Abstract
Root-knot nematodes establish long-term parasitic relationships with diverse hosts by inducing specialized feeding cells. However, the molecular mechanisms by which nematodes manipulate this developmental reprogramming process remain largely unknown. Here we identify a class of ROOT MERISTEM GROWTH FACTOR (RGF)-like peptide effectors conserved in root-knot nematodes. MgRGF from Meloidogyne graminicola and MiRGF1 from M. incognita are expressed in subventral gland cells during early infection and secreted into the host apoplast. Functional analysis reveals that nematode RGFs are critical for feeding site development. Intriguingly, these peptides elicit host-specific outcomes in Arabidopsis and rice, involving both cell proliferation and expansion—two processes essential for establishing feeding cell identity. Further genetic and biochemical evidence demonstrates that nematode RGF peptides functionally mimic plant endogenous RGFs by hijacking the host RGI-receptor-mediated signalling pathway to regulate root growth and promote parasitism. Beyond PLT transcription factors, PSY peptide genes were identified as key downstream components of this RGF signalling cascade in rice. Functional characterization of OsPSY5 suggests its positive role in promoting cell elongation and facilitating nematode parasitism. Our findings unveil a cross-kingdom mimicry strategy whereby root-knot nematode-secreted RGF peptides co-opt host RGF signalling to orchestrate feeding cell formation, highlighting potential targets for engineering nematode resistance in crops.
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Acknowledgements
We thank X. Wang (Zhejiang Academy of Agricultural Sciences, China) for providing the pOsCYCB1.1::GUS transgenic lines, L. Liu (Beijing Normal University) for providing the rgf1,2,3 mutant, W. Yin (HZAU, China) for providing the vectors used in the secretion assays, Y. Zhao (HZAU, China) for critical discussions and G. Li (South China Agricultural University, China) for field management of rice materials. We thank the National Key Laboratory of Agricultural Microbiology Core Facility for assistance in microscopy imaging. The study was supported by grants to Xiaoli Guo from the National Key Research and Development Program of China (no. 2023YFD1400400), the National Natural Science Foundation of China (no. 32472508) and the Huazhong Agricultural University Scientific and Technological Self-Innovation Foundation (no. 2662024ZKPY002).
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Xiaoli Guo conceived and supervised the project. Xiaoli Guo, W.L. and J.M. designed the experiments. Xiaoli Guo and W.L. wrote the paper with input from all coauthors. W.L. performed most of the experiments and data analysis. J.M. constructed most of the transgenic materials and contributed to the root growth and disease assay. X.S., Y.W. and Xiaolin Guo built the MgRGF transgenic materials, assisted with nematode isolation and contributed to the peptide treatment experiments. D.D. contributed to nematode sequencing analysis. J.L., C.C. and K.X. provided some reporter lines and Arabidopsis mutants. D.P. and G.W. provided nematode materials and critical feedback. All authors approved the submitted version of the paper.
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Extended data
Extended Data Fig. 1 MgRGF peptides promotes root development in Arabidopsis.
a, b, Root growth phenotype of Col-0 seedlings on vertical plates supplemented with or without 100 nM of the indicated peptides. Seedlings were photographed (a) and measured (b) at 7 days after germination (n = 15). Ctrl indicate no peptide added. Scale bars, 1 cm. c, d, Confocal images of Col-0 root tips stained with PI. Four-day-old seedlings were used for the staining (c). Number of meristematic cortex cells (d) was measured. n = 12, 11, 10, 11, 11 in d from left to right. Scale bars, 100 μm. e, Seven-day-old seedlings of Col-0 grown on 1.5% agar plates angled at 45°. Seedlings were grown vertically for 2 d on medium supplemented with the indicated peptides, then inclined for an additional 5 d. Representative images are shown, with at least 20 plants observed per treatment in each replicate. Scale bars, 1 cm. f-h, Root meristem phenotypes of tpst-1 grown in the absence or presence of the indicated peptides. Four-day-old seedlings were used for the PI staining and confocal imaging (f). Scale bars, 100 μm. Number of meristematic cortex cells (g) was measured (n = 10). Root length (h) of tpst-1 seedlings was measured at 7 days after germination (n = 15). i, j, Root meristem phenotypes of rgf1,2,3 grown in the absence or presence of indicated peptides. Confocal images showed root meristem of rgf1,2,3 stained with PI (i). Scale bars, 100 μm. Number of meristematic cortex cells (j) was measured (n = 15). Experiments were performed three times with similar results. White arrowheads mark the boundary between meristem and elongation zones and white asterisks indicate the QCs (c,f,i). Data in b, d, g, h and j are presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing the mean of each column with the mean of the control column.
