تغییر بیان ژن‌های مرتبط با اتوفاژی در پاسخ به سرکوبگر خاموشی HC-Pro ویروس ای سیب‌ زمینی (Potato virus A) در گیاه Nicotiana benthamiana

نوع مقاله: مقاله کامل پژوهشی

نویسندگان

1 دانشجویان دکتری، مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز.

2 دانشیار مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز

3 استاد مرکز تحقیقات ویروس شناسی گیاهی، دانشکده کشاورزی، دانشگاه شیراز.

چکیده

در سال­های اخیر نقش اتوفاژی در خاموشی آر­ان­ای القاء شده توسط ویروس از طریق فعالیت پروتئین­های سرکوبگر خاموشی آر­ان­ای به اثبات رسیده است. به منظور درک بهتر نقش احتمالی سرکوبگر خاموشی HC-Pro ویروس ای سیب ­­زمینی در فرآیند اتوفاژی (autophagy)، اثر این سرکوبگر ویروسی روی بیان تعدادی از ژن­های مهم و دخیل در اتوفاژی مورد بررسی قرار گرفت. ژنHC-Pro  ویروس ای سیب ­­زمینی در ناقل بیان pGWB17 با برچسبmyc  در انتهای کربوکسیلی و پروموتور 35S در انتهای آمینی توسط تکنولوژی  gatewayهمسانه­سازی شد. کشت­های اگروباکتریوم حاوی  HC-Proویروس ای سیب ­زمینی با استفاده از سرنگ در ناحیه پشتی برگ­های گیاه Nicotiana benthamianaتزریق شدند. بیان ژن­های ATG2، ATG7،  ATG6و AGO1در پاسخ به سرکوبگر خاموشی HC-Pro ویروس ای سیب ­زمینی، در فاصله زمانی پنج روز پس از تزریق، با استفاده از تکنیک ریل تایم پی­سی­آر (Real-Time PCR) اندازه­گیری شدند. سرکوبگر خاموشیHC-Pro   ویروس ای سیب ­زمینی میزان بیان ژن­های ATG6، ATG2و ATG7را به میزان ۸۹/۵، ۳/۷ و ۶/۷ برابر نسبت به برگ­های تزریق شده با سازه حاوی پلاسمید خالی pGWB افزایش داد. در حالی­که میزان رونوشت  AGO1به­عنوان جزء کلیدی کمپلکس خاموشی القاء شده توسط آر­ان­ای، به میزان ۳۶/۱ برابر نسبت به میزان آن در برگ­های فاقد  HC-Proکاهش نشان داد. نتایج این تحقیق حاکی از دخالت ژن­های ATG2، ATG7، ATG6و AGO1در پاسخ­های دفاعی گیاه  N .benthamianaدر مقابل سرکوبگر خاموشی HC-Pro ویروس ای سیب­ زمینی و امکان استفاده از آن در تدوین روش­های جدید مدیریت ویروس­های گیاهی می­باشد.

کلیدواژه‌ها


عنوان مقاله [English]

Altered expression of autophagy-related genes in Nicotiana benthamiana plants in response to Potato virus A HC-Pro silencing suppressor

نویسندگان [English]

  • A. Tahmasebi 1
  • A. Afsharifar 2
  • S. Rabiee 1
  • K. Izadpanah 3
1 . Ph.D. student of Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran.
3 Professor, Plant Virology Research Center, College of Agriculture, Shiraz University, Shiraz, Iran.
چکیده [English]

