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. Author manuscript; available in PMC: 2015 Dec 10.
Published in final edited form as: Cell Host Microbe. 2014 Nov 26;16(6):748–758. doi: 10.1016/j.chom.2014.10.018

Modulation of RNA polymerase II phosphorylation downstream of pathogen perception orchestrates plant immunity

Fangjun Li 1,2,11, Cheng Cheng 3,11, Fuhao Cui 1,2,11, Marcos V V de Oliveira 2,3,4,11, Xiao Yu 2, Xiangzong Meng 3, Aline C Intorne 2,4, Kevin Babilonia 3,5, Maoying Li 1,3, Bo Li 2, Sixue Chen 6, Xiaofeng Ma 7, Shunyuan Xiao 7, Yi Zeng 8, Zhangjun Fei 8, Richard Metz 9, Charles D Johnson 9, Hisashi Koiwa 10, Wenxian Sun 1, Zhaohu Li 1, Gonçalo A de Souza Filho 4, Libo Shan 2,*, Ping He 3,*
PMCID: PMC4268009  NIHMSID: NIHMS640435  PMID: 25464831

Summary

Perception of microbe-associated molecular patterns (MAMPs) elicits host transcriptional reprogramming as part of the immune response. Although pathogen perception is well studied, the signaling networks orchestrating immune gene expression remain less clear. In a genetic screen for components involved in the early immune gene transcription reprogramming, we identified Arabidopsis RNA polymerase II C-terminal domain (CTD) phosphatase-like 3 (CPL3) as a negative regulator of immune gene expression. MAMP perception induced rapid and transient cyclin-dependent kinase (CDKC)-mediated phosphorylation of Arabidopsis CTD. The CDKCs, which are in-turn phosphorylated and activated by a canonical MAP kinase (MAPK) cascade, represent a point of signaling convergence downstream of multiple immune receptors. CPL3 directly dephosphorylated CTD to counteract MAPK-mediated CDKC regulation. Thus, modulation of the phosphorylation dynamics of eukaryotic RNA polymerase II transcription machinery by MAPKs, CTD kinases and phosphatases constitutes an essential mechanism for rapid orchestration of host immune gene expression and defense upon pathogen attacks.

Introduction

Plants and animals possess pattern recognition receptors (PRRs) to detect the presence of microbes by recognizing microbe-associated molecular patterns (MAMPs) (Boller and Felix, 2009). In plants, MAMPs are usually perceived by cell surface-resident PRRs and elicit pattern-triggered immunity (PTI). Arabidopsis FLS2, a leucine-rich repeat receptor-like kinase (LRR-RLK), recognizes a conserved 22-amino acid peptide (flg22) from bacterial flagellin (Boller and Felix, 2009). Upon flagellin perception, FLS2 rapidly associates with another LRR-RLK BAK1, thereby initiating downstream signaling (Chinchilla et al., 2007; Heese et al., 2007; Sun et al., 2013). BAK1 also heteromerizes with several PRRs, including EFR (receptor for bacterial elongation factor EF-Tu) (Roux et al., 2011) and AtPEPR1 (receptor for endogenous danger signal PEP1) (Postel et al., 2010). BIK1, a plasma membrane-resident receptor-like cytoplasmic kinase in the FLS2/BAK1 complex, is rapidly phosphorylated upon flg22 perception to transduce intracellular signaling (Lin et al., 2014; Lu et al., 2010; Zhang et al., 2010). BIK1 is able to phosphorylate NADPH oxidase family member RBOHD (respiratory burst oxidase homolog D), thereby contributing to the production of reactive oxygen species (ROS) (Kadota et al., 2014; Li et al., 2014). Rapid activation of convergent MAP kinases (MAPKs) and calcium-dependent protein kinases downstream of multiple PRRs is followed with the expression of MAMP responsive genes (Tena et al., 2011). It appears that distinct MAMPs elicit massive overlapping transcriptional reprogramming (Zipfel et al., 2006). Several transcription factors, especially members of WRKY and ERF families, could be directly phosphorylated by MAPKs and are potentially involved in defense gene regulation (Meng and Zhang, 2013). Yet, the molecular signaling networks leading to the rapid reprogramming of immune genes have remained elusive.

Transcription of protein-coding genes in eukaryotes is intricately orchestrated by RNA polymerase II (RNAPII), general transcription factors, mediators and gene-specific transcription factors. The multi-subunit RNAPII is evolutionarily conserved from yeast to human. Its largest subunit Rpb1 contains a carboxyl-terminal domain (CTD) consisting of conserved heptapeptide repeats with the consensus sequence Y1S2P3T4S5P6S7 (Buratowski, 2009). The number of repeats varies from 26 in yeast, 34 in Arabidopsis, to 52 in mammals (Hajheidari et al., 2013). The combinatorial complexity of CTD posttranslational modifications constitutes a “CTD code” that is “read” by CTD binding proteins to regulate the transcription cycle, modify chromatin structure, and modulate RNA capping, splicing, and polyadenylation. In particular, the CTD undergoes waves of serine phosphorylation and dephosphorylation events regulated by various CTD kinases, often members of cyclin-dependent kinases (CDKs), and phosphatases during transcription initiation, elongation and termination. The interplay between different CTD kinases and phosphatases provides a means for coupling and coordinating specific stages of transcription by recruiting other factors required for proper gene expression (Buratowski, 2009).

