An MKP-MAPK protein phosphorylation cascade controls vascular immunity in plants

Abstract

Global crop production is greatly reduced by vascular diseases. These diseases include bacterial blight of rice and crucifer black rot caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas campestris pv. campestris (Xcc). The molecular mechanisms that activate vascular defense against such pathogens remains underexplored. Here, we show that an Arabidopsis MAPK phosphatase 1 (MKP1) mutant has increased host susceptibility to the adapted pathogen Xcc and is compromised in nonhost resistance to the rice pathogen Xoo. MKP1 regulates MAPK-mediated phosphorylation of the transcription factor MYB4 that negatively regulates vascular lignification through inhibiting lignin biosynthesis. Induction of lignin biosynthesis is, therefore, an important part of vascular-specific immunity. The role of MKP-MAPK-MYB signaling in lignin biosynthesis and vascular resistance to Xoo is conserved in rice, indicating that these factors form a tissue-specific defense regulatory network. Our study likely reveals a major vascular immune mechanism that underlies tissue-specific disease resistance against bacterial pathogens in plants.

Fig. 1.. MKP1 mutants have reduced vascular resistance to Xoo and Xcc.

(A) PXO99A-GUS populates the leaf veins of ntx1 plants. Disease symptoms, GUS staining, and Xoo levels were measured in wild-type Col-0 and ntx1/mkp1 mutant plants 7 days after infiltration with Xoo strain PXO99A and GUS-labeled PXO99A-GUS [2 × 10⁷ colony-forming unit (CFU) ml⁻¹]. (B) Disease symptoms, green fluorescent protein (GFP) fluorescence, and growth of Xcc in Col-0 and ntx1 at 4 dpi with GFP-labeled Xcc8004-GFP (1 × 10⁷ CFU ml⁻¹). (C) Genetic complementation of MKP1 in ntx1 plants. Disease symptoms and bacterial populations were recorded at 7 days post inoculation (dpi) with PXO99A in Col-0, ntx1, CR-mkp1, and pMKP1::MKP1/ntx1. (D) GUS staining of pMKP1::GUS transgenic plants demonstrated that MKP1 was mainly expressed in the vascular bundles. Scale bar, 1 cm. (E) The nonhost Xoo susceptibility phenotype of mkp1 was restored by a mutation in MPK3 but not MPK6, showing that the double-mutant mkp1 mpk3 restored the wild-type NHR to Xoo, 7 dpi with PXO99A. Data were shown as means ± SD (n = 3). Letters indicate significant differences (P < 0.05) determined by two-way analysis of variance (ANOVA) with Tukey’s test. Scale bar, 1 cm. Experiments were repeated three times with similar results. (F) Kinase activation of MPK3 but not MPK6 was induced by Xoo inoculation. Proteins were prepared from leaf samples for in-gel kinase assay at the indicated times. hpi, hours post inoculation.

Fig. 2.. MPK3 phosphorylates MYB4 to regulate lignin biosynthesis.

(A) Differential expression of lignin biosynthetic genes induced by Xoo in wild-type Col-0 and mkp1 as revealed by RNA sequencing (RNA-seq). Many lignin biosynthesis genes were less induced in mkp1 as compared with Col-0. FPKM, Fragments per kilobase of exon per million reads mapped. (B) Lignin quantification in leaves at 12 hours infiltrated with Xoo and Xcc. Data were shown as means ± SD (n = 3). Asterisks represented statistical significance (***P < 0.001, Student’s t test). ns, not significant. Experiments were repeated three times. (C) Cell wall autofluorescence and relative fluorescence intensity of Col-0 and mkp1 infiltrated with Xoo. Note that less lignification of the leaf vessel was induced by Xoo in mkp1 as compared with Col-0. Scale bar, 50 μm. Data were shown as means ± SD (n = 10). Letters indicated significant differences (P < 0.05) determined by two-way ANOVA with Tukey’s test. Experiments were repeated three times. (D and E) MPK3 interacts with MYB4, as revealed by Y2H screen (D) and coimmunoprecipitation (coIP) assay (E). SD/-Trp-Leu-His, synthetic dropout (SD) media lacking leucine, tryptophan, and histidine; SD/-Leu -Trp, SD media lacking leucine and tryptophan; 3AT, 3-aminotriazole. MPK3-GFP and MYB4-MYC in transgenic Arabidopsis plants were purified and immunodetected using anti-GFP or anti-MYC antibody. (F) MPK3 phosphorylates MYB4 in vitro. MPK3-MBP (myelin basic protein), MYB4-His, MKK4^DD-MBP (T224D/S230D), and MKK5^DD-MBP (T215D/S221D) were expressed in E. coli and purified for in vitro phosphorylation assays. MYB4-His was incubated at 30°C for 1 hour then separated on a Phos-tag gel. CBB, Coomassie brilliant blue staining for loading control.

