Characterization of the Hsp90 Gene in the White Tip Nematode(Aphelenchoides besseyi) and Its Expression in Response to Environmental Stresses, Early Infection and Feeding
Abstract：Hot shock protein 90 (Hsp90) regulates multitudes of biological processes, such as cell metabolism, normal development and environmental stress tolerance. Herein the full-length cDNA and the corresponding gene of the heat shock protein 90, named Ab-hsp90, were isolated and characterized in the plant parasitic nematode Aphelnchoides besseyi, the agent of the rice white-tip disease. The full-length Ab-hsp90 cDNA contained a 5' UTR of 69, an ORF of 2 139 bp encoding a polypeptide of 712 amino acids and a 3' UTR of 137 bp. The deduced amino acid sequence of Ab-hsp90 showed that high similarity with other known Hsp90s, and the Maximum Likehood tree indicates Ab-hsp90 was located in the clade of plant nematodes. Five conserved amino acid signature indicated that Ab-hsp90 was a cytosolic member of the Hsp90 family. The gene consisted of 9 exons and 8 introns. Expression levels of Ab-hsp90 were determined by quantitative real-time PCR. Ab-hsp90 mRNA was constitutively expressed in all developmental stages of A. besseyi, but the highest transcript abundance existed in egg and the adult nematodes. Exposing the nematode to desiccation condition, relatively low osmotic stress and short-term abamectin solution promote Ab-hsp90 transcription. Ab-hsp90 was also induced in the nematodes under the low temperature (4 ℃) at 1.5 h but continuously expressed in high temperature (40 ℃) in 18 h. Interestingly, differential expression of Ab-hsp90 was detected in A. besseyi infecting the resistant and rice seedlings in 72 h. A burst of expression of Ab-hsp90 was present in the inoculated nematodes on the resistant rice seedlings, while its expression was upregulated in 48 h and then dropped at 72 h in the susceptible rice. Moreover, although the differential expression along with the developmental stages of A. besseyi, the mRNA level of Ab-hsp90 was also largely induced in the nematodes feeding on the callus of Daucus carota and hyphae of Botrytis cinerea in 9 d. Our data suggested that Ab-hsp90 can assist A. besseyi to cope with environmental stresses, infect plants and feeding. The results may provide a new perspective to dissecting the functions of hsp90 proteins in plant parasitic nematodes, which thus is conducive to the further study of nematode and plant interactions.
冯辉 陈曦 束兆林 姚克兵 魏利辉. 水稻干尖线虫Hsp90基因克隆及在不同逆境、侵染早期和取食过程的表达差异[J]. , 2016, 24(11): 1741-1753.
Hui Feng Li-Hui Wei. Characterization of the Hsp90 Gene in the White Tip Nematode(Aphelenchoides besseyi) and Its Expression in Response to Environmental Stresses, Early Infection and Feeding. , 2016, 24(11): 1741-1753.
[1]Aljanabi, S M and Martinez, I.1997. Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques[J]. Nucleic Acids Research, 25(22): 4692-4693. [2]Angel, S O, Figueras, M J, Alomar, M L, et al.2014. Toxoplasma gondii Hsp90: potential roles in essential cellular processes of the parasite[J]. Parasitology, 141(9): 1138-1147. [3]Arena, J P, Liu, K K, Paress, P S, et al.1995. The Mechanism of Action of Avermectins in Caenorhabditis Elegans: Correlation between Activation of Glutamate-Sensitive Chloride Current, Membrane Binding, and Biological Activity[J]. The Journal of Parasitology, 81(2): 286-294. [4]Birnby, D A, Link, E M, Vowels, J J, et al.2000. A transmembrane guanylyl cyclase (DAF-11) and Hsp90 (DAF-21) regulate a common set of chemosensory behaviors in Caenorhabditis elegans[J]. Genetics, 155(1): 85-104. [5]Burkewitz, K, Choe, K P, Lee, E C-H, et al.2012. Characterization of the proteostasis roles of glycerol accumulation, protein degradation and protein synthesis during osmotic stress in C. elegans[J]. PLoS One, 7(3): e34153. [6]Burnell, A and