Although both immune signaling mechanisms and CTD phosphorylation cycles in regulating gene transcription have been studied separately, whether and how they are connected remains enigmatic. Here, we demonstrate that CTD phosphorylation dynamics plays a key role in regulating host immunity. Arabidopsis RNAPII CTD exhibits rapid and transient phosphorylation dynamics upon perception of different MAMPs. Moreover, biochemical and genetic analyses uncovered an immune gene regulation circuit in Arabidopsis by MAPK-mediated phosphorylation of CTD kinases CDKCs upon flagellin perception. A genetic screen for components involved in the early immune gene transcription reprogramming identified Arabidopsis CPL3 (CTD phosphatase-like 3) as a negative regulator of immune gene expression and immunity to pathogen infections. Arabidopsis CPL3 is a homolog of yeast Fcp1 (TFIIF-associating CTD phosphatase) (Koiwa et al., 2002). The Arabidopsis genome encodes 4 CPLs, and CPL1 and CPL3 were uncovered from a genetic screen for hyper-induction of plant abiotic stress response gene RD29A promoter (Koiwa et al., 2002). Our biochemical analyses indicate that CPL3 interacts with and preferentially dephosphorylates Ser2 of RNAPII CTD to counter-regulate MAPK/CDKC-mediated CTD phosphorylation. Thus, our study suggests that modulation of general transcription machinery phosphorylation is a key feature of host immune response.

Results

Enhanced immune gene activation and disease resistance in aggie1 and aggie3 mutants

To elucidate the signaling networks regulating immune gene activation, we developed a genetic screen with an ethyl methanesulfonate (EMS)-mutagenized population of Arabidopsis transgenic plants expressing a luciferase reporter gene under the control of the FRK1 promoter (pFRK1∷LUC). FRK1 (flg22-induced receptor-like kinase 1) is a specific and early marker gene activated by multiple MAMPs, likely downstream of MAPKs (Asai et al., 2002; He et al., 2006). A series of mutants with altered pFRK1∷LUC activity upon flg22 treatment were identified and named as Arabidopsis genes governing immune gene expression (aggie). Two aggie mutants, aggie1 and aggie3 (aggie3 was found to be allelic with aggie1 after map-based cloning), exhibited enhanced FRK1 promoter activity compared to wild-type (WT) pFRK1∷LUC transgenic plants at various time points after flg22 treatment (Fig. 1A & 1B). Notably, the aggie1 and aggie3 mutants did not significantly activate the FRK1 promoter (0.8∼2 fold) in the absence of flg22, suggesting specific regulation of FRK1 expression by Aggie in immune signaling. The aggie1 mutants also potentiated pFRK1∷LUC activity in response to elf18, an 18-amino acid peptide of bacterial EF-Tu, and fungal chitin (Fig. 1C & S1A), suggesting that the mutation in aggie1 likely occurs in a convergent component downstream of multiple MAMP receptors. In addition, the pFRK1∷LUC activation by a nonpathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) hrcC, which is deficient in delivery of type III effectors, and by a non-adaptive bacterium P. syringae pv. phaseolicola NPS3121 was also enhanced in aggie1 and aggie3 mutants (Fig. 1D). The aggie1 and aggie3 mutants were more resistant to virulent Pst and P. syringae pv. maculicola ES4326 (Psm) infections (Fig. 1E, S1B & S1C). The bacterial population in aggie1 and aggie3 mutants was about five to ten-fold less than that in WT plants 4 days post inoculation (dpi) with Psm (Fig. 1E). A ten-fold reduction in bacterial growth was also observed in aggie1 mutant when Pst was inoculated upon dipping inoculation (Fig. S1B). The disease symptom development was less pronounced in aggie1 mutant than WT plants (Fig. S1C). However, the flg22-induced MAPK activation detected by an α-pERK antibody was not affected in aggie1 mutant compared to WT plants (Fig. 1F). Similarly, the aggie1 mutant exhibited normal oxidative burst and BIK1 phosphorylation in response to flg22 treatment (Fig. 1G & S1D). These results suggest that Aggie1 functions either downstream or independently of MAPK activation and ROS production in FLS2 signaling.

Figure 1. Elevated pFRK1∷LUC expression and disease resistance in aggie1 and aggie3 mutants.

