WELCOME MPP 602 ADVANCE VIRIOLOGY Autophagy may be

WELCOME MPP 602 ADVANCE VIRIOLOGY

Autophagy may be divided into several major steps, including induction, nucleation and expansion of the phagophore, maturation of the autophagosomes, and docking/fusion with the vacuole or lysosome, followed by degradation and breakdown.

ü Potyviruses represent the largest group of known plant viruses with a positive-sense, singlestranded RNA as its genome and include many agriculturally important viruses such as Turnip mosaic virus (Tu. MV), Tobacco etch virus (TEV), Plum pox virus (PPV), and Soybean mosaic virus (SMV). A typical potyviral genome encodes a long open reading frame (ORF) and another relatively short ORF that results from RNA polymerase slippage in the P 3 coding sequence. ü Two polyproteins are proteolytically processed by three viral protease domains into 11 mature viral protein. ü NIb is the only viral RNA dependent RNA polymerase (Rd. Rp), and HC-Pro and VPg are two known viral suppressors of RNA silencing (VSRs). Beclin 1 (ATG 6) + ATG 8 a (Key autophagic protein ATG 8 a) P 1 Pro P 3 PIPO 6 K 1 CI 6 K 2 VPg Pro Nlb- Rd. RP Cp

Material and methods Plant materials and growth conditions: N. benthamiana and Arabidopsis ecotype Col-0 seedlings were potted in soil and placed in an insect-free growth chamber under constant conditions of 60% relative humidity and a day/night regime of 16 h in the light at 22 °C followed by 8 h at 18 °C in the dark. Transgenic H 2 B-RFP line-USA Arabidopsis mutants : atg 6 (SALK_109281), atg 8 a (SALK_045344 c), vps 15 (SALK_004719), pi 3 k (CS 355912), atg 2 (SALK_006994 C), atg 5 (CS 39993), and atg 7 (CS 39995). At. ATG 6 overexpression (35 S: Myc-At. ATG 6) and At. ATG 8 a overexpression (35 S: Myc-At. ATG 8 a) transgene Arabidopsis seeds were obtained by using the flower-dip method. Viral strains. The recombinant viruses Tu. MV, Tu. MV-GFP, Tu. MV-ΔGDD, Tu. MV-CFP-NIb-6 K 2 m. Cherry, Tu. MV-6 K 2 -m. Cherry, and PPV-GFP-m. Cherry. Pe. PMV and CGMMV were isolated from tomato and cucumber in a greenhouse in Ontario, Canada. Plasmid construction: Nb. Beclin 1 (AY 701316), Nb. PI 3 K (KX 120977), Nb. VPS 15 (KU 561371), Nb. ATG 2 (KU 561373), Nb. ATG 9 (KX 369399), Nb. ATG 3 (KX 369396), Nb. ATG 5 (KX 369397), Nb. ATG 7 (KX 369398), Nb. ATG 8 a (KX 120976), Nb. ATG 8 f (KU 561372), At. ATG 6 (NM_202746), Tu. MV (NC 002509), TEV (NC 001555), SMV (EU 871724), PPV (KP 998124), Nb. Actin (AY 179605), and At. Actin. II (AT 3 G 18780). Gateway technology (Canada) was used to generate all the plasmid constructs used in this study, unless otherwise stated. Gene sequences were amplified by PCR using Phusion® High-Fidelity DNA Polymerase (Canada) for cloning purposes. Tu. MV P 1, HC-Pro, P 3 N-PIPO, 6 K 1, CI, 6 K 2, VPg, NIa-Pro, NIb and CP, PPV NIb, SMV NIb, and TEV NIb were cloned previously. Nb. Beclin 1 -, Nb. ATG 8 a-, and Nb. ATG 8 f-coding sequence were generated from c. DNA- N. benthamiana leaves At. ATG 6 -coding sequence was generated from c. DNA - Arabidopsis leaves.

