A Meloidogyne graminicola Pectate Lyase Is Involved in Virulence and Activation of Host Defense Responses

Abstract

Plant-parasitic nematodes secrete an array of cell-wall-degrading enzymes to overcome the physical barrier formed by the plant cell wall. Here, we describe a novel pectate lyase gene Mg-PEL1 from M. graminicola. Quantitative real-time PCR assay showed that the highest transcriptional expression level of Mg-PEL1 occurred in pre-parasitic second-stage juveniles, and it was still detected during the early parasitic stage. Using in situ hybridization, we showed that Mg-PEL1 was expressed exclusively within the subventral esophageal gland cells of M. graminicola. The yeast signal sequence trap system revealed that it possessed an N-terminal signal peptide with secretion function. Recombinant Mg-PEL1 exhibited hydrolytic activity toward polygalacturonic acid. Rice plants expressing RNA interference vectors targeting Mg-PEL1 showed an increased resistance to M. graminicola. In addition, using an Agrobacterium-mediated transient expression system and plant immune response assays, we demonstrated that the cell wall localization of Mg-PEL1 was required for the activation of plant defense responses, including programmed plant cell death, reactive oxygen species (ROS) accumulation and expression of defense-related genes. Taken together, our results indicated that Mg-PEL1 could enhance the pathogenicity of M. graminicola and induce plant immune responses during nematode invasion into plants or migration in plants. This provides a new insight into the function of pectate lyases in plants-nematodes interaction.

Keywords: Meloidogyne graminicola; RNAi; enzymatic activity; pectate lyase; plant immunity elicitation.

FIGURE 1

Multiple sequence alignment of the predicted Mg-PEL1 protein with other pectate lyase sequences from plant-parasitic nematodes. Hg-PEL2 (AAM74954), Hg-PEL5 (ADW77536), and Hg-PEL1 (AAK08974) from Heterodera glycines; Hs-PEL1 (ABN14273) and Hs-PEL2 (ABN14272) from H. schachtii; Gts-PEL1 (AEA08834) and Gt-PEL2 (ACU64859) from Globodera tabacum; Gv-PEL1 (AEA08828) from G. virginiae; Gr-PEL1 (AAF80746) and Gr-PEL2 (AAM21970) from G. rostochiensis; Gp-PEL2 (ACU64845) and Gp-PEL1 (AEA08862) from G. pallida; Mi-PEL1 (AAQ09004), Mi-PEL2 (AAQ97032) and Mi-PEL3 (AAW56829) from Meloidogyne incognita; Me-PEL1 (ADN87334) and Me-PEL2 (ALB38961) from M. enterolobii; Mj-PEL1 (AAL66022) from M. javanica; Mg-PEL1 (MW266124) from M. graminicola; Bm-PEL1 (BAE48374) and Bm-PEL2 (BAE48375) from Bursaphelenchus mucronatus; Bx-PEL1 (BAE48371) and Bx-PEL2 (BAE48372) from B. xylophilus; Aa-PEL1 (BAI44499) and Aa-PEL2 (BAI44497) from Aphelenchus avenae. Black bars (I–IV) indicate the conserved regions. Black stars indicate the conserved cysteine residues.

FIGURE 2

Subventral gland localization of Mg-PEL1 in the pre-parasitic second stage juveniles of Meloidogyne graminicola. Fixed nematodes were hybridized with digoxigenin-labeled Mg-PEL1 sense cDNA probe (left) and antisense cDNA probe (right). Scale bars, 20 μm.

FIGURE 3

Secretion function analysis of Mg-PEL1 signal peptide. The predicted signal peptide of Mg-PEL1 was cloned into the yeast vector pSUC2 to generate pSUC2:Mg-PEL1(SP)-invertase construct, and pSUC2:Avr1b (SP)-Invertase was used as a positive control. The YTK12 strain and YTK12 carrying the empty pSUC2 vector were used as negative controls. YTK12 can grow on YPDA plates. CMD-W media was used to ensure the expression of pSUC2-derived plasmids. Yeast transformants were grown on the YPRAA media to detect invertase secretion. Meanwhile, enzymic activity of invertase was further confirmed by the reduction of the dye 2, 3, 5-triphenyltetrazolium chloride (TTC) to the insoluble red-colored triphenylformazan. The experiment was repeated three times independently.

FIGURE 4

Expression of Mg-PEL1 in Meloidogyne graminicola at different life stages. The fold change values were presented as the change in expression relative to the egg. Data shown are the means of three repeats plus standard deviation (± SD). Three independent experiments were performed with similar results. dpi, day post-infection; pre-J2s, pre-parasitic second-stage juveniles.