Figure 1

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(A) Luciferase activity in 10-day-old pFRK1∷LUC (WT), aggie1 and aggie3 seedlings. Seedlings were treated with or without 10 nM flg22 for 12 hr and photographed with an EMCCD camera. (B) Time-course of pFRK1∷LUC activity. The seedlings were treated with 100 nM flg22 over 84 hr. The data are shown as means ± se from at least 6 seedlings for each time point. (C) The pFRK1∷LUC activity triggered by different MAMPs. Ten-day-old seedlings were treated with 100 nM flg22 or elf18 for 15 hr. (D) The pFRK1∷LUC activity triggered by different bacteria. Four-week-old plants were hand-inoculated with hrcC or Psph at OD600 = 0.5. The data are shown as means ± se from at least 8 leaves for each treatment 15 hpi. (E) Elevated disease resistance to Psm infection in aggie mutants. Four-week-old plants were hand-inoculated with Psm at OD600=5 ×10-4. The data are shown as means ± se with Student's t-test. * indicates p<0.05 compared to WT. (F) flg22-induced MAPK activation. Twelve-day-old seedlings were treated with 100 nM flg22 for different time points. MAPK activation was analyzed with an α-pERK antibody (top panel), and protein loading was shown by Coomassie brilliant blue (CBB) (bottom panel) staining for RuBisCO (RBC). (G) flg22-trigered ROS burst. Leaf discs were treated with H2O or 100 nM flg22 over 30 min. The data are shown as means ± se from 40 leaf discs.

The above experiments were repeated 4 times with similar results (see also Figure S1).

Aggie1 and Aggie3 encode a plant CTD phosphatase-like protein

Genetic analysis of F1 plants from a backcross of aggie1 to pFRK1∷LUC plants indicated that aggie1 is a recessive mutation (Fig. 2A). We crossed the aggie1 mutant (in the Col-0 background) with the Ler accession and mapped aggie1 to Chromosome 2 between markers F4P9-3 and T1B8-2 that are 110 kb apart (Fig. S2A). Sequencing the individual genes within this region identified a G to A mutation located 1294 bp downstream of the predicted start codon of At2g33540. The mutation occurred at the 3′ splice site of the 4th intron, resulting in a potential alternative 3′ acceptor site located 43 bp downstream (Fig. 2B & S2B). The mutation in aggie3 was identified by map-based cloning coupled with next generation sequencing. The aggie3 carries a G to A mutation at position 962 bp of At2g33540, located at the 3′ splice site of the 2nd intron, which results in one base pair shift of the 3′ acceptor site. The predicted transcripts of aggie1 and aggie3 were confirmed by Sanger sequencing of cDNA products (Fig. S2B). At2g33540 encodes CPL3 with an N-terminal domain of unknown function, an FCPH (Fcp homology) domain and a BRCT (breast cancer 1 C terminus) domain (Bang et al., 2006; Koiwa et al., 2002). The aggie1 and aggie3 mutants were also named as cpl3-5 and cpl3-6, respectively. The mutations in aggie1/cpl3-5 and aggie3/cpl3-6 resulted in truncated proteins lacking both FCPH and BRCT domains (Fig. 2B). To confirm that the aggie1 phenotypes were caused by mutation in CPL3, we introduced CPL3 under the control of its native promoter into aggie1. The FRK1 promoter activity in response to flg22 treatment was restored to the WT level in three independent complementation lines (Fig. 2C). In addition, similar to aggie1 mutants, two CPL3 T-DNA insertion lines cpl3-3 and cpl3-4, but not cpl2-2 (Ueda et al., 2008), were more resistant to Psm infection than WT plants (Fig. 2D). Interestingly, aggie1/cpl3-5 and cpl3-4 plants also showed enhanced resistance to an obligate biotrophic fungal pathogen, Golovinomyces cichoracearum UCSC1, with 5-7 fold reduction in mildew spores on the inoculated leaf surface compared to WT plants (Fig. 2E). Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that endogenous FRK1 expression was elevated in cpl3-4 and aggie1/cpl3-5 mutants (Fig. 2F). The expression of another MAMP marker gene WRKY30 was also enhanced in cpl3-4 and aggie1/cpl3-5 mutants (Fig. 2F). Taken together, the results indicate that CPL3 plays a negative role in immune gene regulation and plant immunity.

Figure 2. Aggie1 and Aggie3 encode CPL3, a regulator of plant immunity.

Figure 2

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(A) aggie1 is a recessive mutation. Ten-day-old F1 seedlings derived from a cross between aggie1 and WT pFRK1∷LUC were treated with 100 nM flg22 for 12 hr. The data are shown as means ± se from at least 5 seedlings. (B) The scheme of the CPL3 genomic DNA and deduced protein domains. The top panel is a schematic illustration of the CPL3 genomic DNA with exons (solid box), intron (lines) and 3′ untranslated region (open box). The stars indicate the mutations in aggie1/cpl3-5 and aggie3/cpl3-6. The other panels illustrate the protein domain structures of CPL3 and four truncated mutants. (C) Complementation analysis. C1, C2 and C3 are three independent transgenic lines of aggie1 complemented with pCPL3∷CPL3. Ten-day-old seedlings were treated with 100 nM flg22 for 12 hr. The data are shown as means ± se from at least 10 seedlings. (D) The cpl3 mutants are more resistant to bacterial infection. Four-week-old plants were hand-inoculated with Psm at OD600=5 × 10-4. The data are shown as means ± se with Student's t-test. * indicates p<0.05 compared to WT. (E) The cpl3 mutants are more resistant to powdery mildew Golovinomyces cichoracearum UCSC1. The pictures were taken at 10 dpi on six-week-old plants. The numbers of spores per mg tissue (×1000) are shown on the top of the pictures. (F) Endogenous FRK1 and WRKY30 expression. The 12-day-old seedlings were treated with 100 nM flg22 for qRT-PCR analysis. The data are shown as means ± se from three biological repeats with Student's t -test. * indicates p<0.05 compared to WT.

