Isolation of Beta-1,4-Endoglucanase Genes from Globodera Tabacumand Their Expression During Parasitism M

Plant Pathology and Microbiology Publications Plant Pathology and Microbiology 6-2000 Isolation of Beta-1,4-Endoglucanase Genes from Globodera tabacumand their Expression During Parasitism M. Goellner North Carolina State University G. Smant Wageningen University J. M. De Boer Iowa State University T. J. Baum Iowa State University, [email protected] E. L. Davis NForltloh wCa thirolisn aa ndStat ae ddUnitiverionsitaly works at: http://lib.dr.iastate.edu/plantpath_pubs Part of the Agricultural Science Commons, Agriculture Commons, Genetics and Genomics Commons, Plant Breeding and Genetics Commons, and the Plant Pathology Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ plantpath_pubs/188. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html. This Article is brought to you for free and open access by the Plant Pathology and Microbiology at Iowa State University Digital Repository. It has been accepted for inclusion in Plant Pathology and Microbiology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Journal of Nematology 32(2):154–165. 2000. © The Society of Nematologists 2000. Isolation of Beta-1,4-Endoglucanase Genes from Globodera tabacum and their Expression During Parasitism1 M. Goellner,2 G. Smant,3 J. M. De Boer,4 T. J. Baum,4 and E. L. Davis2 Abstract: Two beta-1,4-endoglucanase (EGase) cDNAs were isolated from Globodera tabacum, the tobacco cyst nematode, and have been designated as GT-eng-1 and GT-eng-2. GT-eng-1 and GT-eng-2 encode precursor proteins with a predicted secretion signal sequence, cellulolytic catalytic domain, and a linker domain. The protein product GT-ENG-1 contains an additional 95 amino acid carboxy terminal sequence with strong similarity to type II cellulose binding domains. Riboprobes and polyclonal antibodies raised to recombinant cyst nematode EGases were used to follow expression patterns of EGase transcripts and proteins throughout the nematode life cycle. EGase transcripts and proteins were specifically detected within the subventral esophageal gland cells of G. tabacum second-stage juveniles (J2) within eggs prior to hatching, in preparasitic J2, and in parasitic J2 that had invaded tobacco roots. EGase transcripts and proteins were not detected in G. tabacum after the molt to the sedentary J3, J4, and adult female life stages. Interestingly, EGase transcription and translation resumed in the subventral esophageal glands of late J4 males. It is hypothesized that secreted EGases play a major role to facilitate intracellular migration of G. tabacum within tobacco roots. Key words: beta-1,4-endoglucanases, EGase, esophageal gland cells, Globodera tabacum, Nicotiana tabacum, stylet secretions, tobacco, tobacco cyst nematode. Beta-1,4-endoglucanases (EGases) are a plant cell contents (Barras et al., 1994; Rob- class of hydrolytic enzymes produced by a erts et al., 1988). diverse group of organisms including bacte- Until recently, no direct evidence existed ria, fungi, insects, and plants (Brummel et for the endogenous production of cellulo- al., 1994; Davis et al., 2000; Henrissat and lytic enzymes in animals. Smant et al. (1998) Bairoch, 1993). EGases function in the hy- isolated and cloned the first EGases of ani- drolysis of the beta-1,4-glucan backbone of mal origin from the plant-parasitic cyst polymers such as cellulose and xyloglucan, nematodes Globodera rostochiensis and Het- which are two of the major carbohydrate erodera glycines. This discovery is the first di- components of the plant cell wall (McNeil et rect evidence in support of earlier biochemi- al., 1984). An ensemble of endogenous hy- cal studies that suggested that nematodes drolytic enzymes, including EGases, pro- produced cellulolytic enzymes (Bird et al., motes the degradation of cell walls to serve 1975; Deubert and Rohde, 1971). Genes for as a primary carbon source for many micro- EGases with and without a predicted cellu- organisms (Beguin and Aubert, 1994). Ad- lose-binding domain (CBD) were cloned ditionally, cellulolytic enzymes function as from both species of cyst nematode and pathogenicity factors for many phytopatho- named eng-1 and eng-2, respectively (Smant genic fungi and bacteria by facilitating entry et al., 1998). It was demonstrated that the into host tissue and enhancing access to EGases are produced endogenously within the two subventral esophageal gland (SvG) cells of cyst nematodes and are secreted through the nematode stylet into plant tis- Received for publication 9 January 2000. sue (De Boer et al., 1999; Smant et al., 1997; 1The research reported in this publication was supported by funding provided by the North Carolina Agricultural Research Smant et al., 1998; Wang et al., 1999). Ex- Service, and North Carolina Tobacco Research Commission pression of EGases in the SvG of H. glycines under grant 6-64855. 