Accumulating data have revealed the role of autophagy in virus-induced RNA silencing via viral RNA silencing suppressor activity. To extend our understanding for the possible role of Potato virus A-HC-Pro (PVA-HC-Pro) suppressor protein in autophagy process, we investigated the effect of PVA-HC-Pro on expression of some important genes involved in autophagy. The cDNA of ORF PVA HC-Pro was cloned in pGWB17 vector with a C-terminal myc tag and N-terminal 35S promoter using Gateway technology. Agrobacterium cultures harboring PVA-HC-Pro were infiltrated into the abaxial side of N. benthamiana leaves. The expression of ATG6, ATG2, ATG7 and AGO1 genes were measured at 5 days post infiltration in response to PVA-HC-Pro using qRT-PCR technique. PVA HC-Pro as a suppressor of RNA silencing increased the expression level of ATG6, ATG2 and ATG7 5.89, 7.3 and 7.6 fold, respectively, compared to corresponding values in leaves infiltrated with empty plasmid (EP) as a control. In contrast, the transcript level of Argonaute1 (AGO1), a key component of RNA-induced silencing complex (RISC), was decreased by 1.36-fold compared to the level of AGO1in infiltrated leaves without PVA-HC-Pro. Results of this study indicated that PVA HC-Pro can alter the expression of autophagy related genes in N. benthamiana plant. These findings suggest the involvement of ATG6, ATG2, ATG7 and AGO1 in defense responses of N. benthamiana against PVA-HC-Pro, which might be considered in plant breeding programs as a novel approach to the control of plant viruses.

کلیدواژه‌ها [English]

  • : Autophagy
  • HC-Pro
  • N. benthamiana
  • RNA silencing
  • Suppressor of RNA Silencing
Agius C., Eamens A. L., Millar A. A., Watson J. M. and Wang M. B. 2012. RNA silencing and antiviral defense in plants. Antiviral Resistance in Plants. Methods and Protocols 894: 17-38.

 

Alvarado V. Y. and Scholthof H. B. 2012. AGO2: a new Argonaute compromising plant virus accumulation. Frontiers in Plant Science 2:112.

 

Ascencio-Ibáñez J. T., Sozzani R., Lee T.J., Chu T. M., Wolfinger R. D., Cella R. and Hanley-Bowdoin L. 2008. Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection. Plant Physiology 148(1): 436-454.

 

Azevedo J., Garcia D., Pontier D., Ohnesorge S., Yu A., Garcia S. and Voinnet O. 2010. Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes and Development 24(9): 904-915.

 

Ballut L., Drucker M., Pugniere M., Cambon F., Blanc S., Roquet F. and Badaoui S. 2005. HC-Pro, a multifunctional protein encoded by a plant RNA virus, targets the 20S proteasome and affects its enzymic activities. Journal of General Virology 86(9): 2595-2603.

 

Bassham D. C., Laporte M., Marty F., Moriyasu Y., Ohsumi Y., Olsen L. J. and Yoshimoto K. 2006. Autophagy in development and stress responses of plants. Autophagy 2(1): 2-11.

 

Boya P., Reggiori F. and Codogno P. 2013. Emerging regulation and functions of autophagy. Nature cell biology 15(7): 713-720.

 

Brigneti G., Voinnet O., Li W. X., Ji L. H., Ding S. W. and Baulcombe D. C. 1998. Retracted: Viral pathogenicity determinants are suppressors of transgene silencing in Nicotiana benthamiana. The EMBO journal 17(22): 6739-6746.

 

Carbonell A. and Carrington J. C. 2015. Antiviral roles of plant ARGONAUTES. Current Opinion in Plant Biology 27: 111-117.

 

Cheng X. and Wang A. 2017. The potyvirus silencing suppressor protein VPg mediates degradation of SGS3 via ubiquitination and autophagy pathways. Journal of virology 91(1): e01478-16.

 

Chiera J. M., Bouchard R. A., Dorsey S. L., Park E., Buenrostro-Nava M. T., Ling P. P. and Finer J. J. 2007. Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Reports 26(9): 1501-1509.

 

Chiera J. M., Lindbo J. A. and Finer J. J. 2008. Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing. Transgenic Research 17(6): 1143-1154.

 

Chiu M. H., Chen I. Baulcombe D. C. and Tsai C. H. 2010. The silencing suppressor P25 of Potato virus X interacts with Argonaute1 and mediates its degradation through the proteasome pathway. Molecular plant pathology 11(5): 641-649.