The above experiments were repeated 3 times with similar results (see also Figure S2).

Global regulation of immune gene expression by CPL3

To determine transcriptome dynamics regulated by CPL3 during elicitation of immune responses, we performed RNA sequencing (RNA-seq) analysis of Col-0 WT and cpl3-3 mutant treated without or with flg22 for 30 min. The correlation coefficient (r) for the expression profile of each detectable transcript in WT and cpl3-3 seedlings without flg22 treatment is close to linear (0.98) (Fig. 3A). Among 24074 detectable transcripts, 93 showed reduced and 227 showed enhanced expression [fold change ≥ 2 and false discovery rate (FDR) < 0.05] in cpl3-3 mutant compared to that in WT plants without treatment (Table S1), suggesting that CPL3 does not appear to control general gene transcription. Using a cut-off of fold change ≥ 2 and FDR < 0.05, we identified 964 flg22-induced genes in WT and 1128 in cpl3-3 with 918 genes induced in both WT and cpl3-3, 46 induced only in WT and 210 induced only in cpl3-3 (Fig. 3B & Table S2). Hierarchical clustering analysis of differential flg22-induced genes in WT and cpl3-3, which were defined as CPL3-dependent flg22-induced genes, classified them into four groups, Group I: CPL3-required flg22-induced genes (genes induced in WT, but not in cpl3-3, 46 genes); Group II: CPL3-potentiated flg22-induced genes (genes induced in both WT and cpl3-3 with at least 1.5 fold higher induction in WT than cpl3-3, 32 out of 918 genes); Group III: CPL3-attenuted flg22-induced genes (genes induced in both WT and cpl3-3 with at least 1.5 fold higher induction in cpl3-3 than WT, 180 out of 918 genes); Group IV: CPL3-suppressed flg22-induced genes (genes induced in cpl3-3, but not in WT, 210 genes) (Fig. 3C & Table S3). Thus, among 468 CPL3-dependent flg22-induced genes, 390 (83%) showed increased flg22-induction (Group III & IV) whereas 78 (17%) exhibited reduced flg22-induction (Group I & II) in cpl3-3 compared to WT. Notably, a large portion of CPL3-dependent flg22-induced genes are classified to be associated with defense responses (Table S3 & S4). Enrichment analysis of Gene Ontology (GO) indicates that among 180 CPL3-attenuted flg22-induced genes (Group III), the frequency of genes associated with biotic stress, innate immune response, response to bacterium and fungus, and salicylic acid (SA)-mediated signaling pathway was significantly enriched compared to the predicated frequency in the genome (Fig. 3D & Table S4). Many defense-related transcription factors, such as WRKYs and ERFs, and RLKs were also overrepresented in Group III genes (Table S3). The genes encoding flg22-activated MKK4 and MPK11 were highly induced in cpl3-3 compared to WT. Interestingly, cpl3-3 also displayed the increased expression of genes encoding PROPEP1 and PROPEP3, the precursors of elicitor peptide PEPs, which function as endogenous damage-associated molecular pattern to amplify danger signals during pathogen infection (Liu et al., 2013). The elevated expression of several flg22-induced genes, including At1g07160, At1g51920, At1g59860, and At2g17740 in cpl3-3 and aggie1/cpl3-5 mutants was confirmed with qRT-PCR analysis (Fig. 3E). Collectively, the data suggest that CPL3 plays a negative role in regulating a large subset of flg22-induced genes. Apparently, CPL3-regulated genes did not show significant correlation with SA, ethylene, methyl jasmonate (MeJA) and ABA-responsive genes (Fig. S3). Among 101 flg22 down-regulated genes in WT and cpl3-3 (fold change ≥ 2 and FDR < 0.05), only 2 genes showed > 2-fold difference between WT and cpl3-3, suggesting that CPL3 does not appear to significantly control flg22-reduced genes (Table S5).

Figure 3. CPL3 globally regulates flg22-induced gene expression.

Figure 3

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(A) Scatter plot with the expression of whole genome transcripts between Col-0 (WT) and cpl3-3 mutant. Twelve-day-old seedlings without treatment were used for analysis. y axis indicates gene expression in cpl3-3, and x axis indicates gene expression in WT. (B) Venn diagram of flg22-induced genes (fold change ≥ 2 and FDR < 0.05) in WT and/or cpl3-3 seedlings 30 min after 100 nM flg22 treatment. (C) Heatmaps of CPL3-dependent flg22-induced genes. The four clusters are defined in the text. (D) Enrichment of defense-related genes in CPL3-attenuted flg22-induced genes (Group III). The fold enrichment was calculated based on the frequency of genes annotated to the term in Group III compared to their frequency in the genome. (E) qRT-PCR analysis of CPL3-regulated genes. The data are shown as means ± se from three biological replicates with Student's t-test. * indicates p<0.05, ** indicates p<0.01 compared to WT. (see also Figure S3, Table S1-S5).