Extended Data Fig. 2 Nematode RGF effector regulates root growth and promotes nematode parasitism in tomato.
a, b, Root growth phenotypes under 100 nM synthetic peptide treatment. Tomato seedlings were photographed (a) at 12 h after treatment, and root length (b) was measured (n = 15). Ctrl indicate no peptide added. Scale bars, 1 cm. c, d, Longitudinal observation of EdU-labelled cells in the root meristem. After 12 h of MgRGF1-Y treatment, roots were imaged with confocal microscopy (c), and the size of EdU-labelled root meristems (d) was measured (n = 15). Scale bars, 100 μm. Red arrowheads in c mark the boundary region of EdU-labelled cells and white asterisks indicate the QC region. e, Silencing efficiency of MiRGF1 detected by RT-qPCR. The experiments were performed using MiGAPDH and Mi18S as internal controls. f, Effect of MiRGF1 silencing on nematode infection. Root galls were counted at 30 dpi with dsRNA-treated M. incognita (n = 10). g, h, Effect of MiRGF1 silencing on feeding site establishment. Feeding sites (g) were observed using BABB clearing at 10 dpi (n = 10). Representative images of a single gall are shown. The number of feeding sites per gall (h) was counted. Ten galls were analyzed per treatment. Scale bar in g, 100 μm; N: nematode; Asterisks: feeding site. Experiments were performed three times with similar results. Data in e are presented as mean ± s.e.m. with individual data point of three independent biological replicates (n = 3). Data in b, f, d and h are presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 3 Overexpression of MgRGF promotes root development in Arabidopsis.
a, Root phenotypes of Col-0 and two independent transgenic lines overexpressing full-length or signal peptide-deleted version of MgRGF. Seedlings grown on 0.7% agar or 1.5% agar were photographed at 7 days after germination. Representative images are shown from three independent experiments, with at least 15 plants examined per treatment in each replicate. Scale bar, 1 cm. b, Relative expression levels of MgRGF by RT-qPCR using AtActin2 and AtTUB2 as internal controls. c, Measurements of primary root length (n = 16). d, Confocal images of root meristem of Col-0 and transgenic lines. Seven-day-old seedlings were stained with PI for imaging. White arrows indicate the boundary between root meristem and elongation zones. White asterisks indicate the QCs. Representative images are shown from three independent experiments, with at least 10 roots examined per treatment in each replicate. Scale bars, 100 μm. e, Measurements of the number of meristematic cortex cells in Col-0 and transgenic lines. n = 16, 13, 13, 13, 14 from left to right. f, Measurements of lateral root number. n = 16, 10, 10, 11, 12 from left to right. g-j, Nano-LC-MS/MS spectra of tyrosine-sulfated mature peptides. MgRGF1-Y (g,i) and MgRGF2-Y (h,j) were identified in apoplastic extraction of the transgenic lines expressing full-length MgRGF (g,h) or its signal peptide-deleted variant (i,j). Red and blue lines indicate matched y-ions and b-ions, respectively. Experiments were performed three times with similar results. Data in b are presented as mean ± s.e.m.with individual data point of three independent biological replicates (n = 3). Data in c, e and f are presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing the mean of each column with the mean of the control column.
