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United States Department of Agriculture

Agricultural Research Service

Research Project: Germplasm Enhancement and Genetic Improvement of Cotton

Location: Genetics and Precision Agriculture Research

Title: Isolation and characterization of a GHF5 b-1,4-endoglucanase from the reniform nematode (Rotylenchulus reniformis)

Authors
item Wubben, Martin
item Callahan, Franklin

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 23, 2010
Publication Date: July 11, 2010
Citation: Wubben, M., Callahan, F.E. 2010. Isolation and characterization of a GHF5 b-1,4-endoglucanase from the reniform nematode (Rotylenchulus reniformis). Proceedings of the 2010 Society of Nematology Conference. p. 118-119.

Technical Abstract: The reniform nematode (Rotylenchulus reniformis) is a semi-endoparasitic root pathogen of >300 plant species, including cotton, soybean, and pineapple. Plant-parasitic nematode (PPN) penetration of the root epidermis is facilitated by a collection of cell wall degrading enzymes that are secreted from the esophageal gland cells. Glycosyl hydrolase family 5 (GHF5) ß-1,4-endoglucanses, a.k.a. cellulases, comprise a significant portion of this collection. Recently, R. reniformis cDNAs were identified as part of an expressed sequence tag project that showed similarity to a Heterodera glycines cellulase cDNA. In this report, we describe the isolation and characterization of a predicted GHF5 ß-1,4-endoglucanse gene from R. reniformis. The full-length Rr-eng-1 cDNA was 1,341 nucleotides (nt) long and was comprised of a 19 nt 5'-untranslated region (UTR), a 1,242 nt open reading frame (ORF), and an 80 nt 3'-UTR. Forward and reverse PCR primers specific to the 5'- and 3'-UTRs, respectively, amplified an Rr-eng-1 genomic sequence of 2,325 nt. 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 to the H. glycines cellulase Hg-eng-6 (E=5.0e-121). The Radopholus similis cellulase precursor eng1A was the next most homologous sequence at the DNA level with an E value of 2.0e-18. 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 and to cellulases from migratory PPNs. Quantitative reverse-transcription PCR indicated that Rr-eng-1 expression was highest in later juvenile stages and vermiform infective females; however, Rr-eng-1 transcript was detected in total RNAs isolated from R. reniformis eggs, second-stage juveniles (J2), and sedentary parasitic females.

Last Modified: 4/20/2014