CTD phosphorylation dynamics in PTI signaling

With the FCPH domain, CPLs are hypothesized to regulate gene transcription via modulating the phosphorylation status of RNAPII CTD in the nucleus. When expressed in Arabidopsis protoplasts, CPL3-GFP was observed in the nucleus, which is likely mediated by a nuclear localization signal (NLS) at its N terminus (Fig. S4A). To reveal the potential involvement of CTD phosphorylation in plant immunity, we cloned CTD of Arabidopsis RNAPII and expressed it in protoplasts. Significantly, flg22 treatment induced a rapid mobility shift of CTD as early as 2 min post-treatment (Fig. 4A). The flg22-induced mobility shift could be removed by calf alkaline intestinal phosphatase (CIP) treatment (Fig. 4B), suggesting the involvement of phosphorylation in flg22-induced CTD modification. The elf18 and chitin treatments also induced CTD mobility shift (Fig. 4C). The flg22 treatment also induced the mobility shift of CTD fused with NLS (NLS-CTD) (Fig. S4B).

Figure 4. MAMPs induce CTD phosphorylation.

Figure 4

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(A) flg22 induces the mobility shift and phosphorylation of CTD in protoplasts. Protoplasts were expressed with CTD-HA, and stimulated with 100 nM flg22 for 2∼60 min. The proteins were analyzed by Western blot (WB) with α-HA, α-pSer2, α-pSer5, α-pSer7 or α-pERK antibody. The protein loading is shown by WB with α-Histone H3 antibody. (B) CIP treatment removes CTD-HA mobility shift. The flg22-stimulated protoplasts expressing CTD-HA were treated with 30 units of CIP at 37°C for 1 hr. (C) Different MAMPs induce CTD mobility shift in protoplasts. The CTD-HA transfected protoplasts were treated with 100 nM flg22 or elf 18 or 50 μg/ml chitin for 5 min. (D) flg22 induces endogenous CTD phosphorylation in Arabidopsis seedlings. Twelve-day-old seedlings were treated with 100 nM flg22 for indicated times, and the phosphorylation was detected with specific antibodies. The protein loading was shown by WB with α-CTD antibody.

The above experiments were repeated at least 3 times with similar results (see also Figure S4).

Interestingly, flg22 treatment was able to induce CTD phosphorylation at Ser sites as detected by specific antibodies recognizing pSer2, pSer5 or pSer7 (Fig. 4A). In addition, flg22 treatment induced Ser2, Ser5 and Ser7 phosphorylation of endogenous CTD of RNAPII in Arabidopsis seedlings treated with flg22 for different lengths of time (Fig. 4D). The phosphorylation intensity peaked between 10 and 30 min and gradually declined 60 min after treatment. The phosphorylation modification was confirmed by CIP treatment which diminished the signal detected with α-pSer2 antibody (Fig. S4C). Together, these data indicate that the rapid and transient phosphorylation of CTD upon flg22 perception may constitute an important step in plant immune signaling.

MAPKs phosphorylate CDKCs in flg22 signaling

CPL3 is predicted to be a CTD Ser2 phosphatase since its yeast homolog Fcp1 preferentially dephosphorylates Ser2 (Koiwa et al., 2002). CTD Ser2 is phosphorylated by cyclin-dependent kinases CDK9 and CDK12 in mammals (Bartkowiak et al., 2010). We examined the potential involvement of Arabidopsis orthologs CDKC;1 and CDKC;2 in CTD phosphorylation (Cui et al., 2007). The flg22 treatment induced a mobility shift of CDKC;1 (Fig. 5A), which could be removed by phosphatase CIP treatment (Fig. 5B), suggesting that CDKC;1 was phosphorylated upon flg22 treatment. CDKC;2 exhibited multiple bands in the absence of flg22 treatment (Fig. 5A). Consistent with the activation of CDKs by cyclins, co-expression of CYCT1;3, the partner of CDKCs (Cui et al., 2007), also resulted in two bands of CDKC;1, similar to that after flg22 treatment (Fig. S5A). The data suggest that Arabidopsis CDKCs are activated upon flg22 perception.

Figure 5. MAPKs phosphorylate CDKCs in flagellin signaling.

