CM Virus integrate Plant stem cells .pptx

Cucumber mosaic virus

1. WUSCHEL triggers innate antiviral immunity in plant stem cells Haijun Wu1 , Xiaoya Qu1 , Zhicheng Dong2 , Linjie Luo1 , Chen Shao1 , Joachim Forner3 *, Jan U. Lohmann3 , Meng Su1 , Mengchu Xu1 , Xiaobin Liu2 , Lei Zhu4 , Jian Zeng1 , Sumei Liu1 , Zhaoxia Tian1 †, Zhong Zhao1 †

2. Description Stem cells in plants constantly supply daughter cells in the shoot apical meristem (SAM) form new organs and are expected to safeguard the integrity of the cells from biological invasion. The purpose of this study to show how stem cells of the Arabidopsis shoot apical meristem and their nascent daughter cells suppress infection by cucumber mosaic virus (CMV ). And also analyzed the broad-spectrum antiviral mechanism that safeguards pluripotent cells in the SAM from viral invasion.

3. Plant stem cells and their progeny are immune to CMV infection To test the hypothesis that the top of the SAM is virus free. Followed virus distribution in the Arabidopsis SAM after inoculating the leaves with cucumber mosaic virus (CMV ) (Fig. A). CMV was inoculated in leaves to trigger systemic infection in Arabidopsis

4. CMV distributions in mock (B), 4 dpi (C), and 9 dpi (D) plants. Red box indicates the SAM. Col-0, Columbia-0 At 9 days post inoculation (dpi), we observed CMV accumulations in the shoot and the rib zone of the SAM, but not in the central zone (CZ), peripheral zone (PZ), or floral primordia (Fig. B to D).

5. CMV (E) and WUS (F) distributions in the SAM at 9 dpi Observed CMV accumulation just below the WUSCHEL (WUS) expression domain (Fig. E and F).

6. In the clv3-7 mutant that ectopically expressed WUS in the L2 cell layer (Fig. 1G), CMV moved upward and remained beneath the WUS protein–containing domain (Fig. H). WUS and CMV accumulation patterns in the clv3-7 mutant

7. Then, investigated whether the WUS expression domain blocks virus movement or if WUS inhibits virus accumulation . And generated an mp clv3-7 double mutant with an enlarged SAM, which allowed us to inoculate CMV directly into and below the WUS expression domain (Fig. I). Red lines indicate the surgical incision in the SAM. Red arrows indicate the location of CMV inoculation

8. Although the virus was directly inoculated into stem cells in the L1 cell layer and the organizing center (OC) in the L2 cell layer, where the WUS protein is located, we observed that the CMV only spread under the L2 layer (Fig. J and K), suggesting that the WUS protein–containing regions, including stem cells and the OC, are resistant to CMV invasion. WUS (J) and CMV (K) distributions in the mp clv3-7 double mutant with viral infection. Red lines indicate the surgical incision in the SAM. Red arrows indicate the location of CMV inoculation.

9. WUS protein responds to CMV and inhibits viral infection To test whether WUS proteins are involved in the host defense against viruses , Examined WUS protein distribution patterns during CMV infection at 9 dpi using wus /pCLV3:: mCherry -NLS; pWUS ::WUS-GFP rescue plants. In virus-free plants, WUS protein expression overlapped with CLV3 transcript expression in stem cells (Fig. A to D). WUS::WUS:GFP CLV3:: mCherry Merge WUS protein distribution and CLV3 expression patterns in the wus / pWUS :: WUS-GFP; pCLV3 :: mCherry -NLS rescue plant without [(A) to (D)] CMV infection at 9 dpi (n = 15 )

10. Upon CMV invasion, WUS protein expression ( Fig. E , G, and H), but not transcript expression increased. WUS protein distribution and CLV3 expression patterns in the wus / pWUS :: WUS-GFP; pCLV3 :: mCherry -NLS rescue plant with [(E) to (H)] CMV infection at 9 dpi (n = 15 )

11. In plants in which WUS was inducible with dexamethasone (DEX) treatment [pUBQ10::WUS-GR plants, we induced WUS 1 day after inoculating with CMV. Without induction, 89% of the plants were infected by viruses. With DEX-induced WUS activity, 90% of the plants were free from CMV invasion (Fig. I to L). (I and J) CMV accumulations at 8 dpi in pUBQ10:: WUS-GR plants with (J) or without (I) DEX induction for 7 days. (K) Percentage of plants infected by viruses with (n = 159) or without (n = 117) DEX treatment at 8 dpi. (L)

12. To determine whether the WUS protein is required for the antiviral capacity of stem cells,we used wus / pWUS ::WUS-linker-GFP/pCLV3:: AlcR / pAlcA ::NSlmb-vhhGFP4 plants to inducibly degrade WUS protein in stem cells (Fig. M to T). WUS-linker–GFP signals and CMV distributions in wus / pWUS ::WUS-linker-GFP/pCLV3:: AlcR / pAlcA ::NSlmb-vhhGFP4 lines with or without 1% ethanol treatment for 48 hours with CMV infection at 0 dpi [(Q) to (T)] and 8 dpi [(M) to (P )] .

