Aquaporin OsPIP2;2 links the H2O2 signal and a membrane-anchored transcription factor to promote plant defense

Abstract

To overcome pathogen infection, plants deploy a highly efficient innate immune system, which often uses hydrogen peroxide (H2O2), a versatile reactive oxygen species, to activate downstream defense responses. H2O2 is a potential substrate of aquaporins (AQPs), the membrane channels that facilitate the transport of small compounds across plasma membranes or organelle membranes. To date, however, the functional relationship between AQPs and H2O2 in plant immunity is largely undissected. Here, we report that the rice (Oryza sativa) AQP OsPIP2;2 transports pathogen-induced apoplastic H2O2 into the cytoplasm to intensify rice resistance against various pathogens. OsPIP2;2-transported H2O2 is required for microbial molecular pattern flg22 to activate the MAPK cascade and to induce the downstream defense responses. In response to flg22, OsPIP2;2 is phosphorylated at the serine residue S125, and therefore gains the ability to transport H2O2. Phosphorylated OsPIP2;2 also triggers the translocation of OsmaMYB, a membrane-anchored MYB transcription factor, into the plant cell nucleus to impart flg22-induced defense responses against pathogen infection. On the contrary, if OsPIP2;2 is not phosphorylated, OsmaMYB remains associated with the plasma membrane, and plant defense responses are no longer induced. These results suggest that OsPIP2;2 positively regulates plant innate immunity by mediating H2O2 transport into the plant cell and mediating the translocation of OsmaMYB from plasma membrane to nucleus.

Figure 1

OsPIP2;2 mediates H₂O₂ transport in yeast. A, Yeast colonies grown on SD-Ura medium with different concentrations of H₂O₂. The expression levels of OsPIPs in yeast cells were evaluated by western blot using an antibody against His tag, and Coomassie brilliant blue (CBB) staining was used to show protein loading. Photographs were taken at 3 d postincubation. In this figure and in yeast-involved figures provided hereafter, “Control” indicates the empty yeast binary vector without any insert of tested genes, in contrast to the recombinant vector carrying each of the OsPIPs. B, C, and D, Chronological changes of H₂O₂ content in yeast cells after treatment with 0 or 300 μM H₂O₂ detected by H₂DCF-DA, AR, and AUR probes respectively. The symbol “+” indicates supplied with 300 μM H₂O₂, and “−” indicates supplied with H₂O as control. Data are shown as means ± SEM (n = 8). E and F, H₂DCF-DA (E) and AR (F) probing of yeast cells 45 min after treatment with 300 μM H₂O₂.

Figure 2

OsPIP2;2 confers resistance to X. oryzae pv. oryzae (Xoo) strain PXO99^A, X. oryzae pv. oryzicola (Xoc) strain RS105, and M. oryzae strain HB3. A, Bacterial blight symptoms in leaves 15 d postinoculation with PXO99^A using the clipped-end method. In this figure and in plant-involved figures provided hereafter, “NPB” refers to the rice variety NPB, “OsPIP2;2OE” means an OsPIP2;2-overexpressing rice (NPB) line coded with a number during characterization, and “Ospip2;2” indicates an OsPIP2;2-defected NPB line generated by CRISPR/Cas9 and coded with a number. B, Lesion length in (A). Data are shown as means ± SEM (n = 20). C, Titers of PXO99^A 3 d postinoculation. Data are shown as means ± SEM (n = 4). D, Bacterial leaf stripe symptoms in leaves 5 d after RS105 infiltration. E, Lesion length in (D). Data are shown as means ± SEMs (n = 20). F, Titers of RS105 3 d postinoculation. Data are shown as means ± SEM (n = 3). G, Rice blast symptoms in leaves 5 d after spray inoculation with HB3 conidia. H, Relative fungal biomass in (G). The fungal biomass was determined using qPCR of the M. oryzae Pot2 gene against the rice OsActin gene. Data are shown as means ± SEM (n = 3). Lowercase letters indicate significant differences at by one-way ANOVA and Duncan’s multiple range tests (P ≤ 0.01).

Figure 3

OsPIP2;2 links PAMP-induced apoplastic H₂O₂ to the PTI pathway. A, Chronological changes in the H₂O₂-probing AR fluorescence densities in leaves of 2-week-old rice seedlings after flg22 treatment (means ± SEM, n = 8). OE#1, OsPIP2;2OE#1; OE#2, OsPIP2;2OE#2; KO#41, Ospip2;2#41; KO#279, Ospip2;2#279. B, Cell imaging of the DCF fluorescence by laser confocal microscopy. Leaves were preloaded with H₂DCF-DA 45 min before treatment with 300 μM H₂O₂. C, flg22-induced callose deposition in rice. The leaves of 2-week-old rice seedlings were treated with 10 μM flg22 for 16 h and then stained by aniline blue. B and C, The scale bar representing 100 μm applies to all the images. D, Quantification of callose deposition in leaves in C by ImageJ. Data were shown as means ± SEM (n = 3). E, MAPK activation was detected after flg22 treatment by western blot. The expected identities of the respective bands are marked on the right. The experiment was performed twice with similar results. F–H, Expression levels of pathogenesis-related genes OsPR1a and OsPR10 expression levels in plants with PXO99^A, RS105, or HB3 24 h postinoculation. Data are shown as means ± SEM (n = 4). Lowercase letters indicate significant differences by one-way ANOVA and Duncan’s multiple range tests (at P ≤ 0.01).