Tunnacliffe, A.2011. Gene induction and desiccation stress in nematodes. In: Perry, R N, and Wharton, D A, (Eds.), Molecular and Physiological Basis of Nematode Survival. CABI Press, Wallingford, UK, pp. 126-156. [7]Cheng, X, Xiang, Y, Xie, H, et al.2013. Molecular characterization and functions of fatty acid and retinoid binding protein gene (Ab-far-1) in Aphelenchoides besseyi[J]. PLoS One, 8(6): e66011. [8]Cuc, N T T, Son, N T, Trung, T M, et al.2010. Hot water treatment prevents Aphelenchoides besseyi damage to Polianthes tuberosa crops in the Mekong Delta of Vietnam[J]. Crop Protection, 29(6): 599-602. [9]De Luca, F, Di Vito, M, Fanelli, E, et al.2009. Characterization of the heat shock protein 90 gene in the plant parasitic nematode Meloidogyne artiellia and its expression as related to different developmental stages and temperature[J]. Gene, 440(1-2): 16-22. [10]Denlinger, D L.2002. Regulation of diapause[J]. Annual Review of Entomology, 47(1): 93-122. [11]Devaney, E.2006. Thermoregulation in the life cycle of nematodes[J]. International journal for parasitology, 36(6): 641-649. [12]Devaney, E, O'Neill, K, Harnett, W, et al.2005. Hsp90 is essential in the filarial nematode Brugia pahangi[J]. International Journal for Parasitology, 35(6): 627-636. [13]EPPO.2004. Diagnostic protocols for regulated pests -Aphelenchoides besseyi[J]. Bulletin OEPP/EPPO Bulletin, 34(2): 303-308. [14]Faske, T R and Starr, J L.2006. Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Abamectin[J]. Journal of Nematology, 38(2): 240-244. [15]Feng, H, Wei, L, Chen, H, et al.2015. Calreticulin is required for responding to stress, foraging, and fertility in the white-tip nematode, Aphelenchoides besseyi[J]. Experimental Parasitology, 155: 58-67. [16]Feng, H, Wei, L, Lin, M, et al.2014. Assessment of rice cultivars in china for field resistance to Aphelenchoides besseyi[J]. Journal of Integrative Agriculture, 13(10): 1429-1438. [17]Gillan, V, Maitland, K, McCormack, G, et al.2009. Functional genomics of hsp-90 in parasitic and free-living nematodes[J]. International Journal for Parasitology, 39(10): 1071-1081. [18]Gokte, N and Mathur, V K.1989. Reproduction and development of Aphelenchoides besseyi[J]. Phytoparasitica, 17(4): 263-267. [19]Haslbeck, V, Kaiser, C J and Richter, K.2012[J].Hsp90 in non-mammalian metazoan model systems[J]. Biochimica et Biophysica Acta, 1823, (3):712-721 [20]Jamali, S, Pourjam, E, Alizadeh, A, et al.2008. Reproduction of the white tip nematode in different monoxenic cultures[J]. Journal of Agricultural Science and Technology, 10: 165-171. [21]Jones, O T, Haegeman, A, Danchin, E G J, et al.2013. Top 10 plant-parasitic nematodes in molecular plant pathology[J]. Molecular Plant Pathology, 14(9): 946-961. [22]Kikuchi, T, Cock, P J A, Helder, J, et al.2014. Characterisation of the transcriptome of Aphelenchoides besseyi and identi?cation of a GHF 45 cellulase[J]. Nematology, 16(1): 99-107. [23]Lin, M S, Ding, X F, Wang, Z M, et al.2004. Small grains and erect ears of rice caused by white-tip nematode Aphelenchoides besseyi in China[J]. Rice Science, 12(4): 289-294. [24]Liu, W H, Lin, M S, Li, H M, et al.2008. Dynamic development of Aphelenchoides besseyi on rice plant by artificial inoculation in the greenhouse[J]. Agricultral Science in China, 7(8): 970-976. [25]Lourenco-Tessutti, I T, Souza Junior, J D, Martins-de-Sa, D, et al.2015. Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction[J]. Phytopathology, 105(5): 628-637. [26]McClellan, A J, Xia, Y, Deutschbauer, A M, et al.2007. Diverse cellular functions of the Hsp90 molecular chaperone uncovered using systems approaches[J]. Cell, 131(1): 121-135. [27]Misirlioglu, B, Kocer, H and Ulutas, E, Using of hot water system to control of white tip nematode (Aphelenchoides besseyi christie) on rice.50 Years University of Forestry, Sofia, Sofia, Bulgaria, 2003, pp. 164-166. [28]Mitchum, M G, Hussey, R S, Baum, T J, et al.2013. Nematode effector proteins: an emerging paradigm of parasitism[J]. New Phytologist, 199(4): 879-894. [29]Nicol, J M, Turner, S J, Coyne, D L, et al.2011. Current nematode threats to world agriculture. In: Jones, J, Gheysen, G, and Fenoll, C, (Eds.), Genomics and Molecular Genetics of Plant-Nematode Interactions. Springer, Dordrecht, Netherlands, pp. 21-43. [30]Pratt, W B and Toft, D O.2003. Regulation of signaling protein function and trafficking by the hsp90/hsp70-based chaperone machinery[J]. Experimental Biology and Medicine, 228(2): 111-133. [31]Putter, I, Connell, J G M, Preiser, F A, et al.1981. Avermectins: novel insecticides, acaricides and nematicides from a soil microorganism[J]. Experientia, 37(9): 963-964. [32]Schmittgen, T D and Livak, K J.2008. Analyzing real-time PCR data by the comparative CT method[J]. Nature Protocols, 3(6): 1101-1108. [33]Shigeru, H and Katsumi, T.2000. Effect of water-soaking and air-drying on survival of Aphelenchoides besseyi in Oryza sativa seeds[J]. Journal of Nematology, 32(3): 303-308. [34]Spees, J L, Chang, S A, Snyder, M J, et al.2002. Osmotic induction of stress-responsive gene expression in the lobster Homarus americanus[J]. The Biological Bulletin, 203(3): 331-337. [35]Staheli, J P, Boyce, R, Kovarik, D, et al.2011. CODEHOP PCR and CODEHOP PCR Primer Design, PCR Protocols. Springer, pp. 57-73. [36]Tenente, R V V, Wetzel, M M V S, Manso, E S B G, et al.1994. Survival of Aphelenchoides besseyi in infested rice seeds stored under controlled conditions[J]. Nematologia Brasileira, 18: 85-92. [37]Tsutsumi, S, Mollapour, M, Graf, C, et al.2009. Hsp90 charged-linker truncation reverses the functional consequences of weakened hydrophobic contacts in the N domain[J]. Nature Structural & Molecular Biology, 16(11): 1141-1147. [38]Tulek, A, Kepenekei, I, Cobanoglu, S, et al.2009. A new culturing method for the rice white tip nematode, Aphelenchoides besseyi Christie, 1942, on carrot discs[J]. Russian Journal of Nematology, 17(2): 143-144. [39]van Megen, H, van den Elsen, S, Holterman, M, et al.2009. A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences[J]. Nematology, 11(6): 927-950. [40]Wang, F, Li, D, Wang, Z, et al.2014. Transcriptomic analysis of the rice white tip nematode, Aphelenchoides besseyi (Nematoda: Aphelenchoididae)[J]. PloS one, 9(3): e91591. [41]Wang, F, Wang, Z, Li, D, et al.2012. Identification and characterization of a Bursaphelenchus xylophilus (Aphelenchida: Aphelenchoididae) thermotolerance-related gene: Bx-HSP90[J]. International Journal of Molecular Sciences, 13(7): 8819-8833. [42]Yoshida, K, Hasegawa, K, Mochiji, N, et al.2009. Early embryogenesis and anterior-posterior axis formation in the white-tip nematode Aphelenchoides besseyi (Nematoda: Aphelenchoididae)[J]. Journal of Nematology, 41(1): 17-22. [43]Zhou, T, Gao, C, Du, L, et al.2014. Genetic analysis and QTL detection for resistance to white tip disease in rice[J]. PloS one, 9(8): e106099. [44]耿甜, 彭德良, 李建洪, 等.2011. 腐烂茎线虫热激蛋白新基因的克隆与序列分析[J]. 华中农业大学学报, 30(6): 693-700. (Geng, T, Peng, D L, Li, J H, et al. 2011. Cloning and sequence analysis of a new heat shock protein gene from Ditylenchus destructor[J]. Journal of Huazhong Agricultural University, 30(6): 693-700.) [45]孙洋, 盛洋, 柏立新, 等.2014. 取食不同寄主植物对绿盲蝽热激蛋白AlHSP90表达的影响[J]. 棉花学报, 26(2): 153-160. (Sun Y, Sheng Y, Bai L X, et al.2014. Expression analysis of heat shock protein 90 gene AlHSP90 in Apolygus lucorum (Meyer-Dür) after being fed on different host plants. Cotton Science, 26(2): 153-160.) [46]王胜君, 段玉玺, 靳莹莹, 等.2008. 水稻干尖线虫(Aphelenchoides besseyi) 人工培养研究[J]. 植物保护, 34(3): 46-48. (Wang S J, Duan Y X, Jin Y Y, et al. 2008 Artificial rearing of the rice white-tip nematode (Aphelenchoides besseyi). Plant Protection, 34(3): 46-48.)
