臺灣博碩士論文加值系統

土壤中根圈微生物分泌的各種代謝物對植物有不同程度的影響，其中一群代謝物以氣態方式存在於環境中。這些由微生物分泌出的氣態代謝物稱為微生物揮發性有機物(microbial volatile organic compounds, mVOCs)，前人研究多以微生物釋出的混合mVOCs作為探討的對象，甚少人深入研究其中的成分影響。本研究主要分析植物抑制生長細菌Enterobacter aerogenes釋出的有機揮發化合物，發現主要抑制菸草生長的化合物為混合mVOCs中含量第二高的C2，且透過次世代定序(NGS)分析發現當菸草暴露在E.aerogenes揮發物或C2揮發性化合物時，自噬作用、囊泡運輸、氮運輸關鍵基因表現量上調，推測當菸草處於E.aerogenes揮發物或C2揮發性化合物時會啟動自噬作用、囊泡運輸、氮運輸等反應，以抵禦mVOCs的侵害。為更進一步確認囊泡運輸在防禦上的作用，運用病毒誘導基因靜默技術(virus-induced gene silence, VIGS)將參與自噬作用中的囊泡運輸基因Exo70靜默。發現NbExo70靜默植株對E.aerogenes揮發物及C2揮發性化合物較為敏感，且相較於野生型，NbExo70靜默植株經過E.aerogenes揮發物及C2揮發性化合物處理後過氧化物的累積情況較不顯著。由上述結果可推測Exo70在逆境下會誘導過氧化物的累積，當過氧化物累積時會啟動過敏反應引起的細胞程序性死亡(HR-PCD)反應，然而為了避免累積過多時造成HR-PCD反應失控，因此Exo70會在過氧化物累積過多時協助自噬體，將產生過氧化物訊號的蛋白質運送至液泡中降解。而當NbExo70靜默時不會有過氧化物累積現象，亦不存在HR-PCD反應失控的問題。另外細胞壁沉積程度在未處理mVOCs時，NbExo70靜默植株較野生型薄，但經mVOCs處理後NbExo70靜默植株卻比野生型厚，推測Exo70雖然會參與細胞壁沉積，但在逆境下為加強防禦植物會啟動其他機制促使細胞壁沉積。

Microbes affect plant growth through several mechanisms. One of the mechanisms affected by microbial volatile organic compounds (mVOCs) are gaseous molecules released by microbes. There are lots of reports that focus on the influence by mixing mVOCs. Just a few researches have analyzed the compositions of mVOCs. Here I analyzed two volatile organic compounds, which are released by Enterobacter aerogenes, a plant growth-inhibiting bacteria. I found the major inhibiting compound, C2, the second large compound in E. aerogenes mVOCs. Using next generation sequencing technique, I found that the key genes of autophagy, exocyst and asparagines synthesis were up regulated when tobacco was exposed to mVOCs of E. aerogenes or C2 compound. To further confirm how vesicle trafficking results in tobacco defense mechanism, virus-induced gene silence (VIGS) was performed to silence Exo70, an important gene participates in autophagy process. NbExo70 silenced tobacco was sensitive to mVOCs of E. aerogenes and C2 compound. H2O2 was not accumulated in leaves when NbExo70 silenced tobacco exposed to mVOCs of E. aerogenes and C2 compound. According to the results I can conclude that Exo70 induces the accumulation of H2O2. The accumulation of H2O2 would induce hypersensitive response programmed cell death(HR-PCD). When the level of H2O2 was abnormal accumulation, it would disturbe the HR-PCD of plant. Thus, Exo70 would help the autophagosomal degradation of H2O2 signaling related protein. NbExo70 silenced tobacco has less deposition in callose, but when it is exposed to mVOCs of E. aerogenes and C2 compound the deposition in callose will be higher than wild-type. It seems that Exo70 is involved in callose deposition, but when under the stress tobacco will turn on another mechanism.