 

Chung T., Suttangkakul A. and Vierstra R. D. 2009. The ATG autophagic conjugation system in maize: ATG transcripts and abundance of the ATG8-lipid adduct are regulated by development and nutrient availability. Plant physiology 149(1): 220-234.

 

Deretic V. 2012. Autophagy as an innate immunity paradigm: expanding the scope and repertoire of pattern recognition receptors. Current Opinion in Immunology 24(1): 21-31.

 

Derrien B., Baumberger N., Schepetilnikov M., Viotti C., De Cillia J., Ziegler-Graff V. and Genschik P. 2012. Degradation of the antiviral component ARGONAUTE1 by the autophagy pathway. Proceedings of the National Academy of Sciences 109(39): 15942-15946.

 

Ding S. W. 2010. RNA-based antiviral immunity. Nature Reviews Immunology 10(9): 632-644.

 

Duncan D. B. 1951. A significance test for differences between ranked treatments in an analysis of variance. Virginia journal of science 2: 171–189.

 

Garcia-Ruiz H., Carbonell A., Hoyer J. S., Fahlgren N., Gilbert K. B., Takeda A. and Baladejo M. T. M. 2015. Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection. PLoS Pathogens 11(3): e1004755.

 

Germundsson A., Savenkov E. I., Ala-Poikela M. and Valkonen J. P. 2007. VPg of Potato virus A alone does not suppress RNA silencing but affects virulence of a heterologous virus. Virus genes 34(3): 387-399.

 

Giner A., Lakatos L., García-Chapa M., López-Moya J. J. and Burgyán J. 2010. Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs. PLoS Pathogens 6(7): e1000996.

 

Gursinsky T., Pirovano W., Gambino G., Friedrich S., Behrens S. E. and Pantaleo V. 2015. Homeologs of the Nicotiana benthamiana antiviral ARGONAUTE1 show different susceptibilities to microRNA168-mediated control. Plant Physiology 168(3): 938-952.

 

Han T., Chen Q. and Liu H. 2016. Engineered photoactivatable genetic switches based on the bacterium phage T7 RNA polymerase. ACS Synthetic Biology.

 

Harvey J. J., Lewsey M. G. Patel K. Westwood J. Heimstädt S. Carr J. P. and Baulcombe D. C. 2011. An antiviral defense role of AGO2 in plants. PloS one 6(1): e14639.

 

Harvey J. J., Lewsey M. G., Patel K., Westwood J., Heimstadt S., Carr J. P. and Baulcombe D. C. 2011. An antiviral defense role of AGO2 in plants. PLoS ONE 6: e14639.

 

Havelda Z., Várallyay É., Válóczi A. and Burgyán J. 2008. Plant virus infection‐induced persistent host gene downregulation in systemically infected leaves. The Plant Journal 55(2): 278-288.

 

Inoue Y., Suzuki T., Hattori M., Yoshimoto K., Ohsumi Y. and Moriyasu Y. 2006. At ATG genes, homologs of yeast autophagy genes, are involved in constitutive autophagy in Arabidopsis root tip cells. Plant and Cell Physiology 47(12): 1641-1652.

 

Jauber M., Bhattacharjee S., Mello A. F., Perry K. L. and Moffett P. 2011. ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis. Plant Physiology 156(3): 1556-1564.

 

Johansen L. K. and Carrington J. C. 2001. Silencing on the spot. induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiology126(3): 930-938.

 

Kasschau K. D. and Carrington J. C. 1998. A counter defensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95(4): 461-470.

 

Klionsky D. J. 2005. The molecular machinery of autophagy: unanswered questions. Journal of Cell Science 118(1): 7-18.

 

Levine B., Mizushima N. and Virgin H. W. 2011. Autophagy in immunity and inflammation. Nature 469(7330): 323-335.

 

Li F. and Ding S. W. 2006. Virus counter defense: diverse strategies for evading the RNA-silencing immunity. Annual Review of Microbiology 60: 503.

 

Liu Y. and Bassham D. C. 2012. Autophagy: pathways for self-eating in plant cells. Annual Review of Plant Biology 63: 215-237.

