Evaluation of an antibacterial weapon on Agrobacterium tumorigenesis and crown gall microbiota

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：19

、訪客IP：3.133.149.244

姓名

王思翀(Si-Chong Wang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

(Evaluation of an antibacterial weapon on Agrobacterium tumorigenesis and crown gall microbiota)

相關論文

★ 建立TurboID鄰近標記方法以鑑定根癌農桿菌TssC40的交互作用蛋白質

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

至系統瀏覽論文 ( 永不開放)

摘要(中)

細菌之第六型分泌系統（Type VI secretion system, T6SS）廣泛存在於變型菌門（Proteobacteria）之細菌中，這些細菌透過第六型分泌系統可將其效應子蛋白直接分泌至體外或與其接觸的真核或原核細胞內，因此，第六型分泌系統常與細菌之致病性或細菌間競爭有關。本實驗室前人實驗顯示，於多種雙子葉植物上造成冠癭病（crown gall disease）之土棲性與土傳性植物病原細菌——農桿菌（Agrobacterium tumefaciens），可經由其T6SS於生體外（in vitro）或植物體內（in planta）攻擊種內或種外之細菌，從而提升其在特定生態棲位之競爭力。此外，前人亦利用直接接種於植物(例如番茄)莖上傷口或馬鈴薯塊腫瘤定量分析之實驗顯示，失去T6SS並不會影響農桿菌之致病能力。然而，我們對農桿菌之T6SS是否對自土壤入侵植物傷口從而造成冠癭的過程、或對冠癭內之植物內生微生物相（microbiota）造成影響等所知甚少。因此，本研究以農桿菌之模式菌株A. tumefaciens C58為材料，建立土壤接種番茄幼苗實驗，觀察A. tumefaciens C58野生株及T6SS突變株ΔtssB及ΔtssL之致病能力是否有不同；同時，由土壤接種實驗所造成的番茄冠癭組織萃取DNA，經由16S核醣體核酸基因擴增子定序（16S rRNA gene amplicon sequencing），調查及比較不同菌株造成的番茄冠癭內生細菌菌相，以得知農桿菌之T6SS是否對冠癭內生菌菌相造成影響。七次土壤接種實驗之結果表明，接種A. tumefaciens C58 T6SS突變株ΔtssB及ΔtssL，使番茄產生冠癭之罹病率比野生株顯著較低，說明農桿菌之T6SS可能經由間接的方式對其致病能力造成影響。由初步16S核醣體核酸基因擴增子定序實驗發現，番茄冠癭DNA的定序資料中含有大量來自葉綠體及粒線體之16S核醣體核酸基因序列，因而大幅度降低對內生細菌菌相之定序深度及解析度。為解決該問題，本研究針對16S核醣體核酸基因之不同高度變異區域（variable regions）設計共四對引子對，並同時針對番茄之葉綠體及粒線體16S核糖核酸基因設計相應之聚合酶連鎖反應阻斷性引子（polymerase-chains-reaction blocker），以增加對細菌族群之16S核糖核酸基因的增幅。結果顯示藉由阻斷性引子的運用，成功提高對冠癭內細菌16S核糖核酸基因之定序深度，而最佳化之16S核糖核酸基因增幅條件便可被應用於大規模調查冠癭內生菌相實驗的分析比較。對土壤接種實驗產生的共53個冠癭之內生細菌菌相進行之主座標分析（principle coordinate analysis）後得知A. tumefaciens C58野生株及T6SS突變株ΔtssB及ΔtssL造成之冠癭內生細菌菌相並無顯著差異，但不同月份進行土壤接種實驗造成之冠癭菌相則有顯著的差異。此外，我們發現冠癭之重量與A. tumefaciens C58在內生菌相中的相對豐度有正相關。綜上所述，本研究經由土壤接種番茄幼苗之實驗顯示，農桿菌T6SS突變株之致病力比野生株顯著較低，但對造成之番茄冠癭內之內生菌相則無顯著影響，此結果可能表示農桿菌的T6SS並不是造成冠癭內內生菌相的主因。未來方向可探索農桿菌A. tumefaciens C58之T6SS是否對其於根部的纏據（colonization）或根圈微生物菌相（rhizospheric microbiota）造成影響，以解釋為何T6SS突變株於經由土壤接種番茄幼苗時有較低的致病率。

