Title: Identification and characterization of C-type lectin genes from the reniform nematode, Rotylenchulus reniformis Authors
Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 19, 2013
Publication Date: N/A
Interpretive Summary: The reniform nematode (RN) is an important pathogen of many crops that are vital to the U.S. economy such as cotton, soybean, and pineapple. In order to successfully reproduce on a host plant, RN must establish a complex feeding site within the host root from which the nematode is able to withdraw nutrients from the plant. In addition to feeding site formation, the RN parasitic life-stage must protect itself from various plant defense responses and from microbial pathogens that reside in the soil microenvironment. Our research had previously identified a number of RN genes that were expressed at high levels during the RN parasitic life-stage. One gene in particular, a putative C-type lectin (CTL), was further examined in the present study. CTL proteins bind carbohydrate molecules such as mannose and are found in organisms throughout the tree of life (humans, plants, fungi, insects, etc.). CTL proteins have been shown to play an important role in the immune response of invertebrate organisms to microbial pathogens. We discovered that RN expresses a total of 11 CTL genes. The majority of these genes were expressed in only a few life-stages but three were found to be expressed in every RN life-stage. When the total amount of CTL expression was measured in each RN life-stage, the parasitic life-stage show almost 900-fold more expression than the next highest life-stage; therefore, CTLs appear to serve an important function during plant parasitism. Next, we determined that CTL expression in parasitic nematodes was confined to the hypodermis of those parts of the nematode exposed to the soil during parasitism, i.e., the ‘head’ and ‘neck’ of the nematode, which are embedded within the root, did not show CTL expression. When the RN CTL protein sequences were analyzed, we discovered a unique motif that has not been reported for other CTL proteins. This motif was similar to that found in bacterial proteins that are responsible for assembling water molecules into scaffolds that initiate ice formation. We also determined that CTL proteins expressed by parasitic nematodes have diverged from those expressed by non-parasitic species. This study represents the first in-depth characterization of a CTL gene family in plant-parasitic nematodes and suggests that RN CTLs play a role in protecting the parasitic life-stage from the soil microenvironment during feeding.
Technical Abstract: The reniform nematode, Rotylenchulus reniformis, is a damaging semi-endoparasitic pathogen of more than 300 plant species. As a sedentary obligate biotroph, R. reniformis must establish a complex feeding site within the root vasculature that functions as a continuous supply of nutrients. It was recently shown that the transcriptome of R. reniformis parasitic females was enriched for sequences homologous to C-type lectins (CTLs). CTLs are an evolutionarily ancient family of Ca+2-dependent carbohydrate- binding proteins that constitute an important component of the innate immune response by serving as pattern recognition receptors. Due to its high level of expression in the parasitic life-stage and to gain further insight as to the potential role of CTLs in facilitating plant parasitism by R. reniformis, we performed a comprehensive assessment of the R. reniformis CTL gene family. 5'- and 3'-RACE experiments using life-stage specific cDNAs identified a total of 11 R. reniformis CTL transcripts (Rr-ctl-1 through Rr-ctl-11). Rr-ctl cDNAs ranged in length from 1083-1194 bp and showed 93-99% identity with one another. An alignment of cDNA and genomic sequences revealed three introns with the first intron residing within the 5'-untranslated region. BLAST analyses showed the closest homologs belonging to the parasitic nematodes Heligmosomoides polygyrus and Heterodera glycines. Rr-ctl-1, -2, and -3 were expressed throughout the R. reniformis life cycle; whereas, the remaining Rr-ctl genes showed life stage-specific expression. Quantitative RT-PCR determined that Rr-ctl transcripts were most abundant in sedentary female nematodes, being 192-fold higher than the next most abundant life stage. Predicted Rr-CTL peptides ranged from 301-338 amino acids long, possessed an N-terminal signal peptide for secretion, and contained a conserved CLECT domain having the mannose-binding motifs EPN and EPD and the conserved WND motif that is required for binding Ca+2. We also determined that Rr-CTL peptides harbored repeats of a novel 17-mer motif within their C-terminus that showed similarity to motifs normally associated with bacterial ice nucleation proteins. In situ hybridization of Rr-ctl transcripts within sedentary female nematodes showed specific accumulation within the cuticle and hypodermis of the body regions exposed to the soil environment; those structures normally embedded within the root during parasitism did not show Rr-ctl expression. A phylogenetic analysis of the Rr-CTL CLECT domain with homologous domains from a variety of nematode species suggested that CTLs from animal and plant-parasitic genera may have evolved in order to play an active role in the parasitic process. The results presented here indicate that the R. reniformis CTL gene family (i) shows differential expression between life-stages, (ii) possesses an unusual, and, heretofore unreported for CTL proteins,17-mer amino acid motif with similarity to motifs involved in ice nucleation, and (iii) plays an important role in mediating the interaction between the sedentary female and the soil microenvironment.