Dataset Information

Pratylenchus penetrans PME - Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans

ABSTRACT: Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, showing the presence of four introns that excluded a potential bacterial contamination. Expression of the Pp-pme gene was localized in the esophageal glands of P. penetrans as determined by in situ hybridization. Temporal expression of Pp-pme in planta was validated for early time points of infection. The possible function and activity of the gene were assessed by transient expression of Pp-pme in N. benthamiana plants via a Potato virus X-based vector. To our knowledge, this is the first report on identification and characterization of a PME gene within the phylum Nematoda.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Nicotiana Tabacum (common Tobacco)

TISSUE(S): Leaf

SUBMITTER: Cláudia Vicente

LAB HEAD: Paulo Vieira and Len Nemchinov

PROVIDER: PXD012419 | Pride | 2019-02-28

REPOSITORIES: Pride

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Publications

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Vicente Cláudia S L CSL Nemchinov Lev G LG Mota Manuel M Eisenback Jonathan D JD Kamo Kathryn K Vieira Paulo P

PloS one 20190222 2

Similar to other plant-parasitic nematodes, root lesion nematodes possess an array of enzymes that are involved in the degradation of the plant cell wall. Here we report the identification of a gene encoding a cell wall-degrading enzyme, pectin methylesterase PME (EC 3.1.1.11), in the root lesion nematode Pratylenchus penetrans. Both genomic and coding sequences of the gene were cloned for this species, that included the presence of four introns which eliminated a possible contamination from bac ...[more]

PMID: 30794636

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Project description:Comparison of gene expression from subjects who resolved or formed pustules to H.ducreyi. In human inoculation experiments, the cutaneous immune response to Haemophilus ducreyi consists of serum, PMN, macrophages, T cells and myeloid DC. In reinfection experiments, some subjects form pustules twice (PP group) or resolve infection twice (RR group). Although pustule formation is associated with serum resistance and phagocytic failure, there are no differences in the ability of isolated phagocytes or serum obtained from PP and RR subjects to ingest or kill H. ducreyi. To identify the basis for differential host susceptibility to H. ducreyi, we used microarrays to profile gene expression in infected and uninfected tissue and monocyte-derived DC obtained from PP and RR subjects. In infected tissue, both groups had a core response to H. ducreyi. Many additional transcripts that signify active immune function were upregulated exclusively in RR tissue, while PP tissue exclusively contained differentially regulated transcripts consistent with immune dysregulation. The core response of infected DC from both groups was typical of a DC1 response. RR DC exclusively expressed many additional transcripts indicative of DC1 function, while PP DC uniquely expressed differentially regulated transcripts characteristic of both DC1 and DCreg. The data suggest that DC from PP and RR subjects are prewired to respond differentially to H. ducreyi. Experiment Overall Design: Six healthy adult volunteers (5 females, 1 male, 36 Â± 13 yrs, mean age Â± SD) who had been infected with H. ducreyi twice were inoculated a third time. Each volunteer was inoculated at 3 sites on the upper arm with live H. ducreyi 35000HP (a human passaged isolate of strain 35000) and at 1 site with sterile PBS. Forty-eight hours after inoculation, lesion size was measured and RNA was isolated from the infected site that had the largest diameter. RNA was also obtained from the PBS control site (uninfected site). We obtained peripheral blood 6 to 12 months after inoculation of the 6th subject and derived myeloid DC in vitro. In brief, CD14+ peripheral blood monocytes were isolated from PBMC by positive selection magnetic beads (Miltenyi Biotec, Auburn, CA) and grown in the presence of recombinant human (rh) IL-4 (1 ng/ml) and rhGM-CSF (0.2 ng/ml) (R&D Systems, Minneapolis, MN). The cells were HLA-DR+, CD86+ CD40+, CD3-, CD14-, and CD19- by flow cytometry. The experiment was done in pairs so that DC from 1 RR subject were exposed to the same inoculum as DC from 1 PP subject. DC were incubated with nonopsonized H. ducreyi for 90 minutes at an MOI of 30:1. The DC were washed to remove non-associated bacteria, and incubated an additional 22.5 hours. Cells were collected and used for microarray analysis. Supernatants were collected and analyzed for cytokines using the Human Th1/Th2 II Cytometric Bead Array Kit per manufacturerâs instructions (BD Biosciences).

Dataset Information

Pratylenchus penetrans PME - Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans

Publications

Identification and characterization of the first pectin methylesterase gene discovered in the root lesion nematode Pratylenchus penetrans.

Similar Datasets

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