水稻徒長病(bakanae disease)是由Fusarium fujikuroi所引起的真菌性病害，普遍發生於全球各水稻產區。目前對於水稻徒長病的防治多以化學藥劑為主，然而此方法可能會對環境造成不良影響或導致病原菌抗藥性的產生。寄主誘導基因靜默(host-induced gene silencing, HIGS)是近年來開發之生物技術為背景的防治方法，即利用寄主產生的小片段RNA (siRNA)作為分子標靶，針對病原菌或有害生物的特定基因進行RNA干擾(RNAi)，導致該基因靜默。鈣調磷酸酶(calcineurin)是一種由催化(catalytic, Cna1)及調控(regulatory, Cnb1)次單元所組成的蛋白質二聚體複合體。研究指出，鈣調磷酸酶會調控真菌的鈣離子訊息傳遞，並在生長、逆境耐受性及毒力上扮演重要角色。因此本研究首先利用實驗室已建立之番茄萎凋病菌Cna1及Cnb1兩個次單元的單基因突變菌株(single deletion mutant)進行基因功能分析，探討鈣調磷酸酶是否也在重要植物病原真菌之一的Fusarium屬中扮演重要角色。經由菌絲型態構造分析、酵素活性試驗(calcineurin phosphatase activity test)、菌株產孢及接種試驗，初步證明鈣調磷酸酶活性對於番茄萎凋病菌的生長、厚膜孢子形成及致病力是必需的。經實驗發現，鈣調磷酸酶抑制劑FK506及Cyclosporin A能夠抑制徒長病菌的生長。接著本研究進一步利用HIGS策略抑制F. fujikuroi CNA1及CNB1基因表現，促進水稻對徒長病的抗性。利用農桿菌基因轉殖的方式將徒長病菌鈣調磷酸酶的專一性RNA干擾 (RNAi)構築導入水稻中，建立轉殖株並以南方墨漬法(Southern blot) 篩選出單套轉基因之水稻。根據接種試驗及病原菌質量測定結果顯示，帶有FfCNA1-Ri 及FfCNB1-Ri表現載體的水稻轉殖株皆能提高對徒長病之抗性。本研究證明HIGS策略能有效靜默病原真菌鈣調磷酸酶表現，進而抑制徒長病菌的生長，同時提供未來水稻或其他重要作物病害防治上的新方向。

Bakanae or foolish seedling disease of rice is caused by the ascomycetous fungus Fusarium fujikuroi, of which is widespread in many rice growing areas. Current protection strategies rely on the fungicides, but this results in chemical-resistant F. fujikuroi and other undesirable environmental effects. An alternative approach involving RNAi, termed host-induced gene silencing (HIGS), of which generating siRNA molecules in plants that target to the specific mRNAs of pathogens and thereby resulted in their degradation. Calcineurin belongs to a Ca2+/calmodulin-dependent phosphatase, which is comprised of heterodimer with a catalytic (Cna1) and a regulatory (Cnb1) subunits. There have been known that calcineurin mediates Ca2+ signaling that regulates the growth, stress responses and virulence in fungal kingdom. Based on pharmacological inhibition of calcineurin in F. fujikuroi, we found that calcineurin inhibitor FK506 or cyclosporinA can strongly inhibit the growth of F. fujikuroi. We therefore aim to determine whether repressing the expression of CNA1 or CNB1 gene in F. fujikuroi by the HIGS strategy in rice can enhance resistance against bakanae disease. By using Agrobacterium-mediated gene transformation strategy, the FfCNA1-Ri and FfCNB1-Ri constructs have been introduced into rice plants, respectively, and the copy number of transgenes were analyzed by Southern blot hybridization in the transgenic lines. Based on the pathogen inoculation assay and fungal biomass quantification, results showed that the transgenic rice plants that carry the FfCNA1-Ri or FfCNB1-Ri constructs increased resistance against bakanae disease, promising its potential use in the future.