Figure 4

Phosphorylation of OsPIP2;2 at S125 is required for H₂O₂ transport. A, Yeast transformants grown in SD-Ura medium with H₂O₂ at 28°C. Photographs were collected at 3 d postincubation. B, Imaging of the DCF fluorescence in yeast cells by laser confocal microscopy using H₂DCF-DA dye after treatment with 300 μΜ H₂O₂. C, Kinetic variations of AR fluorescence densities in yeast transformants after application of 300 μΜ H₂O₂ over 40 min. Data are shown as means ± SEM (n = 8). D, Kinetic variations of AR fluorescence densities in N. benthamiana expressing OsPIP2;2 or its mutants after H₂O₂ treatment. Data are shown as means ± SEM (n = 8). E, Immunoblot analysis of OsPIP2;2 phosphorylation at S125 by using His antibody after Phos-tag gel separation. OsPIP2;2 or OsPIP2;2 S125A were expressed in leaves of N. benthamiana, and then the infiltrated leaves were treated with 1 μM flg22 for 30 min before protein extraction. Protein samples treated with (+) or without (–) calf intestinal alkaline phosphatase were separated by a Phos-tag gel. CBB staining was used to show protein loading (bottom panel). F, Subcellular localization of OsPIP2;2 and its mutants in N. benthamiana observed under lasers intensity as in (B).

Figure 5

OsPIP2;2 associates with OsmaMYB. A, Associations between OsPIP2;2 and OsmaMYB in the split-ubiquitin yeast two-hybrid system. Yeast cells co-transformed with bait and prey vectors were grown on medium supplemented with 10 mM of 3-amino-1,2,4-triazole. B, Co-IP of OsPIP2;2 and OsmaMYB after co-expression in N. benthamiana. The indicated constructs were co-expressed in N. benthamiana by A. tumefaciens. Total proteins were extracted 2 d postinfiltration, and subjected to anti-FLAG immunoprecipitations. C and D, Association between OsPIP2;2 and OsmaMYB indicated by the split-LUC complementation assay. The indicated constructs were co-expressed in N. benthamiana leaves and then LUC activities were examined in both qualitative (C) and quantitative assays (D). Cluc-CPR5 and BIK1-Nluc were used as the negative control. Cluc-XLG2 and BIK1-Nluc served as the positive control. Data are shown as means ± SEM (n = 8). Lowercase letters indicate significant differences at P ≤ 0.01, Duncan’s multiple range tests and one-way ANOVA.

Figure 6

OsmaMYB contributes to plant immunity and is activated by phosphorylation of OsPIP2;2 at S125. A, Titers of DC3000 ΔhopQ1-1 3 d post-inoculation. Agrobacterium tumefaciens was used to transiently express OsmaMYB on one-half of an N. benthamiana leaf and LTI6b control on the other. Data are shown as means ± SEM (n = 6). Asterisks indicate significant differences compared to control by Student’s t test (**P ≤ 0.01). The experiment was performed twice with similar results. B, Subcellular localization of OsmaMYB by confocal laser microscopy. OsmaMYB-GFP fusion was transiently expressed in N. benthamiana leaves, and the infiltrated leaves were treated with H₂O or flg22. C, Immunoblot analysis of OsmaMYB. The infiltrated leaves were treated with flg22, and samples were collected at three different time points for immunoblot analysis. The OsmaMYB was detected using an anti-GFP antibody. CBB staining is used to show protein loading (bottom panel). D, Subcellular localization of OsmaMYB-GFP after co-expression with OsPIP2;2 S125A or OsPIP2;2 S125D. B and D, orange scale bar representing 100 μm applies to all the images. E, Immunoblot analysis of OsmaMYB-GFP co-expressed with the OsPIP2;2 phosphorylation mutants. F, Subcellular fractionation analysis of the distribution of OsmaMYB. Histone H3 was used as a nuclear marker, Na⁺/K⁺-ATPase was used as a membrane marker. G, Quantification of LUC activity. The indicated Nluc and Cluc constructs were co-expressed in N. benthamiana leaves for LUC complementation assay. The relative luminescence unit indicates the strength of protein–protein interaction. Data are shown as mean ± SEM (n = 8). S125A, phosphor-deficient form; S125D, phosphomimetic form. Lowercase letters indicate significant differences at P ≤ 0.01 by Duncan’s multiple range tests and one-way ANOVA. These experiments were performed at least twice with similar results.

Figure 7

Model for the modulation of plant immunity by OsPIP2;2 through H₂O₂ transport and OsmaMYB. At a resting state, OsPIP2;2 associates with the membrane-anchored OsmaMYB, and plant defense cannot be activated. Upon perception of microbial pattern flg22, the membrane-associated enzyme RBOHA/B is activated to catalize H₂O₂ generation in the apoplast. Upon flg22 perception, moreover, the AQP OsPIP2;2 is phosphorylated at S125 and therefore functions to mediate the H₂O₂ transport into the cytoplasm. The transported H₂O₂ triggers a series of defense responses including MAPK cascade, callose deposition, and upregulation of defense-related genes. Meanwhile, OsmaMYB is processed by an unknown protease, and the C terminus is released from the plasma membrane and de-associates with the OsPIP2;2 complex. Thereafter OsmaMYB is translocated into the nucleus and activates the expression of defense-related genes. In this way, OsPIP2;2 enhances plant resistance by linking H₂O₂ transport and OsmaMYB.