 

Liu L., Chung H. Y., Lacatus G., Baliji S., Ruan J. and Sunter G. 2014. Altered expression of Arabidopsis genes in response to a multifunctional geminivirus pathogenicity protein. BMC Plant Biology 14(1): 302.

 

Liu Y., Schiff M., Czymmek K., Tallóczy Z., Levine B. and Dinesh-Kumar S. P. 2005. Autophagy regulates programmed cell death during the plant innate immune response. Cell 121(4): 567-577.

 

Livak K. J. and Schmittgen T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods 25(4): 402-408.

 

Lukhovitskaya N. I., Solovieva A. D., Boddeti S. K., Thaduri S., Solovyev A. G. and Savenkov E. I. 2013. An RNA virus-encoded zinc-finger protein acts as a plant transcription factor and induces a regulator of cell size and proliferation in two tobacco species. The Plant Cell 25(3): 960-973.

 

Lukhovitskaya N. I., Vetukuri R. R., Sama I., Thaduri S., Solovyev A. G. and Savenkov E. I. 2014. A viral transcription factor exhibits antiviral RNA silencing suppression activity independent of its nuclear localization. Journal of General Virology 95(12): 2831-2837.

 

Miozzi L., Napoli C., Sardo L. and Accotto G. P. 2014. Transcriptomics of the interaction between the monopartite phloem-limited geminivirus tomato yellow leaf curl Sardinia virus and Solanum lycopersicum highlights a role for plant hormones, autophagy and plant immune system fine tuning during infection. PloS one 9(2); e89951.

 

Morel J. B., Godon C., Mourrain P., Béclin C., Boutet S., Feuerbach F. and Vaucheret H. 2002. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. The Plant Cell 14(3): 629-639.

 

Nakagawa T., Kurose T., Hino T., Tanaka K., Kawamukai M., Niwa Y., Toyooka K., Matsuoka K. and Jinbo T. 2007. Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. Journal of Bioscience and Bioengineering 104: 34–41.

 

Nakahara K. S., Masuta C., Yamada S., Shimura H., Kashihara Y., Wada T. S. and Sekiguchi T. 2012. Tobacco calmodulin-like protein provides secondary defense by binding to and directing degradation of virus RNA silencing suppressors. Proceedings of the National Academy of Sciences 109(25): 10113-10118.

Pirone T. P. and Blanc S. 1996. Helper-dependent vector transmission of plant viruses. Annual review of phytopathology 34(1): 227-247.

 

Qu F., Ye X. and Morris T. J. 2008. Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proceedings of the National Academy of Sciences 105(38): 14732-14737.

 

Rajamaki M. L., Maki-Valkama T., Makinen K. and Valkonen J. P. 2004. Infection with potyviruses. In Plant-Pathogen Interactions. N. Talbot, ed (Oxford, UK: Blackwell Publishing), pp. 68–91.

 

Redondo E., Krause-Sakate R., Yang S. J., Lot H., Le Gall O. and Candresse T. 2001. Lettuce mosaic virus pathogenicity determinants in susceptible and tolerant lettuce cultivars map to different regions of the viral genome. Molecular Plant-Microbe Interactions 14(6): 804-810.

 

Rojas M. R., Zerbini F. M., Allison R. F., Gilbertson R. L., Lucas Sáenz P., Salvador B., Simón-Mateo C., Kasschau K. D., Carrington J. C. and García J. A. 2002. Host-specific involvement of the HC protein in the long-distance movement of potyviruses. Journal of Virology 76(4): 1922-1931.

 

Sahana N., Kaur H., Tena F., Jain R. K., Palukaitis P., Canto T. and Praveen S. 2012. Inhibition of the host proteasome facilitates papaya ringspot virus accumulation and proteosomal catalytic activity is modulated by viral factor HC-Pro. PLoS One 7(12): e52546.

 

Scholthof K. B. G., Adkins S., Czosnek H., Palukaitis P., Jacquot E., Hohn T. and Hemenway C. 2011. Top 10 plant viruses in molecular plant pathology. Molecular Plant Pathology 12(9): 938-954.