摘要(英)

The type VI secretion system (T6SS) is a widespread nanomachine deployed by many proteobacteria to secrete effector proteins into prokaryotic or eukaryotic cells. This system engages in pathogenesis or interbacterial competition, which increases the fitness of the T6SS-containing bacteria. Previous studies demonstrated that Agrobacterium tumefacien, a soil-inhabiting and soil-borne phytopathogen causing crown gall disease on various plant species, deploys T6SS as an antibacterial weapon to attack closely- and distantly-related bacterial species in vitro and in planta. Additionally, tumorigenesis was not affected by the loss of T6SS when A. tumefaciens was inoculated in sterile condition using potato disc tumor assays or directly inoculated on the stems of various plant species including tomato plant. However, it remains unknown whether T6SS influences tumorigenesis in natural infection process, or affects the composition of bacterial community inside crown galls. Here, we established a soil inoculation method on wounded tomato seedlings and performed 16S rRNA gene amplicon sequencing to address these questions through the comparison of the A. tumefaciens C58 wild-type strain and two T6SS deficient mutants (i.e., ΔtssL and ΔtssB). Based on seven inoculation trials, all three strains could induce tumors through this method. Although the mutants have significantly lower disease incidences, for those successful infections there was no significant difference in crown gall weights. For the 16S amplicon sequencing, the first attempt based on a commonly used protocol for plant-associated microbiota suffered high levels of host DNA contaminations. Hence, we evaluated four newly designed primer pairs and their cognate PCR blockers against tomato chloroplasts and mitochondria. The optimized protocol targeting the V5-7 regions was utilized to survey 53 crown gall samples. Based on the principal coordinate analysis (PCoA) of bacterial composition, a functional T6SS did not significantly affect the bacterial compositions of crown gall, but the season of inoculation time did. A positive correlation was found between the relative abundance of A. tumefacien and the crown gall weight. In summary, the T6SS-dependant interbacterial competitions affected the success of A. tumefaciens infection of host plants through invasion from soil, but may not be an important factor in shaping the crown gall microbiota. Future works are necessary to investigate whether the T6SS enhances Agrobacterium colonization in rhizosphere or on wounded site, and consequently influences the initial stages of infection.

關鍵字(中)

★ 農桿菌
★ 冠癭病
★ 第六型蛋白質分泌系統
★ 植物內生菌相
★ 16S擴增子定序

關鍵字(英)

★ Agrobacterium tumefaciens
★ crown gall
★ type VI secretion system
★ endophytic microbiota
★ 16S rRNA gene amplicon sequencing

論文目次

Table of contents
中文摘要 i
Abstract iii
誌謝 v
List of figures vii
List of tables viii
Introduction 1
1 Crown gall disease and Agrobacterium tumefaciens 1
2 Plant associated microbiota under disease environment. 3
3 Type VI secretion system (T6SS) 4
4 T6SS of A. tumefaciens 6
Materials and methods 8
1. Bacterial strains and incubation condition 8
2. Plant material and growth condition 8
3. Procedure of soil inoculation 8
4. Harvest, surface sterilization and storage of crown gall samples 9
5. Crown gall disease incidences 9
6. DNA extraction form crown galls 9
7. 16S rRNA gene amplification and sequencing 10
Amplicon sequencing run I: protocol test 10
Amplicon sequencing run II: Testing the effect of blockers for optimization 12
Amplicon sequencing run III: Formal study of 53 crown gall samples using the optimized method. 14
8. Sequencing data analysis 14
9. Statistics and visualization 15
Results 16
1. Disease incidences of soil inoculation trial 16
2. Weight of crown galls induced by soil inoculation 17
3. Result of 16S rRNA amplicon sequencing run I for protocol test 17
4. Testing the effect of 16S rRNA gene primers and blockers for optimization 18
5. Crown gall microbiota induced by wild type and T6SS mutant strains 20
Discussion 23
Appendixes 62

