Abstract
Microbial pathogens deploy effector proteins to manipulate host cell innate immunity, often using poorly understood unconventional secretion routes. Transfer RNA (tRNA) anticodon modifications are universal, but few biological functions are known. Here, in the rice blast fungus Magnaporthe oryzae, we show how unconventional effector secretion depends on tRNA modification and codon usage. We characterized the M. oryzae Uba4–Urm1 sulfur relay system mediating tRNA anticodon wobble uridine 2-thiolation (s2U34), a conserved modification required for efficient decoding of AA-ending cognate codons. Loss of s2U34 abolished the translation of AA-ending codon-rich messenger RNAs encoding unconventionally secreted cytoplasmic effectors, but mRNAs encoding endoplasmic reticulum–Golgi-secreted apoplastic effectors were unaffected. Increasing near-cognate tRNA acceptance, or synonymous AA- to AG-ending codon changes in PWL2, remediated cytoplasmic effector production in Δuba4. In UBA4+, expressing recoded PWL2 caused Pwl2 super-secretion that destabilized the host–fungus interface. Thus, U34 thiolation and codon usage tune pathogen unconventional effector secretion in host rice cells.
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Data availability
The M. oryzae UBA4 and URM1 gene sequences are available at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov) under the accession numbers MGG_05569 and MGG_03978, respectively. Other gene sequences used in the course of this study can be found at the National Center for Biotechnology Information under the following accession numbers: MGG_13863 for PWL2, MGG_10914 for BAS4, MGG_15370 for AVR-Pita, MGG_10097 for SLP1, FJ807764 for BAS1, MGG_18041 for AvrPiz-t, MGG_10020 for BAS107, MGG_05785 for BAS113, JN035619.1 for AVR-Pik, AB434708 for Avr-Pia, AF463528.1 for AVR1-CO39, and AAK31287.1 for TOXB. Strains generated during the course of this study are available from the corresponding author upon request and with an appropriate Animal and Plant Health Inspection Service permit. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Science Foundation (IOS-1758805 and IOS-2106153). Z.G. was supported by funding from the China Scholarship Council (file number 201903250119). Bioinformatic analyses were completed using the supercomputing resources of the Holland Computing Center at the University of Nebraska-Lincoln, which receives support from the Nebraska Research Initiative.
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R.A.W. conceived the project and obtained funding. R.A.W. designed the experiments. R.A.W. and G.L. interpreted the data. G.L., N.D. and Z.G. generated the strains and performed the experiments. G.L. performed the bioinformatic analyses. R.A.W. wrote the paper, with contributions from all authors.
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Extended data
Extended Data Fig. 1 Loss of UBA4 or URM1 in M. oryzae impairs biotrophic growth in host rice cells.
a-e, Three independent transformants carrying deletions in UBA4 had identical phenotypes. Compared to WT, Δuba4 strains were reduced in radial growth (a) and sporulation (b) on complete media (CM) by 10 days post inoculation (dpi), but appressorium formation rates on detached rice leaf sheath surfaces at 24 hpi (c), and penetration rates of appressoria on host rice leaf sheath surfaces at 30 hpi (d), were not affected by the loss of UBA4; however, cell-to-cell movement of Δuba4 invasive hyphae (IH) from the first infected rice cell into neighbouring cells at 48 hpi was reduced (e). f, Compared to WT and the Δurm1 URM1-GFP complementation strain, loss of URM1 abolished M. oryzae pathogenicity on rice leaves. Left, Leaves were imaged at 5 dpi. Images are representative of at least 5 leaves from three biological replicates. Right, 10 cm lengths of infected rice leaves were imaged at 5 dpi. Disease lesion areas were quantified using Image J and analyzed according to one-way ANOVA followed by a Tukey HSD test for multiple comparisons. Values are the means ± s.d. from three biological replicates. Different letters indicate significant differences (p < 0.05). g-k, Three independent transformants carrying deletions in URM1 had identical phenotypes, and also strongly resembled the Δuba4 strains in growth (g), sporulation rates (h), appressorium formation (i) and function (j), and cell-to-cell movement rates (k), compared to WT. a,g, Colonies of each strain were imaged and measured after 10 days growth on CM. Images are representative of three independent experiments. a-e, g-k, Values are means ± s.d. from three biological replicates. Bars with different letters indicate significant differences at P ≤ 0.05. P-values were determined by one-way ANOVA with Tukey’s multiple comparisons test (a, c-g, i-k) or by Welch ANOVA with Games-Howell post hoc test (b,h). Exact P values are given in Source Data Extended Data Fig. 1. c,i, Spores of the indicated strains were inoculated onto detached rice leaf sheaths at a rate of 0.5 ×105 spores ml−1.
Extended Data Fig. 2 Uba4 is localized to the cytoplasm in invasive hyphae.
Live-cell imaging of the Δuba4 UBA4-GFP complementation strain in detached rice leaf sheath epidermal cells at 38 hpi and 42 hpi shows that the Uba4-GFP fusion protein localizes to the cytoplasm of fungal invasive hyphae. White arrowheads indicate penetration sites, stars indicate movement of invasive hyphae into neighbouring cells. Scale bars, 10 μm. Representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate.
Extended Data Fig. 3 Bas4-mCherry:NLS but not Pwl2-GFP is secreted by Δuba4.
Live-cell imaging at 36 hpi of the indicated strains expressing PWL2-GFP and BAS4-mCherry:NLS in detached rice leaf sheath epidermal cells shows how in 100 % of observed infected cells, Pwl2-GFP accumulated in BICs of the UBA4+ strain but not in Δuba4 BICs, while Bas4-mCherry:NLS accumulated in the apoplast of both strains. Calculations and representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate. White arrowheads indicate penetration sites; red arrows indicate Pwl2 in BICs. Scale bars, 10 μm.
