Figure 1.
A. Primary and secondary structure analysis of predicted Mj-FAR-1. A putative hydrophobic leader/signal peptide, as predicted by the SignalP program is shown at the N terminus along with a cleavage site between two alanine residues at position 20–21. Consensus N-linked glycosylation site (N-gly) and the consensus casein kinase II phosphorylation sites (CK2) are indicated. Secondary structure analysis through Jpred predicts a predominantly alpha-helical conformation (gray cylinder) with coiled-coil structures (gray arrow) as shown. B. Phylogenetic tree of Mj-FAR-1 with other nematode FAR proteins. The tree was constructed with FAR protein sequences of: C. elegans (Ce-FAR-1, Ce-FAR-2 and Ce-FAR-6) belong to group A, human parasitic nematodes O. volvulus (Ov-FAR-1) and W. bancrofti (Wb-FAR), animal parasitic nematodes A. suum (As-FAR) and T. canis (Tc-FAR), and plant-parasitic nematodes, M. javanica (Mj-FAR-1), M. incognita (Mi-FAR), M. arenaria (Ma-FAR), M. hapla (Mh-FAR) and M. chitwoodi (Mc-FAR), cyst forming nematodes G. pallida (Gp-FAR-1), G. rostochiensis (Gr-FAR), H. glycines (Hg-FAR), H. schachtii (Hs-FAR) and migratory endoparasites P. vulnus (Pv-FAR) and R. similis (Rs-FAR). Bootstrap values are shown at each node. 1,000 bootstrap replicates were obtained, with nearly the same results, and only a single tree retrieved from the phylogenetic relationship analysis is shown. Five clusters in the phylogenetic tree have been arbitrarily assigned the names A, B, C, D and E.
Figure 2.
Kinetics of mj-far-1 gene transcripts through M. javanica life cycle stages.
mj-far-1 expression was detected for non-parasitic stages: eggs and pre-parasitic infective juvenile J2s; and within tomato roots, for migratory parasitic J2s (6 h and 48 h after infection), J2s and J3/J4 juveniles, and mature females (15 and 28 DAI, respectively). mj-far-1 transcripts were normalized using the normalisation factor calculated as the geometric mean of the expression levels of two most stable nematode endogenous reference genes, 18S and EF-1α. Each reaction was performed in triplicate and the results represented the mean of two independent biological replicates. Data represent the mean relative expression and standard error obtained from two independent biological experiments. Different letters above the bars denote a significant difference (P≤0.05, analysis of variance) between samples as analyzed by Tukey-Kramer multiple comparison test. The experiment was repeated three times and similar results were obtained.
Figure 3.
Immunodetection of FAR proteins in Meloidogyne incognita pre-parasitic J2 and during parasitism of Arabidopsis thaliana roots.
(A) Cross nematode sections of pre-parasitic J2 displaying the protein localization at the nematode cuticle surface and circular granules structures within the posterior nematode body. (B) Localization of FAR proteins during nematode migration (B-B′), and nematode sedentary stages at 10 DAI (C-C′) and 21 DAI (D-D′) within the roots of A. thaliana. Arrows point out the accumulation of FAR along the nematode cuticle and adjacent cells surrounding the nematode body at 10 and 21 DAI. Micrographs on the left are overlays of Alexa-488 fluorescence (green) and DAPI-stained nuclei (blue). Micrographs on the right are overlays of an Alexa-488 fluorescence (green), DAPI-stained nuclei (blue) and differential interference contrast (grey). c, cuticle, g, granules, n, nematode, m, metacorpus, * giant cell. Bars = 10 µm.
Figure 4.
Constitutive expression of mj-far-1 in tomato hairy roots increases roots susceptibility to infection by the RKN M. javanica.
A. RT-PCR confirmation of mj-far-1 (upper gel) and kanR (lower gel) expression in tomato hairy roots lines far-1 E1.1, far-1 E 7.1, far-1 RNAi3.2 and far-1 RNAi11.5 compared with the respective control Vector 1.1 and WT 870. The expected size of the PCR product is 96 bp for the mj-far-1 and 81 for kanR. RT-PCR was performed on total RNA isolated from non infected transformed tomato hairy roots and WT 870 roots. B. Increased susceptibility of tomato hairy roots expressing mj-far-1 (far-1E1.1 and far-1E7.1) is accompanied by expanded galling production compared with mj-far-1 dsRNA-expressing tomato hairy root lines (far-1 RNAi3.2 and far-1 RNAi11.5) and vector control (Vector 1.1. and Vector 11.5). C. Meloidogyne susceptibility/resistance of vector transformed roots and transgenic tomato roots expressing mj-far-1, or mj-far-1 dsRNA-expressing lines, was measured as nematode developmental stages counted at 15 and 28 DAI. Roots were inoculated with 200 sterile pre-parasitic J2s and then assessed for juveniles, young females and mature females under the dissecting scope following staining with acid fuchsin dye. Note the significant (P≤0.05) increase in percentage of young females at 15DAI and increase in percentage of mature female at 28DAI in roots overexpressing mj-far-1 in comparison with vector control roots. Data are expressed as means of 25 plants from each line; the experiment was repeated three times, giving consistent results. The percentage of each developmental stage is represented by a mean ± standard error. Different letters above the bars denote a significant difference (P≤0.05, analysis of variance) between tomato roots lines analyzed by Tukey-Kramer multiple comparison tests.
Figure 5.
Longitudinal sections of M. javanica feeding sites in inoculated transgenic roots.
Histological analysis of roots expressing mj-far-1 (C, D) and mj-far-1RNAi roots (E, F) compared with vector control (A, B) were conducted at 15 and 28 DAI. At 15DAI giant cell systems were more developed and consists of more giant cells in roots where an excess of Mj-FAR-1 (G, H). Sections were stained with toluidine blue. * giant cell; n, nematode; Bar = 100 µm. The average of giant cell number at 15 and 28 DAI of 60 gall cross sections for each tomato hairy root line is presented (G). Giant cell area was measured on 50 giant cells. Measurements are represented by a mean ± standard error. Statistically significant differences (P≤0.05, analysis of variance) were determined by Tukey-Kramer multiple comparison tests. Different letters above the bars denote a significant difference.
Figure 6.
Analyzing Mj-FAR-1 functions in manipulating plant defense pathways.
Expression level of 11 defence-related target genes in mj-far-1 expressing root line far-1 E1.1 compared with vector control prior and 5 DAI with Meloidogyne javanica. Total RNA was prepared from Vector 11.5 transformed control roots and roots expressing mj-far-1 with/without infection. The graph shows the mean and standard error of the relative amount of transcripts of these genes in mj-far-1 expressing roots (far-1 E1.1) in comparison with vector transformed control roots (Vector 11.5) growing under the same conditions (vector control expression level set at zero). All target genes were normalized using the normalisation factor calculated as the geometric mean of the expression levels of three tomato endogenous reference genes 18S, actin and β-tubulin. Each reaction was performed in triplicate and the results represented the mean of two independent biological replicates. Statistical significance of the differences between far-1 E1.1 and Vector 11.5 transformed control roots were determined by Tukey-Kramer multiple comparison test, and significant differential expression (P≤0.05) is indicated with asterisks. The experiments were repeated three times with similar results.