參考文獻

1. Smith, E.F. and C.O. Townsend, A plant-tumor of bacterial origin. Science, 1907. 25(643): p. 671-673.
2. Tierney, M. and K. Lamour, An introduction to reverse genetic tools for investigating gene function. The Plant Health Instructor, 2005.
3. De Cleene, M. and J. De Ley, The host range of crown gall. The Botanical Review, 1976. 42(4): p. 389-466.
4. Barton, I.S., C. Fuqua, and T.G. Platt, Ecological and evolutionary dynamics of a model facultative pathogen: Agrobacterium and crown gall disease of plants. Environmental microbiology, 2018. 20(1): p. 16-29.
5. Agrios, G., Plant pathology. 2012: Elsevier.
6. Stachel, S.E. and P.C. Zambryski, virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens. Cell, 1986. 46(3): p. 325-333.
7. Shimoda, N., et al., Genetic evidence for an interaction between the VirA sensor protein and the ChvE sugar-binding protein of Agrobacterium. Journal of Biological Chemistry, 1993. 268(35): p. 26552-26558.
8. Escobar, M.A. and A.M. Dandekar, Agrobacterium tumefaciens as an agent of disease. Trends in plant science, 2003. 8(8): p. 380-386.
9. Harris, M.O. and A. Pitzschke, Plants make galls to accommodate foreigners: some are friends, most are foes. New Phytologist, 2020. 225(5): p. 1852-1872.
10. Lang, J., et al., Agrobacterium uses a unique ligand-binding mode for trapping opines and acquiring a competitive advantage in the niche construction on plant host. PLoS Pathog, 2014. 10(10): p. e1004444.
11. Lederberg, J. and A.T. McCray, Ome SweetOmics--A genealogical treasury of words. The scientist, 2001. 15(7): p. 8-8.
12. Peterson, J., et al., The NIH human microbiome project. Genome research, 2009. 19(12): p. 2317-2323.
13. Huttenhower, C., et al., Structure, function and diversity of the healthy human microbiome. nature, 2012. 486(7402): p. 207.
14. Müller, D.B., et al., The plant microbiota: systems-level insights and perspectives. Annual review of genetics, 2016. 50: p. 211-234.
15. Dastogeer, K.M., et al., Plant microbiome–an account of the factors that shape community composition and diversity. Current Plant Biology, 2020: p. 100161.
16. Tian, B.-Y., Y. Cao, and K.-Q. Zhang, Metagenomic insights into communities, functions of endophytes and their associates with infection by root-knot nematode, Meloidogyne incognita, in tomato roots. Scientific reports, 2015. 5(1): p. 1-15.
17. Cui, Z., et al., Temporal and spatial dynamics in the apple flower microbiome in the presence of the phytopathogen Erwinia amylovora. BioRxiv, 2020.
18. Faist, H., et al., Grapevine (Vitis vinifera) crown galls host distinct microbiota. Applied and environmental microbiology, 2016. 82(18): p. 5542-5552.
19. Gan, H.M., et al., Insight into the microbial co-occurrence and diversity of 73 grapevine (Vitis vinifera) crown galls collected across the Northern Hemisphere. Frontiers in microbiology, 2019. 10: p. 1896.
20. Li, Q., et al., Insight into the bacterial endophytic communities of peach cultivars related to crown gall disease resistance. Applied and environmental microbiology, 2019. 85(9).
21. Wu, D., et al., A plant pathogen type III effector protein subverts translational regulation to boost host polyamine levels. Cell Host & Microbe, 2019. 26(5): p. 638-649. e5.
22. Pukatzki, S., et al., Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proceedings of the National Academy of Sciences, 2006. 103(5): p. 1528-1533.
23. Ho, B.T., T.G. Dong, and J.J. Mekalanos, A view to a kill: the bacterial type VI secretion system. Cell host & microbe, 2014. 15(1): p. 9-21.
24. Costa, T.R., et al., Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nature Reviews Microbiology, 2015. 13(6): p. 343-359.
25. Wang, J., M. Brodmann, and M. Basler, Assembly and subcellular localization of bacterial type VI secretion systems. Annual review of microbiology, 2019. 73: p. 621-638.
