE colleagues during the earlier days of my studies.
As I look back now, I can see that those sessions have genuinely helped me get accustomed to the field of plant virus research and its diversities, in a really short duration of time.
This work was carried out at the Department of Microbiology, Faculty of Agriculture and Forestry, University of Helsinki during the years 2014-2019. I would like to thank all the members of the Department for providing me a wonderful atmosphere and facilities to work.
I would also like to thank BRAVE (Erasmus Mundus) scholarship program and Kristiina for providing me financial support to carry out this work. I have been enrolled under Doctoral Programme in Plant Sciences (DPPS). I would like to avail myself of the opportunity to thank DPPS and Viikki Plant Science Centre (ViPS) for arranging countless excellent meetings, retreats and miscellaneous events, which gave me exposure to all the cutting edge
56
researches going on in this field. It helped me increase my visibility in the scientific community and also develop important connections. Most importantly, I would like to express my sincere gratitude to Dr. Karen Sims-Huopaniemi (Doctoral Education Planning Officer). She has been always very friendly and easy to approach, and one-step-solution to all big and small problems during the entire period.
I am also deeply grateful to Professor Santiago Elena for hosting me in his lab for two months. Working in his lab was really an eye opener for me, tools and techniques on visualization and analysis of interaction networks helped me see the bigger picture. I will treasure the experience of this visit forever.
I am deeply indebted to my colleagues Dr. Maija Pollari, Dr. Andres Lõhmus, Ms. Shreya Saha and Ms. Pinky Dutta for providing me a really friendly working atmosphere. I learnt many things from them during all these years of working together and I consider these, as assets for lifetime. I thank Maija for being a constant source of inspiration. I always looked up to her and considered her as an embodiment of efficiency and organization skills. I would also like to thank Andres specially for all the interesting scientific and non-scientific discussions we used to have. Perhaps he was the one whom I disturbed most with all my questions. He was always there to listen everything patiently and ready to help. Shreya had always been more of a friend than a colleague. I want to thank her for all her helps throughout the entire period and brightening my days both in and out of the lab. Pinky is the latest addition to the cadre. I really value her presence of mind and I thank her for all the discussions we had, especially during the last year. I warmly thank all the former lab-mates and trainees: Dr. Jane Besong-Ndika, Dr. Gabriella Chavez-Calvillo, Ms. Marta Bašić, Ms.
Brenda Felix, Mr. Ville Veijalainen, and Mr. Saad Bin Eusuf. I deem it as a privilege to work beside them. I thank them all for their company during lunch and coffee breaks.
I sincerely thank Ms. Säde Virkki for her excellent work in the green-house. She was the unsung hero behind all the successful experiments. I would also like to thank Ms. Mervi Lindman (EM-unit) and Ms. Sini Miettinen (LC-MS facility) for their excellent technical help. A special thanks is reserved for Mr. Matti Wahlsten. I thank him for taking the lead in developing the LC-MS protocol for GSH quantification and for all his inputs in the synergism paper. I would also like to thank Mr. Mika Kalsi and Mr. Pekka Oivanen for kindly lending their helping hands whenever needed.
I earnestly thank all my friends in Helsinki and outside for sharing their life with me during this period. I thank them for reminding me that there is also a life outside the lab.
Finally, I would like to express my warmest gratitude to all my family members. I thank them for all the love, care and tireless support they had given to me. I thank them for keeping faith in me, encouraging me during the hard times and cheering me up when I felt let down.
Without their unconditional love and endless sacrifices completion of this endeavour would not have been possible.
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