Extended Data Fig. 4 Overexpression of MgRGF promotes nematode parasitism in Arabidopsis.
a, Nematode penetration analysis by acid fuchsin staining at 1 dpi with M. incognita. Representative images are shown from three independent experiments (n = 15 roots per replicate). Scale bars, 100 μm. b, Number of second-stage juveniles penetrated into the roots at 1 dpi (n = 15). c, Representative images of root galls and feeding sites visualized by acid fuchsin staining (upper panel) and BABB clearing (lower panel) at 15 dpi with M. incognita. Three independent experiments were performed with similar results (n = 15 roots per replicate). Scale bars, 200 μm (upper panel) and 100 μm (lower panel). N: nematode; Asterisks: giant cells. d, e, Nematode infection phenotypes in Arabidopsis lines overexpressing MgRGF. Root galls and egg masses (d) were stained with acid fuchsin and counted at 15 dpi and 30 dpi with M. incognita (n = 15). Feeding site number (e) was quantified at 15 dpi by BABB clearing (n = 10). Representative data from one of three biological replicates are shown. Data in b, d and e are presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing the mean of each column with the mean of the control column.
Extended Data Fig. 5 Expression of AtRGI1-5 is activated in galls induced by M. incognita in Arabidopsis.
a, GUS staining of pRGI1::GUS, pRGI2::GUS, pRGI3::GUS, pRGI4::GUS, and pRGI5::GUS lines infected with M. incognita. Twelve-day-old seedings were used for inoculation. Twenty roots were stained and representative images are shown. Strong GUS activity was observed in galls at 3 and 5 dpi, which diminished by 12 dpi. The experiments were performed three times with similar results. Scale bars, 500 μm. b, Histological sections of galls at 5 dpi. Ten galls were observed with similar results. Scale bars, 50 μm. N: nematode; Asterisks: giant cells.
Extended Data Fig. 6 Arabidopsis rgi1,2,3,4,5 mutant shows insensitivity to nematode RGF and delayed development of M. incognita.
a, Phenotypes of Col-0 and the rgi1,2,3,4,5 mutant grown on medium for 7 days without or with 100 nM indicated peptides. Scale bars, 1 cm. b, Measurements of primary root length under 100 nM MgRGF1-Y treatment (n = 10). c, Confocal images of root tips from Col-0 and rgi1,2,3,4,5. Seven-day-old seedlings grown with 100 nM MgRGF1-Y were stained and imaged (n = 10). White arrows indicate the boundary between root meristem and transition zones. White asterisks indicate the QC. Scale bars, 100 μm. d, Measurements of the number of meristematic cortex cells in Col-0 and mutant plants (n = 16). e, Measurements of lateral root number. n = 13, 23, 13, 23 from left to right. f, Nematode infection phenotypes in Col-0 and the rgi1,2,3,4,5 mutant. The numbers of root galls per root and per gram of root were counted and quantified (n = 15). g, h, The numbers of egg masses per root (g) and per gram of root (h) were counted and quantified (n = 15). i, Nematode penetration phenotype of wild-type (Col-0) and rgi1,2,3,4,5 mutant by acid fuchsin staining at 1 dpi with M. incognita. Scale bars, 200 μm. j, Average number of M. incognita juveniles per plant at 1 dpi (n = 20). k, Nematode infection phenotypes of wild-type (Col-0) and rgi1,2,3,4,5 mutant by acid fuchsin staining at 7 dpi. Scale bars, 200 μm. l, Proportion of different nematode developmental stages at 7 dpi. n = 10, 9 for Col-0 and rgi1,2,3,4,5 mutant. m, Nematode infection phenotypes of wild-type (Col-0) and rgi1,2,3,4,5 plants by acid fuchsin staining at 15 dpi. Scale bars, 200 μm. n, Proportion of different nematode developmental stages at 15 dpi (n = 8). o, Gall imaging by BABB clearing in wild-type (Col-0) and rgi1,2,3,4,5 plants at 15 dpi. Scale bars, 200 μm. p, Single giant cell size quantification. n = 20, 19 for Col-0 and rgi1,2,3,4,5 mutant. Scale bar, 200 μm, N: nematode; Asterisks: giant cells. Representative images (a,c,i,k,m,o) and data (b,d-h,j,l,n,p) from one of three biological replicates are shown. Data in b, d, e, f, g, h, j and p are presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 7 The size of feeding sites is not significantly affected in rice rgi mutants.
