Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi

RESEARCH ARTICLE Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi Hong-Le Wang1☯, Xi Cheng1,2☯, Shan-Wen Ding1, Dong-Wei Wang1, Chun Chen1, ChunLing Xu1, Hui Xie1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Laboratory of Plant Nematology and Research Center of Nematodes of Plant Quarantine, Department of Plant Pathology / Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Agriculture, South China Agricultural University, Guangzhou, People’s Republic of China, 2 Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, People’s Republic of China ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Wang H-L, Cheng X, Ding S-W, Wang DW, Chen C, Xu C-L, et al. (2018) Molecular identification and functional characterization of the cathepsin B gene (Ab-cb-1) in the plant parasitic nematode Aphelenchoides besseyi. PLoS ONE 13 (6): e0199935. https://doi.org/10.1371/journal. pone.0199935 Editor: Vitaly Citovsky, Stony Brook University, UNITED STATES Received: February 26, 2018 Accepted: June 15, 2018 Published: June 29, 2018 Copyright: © 2018 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. The rice white tip nematode, Aphelenchoides besseyi, is widely distributed in rice planting areas worldwide and causes serious economic losses. Cathepsin genes have been demonstrated to have importance in studying the reproduction, development, pathogenicity, and control methods of plant nematodes. In this paper, a novel cathepsin B gene, Ab-cb-1, was found and cloned. The Ab-cb-1 gene was 1347 bp in length and encodes 369 amino acids. The Ab-CB-1 protein contains characteristic occluding loops but no signal peptide. A homology analysis showed that Ab-CB-1 had the highest identity value (64%) to the known amino acid sequence of cathepsin B-like cysteine protease 6 from Toxocara canis. When Ab-cb-1 was expressed in a prokaryotic system, the protein massed approximately 45 kDa and could decompose carrot callus. Ab-cb-1 mRNA was localized in the nematode intestine. The relative expression level of Ab-cb-1 in the A. besseyi Ab-S24 population, which had high reproductivity, was approximately 6.9 times that in the Ab-N10 population, which had low reproductivity, and the difference was significant (p<0.05). The Ab-cb-1 expression level was highest in females; the expression levels in males, juveniles and eggs were 30%, 12.2% and 5% of that in females, respectively, and the differences were significant among all four stages (p<0.05). Nematodes of the Ab-S24 population were treated with Ab-cb-1 dsRNA for 12 h, 24 h, 36 h and 48 h, and their reproduction decreased with increasing time. These results demonstrated that Ab-CB-1 was a digestive enzyme with hydrolytic protease properties and that Ab-cb-1 played an important role in the reproduction of A. besseyi. Funding: This work was funded by National Natural Science Foundation of China (No. 31371920 and 31071665 to H.X.). Competing interests: The authors declare they have no competing interests. PLOS ONE | https://doi.org/10.1371/journal.pone.0199935 June 29, 2018 1 / 15 Molecular identification and functional characterization of the cathepsin B gene in Aphelenchoides besseyi Background Aphelenchoides besseyi is a migratory plant parasitic nematode with more than 200 host plants in over 35 genera. The main hosts are rice (Oryza sativa) and strawberry (Fragaria ananassa) [1]. A. besseyi is distributed over almost all the world’s rice planting areas, and it is one of the major rice seed-borne diseases that cause serious economic losses [2, 3, 4]. Cathepsins belong to the cysteine protease family and are commonly found in parasites as digestive enzymes. Cathepsins have hydrolytic protease properties [5, 6] and play important physiological and biochemical roles. In recent years, studies have shown that cathepsin genes are essential in the physiological and biochemical processes of parasites and insects, such as hatching, reproduction, development, infection, pathogenicity and immune evasion [7]. Several cathepsins including cathepsin L, cathepsin S, and cathepsin B have been demonstrated to have importance in plant parasitic nematodes, and thus far, cathepsin L has been the subject of greatest interest [8, 9]. So far, several cathepsin L genes have been successfully cloned from a variety of plant nematodes including Heterodera avenae (ACJ13100), H. glycines (Y09498), H. schachtii (ACJ13098), Globodera virginiae (ACJ13094), G. mexicana (ACJ13096) Meloidogyne incognita (CAD89795), Rotylenchulus reniformis (AAY45870), Bursaphelenchus xylophilus (ACH56225), and Ditylenchus destructor (GQ180107). Mi-cpl-1, a cathepsin L gene from M. incognita, has been shown to be correlated with parasite success and to be essential in the interaction between nematodes and plants [10], so this gene could be further developed for root-knot nematode control. Cathepsin S genes (cps) have also been successfully cloned from plant parasite nematodes including H. glycines [9], H. avenae [11] and Radopholus similis (EU659125) [12]. The cps gene in R. similis has been confirmed to be related to reproduction, parasitism and pathogenicity of the nematode [13]. However, cathepsin B genes (cb) in plant parasitic nematodes are seldom reported and have been cloned in only B. xylophilus (GU130153) and R. similis (GU360972). The Rs-cb-1 gene found in R. similis has been reported as a key gene that could affect its development and pathogenicity [14, 15]. In view of the important role and application of the cathepsin gene in the reproduction, development, parasitism and control of nematodes, a novel cathepsin gene was screened from the cDNA library of A. besseyi in this study and successfully cloned. Then, its biological characteristics and functions were identified for the first time. Methods Nematode and cultivation The A. besseyi populations used in this study were the Ab-S24 population collected from strawberry (F. ananassa) in Longgang District, Shenzhen, Guangdong, China, and the Ab-N10 population collected from rice (O. sativa) in Luhe Town, Nanjing, Jiangsu Province, China. Nematodes were isolated, identified, preserved and cultured by the Laboratory of Plant Nematology, South China Agricultural University. The reproductivity was found to be higher in AbS24 than in Ab-N10 [16]. The preservation and cultivation of the nematodes were carried out using the method described by Cheng et al. [1]. Cloning (...truncated)