FIGURE 5

Enzyme activity analysis of recombinant protein Mg-PEL1. (A) Expression and purification of recombinant Mg-PEL1: Strep. Mg-PEL1: Strep was purified by anti-Strep beads and separated by SDS PAGE with Coomassie brilliant blue staining (red arrow). Western blot was used to confirm the correct expression of recombinant protein Mg-PEL1: Strep in culture supernatants. (B) Enzyme activity of Mg-PEL1 was measured using a titrimetric stop reaction assay. Mg-PEL1 possesses enzyme activity to hydrolyze PGA into oligogalacturonic acids, which can bind to free iodine. The rest amount of free iodine was titrated against Na₂S₂O₃ using soluble starch as the indicator. The experiment was repeated three times. Similar results were obtained from three independent experiments. EV, empty vector; PGA, polygalacturonic acid.

FIGURE 6

In planta RNA interference (RNAi) of Mg-PEL1 reduces pathogenicity of Meloidogyne graminicola. (A) Semi-quantitative PCR for the detection of the β-glucuronidase (GUS) intron fragment was used to confirm the expression levels of RNAi construct in roots of transgenic RNAi lines. Three independent samples from different plants of WT, RNAi10 and RNAi28 were used for the analysis. The experiment was repeated three times. OsUBQ (Os03g13170) gene was selected as the reference gene for semi-quantitative PCR. (B) qRT-PCR assays of the expression levels of Mg-PEL1 in M. graminicola collected from RNAi lines and WT at 3 day post-inoculation (dpi). The expression levels of MgCRT and MgExpansin from M. graminicola were used to determine the specificity of the Mg-PEL1-targeting RNAi. Similar results were obtained from three independent experiments. (C) Transgenic RNAi lines showed a significant reduction in the number of adult females compared with the WT. Two-week-old seedlings were inoculated with M. graminicola pre-J2s, and the number of adult females per plant was counted at 15 dpi. Data are presented as the means ± standard deviation (SD) from 9 to 10 plants. Two independent experiments were performed with similar results. All data revealed normal distribution and homogeneity of variance. One-way ANOVA Dunnett’s t-tests, *P < 0.05; **P < 0.01. RNAi10 and 28, different RNAi transgenic rice lines. WT, wild type.

FIGURE 7

Subcellular localization of Mg-PEL1 in plant cells. (A) Schematic diagram showing the protein structures Mg-PEL1:GFP, Mg-PEL1^{– SP}:GFP, Mg-PEL1^PR1a:GFP and GFP. (B) Agrobacterium stain GV3101 carrying fusion constructs Mg-PEL1: GFP, Mg-PEL1^{– SP}: GFP, Mg-PEL1^PR1a:GFP and the GFP control were transiently expressed in Nicotiana benthamiana leaves. (C) N. benthamiana leaves expressing Mg-PEL1: GFP, Mg-PEL1^{– SP}: GFP, Mg-PEL1^PR1a: GFP and GFP were treated with 30% glycerol for plasmolysis. GFP signals were observed at 2 d after infiltration. Red arrows indicate plant cell wall, and white arrows indicate plasma membrane. Scale bar = 100μm. (D) Western blotting confirmed the expression of Mg-PEL1: GFP, Mg-PEL1^{– SP}: GFP, Mg-PEL1^PR1a: GFP and GFP in N. benthamiana leaves. Ponceau S staining was used to show equal loading.

FIGURE 8

Cell wall localization of Mg-PEL1 is required for the activation of plant defenses. (A) Mg-PEL1 triggered cell death in Nicotiana benthamiana. Agrobacterium strain GV3101 carrying constructs Mg-PEL1^PR1a: Flag (a), Mg-PEL1^{– SP}: Flag (b), Mg-PEL1: Flag (c) and Flag (d) were transiently expressed in N. benthamiana leaves. At 5 d after infiltration, infiltrated sites were taken photos for cell death phenotype analysis. (B) Mg-PEL1 induced hydrogen peroxide production in N. benthamiana. Agrobacterium strain GV3101 carrying constructs Mg-PEL1^PR1a: Flag (e), Mg-PEL1^{– SP}: Flag (f), Mg-PEL1: Flag (g) and Flag (h) were transiently expressed in N. benthamiana leaves. At 2 d after infiltration, hydrogen peroxide production in infiltrated leaves was detected by DAB staining. (C) Mg-PEL1 induced the expression of defense related genes in N. benthamiana. The transcript levels of three defense response genes PR-5, PAL and NPR1 were measured in N. benthamiana leaves at 2 d after infiltration with Mg-PEL1^{– SP}:Flag, Mg-PEL1:Flag, Mg-PEL1^PR1a:Flag and Flag constructs. All data revealed normal distribution and homogeneity of variance. One-way ANOVA Dunnett’s t-tests, *P < 0.05; **P < 0.01. (D) Western blotting confirmed the expression of Mg-PEL1^{– SP}: Flag, Mg-PEL1: Flag and Mg-PEL1^PR1a: Flag in N. benthamiana leaves. Ponceau S staining were used to show equal loading.