Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus
Graphical abstract
Southern blot analysis of the Bx-crt-1 gene and effect of RNAi treatment on propagation ability of B. xylophilus 4 d (A) or 8 d (B) after treatment.
Research highlights
► Presently, Calreticulin (CRT) has only been isolated from Meloidogyne incognita, an obligatory sedentary endoparasitism nematode. ► Here, we report the first cloning and characterization of a CRT gene (Bx-crt1) from Bursaphelenchus xylophilus, a migratory parasitism nematode. ► Bx-crt-1 is important for the development of B. xylophilus. ► Such a comparative analysis would provide new insights in understanding the molecular factors involved in two different modes of parasitism.
Introduction
In plant–parasitic nematodes, stylet secretions synthesized in esophageal subventral and dorsal glands are secreted through the stylet during parasitism. Stylet secretions are thought to play a key role throughout parasitism (Jaubert et al., 2005). Recently, many genes of plant-parasitic nematodes expressed in the esophageal gland cells were identified in order to understand the role of these genes in parasitism. In Meloidogyne, an endoparasitic sedentary nematode, more than 20 genes are expressed in the esophageal gland (Liao et al., 2009), including the Mi-crt calreticulin (CRT) gene from M. incognita (Jaubert et al., 2002). CRT is a multifunctional and conserved chaperone calcium-binding protein (Cabezón et al., 2008). CRT has been reported in some animal–parasitic and free-living nematodes such as Necator americanus, Haemonchus contortus, and Caenorhabditis elegans (Kasper et al., 2001, Kamath and Ahringer, 2003, Suchitra and Joshi, 2005). CRT has only been isolated from the plant–parasitic nematodes M. incognita, and Mi-crt may be important in parasitism (Jaubert et al., 2005, Dubreuil et al., 2009).
Bursaphelenchus xylophilus, an endoparasitic migratory nematode, causes severe damage to pines causing pine wilt in China, Japan, and Korea (Mamiya, 1988, Lee et al., 1990, Yang, 2003). β-1,3-endoglucanase, β-1,4-endoglucanase, pectate lyases (Kikuchi et al., 2004, Kikuchi et al., 2006, Kikuchi et al., 2005), and expansin-like genes (Kikuchi et al., 2009) are expressed in B. xylophilus esophageal glands. Kang et al. (2009) established expressed sequence tag databases of the B. xylophilus dispersal and propagative life stages by subtractive hybridization, providing a basis for understanding the pathogenesis molecular mechanisms and new B. xylophilus control strategies. However, CRT has not yet been identified in B. xylophilus. In this study, we report the first cloning and characterization of a CRT gene (Bx-crt1) from a population of B. xylophilus that was strongly pathogenic to Pinus massoniana.
Section snippets
Nematode culture and collection
The pinewood nematodes used in this study were isolated from Pinus massoniana in Ruyuan County, Guangdong Province, China. To obtain a pure B. xylophilus population, a single adult female was selected and cultured on Pestalotiopsis sp. grown on autoclaved potato dextrose agar (PDA) plates at 25 °C in the dark for about one month, in which 100 nematodes were subcultured in another new plate in the same conditions. Nematodes and eggs were collected by washing off plates with sterilized water and
Characterization of the B. xylophilus CRT gene
A 399 bp PCR product was amplified using degenerate primers BxcrtFw and BxcrtRv. The 3′ and 5′ RACE reactions produced a 1620 bp Bx-crt-1 sequence (NCBI GenBank accession GU585910). The full-length cDNA contains a 14 bp 5′-untranslated region upstream of the ATG initiation codon, a 1200 bp ORF that terminates with the TAA stop codon, and a 376 bp untranslated sequence at the 3′ end with a poly(A) tail. The 3′ UTR contained a typical polyadenylation signal (ATAAA). The ORF translated to a 399 amino
Discussion
In this paper, a Bx-crt-1 CRT was cloned from B. xylophilus. To date, only one plant nematode CRT was isolated from M. incognita in Tylenchida (Jaubert et al., 2002), and Bx-crt-1 is the first CRT from a plant Aphelenchida nematode. The hybridization pattern obtained by Southern blot and the assembly of ESTs encoding calreticulin indicated B. xylophilus may have one calreticulin gene and that Bx-crt-1 is probably a single-copy gene.
