Over-expression of GbRac1 Gene Enhances the Tolerance to Phytophthora Root and Stem Rot in Soybean (Glycine max)
WANG Zhong-Wei1,2, ZHANG Yuan-Yu2, LIU Dong-Bo1,2, NIU Lu2, YANG Jing2, YANG Xiang-Dong2, ZHONG Xiao-Fang2,*
1 School of Life Science, Jilin Normal University, Siping 136000, China; 2 Agricultural Biotechnology Institute/Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun 130033, China
Abstract：Phytophthora root and stem rot (PRR) represents one of the destructive diseases in soybean (Glycine max) production, generally causing 10%~50% yield loss every year. Transgenic biotechnology provides an effective strategy for preventing and controlling the occurrence of PRR. The small G protein, known as the 'molecular switch', is not only involved in the process of regulating the adaptation of plants to external environmental stimuli during plant growth and development, but also participates in the establishment of plant defense responses. In this study, Gossypium barbadense Rac family small G protein 1 gene (GbRac1) was introduced into the soybean cultivar 'Williams 82'. Integration and expression of the foreign gene in transgenic soybean plants were confirmed by Southern blot and reverse transcription-PCR (RT-PCR) analysis. The PRR tolerance of the T3 and T4 generations of transgenic soybean lines was evaluated using a hypocotyl inoculation method. At 7 d after inoculation with Phytophthora sojae race 1, typical PRR symptoms such as wilting leaves and plant death were observed in the non-transformed plants. In contrast, most of transgenic plants showed no visible symptoms and developed normally with the survival rate of 70.68%~90.63%, higher than that of the control plants (40.74%~50.85%). However, no significant differences in agronomic traits were observed between the transgenic and non-transformed plants in the field conditions when no P. sojae inoculation involved. Taken together, these results demonstrated that over-expression of GbRac1 significantly enhanced the tolerance of the transgenic soybean to PRR without negative consequences on the agronomic performance. This results also pave the way for broadening disease resistance in soybean and development of the PRR-resistant soybean cultivars.
[1] 沈崇尧, 苏彦纯. 1991. 中国大豆疫霉病菌的发现及初步研究[J].植物病理学报, 21: 298. (Shen C Y, Sun Y C.1991.Discovery and preliminary studies of phytophora megasperma on soybean in China[J]. Acta Phytopathologica Sinica, 21: 298. ) [2] 孙广正. 2019. 番茄与白粉菌互作中的小G蛋白ShROP1与ShROP11和微丝骨架聚合因子ShARPC3与ShARPC5的功能研究[D]. 博士学位论文, 西北农林科技大学, 导师: 马青, pp. 117-122. (Sun G Z.2019. Functional characterization of small GTPase ShROP1 and ShROP11, microfilament skeleton polymerization factor ShARPC3 and ShRPC5 in the interaction between tomato and Oidium neolycopersici[D]. Thesis for Ph.D., Northwest A & F University, Supervisor: Ma Q, pp, 117-122.) [3] 杨静, 赵倩倩, 牛陆, 等. 2020. 转盾壳霉Cmoxdc1基因增强大豆对菌核病抗性的研究[J]. 大豆科学, 39(5): 712-719. (Yang J, Zhao Q-Q, Niu L, et al.2020. Study on the enhancement of soybean resistance to Sclerotinia sclerotiorum with transformation of Cmoxdc1 gene from Coniothyrium minitans[J]. Soybean Science, 39(5): 712-719.) [4] 杨叔青. 2018. 茄科植物小G蛋白ROPs在抗疫霉菌过程中的功能研究[D]. 博士学位论文, 内蒙古农业大学, 导师: 赵君, pp. 71-73. (Yang S Q.2018. The function analyses of Solanaceous small G protein ROPs to Phytophthora pathogens[D]. Thesis for Ph.D., Inner Mongolia Agricultural University, Supervisor: Zhao J, pp. 71-73.) [5] 张晓萝. 2014. 小G蛋白StRac在马铃薯抗晚疫病过程中的功能研究[D]. 硕士学位论文, 内蒙古农业大学, 导师: 赵君, pp. 54-55. (Zhang X L.2014. The study on the function of small G protein in StRac on the resistance establishment to potato late blight[D]. Thesis for M.S., Inner Mongolia Agricultural University, Supervisor: Zhao J, pp. 54-55.) [6] 赵雪, 孙明明, 韩英鹏, 等. 2014. 大豆种质资源耐疫霉根腐病优异等位变异挖掘[J]. 大豆科学, 33(4): 488-491. (Zhao X, Sun M M, Han Y P, et al.2014. Identification of loci underlying tolerance to Phytophthora root rotin soybean germplasm[J]. Soybean Science, 33(4):488-491. ) [7] 左豫虎, 薛春生, 韩文革, 等. 2002. 大豆疫霉菌对大豆幼苗的侵染特性[J]. 