國立臺灣大學碩士學位論文口試委員會審定書 I
致謝 II
中文摘要 IV
Abstract V
目錄 VI
圖表目錄 VIII
1. Introduction 1
2. Materials and Methods 6
2.1 Fungal strain and culture condition 6
2.2 Colony morphology 6
2.3 Plant DNA isolation and Southern blotting 6
2.4 Primers 7
2.5 Plant infection assay 7
2.6 Pathogen biomass quantification in planta 8
2.7 Plant materials and callus induction 8
2.8 Construction of the FfCNA1-Ri and FfCNB1-Ri expression vectors 9
2.9 Rice transformation 9
3. Results 11
3.1 Identification of Cna1 and Cnb1 in F. fujikuroi 11
3.2 In vitro effect of calcineurin inhibitors on against F. fujikuroi 11
3.3 Generation of RNAi constructs and transgenic rice 12
3.4 Molecular analysis of transformants 12
3.5 FfCNA1-Ri and FfCNB1-Ri transgenic rice plants exhibit enhanced resistance against F. fujikuroi 13
3.6 Quantification of F. fujikuroi biomass in transgenic rice plants 14
4. Discussion 16
5. Figures 18
6. Supplementary information 26
Tables 26
Figures 27
7. References 31
8. Appendix 36
The roles of calcineurin in hyphal growth and virulence of Fusarium oxysporum f. sp. lycopersici 36

1.Agrawal N, Dasaradhi PV, Mohmmed A, Malhotra P, Bhatnagar RK, Mukherjee SK (2003) RNA interference: biology, mechanism, and applications. Microbiology and Molecular Biology Reviews 67 (4):657-685. doi:10.1128/mmbr.67.4.657-685.2003
2.Agrios GN (2005) Plant Pathology vol 5th Edition. Elsevier Academic Press, Amsterdam.
3.Bahn Y-S, Jung K-W (2013) Stress signaling pathways for the pathogenicity of Cryptococcus. Eukaryotic Cell 12 (12):1564-1577. doi:10.1128/EC.00218-13
4.Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248-254. doi:10.1006/abio.1976.9999
5.Brewster JL, de Valoir T, Dwyer ND, Winter E, Gustin MC (1993) An osmosensing signal transduction pathway in yeast. Science 259 (5102):1760-1763. doi:10.1126/science.7681220
6.Cervantes-Chavez JA, Ali S, Bakkeren G (2011) Response to environmental stresses, cell-wall integrity, and virulence are orchestrated through the calcineurin pathway in Ustilago hordei. Molecular Plant-Microbe Interactions 24 (2):219-232. doi:10.1094/mpmi-09-10-0202
7.Chaveroche MK, Ghigo JM, d''Enfert C (2000) A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28 (22):E97-E97. doi:10.1093/nar/28.22.e97
8.Chen W, Kastner C, Nowara D, Oliveira-Garcia E, Rutten T, Zhao Y, Deising HB, Kumlehn J, Schweizer P (2016a) Host-induced silencing of Fusarium culmorum genes protects wheat from infection. Journal of Experimental Botany 67 (17):4979-4991. doi:10.1093/jxb/erw263
9.Chen Y-C, Lai M-H, Wu C-Y, Lin T-C, Cheng A-H, Yang C-C, Wu H-Y, Chu S-C, Kuo C-C, Wu Y-F, Lin G-C, Tseng M-N, Tsai Y-C, Lin C-C, Chen C-Y, Huang J-W, Lin H-A, Chung C-L (2016b) The Genetic Structure, Virulence, and Fungicide Sensitivity of Fusarium fujikuroi in Taiwan. Phytopathology 106 (6):624-635. doi:10.1094/phyto-11-15-0285-r
10.Chen Y-L, Brand A, Morrison EL, Silao FGS, Bigol UG, Malbas FF, Nett JE, Andes DR, Solis NV, Filler SG, Averette A, Heitman J (2011) Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis. Eukaryotic Cell 10 (6):803-819. doi:10.1128/ec.00310-10