2Department of Plant Pathology, North Carolina State Uni- ends in the early parasitic J3 stage and, in- versity, 1515 Gardner Hall, Raleigh, NC 27695. terestingly, resumes only in males as they 3Laboratory of Nematology, Wageningen University (WUR), 6709 PD Wageningen, The Netherlands. develop to adults (De Boer et al., 1999). It is 4Department of Plant Pathology, Iowa State University, 351 hypothesized that these enzymes may facili- Bessey Hall, Ames, IA 50011. Email: eric–[email protected] tate penetration and intracellular migration This paper was edited by B. C. Hyman. of root tissue by nematodes during parasit- 154 Globodera tabacum Beta-1,4-Endoglucanases: Goellner et al. 155 ism. In addition, cyst nematode EGases fresh J2 and other life stages of G. tabacum could contribute to the partial cell wall dis- were used for protein and nucleic acid ex- solution that occurs among neighboring tractions, and some were fixed for localiza- cells of developing feeding sites (syncytia; tion analyses. Jones and Dropkin, 1975; Jones, 1981), al- For in situ hybridization and immunolo- though this has not been definitively dem- calization analyses, eggs were fixed in 2% onstrated. paraformaldehyde in M9 buffer (42 mM The monoclonal antibody (MGR48) used Na2HPO4,22mMKH2PO4, 85 mM NaCl, 1 ⅐ to immunopurify EGases from G. rostochien- mM MgSO4 7H2O, pH 7.0) plus 10% su- sis, and H. glycines (Smant et al., 1988) also crose for 5 days at 4 °C. Preparasitic J2 were recognized SvG antigens of other cyst nema- fixed in 2% paraformaldehyde in M9 buffer tode species including G. tabacum, the to- overnight at 4 °C. Mixed parasitic stages of bacco cyst nematode (De Boer et al., 1996). G. tabacum were collected from infected to- Globodera tabacum is a sedentary endopara- bacco roots that were washed, cut into 2.5- site with a host range limited to tobacco and cm pieces, suspended in water, and some solanaceous weeds (Miller and Gray, chopped in a blender for 30 seconds. An 1972). If cellulase production is a common equal volume of 2× fixative (4% paraform- mechanism used by plant-parasitic nema- aldehyde in 2× M9 buffer) was added to the todes to invade roots and to establish feed- root suspension and incubated at room tem- ing sites, then this may be a feasible target perature for 1 hour for initial fixation to for developing transgenic strategies to im- reduce mRNA degradation. The root sus- prove plant disease resistance. The G. taba- pension was poured over a stack of sieves, cum-tobacco interaction provides a geneti- and parasitic nematodes were collected on a cally amenable model system, which can be 25-µm sieve and rinsed in water. Fixed para- used to test transgenic approaches that tar- sitic nematodes were separated from root get nematode cellulases for resistance to debris by mixing the slurry 1:1 with 70% su- plant-parasitic nematodes. In this paper, we crose and overlaid with a 1/4 volume of wa- describe the isolation of two G. tabacum beta- ter. The sucrose suspension was centrifuged 1,4-endoglucanase cDNAs, GT-eng-1 and GT- at 1,000g for 5 minutes, nematodes were pi- eng-2, and monitor their expression during peted from the upper 2/3 of the suspension, the parasitic cycle. and rinsed with water. Distinct nematode developmental stages were identified under Materials and Methods a dissecting microscope, separated by hand, and fixed further in 2% paraformaldehyde Nematode cultures: Globodera tabacum sub- in M9 buffer. Parasitic J2 and adult males species solanacearum (Miller and Gray, 1972) were fixed overnight at 4 °C in 2% para- was propagated on greenhouse-grown to- formaldehyde in M9 buffer. All other para- bacco (Nicotiana tabacum ‘NC95’). Cysts of sitic life stages were fixed overnight at room G. tabacum were extracted from infested soil temperature. Fixed nematodes were col- by flotation in water and collected on a 250- lected, rinsed in M9 buffer, and stored at µm sieve. The cysts were transferred to cen- −80 °C. trifuge tubes and mixed with 70% (w/v) su- Isolation of Globodera tabacum EGase cDNA’s: crose in a 1:1 ratio. Cysts were separated Total RNA was isolated from preparasitic J2 from contaminating debris by centrifugal using the Pharmacia Biotech (Piscataway, flotation at 1,000g for 5 minutes. Subse- NJ) Cs-TFA RNA extraction kit according to quently, the cysts were gently crushed in a the manufacturer’s protocol. Globodera taba- glass homogenizer to release nematode cum EGase cDNAs were amplified using 3Ј eggs, which were collected on a 25-µm sieve. and 5Ј random amplification of cDNA ends To collect preparasitic second-stage juve- (RACE) PCR systems (GibCo BRL, Rock- niles (J2), eggs were hatched over water at ville, MD). Globodera tabacum mRNA was con- 28 °C on a Baermann pan. Some aliquots of verted into cDNA using reverse transcriptase 156 Journal of Nematology, Volume 32, No. 2, June 2000 and the supplied oligo-dT primer.

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