SlWRKY45, nematode-responsive tomato WRKY gene, enhances susceptibility to the root knot nematode; M. javanica infection

Abstract

The fluctuation of tomato's WRKY defense regulators during infection by the root knot nematode Meloidogyne javanica was analyzed: and the spatial and temporal expression of SlWRKY45 was studied in depth with regard to its response to nematode infection, phytohormones, and wounding. Expression of WRKY45 increased substantially within 5 d upon infection and continued through feeding-site development and gall maturation. Histological analysis of nematode feeding sites indicated that WRKY45 was highly expressed within the feeding cells and associated vascular parenchyma cells. Responses of SlWRKY45 promoters to several phytohormones showed that WRKY45 was highly induced by specific phytohormones, including cytokinin, auxin, and the defense-signaling molecule salicylic acid (SA), but not by the jasmonates. Overexpressing tomato lines were generated, and infection tests showed that, significantly, roots over-expressing SlWRKY45 contained substantially increased number of females, indicating that WRKY45 overexpression supported faster nematode development. qRT-PCR tests have shown roots overexpressing WRKY45 suppressed the jasmonic acid and salicylic acid marker genes, proteinase inhibitor (PI), and pathogenesis related protein (PR1), respectively, and also the cytokinin response factors CRF1 and CRF6. Overall, this study indicated SlWRKY45 to be a potential transcription factor whose manipulation by the invading nematode might be critical for coordination of hormone signals supporting favorable condition for nematode development in root tissue.

Keywords: Basal resistance; Meloidogyne javanica; WRKY transcription factor; innate immunity; phytohormones; plant defense signaling; root susceptibility.

Figure 1.

Tomato SlWRKY45 protein structure and phylogenetics. A. Distribution of conserved motifs of the WRKY proteins along WRKY45 protein sequences. The colored boxes placed along the protein sequences are explained in the legend within the bottom of the Figure. A putative nuclear localization signal, as predicted by the SignalP program is shown at positions 83 of WRKY45. B. Unrooted phylogenetic tree representing relationships among WRKYs genes from tomato, Arabidopsis and rice. Members of WRKYs genes from tomato (SlWRKYs), Arabidopsis (AtWRKYs) and rice (OsWRKYs) were subjected to phylogenetic analysis. The amino acid sequences of WRKYs proteins were aligned with aid of ClustalW, and the phylogenetic tree was constructed by means of MEGA 7.0 software by the UPGMA method, with 1,000 bootstrap replicates. The percentage bootstrap scores higher than 50% are indicated on the nodes.

Figure 2.

Microscopic analysis of β-glucuronidase (GUS) expression patterns of root-knot nematode (RKN)-infected tomato roots harboring the SlWRKY45 promoter-GUS fusion construct. (A) Uninoculated root harboring the WRKY45:GUS fusion construct exhibited a basal mild GUS signal in the xylem bundles at the root elongation zone; (B) Roots at 2 dai (C) Uninfected roots at 5 d (D) Infected roots at 5 dai. (E) Uninoculated roots at 15 d (F) Developing galls at 15 dai (G) Uninoculated roots at 28 d (H) Mature galls at 28 dai. Arrows indicate nematode. (A-E, G): micrographs as viewed under light microscope. (F, H): bright-field image of galls photographed through a stereomicroscope. Bars: (A-EG) 50 μm; (FH) 500 μm.

Figure 3.

Activity of WRKY45 promoter within thin sections of galls analyzed by GUS staining in representative transformed tomato lines infected with Meloidogyne javanica. Microscopic analyses of β-glucuronidase (GUS) activity in cross-sections of tomato root gall expressing WRKY45 promoter-GUS constructs at 15 and 28 dai. At 15 and 28 dai all observed giant cells were already mature. ((A, B) Galls sections induced on WRKY45:GUS line at 15 dai. (C, D) Galls sections induced on WRKY45:GUS line at 28 dai. (N) The female body of the nematode can be seen at the edge of the giant cells (*). Bars, 200 μm. GUS staining is observed as blue color in whole mounts, and as a red precipitate in the dark field micrographs of the sections.

Figure 4.

Wounding-induced expression of WRKY45 promoter activity. (A) Intact WRKY45:GUS root line. (B) WRKY45:GUS root line at 9 after wounding (C) at 24 h after wounding (Bars, 100 μm).

Figure 5.

Effect of exogenous phytohormones application on GUS expression in root line WRKY45:GUS. Seven-days-old subcultured roots were transferred to GB as control (a,d,g,j,m,p) or to GB containing SA (1, 5mM) (a-c), IBA (1, 10 µM) (d-f), IAA (1, 5, µM) (g-i), and BA (0.1, 0.5, µM) (j-l) JA (10, 20 µM (m-o), MeJA (0.01, 0.1mM (p-r) for 16 hr before root staining. GUS was detected histochemically and roots were monitored. The figures are representative of at least 5 independent experiments. Scale bar = 0.5 mm.

Figure 6.

Overexpression of SlWRKY45 in tomato hairy roots promote RKN M. javanica development. A. Increased susceptibility of tomato hairy roots expressing SlWRKY45 (oe:wk-03, oe:wk-04) is accompanied by increased galling occurrence compared with pControl line. B. Meloidogyne susceptibility/resistance of transgenic tomato roots expressing SlWRKY45 compared with pControl line, All roots lines were inoculated with 300 sterile pre-parasitic J2s and the infected roots were assessed for J3 and J4 and mature females development at 28 dai through observation under the dissecting microscope following staining with acid fuchsin dye. Note the significant (P < 0.05) increase in percentage of mature females in oe:wk-03 and oe:wk-04 root lines in comparison with vector control roots. Data are expressed as means of 25 plants from each line; the experiment was repeated 3 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 hairy roots lines analyzed by Tukey-Kramer multiple comparison tests. C. Longitudinal sections of Meloidogyne javanica feeding sites developed in root lines oe:wk-03, oe:wk-04 and pControl. Average GC area was measured on 50 GC systems and average number of GCs of each feeding site system as measured on 60 gall cross sections for each root line and measurements are given as mean ± standard error. Different letters above the bars denote statistically significant differences (P ≤ 0.05, analysis of variance) determined by Tukey-Kramer multiple comparison tests. Giant-cell (GC) area of plants carrying the SlWRK45 gene were more extensive compared with pControl line. D. Thin sections of oe:wk-03, oe:wk-04 and control line were stained with toluidine blue; * = GC, N = nematode, bar = 100 μm.

Figure 7.

Analyzing the effect of WRKY45 overexpression in manipulating hormonal pathways response. Expression level of defense-related target genes in oe:wk-03 expressing root line compared with vector control prior and 5 dai with Meloidogyne javanica. Total RNA was prepared from pControl transformed control roots and roots expressing SlWRKY45 with/without infection. The graph shows the mean and standard error of the relative amount of transcripts of these genes in SlWRKY45 expressing roots (oe:wk-03) in comparison with vector transformed control roots (pControl) growing under the same conditions (vector control expression level set at zero). All target genes were normalized using the normalization factor calculated as the geometric mean of the expression levels of 3 tomato endogenous reference genes 18S, β-actin and β-tubulin. Each reaction was performed in triplicate and the results represented the mean of 2 independent biologic replicates. Statistical significance of the differences between oe:wk-03 and pControl 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.