Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Jul;25(7):e13491.
doi: 10.1111/mpp.13491.

The root-knot nematode effector MiEFF12 targets the host ER quality control system to suppress immune responses and allow parasitism

Salomé Soulé  1 , Kaiwei Huang  2 , Karine Mulet  1 , Joffrey Mejias  1 , Jérémie Bazin  3 , Nhat My Truong  1 , Junior Lusu Kika  1 , Stéphanie Jaubert  1 , Pierre Abad  1 , Jianlong Zhao  2 , Bruno Favery  1 , Michaël Quentin  1
Affiliations

The root-knot nematode effector MiEFF12 targets the host ER quality control system to suppress immune responses and allow parasitism

Salomé Soulé et al. Mol Plant Pathol. 2024 Jul.

Abstract

Root-knot nematodes (RKNs) are microscopic parasitic worms able to infest the roots of thousands of plant species, causing massive crop yield losses worldwide. They evade the plant's immune system and manipulate plant cell physiology and metabolism to transform a few root cells into giant cells, which serve as feeding sites for the nematode. RKN parasitism is facilitated by the secretion in planta of effector molecules, mostly proteins that hijack host cellular processes. We describe here a conserved RKN-specific effector, effector 12 (EFF12), that is synthesized exclusively in the oesophageal glands of the nematode, and we demonstrate its function in parasitism. In the plant, MiEFF12 localizes to the endoplasmic reticulum (ER). A combination of RNA-sequencing analysis and immunity-suppression bioassays revealed the contribution of MiEFF12 to the modulation of host immunity. Yeast two-hybrid, split luciferase and co-immunoprecipitation approaches identified an essential component of the ER quality control system, the Solanum lycopersicum plant bap-like (PBL), and basic leucine zipper 60 (BZIP60) proteins as host targets of MiEFF12. Finally, silencing the PBL genes in Nicotiana benthamiana decreased susceptibility to Meloidogyne incognita infection. Our results suggest that EFF12 manipulates PBL function to modify plant immune responses to allow parasitism.

Keywords: Meloidogyne incognita; Nicotiana benthamiana; Solanum lycopersicum; ER quality control; effector; endoplasmic reticulum (ER).