目錄
摘要 Ⅰ
英文延伸摘要 Ⅱ
致謝 Ⅴ
縮寫對照表 Ⅵ
壹、前言 1
一、微生物揮發性氣體 1
二、mVOCs與植物之間的關係 1
三、Enterobacter aerogenes 2
四、植物防禦機制 2
(一)效應因子觸發免疫(effector-triggered immunity, ETI) 3
(二)自噬作用 3
(三)囊泡運輸 4
(四)氮運輸 4
貳、材料方法 5
一、菌種材料 5
(一)土壤採集 5
(二)菌種培養 5
(三)菌種純化與保存 5
(四)菌種篩選 5
(五)菌種DNA萃取 6
(六)菌種鑑定 7
二、Enterobacter aerogenes之揮發物分析 7
(一) Enterobacter aerogenes菌盤製備 7
(二)GC-MS 7
三、植物材料 8
(一)組織培養 8
四、mVOCs對植物之生理生化分析 8
(一)Enterobacter aerogenes揮發物對植物之處理 8
(二)揮發性化合物對植物之處理 8
(三)植株體含菌量確認 8
(四)葉綠素含量測定 9
(五)葉片組織的過氧化物染色 9
(六)植物細胞壁染色 9
五、植物基因表現量分析 9
(一)植物RNA萃取 9
(二)反轉錄聚合酶連鎖反應(RT-PCR) 10
(三)聚合酶連鎖反應(polymerase chain reaction；PCR) 10
(四)即時聚合酶鏈式反應(Real-time polymerase chain reaction；Q-PCR)
10
六、次世代定序(Next Generation Sequencing, NGS) 11
(一)RNA資歷料庫(Library)製備與次世代定序流程 11
七、NbExo70病毒誘導基因靜默轉殖株製作 11
(一) 純化Nb Exo70 B基因片段 11
(二)增殖基因片段 11
(三)構築pTRV2/Nb Exo70 B 11
(四)電穿孔轉型 12
(五)農桿菌接種 12
(六)VIGS 植株栽培條件 13
參、結果 14
一、菌株篩選與微生物揮發性有機物質分析 14
(一)菌株篩選 14
(二) Enterobacter aerogenes揮發物之GC-MS分析 14
二、Enterobacter aerogenes揮發物及C1、C2揮發性化合物影響野生型菸草生長與生理反應 15
(一) E. aerogenes揮發物及C1、C2揮發性化合物抑制野生行菸草的生長 15
(二) E. aerogenes揮發物及C1、C2揮發性化合物影響菸草葉綠素含量 15
(三) E. aerogenes揮發物及C1、C2揮發性化合物影響過氧化氫的累積 16
三、次世代定序(Next Generation Sequencing, NGS)分析E. aerogenes揮發物及C1、C2揮發性化合物處理後之菸草 16
四、E. aerogenes揮發物及C1、C2揮發性化合物處理後作用機制之基因表現量 16
五、E. aerogenes揮發物及C1、C2揮發性化合物對NbExo70靜默菸草的影響 17
(一) E. aerogenes揮發物及C1、C2揮發性化合物對NbExo70 靜默菸草地上部重量及葉綠素含量影響 17
(二) E. aerogenes揮發物及C1、C2揮發性化合物影響NbExo70 靜默菸草之過氧化氫累積 18
(三)Enterobacter aerogenes揮發物及C1、C2揮發性化合物影響NbExo70靜默(TRV-NbExo70)菸草細胞壁沉積 18
肆、討論 20
一、Enterobacter aerogenes釋出之揮發物對菸草之影響 20
(一)Enterobacter aerogenes釋出之揮發性化合物分析 20
(二) Enterobacter aerogenes揮發物與C1、C2揮發性化合物處理下對菸草的生理影響 20
二、Enterobacter aerogenes與C2揮發性化合物引起的植物防禦機制 21
(一)自噬作用 21
(二)囊泡運輸 22
(三) E. aerogenes與C2揮發性化合物對NbExo70靜默植株的影響 22
I. NbExo70靜默植株對E. aerogenes與C2揮發性化合物較為敏感 22
II. 過氧化物的累積與清除 23
III. 細胞壁沉積 23
(四)氮運輸 24
三、結論 25
參考文獻 26
附件
附件一、植株體內含菌量確認 63
附件二、MapMan 分析Enterobacter aerogenes揮發物處理野生型(WT)菸草3天轉錄產物(transcripts) 64

Avila-Ospina, L., Moison, M., Yoshimoto, K., ＆ Masclaux-Daubresse, C. Autophagy, plant senescence, and nutrient recycling. Journal of Experimental Botany. (2014).
Banchio, E., Xie, X., Zhang, H., ＆ Pare, P. W. Soil bacteria elevate essential oil accumulation and emissions in sweet basil. J Agric Food Chem, 57(2): 653-657. (2009).