Extended Data Fig. 4 Paromomycin treatment impairs biotrophic growth in WT but does not prevent effector translation or secretion.
Live-cell imaging at 44 hpi of detached rice leaf sheath epidermal cells colonized with the wild type Guy11 strain expressing PWL2-mCherry:NLS and BAS4-GFP. Compared to the non-treated (NT) control (Top), treatment with paromomycin at 36 hpi (Bottom) impaired biotrophic growth and prevented cell-to-cell spread of invasive hyphae by 44 hpi, but effectors were nonetheless translated and secreted, and the BIC and apoplastic space were intact. White arrowheads indicate penetration sites; red arrows indicate Pwl2 in BICs; stars indicate movement of invasive hyphae into neighbouring cells. Scale bars, 10 μm. Representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate.
Extended Data Fig. 5 Slp1 is secreted into the Δuba4 apoplast.
Live-cell imaging at 44 hpi of detached rice leaf sheath epidermal cells infected with the indicated strains expressing SLP1-GFP shows how in 100 % of observed infected cells, compared to WT lacking SLP1-GFP, Slp1 accumulated in the apoplast of both UBA4+ and Δuba4 strains. Slp1-GFP fluorescence was weak in both strains and can be best seen outlining IH in the blow-up boxes, indicated with green arrow heads. Calculations and representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate. White arrowheads indicate penetration sites. Scale bars, 10 μm.
Extended Data Fig. 6 Location of AA-ending codons in PWL2-mCherry:NLS.
AA-ending codons in the PWL2-mCherry:NLS coding sequence26 that were recoded to synonymous AG-ending codons in PWL2 (–AA → –AG) are shown in red. The first mCherry codon is highlighted in yellow. The start of the NLS sequence is highlighted in green. The linker sequence is highlighted in grey.
Extended Data Fig. 7 Expressing PWL2 (-AA → -AG) in Δuba4 affects BIC integrity.
Live-cell imaging at 40 hpi of detached rice leaf sheath epidermal cells infected with Δuba4 expressing PWL2 (-AA → -AG) and BAS4-GFP. White arrowheads indicate penetration sites; red arrowheads indicate recoded Pwl2 in host nuclei; solid red arrows indicate recoded Pwl2 in enlarged BICs; dashed red arrows indicate recoded Pwl2 in fractured BICs. Scale bars, 10 μm. Representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate.
Extended Data Fig. 8 Expressing BAS4 (-AG → -AA) in Δuba4 does not affect Bas4 secretion.
a, AG-ending codons in the BAS4-GFP coding sequence26 that were recoded to synonymous AA-ending codons in BAS4 (–AG → –AA) are shown in red. The GFP start codon is highlighted in green. The linker sequence is highlighted in grey. b, Live-cell imaging at 36 hpi of detached rice leaf sheath epidermal cells infected with Δuba4 expressing PWL2 (-AA → -AG) and BAS4 (-AG → -AA). Recoded Bas4 and recoded Pwl2 are correctly deployed, albeit sometimes resulting in enlarged or fractured BICs in the latter case. White arrowheads indicate penetration sites. Scale bars, 10 μm. Images are based on the observations of 50 infected rice cells per leaf sheath, repeated in triplicate.
Extended Data Fig. 9 AA- and AG-ending codon counts for apoplastic and cytoplasmic effector mRNAs.
Tables showing frequencies and numbers of synonymous AA-ending (red) and AG-ending (black) codons in all M. oryzae protein coding sequences (∑CDS) (a) and in the indicated effector-encoding mRNAs (a-c). See text for details.
Extended Data Fig. 10 Brefeldin A treatment did not affect the secretion of recoded Pwl2 into the BIC.
Live-cell imaging at 40 hpi of detached rice leaf sheath epidermal cells infected with UBA4+ strains expressing BAS4-GFP and either PWL2 (Top) or recoded PWL2 (-AA → -AG) (Bottom) shows how in both cases, and in 100 % of observed cells, Brefeldin A (BFA) treatment, which inhibits conventional ER-Golgi secretion, led to the retention of Bas4 in IH cytoplasm as expected, but Pwl2 secretion into the BIC was unaffected. This confirms that recoded Pwl2 (-AA → -AG) is, like Pwl2, secreted into the BIC via the unconventional protein secretion pathway and that, moreover, no spillover secretion of Pwl2 (-AA → -AG) through the ER-Golgi pathway contributes to the enlarged, split-BIC phenotype observed for this strain. White arrowheads indicate penetration sites; hatched yellow circles indicate BICs, which are enlarged in the UBA4+ PWL2 (-AA → -AG) strain compared to the UBA4+ PWL2 strain. Scale bars, 10 μm. Calculations and representative images are based on the observations of 50 infected rice cells per leaf sheath per treatment, repeated in triplicate.
Supplementary information
Supplementary Information
Supplementary Tables 2 and 3 and References.
Supplementary Table 1
Raw and normalized peak information datasheet (LC–MS of modified nucleosides).
Source data
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Statistical source data for Fig. 1.
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Li, G., Dulal, N., Gong, Z. et al. Unconventional secretion of Magnaporthe oryzae effectors in rice cells is regulated by tRNA modification and codon usage control. Nat Microbiol 8, 1706–1716 (2023). https://doi.org/10.1038/s41564-023-01443-6
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DOI: https://doi.org/10.1038/s41564-023-01443-6
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