Molecular cloning and characterization of a calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

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Abstract

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. 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.

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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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    These authors contributed equally to the work.

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