26. Vacheron, J., et al., T6SS contributes to gut microbiome invasion and killing of an herbivorous pest insect by plant-beneficial Pseudomonas protegens. The ISME journal, 2019. 13(5): p. 1318-1329.
27. Chien, C.-F., et al., HSI-II gene cluster of Pseudomonas syringae pv. tomato DC3000 encodes a functional Type VI secretion system required for interbacterial competition. Frontiers in Microbiology, 2020. 11: p. 1118.
28. Ceseti, L.M., et al., The Xanthomonas citri pv. citri Type VI Secretion System is Induced During Epiphytic Colonization of Citrus. Current microbiology, 2019. 76(10): p. 1105-1111.
29. Kim, N., et al., Type VI secretion systems of plant‐pathogenic Burkholderia glumae BGR1 play a functionally distinct role in interspecies interactions and virulence. Molecular plant pathology, 2020. 21(8): p. 1055-1069.
30. Wu, H.-Y., et al., Secretome analysis uncovers an Hcp-family protein secreted via a type VI secretion system in Agrobacterium tumefaciens. Journal of bacteriology, 2008. 190(8): p. 2841-2850.
31. Ma, L.-S., J.-S. Lin, and E.-M. Lai, An IcmF family protein, ImpLM, is an integral inner membrane protein interacting with ImpKL, and its walker a motif is required for type VI secretion system-mediated Hcp secretion in Agrobacterium tumefaciens. Journal of bacteriology, 2009. 191(13): p. 4316-4329.
32. Ma, L.-S., et al., Agrobacterium tumefaciens deploys a superfamily of type VI secretion DNase effectors as weapons for interbacterial competition in planta. Cell host & microbe, 2014. 16(1): p. 94-104.
33. Wu, C.-F., et al., Acid-induced type VI secretion system is regulated by ExoR-ChvG/ChvI signaling cascade in Agrobacterium tumefaciens. PLoS Pathog, 2012. 8(9): p. e1002938.
34. Wu, C.-F., et al., Plant-Pathogenic Agrobacterium tumefaciens strains have diverse type VI effector-immunity pairs and vary in In-Planta competitiveness. Molecular Plant-Microbe Interactions, 2019. 32(8): p. 961-971.
35. Lin, J.-S., L.-S. Ma, and E.-M. Lai, Systematic dissection of the agrobacterium type VI secretion system reveals machinery and secreted components for subcomplex formation. PloS one, 2013. 8(7): p. e67647.
36. Hawes, M.C. and L.Y. Smith, Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants. Journal of bacteriology, 1989. 171(10): p. 5668-5671.
37. Yakabe, L., S. Parker, and D. Kluepfel, Role of systemic Agrobacterium tumefaciens populations in crown gall incidence on the walnut hybrid rootstock ‘Paradox’. Plant disease, 2012. 96(10): p. 1415-1421.
38. McPherson, M.R., et al., Isolation and analysis of microbial communities in soil, rhizosphere, and roots in perennial grass experiments. JoVE (Journal of Visualized Experiments), 2018(137): p. e57932.
39. Thijs, S., et al., Comparative evaluation of four bacteria-specific primer pairs for 16S rRNA gene surveys. Frontiers in microbiology, 2017. 8: p. 494.
40. Lefèvre, E., C.M. Gardner, and C.K. Gunsch, A novel PCR-clamping assay reducing plant host DNA amplification significantly improves prokaryotic endo-microbiome community characterization. FEMS Microbiology Ecology, 2020. 96(7): p. fiaa110.
41. Bolyen, E., et al., Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nature biotechnology, 2019. 37(8): p. 852-857.
42. Callahan, B.J., et al., DADA2: high-resolution sample inference from Illumina amplicon data. Nature methods, 2016. 13(7): p. 581-583.
43. Rognes, T., et al., VSEARCH: a versatile open source tool for metagenomics. PeerJ, 2016. 4: p. e2584.
44. Bokulich, N.A., et al., Optimizing taxonomic classification of marker-gene amplicon sequences with QIIME 2’s q2-feature-classifier plugin. Microbiome, 2018. 6(1): p. 90.
45. Yilmaz, P., et al., The SILVA and “all-species living tree project (LTP)” taxonomic frameworks. Nucleic acids research, 2014. 42(D1): p. D643-D648.
