Proteomic profiles of soluble proteins from the esophageal gland in female Meloidogyne incognita Xin-Rong Wang a , Yovany A. Moreno b,1 , Han-Rong Wu a , Chao Ma a , Yun-feng Li a , Jin-ai Zhang a , Chong Yang a , Si Sun a , Wei-jie Ma a , Timothy G. Geary b,⇑ a Laboratory of Plant Nematology, College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China b Institute of Parasitology, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada article info Article history: Received 18 June 2012 Received in revised form 9 October 2012 Accepted 10 October 2012 Available online 8 November 2012 Keywords: Protein extraction LC–MS/MS Meloidogyne incognita Nematode Plant parasite Esophageal gland cells Secretome abstract Meloidogyne incognita can infect multiple plant species. Proteins synthesized in the esophageal glands and secreted through the stylet of plant parasitic nematodes play critical roles in the plant-nematode interactions. Female M. incognita live for approximately 15 days, embedded in a host plant, but their esophageal gland proteins have not yet been comprehensively analyzed. In this study, a new bacterium- contamination-resistant method for collecting soluble proteins from esophageal gland cells (SPEGC) of female M. incognita was established. Approximately 5 lg of freeze-dried proteins could be extracted from 150 female M. incognita. Bands of a one-dimensional SDS–polyacrylamide gel were excised after electrophoresis of 20 lg of protein and were analyzed. Two hundred and forty-six proteins from SPEGC of female M. incognita were identified by LC–MS/MS. Gene Ontology analysis suggests that many of the secreted proteins are involved in protein or carbohydrate metabolism and proteolysis. Some of the SPEGC (46.3%) were predicted to be secreted through classical or non-classical secretory pathways. The described method presents a new approach for the identification of proteins stored in SPEGC of an impor- tant plant parasitic nematode. This global proteomic profile of SPEGC provides a basis for future studies to elucidate the functions of proteins secreted from female M. incognita on plant responses. Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. 1. Introduction Root-knot nematodes in the genus Meloidogyne spp. can infect 1700 plant species (Sasser et al., 1983). These parasites synthe- size secretory proteins in dorsal and subventral esophageal gland cells and release these proteins through a hollow, protrusible stylet (feeding structure) during migration within plant roots. A subse- quent modification of root cells into the elaborate permanent feed- ing cells (called giant cells) ensues (Hussey and Grundler, 1998). During the life cycle of a root knot nematode, only the second juve- nile (J2) and adult female stages possess stylets (Abdel-Rahman and Maggenti, 1987). Even though females live up to 10 days long- er than J2s in host root tissue, most previous studies of the host- parasite interface have focused on how J2 stages initiate giant cells in a host root, and how host gene expression in a giant cell is altered up to 14 days p.i. (Jammes et al., 2005; Dubreuil et al., 2007). Few studies have focused on the adult female – host plant interface. Initial analyses of female root-knot nematode stylet exu- date revealed the presence of at least nine major protein bands. Upon acid hydrolysis, 14 proteins were detected in the stylet exu- date (Veech et al., 1987). In subsequent studies, 26 putative para- sitism genes encoding proteins preceded by a signal peptide for secretion and expressed exclusively within the esophageal gland cells of J3-adult Meloidogyne incognita were characterized by high-throughput in situ hybridization with cDNA clones that included a signal peptide (Huang et al., 2003), but proteins were not isolated. As the biology of host giant cells clearly changes during the growth of adult female root-knot nematodes (Wang et al., 2011), it was of interest to explore the female M. incognita secretome in greater depth. A major drawback to the study of the proteomics of esophageal glands of adult female M. incognita is the small quantity of proteins present in this compartment, requiring a procedure to concentrate the proteins preceding their analysis while precluding the incorpo- ration of contaminating bacterial proteins. For example, Veech et al. (1987) collected female stylet exudates in moisturized chambers exposed to 20 s pulses of 302-nm radiation (>1600 W/cm 2 /s) dur- ing the incubation to suppress bacterial contamination; an individ- ual can collect 50 nl of exudate in 8 h using this method. We developed an alternative time-saving and bacterium-contamination- resistant method to collect soluble proteins from esophageal gland cells (SPEGC) of female M. incognita for proteomic analysis. 0020-7519/$36.00 Ó 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijpara.2012.10.008 ⇑ Corresponding author. Tel.: +1 514 398 7612; fax: +1 514 398 7857. E-mail address: timothy.g.geary@mcgill.ca (T.G. Geary). 1 Current address: Harvard School of Public, Boston, MA 02115, USA. International Journal for Parasitology 42 (2012) 1177–1183 Contents lists available at SciVerse ScienceDirect International Journal for Parasitology journal homepage: www.elsevier.com/locate/ijpara