11.Chen Y-L, Kozubowski L, Cardenas ME, Heitman J (2010a) On the Roles of Calcineurin in Fungal Growth and Pathogenesis. Current Fungal Infection Reports 4 (4):244-255. doi:10.1007/s12281-010-0027-5
12.Cheng W, Song XS, Li HP, Cao LH, Sun K, Qiu XL, Xu YB, Yang P, Huang T, Zhang JB, Qu B, Liao YC (2015) Host-induced gene silencing of an essential chitin synthase gene confers durable resistance to Fusarium head blight and seedling blight in wheat. Plant Biotechnology Journal 13 (9):1335-1345. doi:10.1111/pbi.12352
13.Choi JH, Kim Y, Lee YH (2009) Functional analysis of MCNA, a gene encoding a catalytic subunit of calcineurin, in the rice blast fungus Magnaporthe oryzae. Journal of Microbiology and Biotechnology 19 (1):11-16
14.Cramer RA, Jr., Perfect BZ, Pinchai N, Park S, Perlin DS, Asfaw YG, Heitman J, Perfect JR, Steinbach WJ (2008) Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus. Eukaryot Cell 7 (7):1085-1097. doi:10.1128/ec.00086-08
15.Cyert MS (2003) Calcineurin signaling in Saccharomyces cerevisiae: how yeast go crazy in response to stress. Biochemical and Biophysical Research Communications 311 (4):1143-1150. doi:10.1016/s0006-291x(03)01552-3
16.da Silva Ferreira ME, Heinekamp T, Hartl A, Brakhage AA, Semighini CP, Harris SD, Savoldi M, de Gouvea PF, de Souza Goldman MH, Goldman GH (2007) Functional characterization of the Aspergillus fumigatus calcineurin. Fungal Genetics and Biology 44 (3):219-230. doi:10.1016/j.fgb.2006.08.004
17.de Castro PA, Chen C, de Almeida RSC, Freitas FZ, Bertolini MC, Morais ER, Brown NA, Ramalho LNZ, Hagiwara D, Mitchell TK, Goldman GH (2014) ChIP-seq reveals a role for CrzA in the Aspergillus fumigatus high-osmolarity glycerol response (HOG) signalling pathway. Molecular Microbiology 94 (3):655-674. doi:10.1111/mmi.12785
18.Dinamarco TM, Freitas FZ, Almeida RS, Brown NA, dos Reis TF, Ramalho LNZ, Savoldi M, Goldman MHS, Bertolini MC, Goldman GH (2012) Functional characterization of an Aspergillus fumigatus calcium transporter (PmcA) that is essential for fungal infection. PLoS ONE 7 (5):e37591-e37591. doi:10.1371/journal.pone.0037591
19.Egan JD, Garcia-Pedrajas MD, Andrews DL, Gold SE (2009) Calcineurin is an antagonist to PKA protein phosphorylation required for postmating filamentation and virulence, while PP2A is required for viability in Ustilago maydis. Molecular Plant-Microbe Interactions 22 (10):1293-1301. doi:10.1094/mpmi-22-10-1293
20.Fox DS, Heitman J (2002) Good fungi gone bad: the corruption of calcineurin. BioEssays : news and reviews in molecular, cellular and developmental biology 24 (10):894-903. doi:10.1002/bies.10157
21.Fuller KK, Rhodes JC (2012) Protein kinase A and fungal virulence: a sinister side to a conserved nutrient sensing pathway. Virulence 3 (2):109-121. doi:10.4161/viru.19396
22.Ghag SB (2017) Host induced gene silencing, an emerging science to engineer crop resistance against harmful plant pathogens. Physiological and Molecular Plant Pathology 100:242-254. doi:10.1016/j.pmpp.2017.10.003