Fig. 3.. Phosphorylation of MYB4 suppresses vascular defense by inhibiting lignin biosynthesis.

(A) Disease symptoms (left) and bacterial growth (right) in Col-0, mkp1, CR-myb4, MYB4-OE, MYB4^AA-OE, and MYB4^DD-OE at 7 dpi with PXO99A. The wild-type MYB4 and the site mutation variants (MYB4^DD and MYB4^AA) were ectopically expressed (OE) as a MYC fusion protein. Scale bar, 1 cm. (B) Leaf lignin quantification indicated that lignin biosynthesis was inhibited by overexpression of MYB4 and MYB4^DD but not MYB4^AA. (C) MYB4 also negatively regulates resistance to Xcc. Disease symptoms (left) and bacterial growth (right) of Xcc in Col-0, mkp1, CR-myb4, MYB4-OE, MYB4^AA-OE, and MYB4^DD-OE at 4 dpi with Xcc8004. Scale bar, 1 cm. Data were shown as means ± SD (n = 3). Asterisks represented statistical significance (**P < 0.01 and ***P < 0.001, Student’s t test) (B). Letters indicated significant differences (P < 0.05) determined by two-way ANOVA with Tukey’s test (A to C). Experiments were repeated three times.

Fig. 4.. OsMKP1 mediates Xoo resistance through OsMPK6 dephosphorylation.

(A and B) Population size and distribution of Xoo in leaves of 2-month-old wild-type NIP, CR-Osmkp1, and OsMKP1-OE following clip inoculation. Bacterial populations in 5-cm leaf segments were measured at 0, 8, and 14 dpi. (C) Lesion lengths of NIP, CR-Osmkp1, and OsMKP1-OE inoculated with PXO99A [optical density at 600 nm (OD₆₀₀), 1.0] at 14 dpi. (D) Lesions (left) and statistical analysis (right) of NIP, CR-Osmkp1, and OsMKP1-OE inoculated with Xoc strains RS85. OsMKP1 negatively regulated resistance to Xoc. Scale bar, 1 cm. (E) Lesions and statistical analysis of NIP, CR-Osmkp1, and OsMKP1-OE inoculated with Xcc8004. (F) Disease symptoms of representative leaves (left) and lesion lengths (right) of NIP, CR-Osmkp1, CR-Osmkp1Osmpk3, and CR-Osmkp1Osmpk6 inoculated with PXO99A at 14 dpi. Data were shown as means ± SD; n > 20 (C to F) and n = 3 (B). Asterisks represented statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test) (B to F). Experiments were repeated three times with similar results. (G) OsMPK3 and OsMPK6 activation by flg22 treatment. Two-week-old seedlings of NIP and CR-Osmkp1 were treated with flg22 (1 μM) for the indicated times for protein preparation. The phosphorylated OsMPK3 and OsMPK6 were detected using the anti-p44/42 MPK antibody, with Ponceau S staining as loading control. (H) MPK6 was stronger activated in CR-Osmkp1 infected by PXO99A as compared with NIP.

Fig. 5.. OsMYB102 and OsMYB108 negatively regulate lignin biosynthesis and resistance to Xoo.

(A) OsMPK6 interacts with OsMYB102 in a Y2H assay. (B) coIP assay of the OsMPK6-OsMYB102 interaction in N. benthamiana. Fusion proteins were purified and immunodetected using anti-GFP or anti-FLAG antibody. (C) Lignin quantification in leaves of NIP, CR-Osmyb102, CR-Osmyb108, and CR-Osmyb102Osmyb108, indicating that OsMYB102 and OsMYB108 negatively regulate lignin production. (D) Disease resistance of representative lines (left) and lesion lengths (right) of 2-month-old NIP, CR-Osmyb102, CR-Osmyb108, and CR-Osmyb102Osmyb108 at 14 dpi with PXO99A. (E and F) Phosphorylation of OsMYB102 negatively regulates lignin biosynthesis and Xoo resistance. Lesions and bacterial growth of wild-type NIP, OsMYB102-OE, OsMYB102^AA-OE, and OsMYB102^DD-OE inoculated with PXO99A at 14 dpi. Data were shown as means ± SD; n = 3 (C and E) and n > 20 (D and F). Asterisks represented statistical significance (**P < 0.01 and ***P < 0.001, Student’s t test). Experiments were repeated three times with similar results.

Fig. 6.. A proposed model for the function of the MKP-MPK protein phosphorylation cascade in vascular defense in plants.

MKP1 is induced by vascular bacterial pathogens, such as Xoo and Xcc, which negatively regulate MPK3/6 through dephosphorylation. MPK3/6, in turn, activates the MYB4 transcription factor that negatively regulates lignin biosynthesis. This cascade orchestrates vascular-specific resistance against vascular pathogens and negatively regulates defense against mesophyll cell pathogens through inhibiting SA and ROS biosynthesis.