13. WUS represses CMV protein accumulation in plants To examine the effect of WUS on viral protein synthesis, Fused GFP to the C terminus of each of the CMV proteins 2a, 3a, CP, and 2b . Coinfiltrated these constructs with 35S::WUS in tobacco leaves (Fig. A). p35s::GUS p35s:: 3a-GFP p35s:: 2a-GFP p35s:: CP-GFP p35s:: 2b-GFP p35s ::WUS A

14. Expression of 3a-GFP, 2a-GFP, CP-GFP, and 2b-GFP was reduced by coinfiltration with 35S::WUS as shown by fluorescence and Western blot analysis (Fig. B and C). CP-GFP GUS WUS 2b-GFP GUS WUS a-GFP a-H3 C B (B ) Quantification of the relative fluorescence intensity of GFP in (A) ( C) Western blots of CP and 2b proteins in (A )

15. To determine whether WUS represses CMV protein accumulation in the Arabidopsis SAM, we inoculated pUBQ10::WUS-GR plants with CMV and then induced WUS with DEX treatment. At 8 dpi, 2b protein abundance in the SAM decreased (Fig. D). WUS represses CMV 2b protein accumulation in the SAM of Arabidopsis at 8 dpi. D

16. To elucidate whether WUS-mediated protein inhibition is virus specific or if it has global effects on protein synthesis , Followed incorporation of O- propargyl - puromycin to assess nascent protein levels in pUBQ10::WUS-GR plants(Fig . E and F ). DIPA DIPA OP-P OP-P E F WUS globally inhibits nascent protein synthesis in Arabidopsis (E), and the relative fluorescence intensity is quantified in (F ). 35S::GUS was used as a negative control.

17. MTase mediates WUS-triggered innate antiviral immunity Observed decreases in nop2a/NOP2B RNA interference ( RNAi ) transgenic plants (Fig. A ). Dot-blot assay showing that total m 5 C levels were decreased in nop2a/NOP2B RNAi plants. A

18. RNA interference in plant further confirmed with liquid chromatography–tandem mass spectrometry (LC-MS/MS) quantification (Fig. B). B LC–MS/ MS quantifications of the total m 5 C levels of wild-type (WT ), nop2a (n = 8), and independent nop2a/ NOP2B RNAi T1 plants

19. RNA immunoprecipitation showed m 5 C - modified 25S rRNAs enriched around C2860 in the wild-type plants and less so in nop2a/NOP2B RNAi plants (Fig. C), demonstrating that NOP2A and NOP2B act redundantly in m5C methylation of 25S rRNAs in vivo. m 5 C-RNA immunoprecipitation qRT -PCR of 4-week-old WT, nop2a , and independent nop2a/NOP2B RNAi T1 plants. The region of 25S#2 contains the predicted m 5 C target C2860 of 25S rRNA . The regions of 25S#1 and 18S#1 were used as the negative controls. C

20. Using RNA bisulfite sequencing, we quantified the m 5 C levels of 25S rRNA and observed a 64% increase of unmethylated C2860 in nop2a/NOP2B RNAi plants (Fig. D). D Quantification of the 25S rRNA m 5 C levels of C2860 in WT, nop2a , and nop2a/ NOP2B RNAi plants using RNA bisulfite sequencing. Black box indicates the C2860 site.

21. With polysomic profiling, they observed reduced levels of 80S and 60S ribosomes in plants after activating WUS (Fig. E). E Polysome profile of 10-day-old pUBQ10:: WUS-GR seedlings treated with DEX for 2 days

22. To observed less nascent protein synthesis in stem cells in an area coincident with the WUS protein domain (Fig. F). Inflorescences without puromycin ( Puro ) incorporation or with Puro and CHX treatment were used as the negative controls. Puro -incorporated nascent proteins were mainly observed in the PZ and primordia in areas complementary to the WUS protein domain. F

23. For tested the genetic interaction of WUS and MTases in Arabidopsis during viral infection by overexpressing sixMTasesinpUBQ10::WUS-GR plants and observed increases in viral protein and genomic RNA accumulation upon WUS induction (Fig. G). The shoot apices of MTases overexpression lines in the UBQ10::WUS-GR background with CMV infection at 8 dpi and DEX induction for 7 days were used for detection of the 2b protein with an anti-2b antibody and of the viral genomic RNA with the CMV RNA3 probe. G

24. Conclusion Meristem tip culture has been used to produce virus-free plants of many species against a range of viral infections, which suggests a conserved and broad-spectrum antiviral mechanism in the SAM. Given that WUSCHEL-related homeobox (WOX) genes are widespread in the plant kingdom and have conserved functions in shoot and root meristems, WUS/WOX–mediated antiviral immunity might be a conserved mechanism in plants. As a parasitic strategy, viruses hijack the host protein synthesis machinery to complete their life cycle. Plants in turn have evolved various mechanisms to protect them against viral infection.