46. Eddy, S.R., Accelerated profile HMM searches. PLoS Comput Biol, 2011. 7(10): p. e1002195.
47. Janssen, S., et al., Phylogenetic placement of exact amplicon sequences improves associations with clinical information. Msystems, 2018. 3(3).
48. Matsen, F.A., et al., A format for phylogenetic placements. PLoS One, 2012. 7(2): p. e31009.
49. Matsen, F.A., R.B. Kodner, and E.V. Armbrust, pplacer: linear time maximum-likelihood and Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC bioinformatics, 2010. 11(1): p. 538.
50. Lozupone, C. and R. Knight, UniFrac: a new phylogenetic method for comparing microbial communities. Applied and environmental microbiology, 2005. 71(12): p. 8228-8235.
51. McMurdie, P.J. and S. Holmes, phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PloS one, 2013. 8(4): p. e61217.
52. Anderson, M.J., A new method for non‐parametric multivariate analysis of variance. Austral ecology, 2001. 26(1): p. 32-46.
53. Jari Oksanen, F., et al., Vegan: community ecology package. R package version, 2018. 2(6).
54. Beckers, B., et al., Performance of 16s rDNA Primer Pairs in the Study of Rhizosphere and Endosphere Bacterial Microbiomes in Metabarcoding Studies. Frontiers in Microbiology, 2016. 7(650).
55. Thijs, S., et al., Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys. Frontiers in Microbiology, 2017. 8(494).
56. Yu, M., et al., Agrobacterium tumefaciens Deploys a Versatile Antibacterial Strategy to Increase its Competitiveness. Journal of Bacteriology, 2020.
57. Deeken, R., et al., An integrated view of gene expression and solute profiles of Arabidopsis tumors: a genome-wide approach. The Plant Cell, 2006. 18(12): p. 3617-3634.
58. Willems, A., et al., Comamonadaceae, a new family encompassing the acidovorans rRNA complex, including Variovorax paradoxus gen. nov., comb. nov., for Alcaligenes paradoxus (Davis 1969). International Journal of Systematic and Evolutionary Microbiology, 1991. 41(3): p. 445-450.
59. Finkel, O.M., et al., A single bacterial genus maintains root growth in a complex microbiome. Nature, 2020. 587(7832): p. 103-108.
60. Moore, L.W., W.S. Chilton, and M.L. Canfield, Diversity of opines and opine-catabolizing bacteria isolated from naturally occurring crown gall tumors. Applied and Environmental Microbiology, 1997. 63(1): p. 201-207.
61. Beauchamp, C.J., et al., Physiological characterization of opine-utilizing rhizobacteria for traits related to plant growth-promoting activity. Plant and soil, 1991. 132(2): p. 273-279.
62. Riker, A., Studies on the influence of some environmental factors on the development of crown gall. J. Agric. Res, 1926. 32: p. 83-96.
63. Banta, L.M., et al., Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption. Journal of Bacteriology, 1998. 180(24): p. 6597-6606.
64. Fullner, K.J., J.C. Lara, and E.W. Nester, Pilus assembly by Agrobacterium T-DNA transfer genes. Science, 1996. 273(5278): p. 1107-1109.
65. Lai, E.-M. and C.I. Kado, Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. Journal of Bacteriology, 1998. 180(10): p. 2711-2717.
66. Fitzpatrick, C.R., et al., Chloroplast sequence variation and the efficacy of peptide nucleic acids for blocking host amplification in plant microbiome studies. Microbiome, 2018. 6(1): p. 1-10.
67. Arenz, B.E., et al., Blocking primers reduce co-amplification of plant DNA when studying bacterial endophyte communities. Journal of microbiological methods, 2015. 117: p. 1-3.
68. Vestheim, H. and S.N. Jarman, Blocking primers to enhance PCR amplification of rare sequences in mixed samples–a case study on prey DNA in Antarctic krill stomachs. Frontiers in zoology, 2008. 5(1): p. 1-11.

指導教授

賴爾珉吳少傑(Erh-Min Lai Shaw-Jye Wu)

審核日期

2021-2-25

推文