Ø Ø Ø Agroinfiltration and Viral Inoculation Y 2 H Bi. FC, subcellular localization Protoplast isolation and Tu. MV replication assay Chemical treatments and TEM. Immunoblotting and immunoprecipitation

EXPERIMENTS 1) To determine whether Tu. MV infection induces autophagy ? 2) Nb Beclin 1 interacts with Nlb? 3) Nb Beclin 1 or the Nb. Beclin 1 -Nlb interaction complex co-localizes with the autophagosome marker Nb. ATG 8 a in N. benthamiana ? 4) Nb Beclin 1 or the Nb. Beclin 1 -Nlb interaction complex co-localizes with Tu. MV viral replication complex in autophagosomes in N. benthamiana ? 5) Overexpression of Nb Beclin 1 promotes autophagy-dependent degradation of Nlb and inhibits TMV replication? 6) GDD motif of Nlb is required for the Nb. Beclin 1 -Nlb interaction and Nb Beclin 1 via its AIM, interacts with Nb. ATG 8 a to facilitate the formation of autophagosomes. 7) Silencing of Nb Beclin 1 or Nb. ATG 8 a promotes Tu. MV infection in N. benthamiana.

To determine whether Tu. MV infection induces autophagy ? a) N. benthamiana leaf cells co-infiltrated with Agrobacterium harboring a YFP-Nb. ATG 8 a expression construct and Agrobacterium carrying an empty vector (mock) or a Tu. MV infectious clone (Tu. MV) or a Tu. MV replication defective mutant (Tu. MV-ΔGDD) at 60 h post infiltration (hpi). b) The average number of YFP-Nb. ATG 8 a spots per 10 cells. c) Relative m. RNA expression levels d) Representative TEM images from upper non-inoculated leaves of N. Benthamiana plants infected with mock, Tu. MV, and Tu. MV-ΔGDD at 7 dpi.

2) Nb Beclin 1 interacts with Nlb? • • Yeast- two hybrid assay – activation domain and binding domain 3 days after transformation Bi. FC assays – in the leaves of H 2 B-RFP transgenic N. benthamiana. Co-localization with of Nlb-YFP with Nb Beclin 1 -CFP in the leaf cells of H 2 B-RFP transgenic N. benthamiana Co- immunoprecipitation analysis Nb Beclin 1 -CFP and Myc-Nlb invivo.

3) Nb Beclin 1 or the Nb. Beclin 1 -Nlb interaction complex co-localizes with the autophagosome marker Nb. ATG 8 a in N. benthamiana ?

4) Nb Beclin 1 or the Nb. Beclin 1 -Nlb interaction complex co-localizes with Tu. MV viral replication complex in autophagosomes in N. benthamiana ?

5) Overexpression of Nb Beclin 1 promotes autophagy-dependent degradation of Nlb and inhibits TMV replication ? ü Immunoblotting analysis ü Quantification Tu. MV RNA levels q. RT-PCR ü Effect of autophagy inhibitor – DMSO or 3 -MA treatment

6) GDD motif of Nlb is required for the Nb. Beclin 1 -Nlb interaction and Nb Beclin 1 via its AIM, interacts with Nb. ATG 8 a to facilitate the formation of autophagosomes.

7) Silencing of Nb Beclin 1 or Nb. ATG 8 a promotes Tu. MV infection in N. benthamiana. ----Protoplast transfection assay

Conclusion

Plant virus Autophagy Ca. MV infected Arabidopsis Neighbor of BRCA 1 (NBR 1) interacts with capsid proteins and mediates their degradation CLCu. Mu. V infection induces autophagy in Nicotiana benthamiana Cargo acceptor is Nb. ATG 8 f, an ATG 8 family protein, which interacts with the satellite-encoded protein βC 1, a major pathogenicity factor of CLCu. Mu. V, and the interaction directs βC 1 to autophagosomes for degradation Rd. Rp.