Blom, D., Fabbri, C., Connor, E. C., Schiestl, F. P., Klauser, D. R., Boller, T., Eberl, L., ＆ Weisskopf, L. Production of plant growth modulating volatiles is widespread among rhizosphere bacteria and strongly depends on culture conditions. Environ Microbiol, 13(11): 3047-3058. (2011).
Cheng, C., Gao, X., Feng, B., Sheen, J., Shan, L., ＆ He, P. Plant immune response to pathogens differs with changing temperatures. Nat Commun, 4: 2530. (2013).
Cohen, L. B., ＆ Troemel, E. R. Microbial pathogenesis and host defense in the nematode C. elegans. Curr Opin Microbiol, 23: 94-101. (2015).
Collins, N. C., Thordal-Christensen, H., Lipka, V., Bau, S., Kombrink, E., Qiu, J. L., Huckelhoven, R., Stein, M., Freialdenhoven, A., Somerville, S. C., ＆ Schulze-Lefert, P. SNARE-protein-mediated disease resistance at the plant cell wall. Nature, 425(6961): 973-977. (2003).
de Torres‐Zabala, M., Truman, W., Bennett, M. H., Lafforgue, G., Mansfield, J. W., Rodriguez Egea, P., Bögre, L., ＆ Grant, M. Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic acid signalling pathway to cause disease. The EMBO Journal, 26(5): 1434-1443. (2007).
Du, Y., Mpina, M. H., ＆ Birch, P. R. Phytophthora infestans RXLR Effector AVR1 Interacts with Exocyst Component Sec5 to Manipulate Plant Immunity. 169(3): 1975-1990. (2015).
Farag, M. A., Zhang, H., ＆ Ryu, C. M. Dynamic chemical communication between plants and bacteria through airborne signals: induced resistance by bacterial volatiles. J Chem Ecol, 39(7): 1007-1018. (2013).
Hayward, A. P., ＆ Dinesh-Kumar, S. P. What can plant autophagy do for an innate immune response? Annu Rev Phytopathol, 49: 557-576. (2011).
Hwang, I. S., An, S. H., ＆ Hwang, B. K. Pepper asparagine synthetase 1 (CaAS1) is required for plant nitrogen assimilation and defense responses to microbial pathogens. Plant J, 67(5): 749-762.( 2011).
Kai, M., ＆ Piechulla, B. Plant growth promotion due to rhizobacterial volatiles--an effect of CO2 ? FEBS Lett, 583(21): 3473-3477. (2009).
Kalde, M., Nuhse, T. S., Findlay, K., ＆ Peck, S. C. The syntaxin SYP132 contributes to plant resistance against bacteria and secretion of pathogenesis-related protein 1. Proc Natl Acad Sci U S A, 104(28): 11850-11855. (2007).
Kulich, I., Pecenkova, T., Sekeres, J., Smetana, O., Fendrych, M., Foissner, I., Hoftberger, M., ＆ Zarsky, V. Arabidopsis exocyst subcomplex containing subunit EXO70B1 is involved in autophagy-related transport to the vacuole. Traffic, 14(11): 1155-1165. (2013).
Kulich, I., Vojtikova, Z., Glanc, M., Ortmannova, J., Rasmann, S., ＆ Zarsky, V. Cell wall maturation of Arabidopsis trichomes is dependent on exocyst subunit EXO70H4 and involves callose deposition. Plant Physiol, 168(1): 120-131. (2015).
Liu, Y., Schiff, M., Czymmek, K., Talloczy, Z., Levine, B., ＆ Dinesh-Kumar, S. P. Autophagy regulates programmed cell death during the plant innate immune response. Cell, 121(4): 567-577. (2005).
Miransari, M. Use of microbes for the alleviation of soil stressesSpringer,NY.78-79(2014).
Pérez-García, A., Pereira, S., Pissarra, J., Gutiérrez, A. G., Cazorla, F., Salema, R., De Vicente, A., ＆ Cánovas, F. Cytosolic localization in tomato mesophyll cells of a novel glutamine synthetase induced in response to bacterial infection or phosphinothricin treatment. Planta, 206(3): 426-434. (1998).
Paul, D., ＆ Park, K. S. Identification of volatiles produced by Cladosporium cladosporioides CL-1, a fungal biocontrol agent that promotes plant growth. Sensors (Basel), 13(10): 13969-13977.(2013).
Pecenkova, T., Hala, M., Kulich, I., Kocourkova, D., Drdova, E., Fendrych, M., Toupalova, H., ＆ Zarsky, V. The role for the exocyst complex subunits Exo70B2 and Exo70H1 in the plant-pathogen interaction. J Exp Bot, 62(6): 2107-2116. (2011).