植物保护学报, 12(4): 377-378. (Zuo Y H, Xue C S, Han W G, et al.2002. The infections characteristics of Phytophthora sojae to soybean seedling[J]. Acta Phytophylacica Sinica, 12(4): 377-378.) [8] Akamatsu A, Chilvers M, Stewatr J, et al.2010. Identifacation and function of a polyketide synthase gene responsible for 1,8-dihydroxynaphthalene-melanin pigment biosynthesis in Ascochyta rabiei[J]. Current Genetics, 56(4):349-360. [9] Assmann S M.2002. Heterotrimeric and unconventional GTP binding proteins in plant cell signaling[J]. The Plant Cell, 14: S355-373. [10] Berken A.2006. ROPs in the spotlight of plant signal transduction[J]. Cellular & Molecular Life Sciences, 63(21): 2446-2459. [11] Burnham K, Dorrance A, Van Toai T T, et al.2003. Quantitative trait loci for partial resistance to Phytophthora sojae in soybean[J]. Crop Science, 43: 1610-1617. [12] Du Q, Yang X D, Zhang J H, et al.2018. Over-expression of the Pseudomonas syringae harpin-encoding gene hrpZm confers enhanced tolerance to Phytophthora root and stem rot in transgenic soybean[J]. Transgenic Research, 27(3): 277-288. [13] Edwards K, Johnstone C, Thompson C.1991. A simple and rapid method for the preparation of plant genomic DNA for pcr analysis[J]. Nucleic Acids Research, 19(6): 1349. [14] Ellinger D, Annemarie G, Koch J, et al.2014. Interaction of the Arabidopsis GTPase Rab A4c with its effector PMR4 results in complete penetration resistance to powdery mildew[J]. The Plant Cell, 26(7): 3185-3200. [15] Fan S, Jiang L, Wu J, et al.2015. A novel pathogenesis-related class 10 protein Gly m 4l, increases resistance upon Phytophthora sojae infection in soybean (Glycine max [L.] Merr.)[J]. PLOS ONE, 10(10): e0140364. [16] Fujiwara M, Umemura K, Kawasaki T, et al.2006. Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells[J]. Plant Physiology, 140(2): 734-745. [17] Kawano Y, Chen L, Shimamoto K.2010. The function of Rac small GTPase and associated proteins in rice innate immunity[J]. Rice, 3(2-3): 112-121. [18] Kawano Y, Shimamoto K.2013. Early signaling network in rice PRR-mediated and R-mediated immunity[J]. Current Opinion in Plant Biology, 16(4): 496-504. [19] Kawasaki T, Henmi K, Ono E, et al.1999. The small GTP-binding protein Rac is a regulator of cell death in plants[J]. Proceedings of the National Academy of Sciences of the USA, 16(4): 10922-10926. [20] Kawasaki T, Koita H, Nakatsubo T, et al.2006. Cinnamoyl-Co A reductase, a key enzyme in lignin biosynthesis, is an effector of small GTPase Rac in defense signaling in rice[J]. Proceedings of the National Academy of Sciences of the USA, 103(1): 230-235. [21] Lin F, Zhao M, Baumann D D, et al.2014. Molecular response to the pathogen Phytophthora sojae among ten soybean near isogenic lines revealed by comparative transcriptomics[J]. BMC Genomics, 15: 18. [22] Lin F, Zhao M, Ping J, et al.2013. Molecular mapping of two genes conferring resistance to Phytophthora sojae in a soybean landrace pi 567139b[J]. Theoretical and Applied Genetics, 126(8): 2177-2185. [23] Meyer P.2000.Transcriptional transgene silencing and chromatin components[J]. Plant Molecular Biology, 43(2-3): 221-234. [24] Niu L, Yang J, Zhang J H, et al.2019. Introduction of the harpin Xooc-encoding gene hrf2 in soybean enhances resistance against the oomycete pathogen Phytophthora sojae[J]. Transgenic Research, 28(2): 257-266. [25] Niu L, Zhong X F, Zhang Y Y, et al.2020. Enhanced tolerance to Phytophthora root and stem rot by over-expression of the plant antimicrobial peptide CaAMP1 gene in soybean[J]. BMC Genetics, 21(1): 68. [26] Ono E, Wong H L, Kawasaki T, et al.2001. Essential role of the small GTPase Rac in disease resistance of rice[J]. Proceedings of the National Academy of Sciences of the USA, 98(2): 759-764. [27] Opalski K S, Schultheiss H, Kogel K H, et al.2005. The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f. sp. hordei[J]. The Plant Journal, 41(2): 291-303. [28] Schmitthenner A F, Hobe M, Bhat R G.1994. Phytophthora sojae races in Ohio over 10-year interval[J]. Plant Disease, 78(3): 269-276. [29] Sugimoto K, Fujita S, Miyazawa T, et al.2012a. Pediatric left renal vein entrapment syndrome diagnosed by 99mtc-albumin-conjugate scintigraphy[J]. Nephron Clinical Practice, 122(3-4): 122-126. [30] Sugimoto T, Kato M, Yoshida S, et al.2012b. Pathogenic diversity of Phytophthora sojae and breeding strategies to develop Phytophthora-resistant soybeans[J].Breeding Science, 61(5): 511-522. [31] Tel-zur N, Abbo S, Myslabodski D, et al.1999. Modified CTAB procedure for DNA isolation from epiphytic cacti of the genera Hylocereus and Selenicereus (Cactaceae)[J]. Plant Molecular Biology Reporter, 17: 249-254. [32] Tyler B M.2007. Phytophthora sojae: Root rot pathogen of soybean and model oomycete[J]. Molecular Plant Pathology, 8(1): 1-8. [33] Yang J, Xing G J, Niu L, et al.2018. Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B[J]. Transgenic Research, 27(2): 155-166. [34] Yang S Q, Yan N N, Bouwmeester K, et al.2020. Genome-wide identification of small G protein ROPs and their potential roles in Solanaceous family[J]. Gene, 753: 144809.
