The Meloidogyne graminicola effector Mg16820 is secreted in the apoplast and cytoplasm to suppress plant host defense responses

Abstract

On invasion of roots, plant-parasitic nematodes secrete effectors to manipulate the cellular regulation of the host to promote parasitism. The root-knot nematode Meloidogyne graminicola is one of the most damaging nematodes of rice. Here, we identified a novel effector of this nematode, named Mg16820, expressed in the nematode subventral glands. We localized the Mg16820 effector in the apoplast during the migration phase of the second-stage juvenile in rice roots. In addition, during early development of the feeding site, Mg16820 was localized in giant cells, where it accumulated in the cytoplasm and the nucleus. Using transient expression in Nicotiana benthamiana leaves, we demonstrated that Mg16820 directed to the apoplast was able to suppress flg22-induced reactive oxygen species production. In addition, expression of Mg16820 in the cytoplasm resulted in the suppression of the R2/Avr2- and Mi-1.2-induced hypersensitive response. A potential target protein of Mg16820 identified with the yeast two-hybrid system was the dehydration stress-inducible protein 1 (DIP1). Bimolecular fluorescence complementation resulted in a strong signal in the nucleus. DIP1 has been described as an abscisic acid (ABA)-responsive gene and ABA is involved in the biotic and abiotic stress response. Our results demonstrate that Mg16820 is able to act in two cellular compartments as an immune suppressor and targets a protein involved in the stress response, therefore indicating an important role for this effector in parasitism.

Keywords: Nicotiana benthamiana; Oryza sativa; PAMP-triggered immunity; effector-triggered immunity; parasitism; reactive oxygen species; root-knot nematodes.

Figure 1

Alignment of the protein sequence of Mg16820 with the DNA sequence obtained from MGRAMBASE and possible homologues (Minc05708 and Minc03663) in Meloidogyne incognita. The green line indicates the predicted signal peptide of Mg16820. The red line indicates the predicted nuclear localization signal. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

In situ hybridization of Meloidogyne graminicola pre‐parasitic second‐stage juveniles. (A) Antisense probe of Mg16820. (B) Sense probe (negative control; putative effector Mg9152). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 3

Immunolocalization of Mg16820 secreted by Meloidogyne graminicola in rice. (A–F) Immunolocalization of Mg16820 during the migratory phase of the nematodes [1 day after inoculation (dai)]. (A, B) Secretion of Mg16820 (green fluorescence) by two nematodes with signal seen in the subventral glands in one juvenile nematode. (C, D) Migrating nematode with Mg16820 signal at the anterior part, along the body in the plant apoplasm and in the nematode body. (E, F) Signal at the anterior part and inside the nematode body. (G–J) Immunolocalization of Mg16820 in the feeding sites at 3 dai. (G, H) Secretion of Mg16820 in the plant tissue prior to giant cell development. (I, J) Young giant cells showing signal in the cytoplasm and in the nuclei, apparently excluded from the nucleoli. (K–N) Immunolocalization of Mg16820 during feeding site development (5 dai). (K, L) Accumulation of Mg16820 in the cytoplasm of giant cells. (M, N) Young giant cells showing signal in the cytoplasm and nucleus, apparently excluding the nucleoli. Grey images, differential interference contrast (DIC), 4’,6‐diamidino‐2‐phenylindole (DAPI) and green fluorescent protein (GFP) overlay; black images, DAPI and GFP overlay; n, nematode; asterisks, giant cells; red arrowheads, Mg16820; blue colour, nuclei; white arrow, nucleolus. Scale bars: (A–J) 10 μm; (K–N) 20 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 4

Subcellular localization of Mg16820 after transient expression in Nicotiana benthamiana leaves. (A) Localization of free red fluorescent protein (RFP) in the cytoplasm and nucleus. (B) Localization of Mg16820::RFP in the cytoplasm and nucleus. (C) Localization of SPMg16820::GFP. Scale bars: (A) 20 μm; (B) 10 μm; (C) 20 μm. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 5

Suppression assay of flg22‐induced reactive oxygen species (ROS) production in Nicotiana benthamiana with Mg16820. (A) Weak suppression of ROS with Mg16820 located in the cytoplasm compared with the non‐suppressing effector Mg03015. Mp10 was used as a positive control suppressing ROS. (B) Very strong suppression with Mg16820 localized in the apoplasm (SPMg16820). The effector Mg03015 with signal peptide (SP) was used as a negative control not suppressing ROS. Mp10 was used as a positive control. Bars indicate standard error. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 6

Effector‐triggered immunity (ETI) suppression assays in Nicotiana benthamiana with effector Mg16820 suppressing two ETI pathways. (A) Suppression of the Mi‐1.2^T557S‐induced hypersensitive response (HR) by Mg16820 without signal peptide, targeted to the cytoplasm. The controls empty vector (EV), green fluorescent protein (GFP) and non‐suppressing effector Mg03015 of Meloidogyne graminicola did not result in suppression. (B) Suppression of the R2/Avr2‐induced HR by Mg16820 localized in the cytoplasm with the same controls as in (A). (C) Percentage of necrotic and suppressed spots with Mi‐1.2^T557S and R2/Avr2 co‐infiltrated with Mg16820. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 7

Yeast two‐hybrid (Y2H) co‐transformation assay with GAL4‐DNA‐binding‐domain::Mg16820 and GAL4‐DNA‐activation‐domain::DIP1. As a control for cell growth colonies were grown on medium lacking the autotrophic markers trypophan and leucine. Interaction was tested using medium lacking the auxotrophic markers tryptophan, leucine and histidine (‐TLH) or tryptophan, leucine and uracil (‐TLU). To test β‐galactosidase activity, the X‐gal (5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside) substrate was used. The truncated dehydration stress‐inducible protein 1 (DIP1) isolated from the library showed a weak interaction with Mg16820, whereas the complete DIP1 sequence did not show interaction in the Y2H assay. [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 8

Bimolecular fluorescence complementation assay with Mg16820 and dehydration stress‐inducible protein 1 (DIP1) in Nicotiana benthamiana leaves. Yellow fluorescent protein (YFP) signal was seen in the nucleus with exclusion of the nucleolus. (A) Negative control YFPc::Mg03015 and YFPn::DIP1 with infiltration at an optical density at 600 nm (OD₆₀₀) of 0.3. (B) YFP signal with infiltration of YFPc::Mg16820 and YFPn::DIP1 at OD₆₀₀ = 0.3. (C) YFP signal with infiltration of YFPc::Mg16820 and YFPn::DIP1 at OD₆₀₀ = 0.1. Scale bars: (A) 40 μm; (B) 5 μm; (C) 10 μm. [Colour figure can be viewed at wileyonlinelibrary.com]