PubMed Disclaimer

Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Effector 12 (EFF12) is a conserved effector in root‐knot nematodes. (a) Alignment of the MiEFF12 protein sequences. The green box indicates the position of the signal peptide for secretion. The conserved C‐terminal region is enriched in positively (lysine, K) and negatively (asparagine, D and glutamic acid, E) charged residues. (b) Phylogenetic tree of Meloidogyne spp. EFF12 amino acid sequences. The percentages displayed next to each branch represent the number of tree replicates in which the associated taxa were assembled in 100 bootstraps. The lengths of the branches are not proportional to phylogenetic distance. (c) Pairwise sequence identity matrix for root‐knot nematode EFF12 nucleotide sequences.
FIGURE 2
FIGURE 2
The Meloidogyne EFF12 genes are specifically expressed in the dorsal oesophageal gland. In situ hybridization with specific antisense probes localized EFF12 transcripts exclusively in the dorsal gland cell of preparasitic juveniles of Meloidogyne incognita and Meloidogyne enterolobii. Sense probes for MiEFF12 and MeEFF12 transcripts were used as a negative control and gave no signal. DG, dorsal gland. Bars: 20 μm.
FIGURE 3
FIGURE 3
The silencing of MiEFF12 genes by virus‐induced gene silencing affects Meloidogyne incognita parasitism. (a) Transcript quantification by reverse transcription‐quantitative PCR confirmed the effective silencing of MiEFF12 genes in parasitic nematodes extracted from Nicotiana benthamiana roots infected with TRV2‐MiEFF12 relative to controls (TRV2‐empty and TRV2‐GFP). Normalized relative transcript levels for three independent biological replicates are shown. (b) Infection test on N. benthamiana control plants (TRV2‐empty and TRV2‐GFP) and plants producing siRNA for the silencing of MiEFF12 genes in M. incognita (TRV2‐MiEFF12). Galls were counted 6 weeks after inoculation with 200 M. incognita second‐stage juveniles per plant. Results from two independent experiments are shown (n = 15 and n = 17 plants for tests 1 and 2, respectively). The cross represents average value. Box indicates interquartile range (25th to the 75th percentile). The central line within the box represents mean value. Whiskers indicate the minimum and maximum values for the normal values present in the dataset. Statistical significance was assessed in Student's t tests. Significant differences were observed between controls and TRV‐MiEFF12 plants (*p < 0.05).
FIGURE 4
FIGURE 4
MiEFF12 suppress host defence responses. (a, b) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) in the Arabidopsis MiEFF12a‐expressing line with AgriGO v. 2.0. (a) GO enrichment analysis for the 1103 genes upregulated in the MiEFF12a‐expressing line with log2 fold change ≥1, indicating an enrichment in genes related to the response to decreased oxygen levels. (b) GO enrichment analysis on the 1126 genes downregulated in the MiEFF12a‐expressing line with log2 fold change ≤ −1, indicating an enrichment in genes related to the defence response. (c) MiEFF12 suppresses flg22‐mediated reactive oxygen species (ROS) production in Nicotiana benthamiana. Agrobacterium tumefaciens GV3101 carrying MiEFF12a was used to infiltrate the leaves of N. benthamiana plants. Infiltrated leaf discs were collected 48 h post‐agroinfiltration and assayed for ROS production in response to treatment with the flg22 elicitor. ROS production was monitored for 40 min, and the values shown are the mean relative luminescence units ± SD for 28 leaf discs. (d) BAX‐triggered cell death was not suppressed by MiEFF12a. Photographs for assessment of the cell‐death phenotype were taken 5 days after the last infiltration. (e) Gpa2/RBP‐1‐triggered cell death was suppressed by MiEFF12a. Photographs showing the suppression of cell death were taken 5 days after the last infiltration. Each cell death suppression bioassay was performed at least three times; results from a representative experiment are shown.
FIGURE 5
FIGURE 5
MiEFF12a was localized to the endoplasmic reticulum (ER) of epidermal Nicotiana benthamiana leaf cells. (a) Single‐plane confocal images of N. benthamiana leaf cells infiltrated with Agrobacterium tumefaciens and producing MiEFF12a without its signal peptide, fused to the C‐terminal end of the green fluorescent protein (GFP) reporter (GFP‐MiEFF12a; green signal; left pictures). Overlays of differential interference contrast and fluorescence images are shown (right pictures). (b) Single‐plane confocal images of N. benthamiana leaf cells infiltrated with A. tumefaciens and producing MiEFF12a without the signal peptide, fused to the N‐terminal end of the green fluorescent protein (GFP) reporter (MiEFF12a‐GFP; green signal; left pictures). Overlays of differential interference contrast and fluorescence images are shown (right pictures). (c) The monomeric red fluorescent protein (mRFP) signal (RFP‐ER; red signal) of an ER marker was used to analyse colocalization of the GFP‐MiEFF12a fusion (green signal) and the ER. (d) The mRFP signal (red signal) of the ER marker was used to analyse its colocalization with the MiEFF12a‐GFP fusion (green signal). Both fusions between the GFP and MiEFF12a colocalized with the ER marker in N. benthamiana leaf cells. Asterisk; nucleus. Scale bars: 20 μm.