a, Feeding site observation in rice rgi mutants at 7 dpi with 35 ppJ2s. Representative images of a single gall are shown after BABB clearing. Scale bars, 100 μm (upper panel) and 50 μm (lower panel). N: nematode; Asterisks: giant cells. b, Quantification of feeding cell size (n = 10). c, d, The number of females per root (c) and per gram of root (d) was counted and quantified in rgi mutant (n = 10). Experiments were performed three times with similar results. Data in b, c and d is presented as box-whisker plots with individual data points. The central line indicates the median; box limits represent the first and third quartiles; whiskers extend to the minima and maxima. P values were determined by one-way ANOVA with Dunnett’s multiple comparisons test, comparing the mean of each column with the mean of the control column.
Extended Data Fig. 8 Treatment with MgRGF1 or AtRGF1 peptide activates MAPK cascade and PLT1/2 expression through RGI receptors.
a, b, MPK3 and MPK6 activation upon MgRGF1 or AtRGF1 peptide treatment. Wild-type seedings and the corresponding rgi mutants were tested in Arabidopsis (a) and rice (b). Five-day-old seedlings were treated with 10 μM sulfated peptides for the indicated times. Phosphorylated MPK3/6 was detected using an anti-pERK antibody. Equal loading was shown by anti-actin immunoblot. The experiments were performed three times with similar results. c, Peptide-induced promoter activities and protein levels of PLT1/2 were blocked in the Arabidopsis rgi mutant. Representative confocal images show root meristems of the transgenic seedlings expressing pPLT1::CFP, pPLT2::CFP, pPLT1::PLT1-YFP, or pPLT2::PLT2-YFP in wild-type and rgi mutant backgrounds. The experiments were performed three times with similar results. Scale bars, 100 μm.
Extended Data Fig. 9 Sequence and expression analysis of PSY family genes in rice.
a, Multiple sequence alignment and Web-Logo analysis of rice PSYs. Conserved residues are highlighted in blue box. b, Gene expression levels of OsPSY genes at 1 dpi with M. graminicola. TPM values were retrieved from previously published RNA-seq data66. c, d, Relative expression analysis of OsPSY genes by RT-qPCR in Kitaake root tips after 3 days post M. graminicola inoculation (c) and 12 h MgRGF1-Y treatment (d). e, f, Relative expression levels of PSY family genes in root tips of Arabidopsis (e) and rice (f) after 24 h MgRGF1-Y treatment. Expression levels were quantified using the 2 − ΔΔCt method. AtActin2 and AtTUB2 served as internal controls for Arabidopsis, while OsUBQ10 and OsActin were used for rice. Data in c, d, e and f were presented as mean ± s.e.m.with individual data point of three independent biological replicates (n = 3). P values were determined by unpaired two-tailed Student’s t-test.
Extended Data Fig. 10 A proposed model for nematode RGF signaling during feeding site formation.
RKNs secrete RGF-like peptide mimics that specifically bind to the host RGI receptors and activate RGI receptor-mediated signaling pathway to regulate cell proliferation and expansion–two processes critical for establishing feeding cell identity. Downstream components, including PSY peptide genes and PLT transcription factors, are activated to coordinate giant cell development and promote parasitism. Solid lines indicate regulation tested in this study, whereas dashed lines represent proposed regulation. Based on recent findings regarding the PSY peptide-mediated trade-off between growth and stress responses, the induction of PSYs at nematode feeding sites may further facilitate nematode infection by suppressing PSYR-mediated defense responses73. RKNs: root-knot nematodes; RGFs: ROOT MERISTEM GROWTH FACTORs; MPK3/6: Mitogen-Activated Protein Kinase 3/6; PSY: PLANT PEPTIDES CONTAINING SULFATED TYROSINE; PLT2: PLETHORA 2; NCs: neighboring cells; GCs: giant cells. Figure created in BioRender; Li, W. https://biorender.com/tsfuzix (2026).
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Li, W., Mo, J., Su, X. et al. Root-knot-nematode-derived mimics of RGF peptides hijack host signalling to orchestrate feeding site formation. Nat. Plants (2026). https://doi.org/10.1038/s41477-026-02301-z
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DOI: https://doi.org/10.1038/s41477-026-02301-z