Figure 5

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(A) flg22 induces CDKC mobility shift in protoplasts. Protoplasts were expressed with CDKC; 1-HA or CDKC;2-HA and treated with 100 nM flg22 for 15 min. Total proteins were separated in a regular 10% SDS-PAGE gel (top) or supplemented with 50 μM phos-tag (Wako chemicals USA, Inc.) (middle). The loading control with α-H3 is shown on the bottom. (B) CDKC;1 mobility shift was removed by CIP treatment. Total proteins were separated in a SDS-PAGE gel supplemented with 50 μM phos-tag (top). (C) CDKC;1 mobility shift is blocked by MKK inhibitor PD184161 in protoplasts. 7.5 μM PD184161 was added 1 hr before flg22 treatment. (D) MAPK phosphatase (MKP) blocks CDKC;1 mobility shift. Protoplasts were co-expressed with CDKC;1-HA and MKP. In C & D, top is a regular SDS-PAGE and middle is phos-tag gel. (E) MPK3 phosphorylates GST-CDKC;1 and GST-CDKC;2 fusion proteins. FLAG epitope-tagged MPKs were expressed in protoplasts treated with 100 nM flg22, and immunoprecipitated for in vitro kinase assay with GST-CDKC;1 or GST-CDKC;2 as substrate. The reactions with myelin basic protein and GST protein as substrates are shown as controls. The protein loading of substrates is shown by CBB staining. (F) MPKs phosphorylate CDKCs at S94 in vitro. The recombinant HIS-MPK3 and HIS-MPK6 proteins were activated by constitutively active MKK5DD, and used to phosphorylate GST-CDKCs and their S94A mutants. The phosphorylation was detected by autoradiograph and the protein loading is shown by CBB staining. * in E & F indicates the expected position of GST-CDKC. (G) CDKC;1 and CDKC;2 interact with MPK3 by Co-IP assay. FLAG epitope-tagged CDKC and HA epitope-tagged MPK3 were co-expressed in Col-0 protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immune-blotted with α-HA or α-FLAG antibody. The input of MPK3 and CDKCs is shown by WB. (H) CDKC;1 S94 is phosphorylated by MPK3 as shown with MS analysis. The graph indicates the sequence of a doubly charged peptide ion at m/z 469.72 that matches to EIVTpSPGR of CDKC;1.

The above experiments (except MS assay) were repeated 3 times with similar results (see also Figure S5).

MAPK cascades act as a convergent point mediating multiple MAMP-triggered signaling. The flg22-induced mobility shift of CDKC;1 was blocked by a MAPK inhibitor PD184161 (Fig. 5C & S5B) and by co-expression of MAPK-specific phosphatase MKP (Fig. 5D & S5B), suggesting that activation of MAPKs by flg22 leads to CDKC phosphorylation. MPK3, MPK4, MPK6 and MPK11 have been shown to be activated by flg22 treatment (Meng and Zhang, 2013). We tested whether these MAPKs could directly phosphorylate CDKCs. The FLAG epitope-tagged MAPK was expressed in protoplasts (Fig. S5C), activated by flg22 treatment, and then immunoprecipitated with α-FLAG antibody for an in vitro kinase assay with GST-CDKC as a substrate. The flg22-activated MPK3 complex strongly phosphorylated both GST-CDKC; 1 and GST-CDKC;2 (Fig. 5E). Consistently, the in vitro kinase assay with purified MPK proteins also indicated that activated MPK3 directly phosphorylated GST-CDKC; 1 and GST-CDKC;2 (Fig. 5F & S5D). Activated MPK6 also phosphorylated GST-CDKC; 1 and GST-CDKC;2 in vitro (Fig. 5F), suggesting potential redundancy of MPK3 and MPK6 in controlling CDKC activity. Notably, MPK3 and MPK6 interacted with CDKC;1 and CDKC;2, but not CYCT1;3, in protoplast co-immunoprecipitation (Co-IP) assays (Fig. 5G, S5E & S5F). Sequence analysis of CDKCs identified two potential MAPK phosphorylation sites (Ser/Thr followed by Pro) S94 and S259 (Sorensson et al., 2012). Exchange of S94, but not S259, to Ala, blocked flg22-induced CDKC;1 mobility shift and suppressed CDKC;2 mobility shift (Fig. S5G). Importantly, the CDKC;1S94A and CDKC;2S94A mutants were no longer phosphorylated by activated MPK3 and MPK6 in vitro (Fig. 5F). Furthermore, mass spectrometry (MS) analyses identified the phosphorylation of S94 in both CDKC;1 and CDKC;2 phosphorylated by MPK3 or MPK6 (Fig. 5H, S5H, S5I & S5J). The data support that MPK3 and MPK6 phosphorylate CDKCs at S94.

CDKCs are MAPK-activated CTD kinases

To test whether the flg22-induced MAPK cascade could activate CDKC kinase activity towards CTD, we first activated MBP-CDKCs with MPK3 immunoprecipitated from flg22-treated protoplasts. The activated MBP-CDKC proteins were used for a kinase assay with GST-CTD as a substrate. The phosphorylation of CTD was detected by α-pSer2, α-pSer5 and α-pSer7 antibodies. MPK3-activated CDKC;1 or CDKC;2 phosphorylated CTD at three Ser residues (Fig. 6A & S6A). Both CDKC;1S94A and CDKC;2S94A, which were no longer able to be phosphorylated by MAPKs, were compromised in their ability to phosphorylate CTD (Fig. 6B). Similar with the activation of CDKs by cyclins, immunoprecipitated CDKCs with CYCT1;3 could also phosphorylate CTD at Ser2, Ser5 and Ser7 residues (Fig. 6C & 6D). These data indicate that CDKCs are authentic CTD kinases and their activity is regulated by an flg22-induced MAPK cascade.

Figure 6. CPL3 is a CTD phosphatase.