Ren, C., Liu, J., ＆ Gong, Q. Functions of autophagy in plant carbon and nitrogen metabolism. Autophagy in plants and algae: 98.(2015).
Rudrappa, T., Biedrzycki, M. L., Kunjeti, S. G., Donofrio, N. M., Czymmek, K. J., Pare, P. W., ＆ Bais, H. P. The rhizobacterial elicitor acetoin induces systemic resistance in Arabidopsis thaliana. Commun Integr Biol, 3(2): 130-138. (2010).
Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Kloepper, J. W., ＆ Pare, P. W. Bacterial volatiles induce systemic resistance in Arabidopsis. Plant Physiol, 134(3): 1017-1026. (2004).
Ryu, C. M., Farag, M. A., Hu, C. H., Reddy, M. S., Wei, H. X., Pare, P. W., ＆ Kloepper, J. W. Bacterial volatiles promote growth in Arabidopsis. Proc Natl Acad Sci U S A, 100(8): 4927-4932. (2003).
Santoro, M., Cappellari, L., Giordano, W., ＆ Banchio, E. Production of Volatile Organic Compounds in PGPR. In D. F. Cassán, Y. Okon, ＆ M. C. Creus (Eds.), Handbook for Azospirillum: Technical Issues and Protocols: 307-317. Cham: Springer International Publishing. (2015).
Santoro, M. V., Cappellari, L. R., Giordano, W., ＆ Banchio, E. Plant growth-promoting effects of native Pseudomonas strains on Mentha piperita (peppermint): an in vitro study. Plant Biology, 17(6): 1218-1226. (2015).
Santoro, M. V., Zygadlo, J., Giordano, W., ＆ Banchio, E. Volatile organic compounds from rhizobacteria increase biosynthesis of essential oils and growth parameters in peppermint (Mentha piperita). Plant Physiol Biochem, 49(10): 1177-1182. (2011).
Selosse, M. A., Bessis, A., ＆ Pozo, M. J. Microbial priming of plant and animal immunity: symbionts as developmental signals. Trends Microbiol, 22(11): 607-613. (2014).
Stegmann, M., Anderson, R. G., Ichimura, K., Pecenkova, T., Reuter, P., Zarsky, V., McDowell, J. M., Shirasu, K., ＆ Trujillo, M. The ubiquitin ligase PUB22 targets a subunit of the exocyst complex required for PAMP-triggered responses in Arabidopsis. Plant Cell, 24(11): 4703-4716. (2012).
Stegmann, M., Anderson, R. G., Westphal, L., Rosahl, S., McDowell, J. M., ＆ Trujillo, M. The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death. Plant Signal Behav, 8(12): e27421.(2013).
Teh, O.-K., ＆ Hofius, D. Membrane trafficking and autophagy in pathogen-triggered cell death and immunity. Journal of experimental botany: ert441. (2014).
Utkhede, R. S., ＆ Sholberg, P. L. In vitro inhibition of plant pathogens by Bacillus subtilis and Enterobacter aerogenes and in vivo control of two postharvest cherry diseases. Canadian Journal of Microbiology, 32(12): 963-967.(1986).
van Dam, N. M., ＆ Bouwmeester, H. J. Metabolomics in the rhizosphere: Tapping into belowground chemical communication. Trends in plant science, 21(3): 256-265. (2016).
Vespermann, A., Kai, M., ＆ Piechulla, B. Rhizobacterial Volatiles Affect the Growth of Fungi and Arabidopsis thaliana. Applied and Environmental Microbiology, 73(17): 5639-5641. (2007).
Weise, T., Kai, M., ＆ Piechulla, B. Bacterial ammonia causes significant plant growth inhibition. PLoS One, 8(5): e63538. (2013).
Yoshimoto, K., Jikumaru, Y., Kamiya, Y., Kusano, M., Consonni, C., Panstruga, R., Ohsumi, Y., ＆ Shirasu, K. Autophagy negatively regulates cell death by controlling NPR1-dependent salicylic acid signaling during senescence and the innate immune response in Arabidopsis. Plant Cell, 21(9): 2914-2927. (2009).
Zou, C. G., Ma, Y. C., Dai, L. L., ＆ Zhang, K. Q. Autophagy protects C. elegans against necrosis during Pseudomonas aeruginosa infection. Proc Natl Acad Sci U S A, 111(34): 12480-12485. (2014).