23.Ghag SB, Shekhawat UK, Ganapathi TR (2014) Host-induced post-transcriptional hairpin RNA-mediated gene silencing of vital fungal genes confers efficient resistance against Fusarium wilt in banana. Plant Biotechnology Journal 12 (5):541-553. doi:10.1111/pbi.12158
24.Gordon TR (2017) Fusarium oxysporum and the Fusarium Wilt Syndrome. Annual Review of Phytopathology 55 (1):23-39. doi:10.1146/annurev-phyto-080615-095919
25.Harel A, Bercovich S, Yarden O (2006) Calcineurin is required for sclerotial development and pathogenicity of Sclerotinia sclerotiorum in an oxalic acid-independent manner. Molecular Plant-Microbe Interactions 19 (6):682-693. doi:10.1094/mpmi-19-0682
26.Hartl L, Gastebois A, Aimanianda V, Latgé J-P (2011) Characterization of the GPI-anchored endo β-1,3-glucanase Eng2 of Aspergillus fumigatus. Fungal Genetics and Biology 48 (2):185-191. doi:10.1016/j.fgb.2010.06.011
27.Helliwell C, Waterhouse P (2003) Constructs and methods for high-throughput gene silencing in plants. Methods 30 (4):289-295
28.Hemenway CS, Heitman J (1999) Calcineurin. Structure, function, and inhibition. Cell Biochemistry and Biophysics 30 (1):115-151. doi:10.1007/bf02737887
29.Ho S-L, Tong W-F, Yu S-M (2000) Multiple Mode Regulation of a Cysteine Proteinase Gene Expression in Rice. Plant Physiology 122 (1):57-66. doi:10.1104/pp.122.1.57
30.Juvvadi PR, Fortwendel JR, Pinchai N, Perfect BZ, Heitman J, Steinbach WJ (2008) Calcineurin localizes to the hyphal septum in Aspergillus fumigatus: implications for septum formation and conidiophore development. Eukaryot Cell 7 (9):1606-1610. doi:10.1128/ec.00200-08
31.Juvvadi PR, Fortwendel JR, Rogg LE, Burns KA, Randell SH, Steinbach WJ (2011) Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus. Molecular Microbiology 82 (5):1235-1259. doi:10.1111/j.1365-2958.2011.07886.x
32.Juvvadi PR, Lamoth F, Steinbach WJ (2014a) Calcineurin-mediated regulation of hyphal growth, septation, and virulence in Aspergillus fumigatus. Mycopathologia 178 (5-6):341-348. doi:10.1007/s11046-014-9794-9
33.Juvvadi PR, Lamoth F, Steinbach WJ (2014b) Calcineurin as a Multifunctional Regulator: Unraveling Novel Functions in Fungal Stress Responses, Hyphal Growth, Drug Resistance, and Pathogenesis. Fungal biology Reviews 28 (2-3):56-69. doi:10.1016/j.fbr.2014.02.004
34.Juvvadi PR, Steinbach WJ (2015) Calcineurin Orchestrates Hyphal Growth, Septation, Drug Resistance and Pathogenesis of Aspergillus fumigatus: Where Do We Go from Here? Pathogens 4 (4):883-893. doi:10.3390/pathogens4040883
35.Koch A, Kumar N, Weber L, Keller H, Imani J, Kogel KH (2013) Host-induced gene silencing of cytochrome P450 lanosterol C14alpha-demethylase-encoding genes confers strong resistance to Fusarium species. Proceedings of the National Academy of Sciences of the United States of America 110 (48):19324-19329. doi:10.1073/pnas.1306373110
36.Langner T, Gohre V (2016) Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions. Current Genetics 62 (2):243-254. doi:10.1007/s00294-015-0530-x