Figure 6

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(A) MPK3-activated CDKCs induce GST-CTD phosphorylation in vitro. MPK3-HA was expressed in protoplasts treated with 100 nM flg22, immunoprecipitated with α-HA agarose beads, and incubated with MBP-CDKC proteins. The phosphorylated CDKC proteins were collected by centrifugation as supernatant (MPK3-HA conjugated beads were in pellets) and used to phosphorylate GST-CTD. The CTD phosphorylation was analyzed by WB with specific antibodies. (B) CDKC;1S94A and CDKC;2S94A reduce the ability to phosphorylate GST-CTD. (C) CPL3 dephosphorylates CDKC; 1-activated GST-CTD in vitro. CDKC;1-HA and CYCT1;3-HA were expressed in protoplasts, immunoprecipitated to phosphorylate GST-CTD. The phosphorylated GST-CTD was dephosphorylated by MBP-CPL3N (N), MBP-CPL3C (C) and MBP-FCPH. CTD phosphorylation was detected by WB with specific antibodies. CTD loading is shown by WB with α-GST antibody. (D) CPL3 dephosphorylates CDKC;2-activated GST-CTD in vitro. (E) CPL3 interacts with GST-CTD in vitro. Pull-down assay was performed by incubating MBP-CPL3 together with glutathione beads containing CTD or phosphorylated CTD (pCTD). The HA-tagged CPL3 proteins were detected with an α-HA WB after glutathione bead pull-down (PD). The input control is shown by WB. (F) CTD and CPL3C interact in yeast. The interactions between pGADT7-CTD and pGBDT7-CPL3C, pGBDT7-CPL3N and pGBDT7-FCPH were tested in SD medium without histidine, leucine and tryptophan (SD-H-L-T) supplemented with 1 mM 3AT. EV is the empty vector pGADT7. (G) CPL3CD933A and CPL3CDD1064AA lose phosphatase activity. (H) Enhanced CTD Ser2 phosphorylation in cpl3-4 mutant seedling. One-week-old seedlings of WT and cpl3-4 were treated with or without 1 μM flg22 for 30 min.

The above experiments were repeated 3 times with similar results (see also Figure S6).

CPL3 dephosphorylates CDKC-mediated CTD phosphorylation

We next examined whether CPL3 possesses CTD phosphatase activity. We purified MBP fusion proteins of full-length CPL3, N-terminal CPL3 (CPL3N) and C-terminal CPL3 (CPL3C) and performed in vitro phosphatase assays with GST-CTD that was phosphorylated by CDKC;1/CYCT1;3 (Fig. 6C) or CDKC;2/CYCT1;3 (Fig. 6D & S6B). CPL3 was able to dephosphorylate pSer2, pSer5 and pSer7 in vitro with a preference for pSer2 (Fig. 6C, 6D & S6B). CPL3C with both the FCPH and BRCT domains, but not FCPH alone nor CPL3N, carries the phosphatase activity that completely dephosphorylated CTD pSer2 and partially dephosphorylated pSer5 and pSer7 (Fig. 6C & 6D). Our results suggested that both FCPH and BRCT domains are required for CPL3 phosphatase activity. We further tested the potential interaction between CPL3 and CTD. An in vitro pull-down assay indicated that GST-CTD could pull down CPL3. The interaction between CPL3 and CTD was markedly enhanced when CTD was phosphorylated (Fig. 6E). Further analyses with different domains of CPL3 indicated that both FCPH and BRCT domains were required for the interaction (Fig. S6C & S6D). The interaction between CPL3C and CTD was confirmed with yeast two-hybrid assay (Fig. 6F). These results are consistent with the finding that yeast Fcp1 predominantly dephosphorylates pSer2 and requires both FCPH and BRCT domains for its catalytic activity (Ghosh et al., 2008).

Structural and mutational analyses of yeast Fcp1 defined essential conserved residues within the FCPH domain (Ghosh et al., 2008). We mutated some of these conserved residues in CPL3, including D933 (corresponding to Fcp1D258), K1046 (Fcp1K280) and D1064D1065 (Fcp1D297D298) to Ala, and tested their phosphatase activity. All these mutants were still able to interact with CTD in an in vitro pull-down assay (Fig. S6E). D933A and D1064AD1065A (DD1064AA) rendered a complete loss of CPL3 catalytic activity (Fig. 6G). Similarly, expression of WT CPL3C, but not D933A mutant, reduced flg22-induced pFRK1-LUC induction in cpl3-4 mutant protoplasts, suggesting the functional importance of D933 (Fig. S6F). Surprisingly, the K1046A mutation did not affect CPL3 activity (Fig. 6G). Given that the corresponding residue K280 in Fcp1 was suggested to participate in transition-state stabilization by binding to acylphosphate intermediate during CTD dephosphorylation (Ghosh et al., 2008), our data suggest that K1046 in CPL3 might not be essential for stabilizing the transition state, and that CPL3 has a similar but distinct requirement for catalytic activity compared to Fcp1.

We further compared Ser2, Ser5 and Ser7 phosphorylation level of endogenous CTD in WT and cpl3-4 seedlings. The overall Ser2 phosphorylation level was enhanced in the cpl3-4 mutant compared to WT plants with and without flg22 treatment (Fig. 6H). In contrast, the level of Ser5 and Ser7 phosphorylation appears to be similar in WT and cpl3-4 seedlings (Fig. 6H). This is consistent with in vitro analysis that CPL3 preferentially dephosphorylates Ser2 of CTD.