In C. elegans, the crt-1 CRT is expressed in the pharynx,
Acknowledgments
This research was supported by Genetically Modified Organisms Breading Major Projects with grant No. 2009ZX08009-045B, the Natural Science Foundation of China with grant No. 30871628, Special Scientific Research Funds for Commonweal Section of China with grant No. nyhyzx07-050-4, National Technology R&D Program in the 11th Five year Plan of China with grant No. 2006BAD08A08, Planning Project for Science and Technology in Guangdong Province with grant No. 2005B20801013 and No. 2007B020709008 and
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2015, Experimental ParasitologyCitation Excerpt :The single-copy gene, determined by Southern blotting, is transcribed mainly in adult nematodes and less so in juvenile nematodes. BxCRT-1-silenced B. xylophilus was less fecund in vitro than normal nematodes of the species, suggesting that BxCRT-1 has an important role in the development of B. xylophilus (Li et al., 2011). The white-tip nematode Aphelenchoides besseyi, which is among the top ten plant parasitic nematodes, lowers rice yields by 12%–20%; the loss worldwide is estimated at $16 billion (Jones et al., 2013).
Amblyomma americanum tick calreticulin binds C1q but does not inhibit activation of the classical complement cascade
2015, Ticks and Tick-borne DiseasesCitation Excerpt :CRT was originally identified as a high calcium (Ca2+) binding protein from the sarcoplasmic reticulum of rabbit muscle (Ostwald and MacLennan, 1974; Fliegel et al., 1989). Coding cDNAs have now been identified in multiple organisms including ticks (Jaworski et al., 1995; Ferreira et al., 2002; Xu et al., 2004, 2005; Kaewhom et al., 2008; Gao et al., 2008), fleas (Jaworski et al., 1996), nematodes (Smith, 1992; Huggins et al., 1995; Tsuji et al., 1998; Scott and McManus, 1999; Mendlovic et al., 2004; Cabezón et al., 2008; Li et al., 2011), protozoan parasites (Aguillón et al., 2000; Marcelain et al., 2000; González et al., 2002), fish (Kales et al., 2004; Bai et al., 2012) and plants (Jia et al., 2008; Jin et al., 2009; An et al., 2011). Many more CRT sequences have been deposited in GenBank unpublished as direct submissions.
RNA interference in Pratylenchus coffeae: Knock down of Pc-pat-10 and Pc-unc-87 impedes migration
2012, Molecular and Biochemical ParasitologyCitation Excerpt :Today, RNAi has become an established experimental technique in a number of parasitic nematodes to investigate the function of different genes. The successful application of RNAi as tool for functional genomics has also been demonstrated in plant parasitic nematodes including the sedentary endoparasitic nematodes Globodera pallida, Heterodera glycines and Meloidogyne incognita [10–13] as well as the migratory parasitic nematodes Radopholus similis [14] and Bursaphelenchus xylophilus [15–18]. Research on plant–nematode interactions has been mainly focused on the sedentary endoparasitic nematodes of the genus Globodera, Heterodera and Meloidogyne.
An eye on RNAi in nematode parasites
2011, Trends in ParasitologyCitation Excerpt :These difficulties were further highlighted in a number of reports and reviews that voiced concerns on the applicability of RNAi in animal parasitic nematodes (APNs) [7–10]. In plant parasitic nematodes (PPNs), RNAi has been demonstrated in both sedentary (Glossary) endoparasites (Meloidogyne incognita infective J2 s and eggs [11–18], Meloidogyne artiellia eggs [19], Globodera rostochiensis J2 s [20,21], Globodera pallida J2 s [6,16,22], Heterodera glycines J2 s [6,23–26], Meloidogyne javanica J2 s [27,28]) and in migratory endoparasites (Bursaphelenchus xylophilus L2, L3, L4 and adults [29–31] and Radopholus similis mixed stage larvae [32]) (Box 1). Indeed, published RNAi data for PPNs are overwhelmingly positive with almost unanimous success in the knockdown of genes using dsRNAs or siRNAs.
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These authors contributed equally to the work.