 

Shin J. H., Yoshimoto K., Ohsumi Y., Jeon J. S. and An G. 2009. OsATG10b, an autophagosome component, is needed for cell survival against oxidative stresses in rice. Molecules and cells 27(1): 67-74.

 

Shoji-Kawata S. and Levine B. 2009. Autophagy, antiviral immunity, and viral countermeasures. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research 1793(9): 1478-1484.

 

Sláviková S., Shy G., Yao Y., Glozman R., Levanony H., Pietrokovski S. and Galili G. 2005. The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants. Journal of Experimental Botany 56(421): 2839-2849.

 

Stephan D., Slabber C., George G., Ninov V., Francis K. P. and Burger J. T. 2011. Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging. Plant Methods 7(1): 25.

 

Su W., Ma H., Liu C., Wu J. and Yang J. 2006. Identification and characterization of two rice autophagy associated genes, OsAtg8 and OsAtg4. Molecular Biology Reports 33: 273-278.

 

Tadamura K., Nakahara K. S., Masut C. and Uyeda I. 2012 Wound-induced rgs-CaM gets ready for counterresponse to an early stage of viral infection. Plant Signal Behav 7: 1548–1551.

 

Urcuqui-Inchima S., Haenni A. L. and Bernardi F. 2001. Potyvirus proteins: a wealth of functions. Virus Research 74(1): 157-175.

 

Várallyay É., Válóczi A., Ágyi Á., Burgyán J. and Havelda Z. 2010. Plant virus‐mediated induction of miR168 is associated with repression of ARGONAUTE1 accumulation. The EMBO journal 29(20): 3507-3519.

 

Vaucheret H. 2008. Plant argonautes. Trends in Plant Science 13(7): 350-358.

 

Voinnet O. 2005. Induction and suppression of RNA silencing: insights from viral infections. Nature Reviews Genetics 6(3): 206-220.

 

Voinnet O. 2009. Origin, biogenesis, and activity of plant microRNAs. Cell 136(4): 669-687.

 

Voinnet O., Rivas S., Mestre P. and Baulcombe D. 2003. Retracted: an enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. The Plant Journal 33(5): 949-956.

 

 

Wang X. B., Jovel J., Udomporn P., Wang Y., Wu Q., Li W. X. and Ding S. W. 2011. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana. The Plant Cell 23(4): 1625-1638.

 

Wang Y., Gaba V., Yang J., Palukaitis P. and Gal-On A. 2002. Characterization of synergy between Cucumber mosaic virus and potyviruses in cucurbit hosts. Phytopathology 92(1): 51-58.

 

Wang Y., Yu B., Zhao J., Guo J., Li Y., Han S., Huang L., Du Y., Hong Y., Tang D. and Liu Y. 2013. Autophagy contributes to leaf starch degradation. The Plant Cell 25(4): 1383-1399.

 

Weigel D. and Glazebrook J. 2006. Transformation of agrobacterium using the freeze-thaw method. Cold Spring Harb Protoc doi/10.1101/pdb.prot4665.

 

Xiong Y., Contento A. L., Nguyen P. Q. and Bassham D. C. 2007. Degradation of oxidized proteins by autophagy during oxidative stress in Arabidopsis. Plant Physiology 143(1): 291-299.

 

Zhang X., Yuan Y. R., Pei Y., Lin S. S., Tuschl T., Patel D. J. and Chua N. H. 2006. Cucumber mosaic virus-encoded 2b suppressor inhibits Arabidopsis Argonaute1 cleavage activity to counter plant defense. Genes & Development 20(23): 3255-3268.

 

Zhang Y. H. P., Himmel M. E. and Mielenz J. R. 2006. Outlook for cellulase improvement: screening and selection strategies. Biotechnology Advances 24(5): 452-481.

 

Zhou T., Murphy A. M., Lewsey M. G., Westwood J. H., Zhang H. M., Gonzalez I. and Carr J. P. 2014. Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection. Journal of General Virology 95(6): 1408-1413.