37.Lievens B, Houterman PM, Rep M (2009) Effector gene screening allows unambiguous identification of Fusarium oxysporum f. sp. lycopersici races and discrimination from other formae speciales. FEMS Microbiology Letters 300 (2):201-215. doi:10.1111/j.1574-6968.2009.01783.x
38.Liu S, Hou Y, Liu W, Lu C, Wang W, Sun S (2015) Components of the calcium-calcineurin signaling pathway in fungal cells and their potential as antifungal targets. Eukaryot Cell 14 (4):324-334. doi:10.1128/ec.00271-14
39.Luna-Tapia A, DeJarnette C, Sansevere E, Reitler P, Butts A, Hevener KE, Palmer GE (2019) The Vacuolar Ca2+ ATPase Pump Pmc1p Is Required for Candida albicans Pathogenesis. mSphere 4 (1):e00715-00718. doi:10.1128/mSphere.00715-18
40.Majumdar R, Rajasekaran K, Cary JW (2017) RNA Interference (RNAi) as a Potential Tool for Control of Mycotoxin Contamination in Crop Plants: Concepts and Considerations. Frontiers in Plant Science 8 (200). doi:10.3389/fpls.2017.00200
41.Martinez-Soto D, Ruiz-Herrera J (2017) Functional analysis of the MAPK pathways in fungi. Revista Iberoamericana de Micologia 34 (4):192-202. doi:10.1016/j.riam.2017.02.006
42.Michielse CB, van Wijk R, Reijnen L, Manders EM, Boas S, Olivain C, Alabouvette C, Rep M (2009) The nuclear protein Sge1 of Fusarium oxysporum is required for parasitic growth. PLoS Pathogens 5 (10):e1000637. doi:10.1371/journal.ppat.1000637
43.Moradi S, Sanjarian F, Safaie N, Mousavi A, Bakhshi Khaniki GR (2013) A modified method for transformation of Fusarium graminearum. Journal of Crop Protection 2 (3):297-304
44.Munro CA, Selvaggini S, de Bruijn I, Walker L, Lenardon MD, Gerssen B, Milne S, Brown AJP, Gow NAR (2007) The PKC, HOG and Ca2+ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Molecular Microbiology 63 (5):1399-1413. doi:10.1111/j.1365-2958.2007.05588.x
45.Niehaus EM, Kim HK, Munsterkotter M, Janevska S, Arndt B, Kalinina SA, Houterman PM, Ahn IP, Alberti I, Tonti S, Kim DW, Sieber CMK, Humpf HU, Yun SH, Guldener U, Tudzynski B (2017) Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles. PLoS Pathogens 13 (10):e1006670. doi:10.1371/journal.ppat.1006670
46.Nunes CC, Dean RA (2012) Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies. Molecular Plant Pathology 13 (5):519-529. doi:10.1111/j.1364-3703.2011.00766.x
47.Park HS, Chow EW, Fu C, Soderblom EJ, Moseley MA, Heitman J, Cardenas ME (2016) Calcineurin Targets Involved in Stress Survival and Fungal Virulence. PLoS Pathogens 12 (9):e1005873. doi:10.1371/journal.ppat.1005873
48.Punt PJ, Oliver RP, Dingemanse MA, Pouwels PH, van den Hondel CA (1987) Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli. Gene 56 (1):117-124
49.Qi T, Guo J, Peng H, Liu P, Kang Z, Guo J (2019) Host-Induced Gene Silencing: A Powerful Strategy to Control Diseases of Wheat and Barley. International Journal of Molecular Sciences 20 (1):206. doi:10.3390/ijms20010206
50.Qi T, Zhu X, Tan C, Liu P, Guo J, Kang Z, Guo J (2018) Host-induced gene silencing of an important pathogenicity factor PsCPK1 in Puccinia striiformis f. sp. tritici enhances resistance of wheat to stripe rust. Plant Biotechnology Journal 16 (3):797-807. doi:10.1111/pbi.12829
