Title: Identification and Molecular Characterization of a Glycosyl Hydrolase Family 5 B-1,4-endoglucanase (Rr-eng-1) from the Reniform Nematode, Rotylenchulus reniformis Authors
Submitted to: Journal of Nematology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 23, 2011
Publication Date: December 1, 2010
Repository URL: http://handle.nal.usda.gov/10113/54290
Citation: Wubben, M.J, Ganji, S., Callahan, F.E. 2010. Identification and molecular characterization of a glycosyl hydrolase family 5 B-1,4-endoglucanase (Rr-eng-1) from the reniform nematode, Rotylenchulus reniformis. Journal of Nematology. 42:342-351. Interpretive Summary: In order to infect host plant roots, Rotylenchulus reniformis, the reniform nematode, must be able to penetrate the root epidermis and navigate its way to the central vascular bundle of the root. Penetration of the root epidermis would be facilitated by cellulolytic enzymes, i.e., cellulases, which degrade cellulose, the primary structural component of the plant cell wall. In this report, we present the first description of a cellulase gene (Rr-eng-1) from R. reniformis, which is a serious root pathogen of cotton, soybean, and many other crops. We determined that Rr-eng-1 encodes a predicted cellulase protein (Rr-ENG-1) of 414 amino acids that contains a signal peptide for secretion at its N-terminus. Rr-ENG-1 belongs to the glycosyl hydrolase family 5 (GHF5) group of cellulases, which is the most common group isolated from plant-parasitic nematodes. Rr-eng-1 is comprised of eight exons and seven introns. At the nucleotide and amino acid sequence level, Rr-eng-1 was most similar to Hg-eng-6, a cellulase from the soybean cyst nematode (Heterodera glycines). Rr-ENG-1 was also similar to the root-knot nematode (Meloidogyne incognita) cellulase Mi-ENG-2 in regards to the conservation of the relative positions of many intron splice junctions. GHF5 cellulases are normally composed of three parts: a catalytic domain, a linker sequence, and a cellulose binding module. Interestingly, Rr-ENG-1 showed the catalytic and linker domains but no cellulose binding module was present. We determined that Rr-eng-1 expression was steady throughout the R. reniformis vermiform life-stages; however, transcript levels decreased sharply following feeding site formation. The discovery of Rr-ENG-1 adds to our knowledge of parasitism gene evolution in plant-parasitic nematodes and provides a potential target for host-mediated gene silencing in order to increase host plant resistance to R. reniformis.
Technical Abstract: Glycosyl hydrolase family 5 (GHF5) ß-1,4-endoglucanses, a.k.a. cellulases, are important parasitism genes that facilitate root penetration and migration by plant-parasitic nematodes. The reniform nematode (Rotylenchulus reniformis) is a sedentary semi-endoparasite of >300 plant species for which little molecular data has been collected. In this report, we describe the isolation and characterization of a predicted GHF5 cellulase from R. reniformis that we have named Rr-eng-1. The Rr-eng-1 cDNA was 1,341 bp long and was comprised of a 19 bp 5'-untranslated region (UTR), a 1,242 bp open reading frame (ORF), and an 80 bp 3'-UTR. The corresponding Rr-eng-1 genomic sequence was 2,325 bp. Alignment of the cDNA and genomic sequences revealed 7 introns and 8 exons for Rr-eng-1. BLASTN analysis showed the Rr-eng-1 cDNA was most homologous (E=5/e-121) to the H. glycines cellulase Hg-eng-6. A Southern blot probed with DIG-labeled Rr-eng-1 cDNA suggested a total of three Rr-eng-1-like sequences were present in the R. reniformis genome. Translation of the Rr-eng-1 ORF yielded a 414 amino acid peptide having an N-terminal signal sequence for secretion as determined by SignalP3.0. No cellulose binding domain (CBD) was detected in the Rr-ENG-1 protein; however, a putative CBD linker sequence N-terminal to the GHF5 cellulase domain was present. Rr-ENG-1 was most homologous to Hg-ENG-6 but also shared a number of intron splice positions with Mi-ENG-2. Quantitative reverse-transcription PCR indicated that Rr-eng-1 expression was roughly equivalent among all vermiform life-stages with a sharp decline in expression detected in sedentary parasitic females.