FIGURE 6
FIGURE 6
MiEFF12a physically interacts in planta with SlPBL1 and SlBZIP60. SlPBL1 and SlBZIP60 colocalized with MiEFF12a in the endoplasmic reticulum (ER) of epidermal Nicotiana benthamiana leaf cells. (a) Single‐plane confocal images of N. benthamiana leaf cells infiltrated with Agrobacterium tumefaciens and producing SlPBL1, fused to the C‐terminal end of the green fluorescent protein (GFP) reporter (GFP‐SlPBL1; green signal) and MiEFF12a fused to the C‐terminal end of the red fluorescent protein (RFP) reporter (RFP‐MiEFF12a; red signal). (b) Single‐plane confocal images of N. benthamiana leaf cells infiltrated with A. tumefaciens and producing SlBZIP60, fused to the C‐terminal end of the green fluorescent protein (GFP) reporter (GFP‐SlBZIP60; green signal) and the RFP‐MiEFF12a recombinant protein (RFP‐MiEFF12a: red signal). Overlays of fluorescence images are shown (merge). Scale bars: 20 μm. (c) Schematic representation of the full‐length (tot, total) and truncated (sol, soluble) SlPBL1 and SlBZIP60 proteins used for interactomic assays. (d) Co‐immunoprecipitation (Co‐IP) experiments confirmed that MiEFF12a interacted with the full‐length SlPBL1 and BZIP60. SlPBLtot‐GFP, SlPBLsol‐GFP or SlBZIP60 were transiently co‐expressed with MiEFF12a‐HA or MiCRT in N. benthamiana leaves. The Co‐IP experiment was performed with anti‐HA affinity gel, and the protein isolated was analysed by western blotting (WB) with anti‐GFP antibodies to detect SlPBLtot and SlPBLsol, and with anti‐HA antibodies to detect MiEFF12a and MiCRT. Three independent experiments were performed, with similar results.
FIGURE 7
FIGURE 7
The silencing of PBL genes in Nicotiana benthamiana affects susceptibility to Meloidogyne incognita. (a) Timeline used for virus‐induced gene silencing (VIGS) experiments. (b) Reverse transcription‐quantitative PCR showing the efficient silencing of the NbPBL1a/b, NbPBL2a/b and NbPBL3a/b gene pairs in N. benthamiana control plants (TRV‐GFP) and plants in which NbPBLs were silenced (TRV2‐PBLs). The data shown are normalized relative transcript levels for three independent biological replicates obtained with SatqPCR software. The NbEF1a and NbGADPH housekeeping genes were used for data normalization. Error bars indicate the SEM. (c) Infection test on N. benthamiana TRV‐GFP or TRV2‐PBLs plants. Galls were counted 6 weeks after inoculation with 200 M. incognita second‐stage juveniles (J2s) per plant. Results from three independent experiments are shown (n = 19, n = 15 and n = 21 plants for tests 1, 2 and 3, respectively). The cross represents average value. Box indicates interquartile range (25th to the 75th percentile). The central line within the box represents mean value. Whiskers indicate the minimum and maximum values for the normal values present in the dataset. Statistical significance was determined in Student's t test and significant differences were observed between TRV‐GFP control and TRV‐PBL plants (*p < 0.01).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Abad, P. , Gouzy, J. , Aury, J.M. , Castagnone‐Sereno, P. , Danchin, E.G. , Deleury, E. et al. (2008) Genome sequence of the metazoan plant‐parasitic nematode Meloidogyne incognita . Nature Biotechnology, 26, 909–915. - PubMed
    1. Atabekova, A.K. , Pankratenko, A.V. , Makarova, S.S. , Lazareva, E.A. , Owens, R.A. , Solovyev, A.G. et al. (2017) Phylogenetic and functional analyses of a plant protein related to human B‐cell receptor‐associated proteins. Biochimie, 132, 28–37. - PubMed
    1. Bao, Y. & Howell, S.H. (2017) The unfolded protein response supports plant development and defense as well as responses to abiotic stress. Frontiers in Plant Science, 8, 344. - PMC - PubMed
    1. Berger, A. , Boscari, A. , Horta Araújo, N. , Maucourt, M. , Hanchi, M. , Bernillon, S. et al. (2020) Plant nitrate reductases regulate nitric oxide production and nitrogen‐fixing metabolism during the Medicago truncatula–Sinorhizobium meliloti symbiosis. Frontiers in Plant Science, 11, 1313. - PMC - PubMed
    1. Blanc‐Mathieu, R. , Perfus‐Barbeoch, L. , Aury, J.‐M.M. , Da Rocha, M. , Gouzy, J. , Sallet, E. et al. (2017) Hybridization and polyploidy enable genomic plasticity without sex in the most devastating plant‐parasitic nematodes. PLoS Genetics, 13, e1006777. - PMC - PubMed

Substances