51.Rasmussen C, Garen C, Brining S, Kincaid RL, Means RL, Means AR (1994) The calmodulin-dependent protein phosphatase catalytic subunit (calcineurin A) is an essential gene in Aspergillus nidulans. The EMBO journal 13 (11):2545-2552
52.Rogg LE, Fortwendel JR, Juvvadi PR, Steinbach WJ (2012) Regulation of expression, activity and localization of fungal chitin synthases. Medical Mycology 50 (1):2-17. doi:10.3109/13693786.2011.577104
53.Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiological Reviews 80 (4):1483-1521. doi:10.1152/physrev.2000.80.4.1483
54.Sanglard D, Ischer F, Marchetti O, Entenza J, Bille J (2003) Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence. Molecular Microbiology 48 (4):959-976
55.Song Y, Thomma B (2018) Host-induced gene silencing compromises Verticillium wilt in tomato and Arabidopsis. Molecular Plant Pathology 19 (1):77-89. doi:10.1111/mpp.12500
56.Srinivas C, Devi DN, Murthy KN, Mohan CD, Lakshmeesha TR, Singh B, Kalagatur NK, Niranjana SR, Hashem A, Alqarawi AA, Tabassum B, Abd_Allah E, Nayaka SC (2019) Fusarium oxysporum f. sp. lycopersici causal agent of vascular wilt disease of tomato: Biology to diversity– A review. Saudi Journal of Biological Sciences. doi:10.1016/j.sjbs.2019.06.002
57.Stie J, Fox D (2008) Calcineurin regulation in fungi and beyond. Eukaryotic Cell 7 (2):177-186. doi:10.1128/EC.00326-07
58.Viaud M, Brunet-Simon A, Brygoo Y, Pradier JM, Levis C (2003) Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea. Molecular Microbiology 50 (5):1451-1465
59.Viaud MC, Balhadere PV, Talbot NJ (2002) A Magnaporthe grisea cyclophilin acts as a virulence determinant during plant infection. The Plant Cell 14 (4):917-930. doi:10.1105/tpc.010389
60.Xu J, Wang X, Li Y, Zeng J, Wang G, Deng C, Guo W (2018) Host-induced gene silencing of a regulator of G protein signalling gene (VdRGS1) confers resistance to Verticillium wilt in cotton. Plant Biotechnology Journal. doi:10.1111/pbi.12900
61.Yu S-J, Chang Y-L, Chen Y-L (2015) Calcineurin signaling: lessons from Candida species. FEMS Yeast Research 15 (4). doi:10.1093/femsyr/fov016
62.Zhang H, Guo J, Voegele RT, Zhang J, Duan Y, Luo H, Kang Z (2012a) Functional characterization of calcineurin homologs PsCNA1/PsCNB1 in Puccinia striiformis f. sp. tritici using a host-induced RNAi system. PLoS ONE 7 (11):e49262-e49262. doi:10.1371/journal.pone.0049262
63.Zhang H, Tang W, Liu K, Huang Q, Zhang X, Yan X, Chen Y, Wang J, Qi Z, Wang Z, Zheng X, Wang P, Zhang Z (2011) Eight RGS and RGS-like proteins orchestrate growth, differentiation, and pathogenicity of Magnaporthe oryzae. PLoS Pathogens 7 (12):e1002450. doi:10.1371/journal.ppat.1002450
64.Zhang J, Silao FG, Bigol UG, Bungay AA, Nicolas MG, Heitman J, Chen YL (2012b) Calcineurin is required for pseudohyphal growth, virulence, and drug resistance in Candida lusitaniae. PLoS ONE 7 (8):e44192. doi:10.1371/journal.pone.0044192
65.陳思聿, 黃凱均, 郭彥甫, 賴明信, 陳又嘉, 鍾嘉綾 (2015) 以三種改良之評估方法判定水稻幼苗對徒長病之抗感病性 (Three modified methods for evaluation of bakanae disease resistance in rice seedlings). 植物病理學會刊 24 (3&4):201-210