CDKCs positively regulate plant innate immunity

We tested the potential involvement of CDKCs in CTD phosphorylation and plant immunity. The cdkc;2-1 (SALK_149280) mutant has undetectable full-length transcripts (Fig. S7A & S7B), whereas the available T-DNA insertion lines of cdkc;1 (SALK_148550 & SALK_091405) remained similar transcript levels as WT plants. We generated CDKC;1 RNA interference (RNAi) plants with reduced expression of CDKC;1 transcripts (Fig. S7C). Consistent with CDKCs being CTD kinases, the flg22-induced phosphorylation of Ser2, Ser5 or Ser7 of CTD was reduced in cdkc;1RNAi and cdkc;2 mutant plants (Fig. 7A & S7D). Similarly, the cdkc;1RNAi and cdkc;2 plants showed reduced induction of MAMP marker genes, including FRK1, WRKY30, PP2C (At1g07160) and At1g51920 upon flg22 treatment compared to WT plants (Fig. 7B). In addition, the cdkc;1RNAi and cdkc;2 mutant plants were more susceptible to virulent Pst infection than WT plants as indicated by 5-8 fold increase of bacterial growth in the mutants (Fig. 7C). The bacterial growth of type III deficient Pst hrcC was also increased in the cdkc;1RNAi or cdkc;2 mutants (Fig. 7C). The increased growth of Psm was also observed in cdkc;2 mutant (Fig. S7E). CDKC;1 and CDKC;2 play redundant roles in plant growth and development, and resistance to virus infections (Cui et al., 2007). Therefore, we generated double mutants by silencing CDKC;1 in the cdkc;2 mutant using virus-induced gene silencing (VIGS). The cdkc;2/cdkc;1VIGS plants displayed wrinkled leaves 3 weeks after VIGS and exhibited retarded growth 4 weeks after VIGS (Fig. S7F). We performed the pathogen infection assays before the growth defects clearly appeared. Similar with RNAi plants, the cdkc;1VIGS plants showed reduced induction of FRK1 and WRKY30 upon flg22 treatment, and the reduction seemed to be further exacerbated in cdkc;2/cdkc;1VIGS plants compared to plants silenced with a vector control (Fig. S7G). Consistently, the cdkc;1VIGS mutant plants were more susceptible to Pst infection with about 5-10 fold increase of bacterial growth than control plants (Fig. 7D). Notably, bacterial population in the cdkc;2/cdkc;1VIGS plants was about 100-fold higher than that in control plants (Fig. 7D). Together, these data indicate that CDKC;1 and CDKC;2 are positive regulators in plant immunity to bacterial infections.

Figure 7. CDKCs positively regulate plant immunity.

Figure 7

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(A) CTD phosphorylation in cdkc mutants. One-week-old seedlings of WT, cdkc;1RNAi (cdkc; 1) and cdkc;2 were treated with or without 1μM flg22 for 30 min. (B) flg22-induced gene expression. WT and cdkc seedlings were treated with 1 μM flg22 for 1 hr. (C) Bacterial growth in cdkc plants. WT and cdkc plants were hand-inoculated with Pst or hrcC at OD600 = 5 × 10-4. (D) Bacterial growth in CDKC VIGS-silenced plants. The data in B, C and D are shown as mean ± se from three independent repeats with Student's t-test. * indicates p<0.05, and ** indicates p<0.01 compared to WT. (E) MPK3/6 are required for flg22-induced CTD phosphorylation. Protoplasts isolated from WT and mpk3/MPK6RNAi plants pretreated with 5 μM estradiol were treated with 100 nM flg22 for 15 min. (F) CDKC;1 mobility shift was partially blocked in mpk3/MPK6RNAi plants. Protoplasts were expressed with CDKC; 1-HA and treated with 100 nM flg22 for 15 min. Total proteins were separated in a 8% SDS-PAGE gel supplemented with 50 μM phos-tag. (G) A model of RNAPII CTD phosphorylation dynamics in plant immunity. Upon flg22 perception by the FLS2/BAK1/BIK1 receptor complex, rapid activation of MAPK cascade phosphorylates and activates CDKCs which further phosphorylate the tail of RNAPII CTD heptapeptide. The phosphorylation status of CTD is counter-regulated by CPL3 (Aggie 1) through direct dephosphorylation of the Ser residues on CTD. The phosphorylation dynamics of CTD serves as a “regulatory code” to recruit gene specific transcription factors (TFs) and orchestrate immune gene transcription.

The above experiments were repeated 3 times with similar results (see also Figure S7).

To further investigate the involvement of MPK3 and MPK6 in flg22-induced CTD and CDKC phosphorylation, we generated estradiol inducible MPK6 RNAi transgenic plants in mpk3 mutant. The protein level of MPK6 was markedly reduced in mpk3/MPK6RNAi plants after estradiol treatment (Fig. S7H). Importantly, the flg22-induced CTD Ser2, Ser5 and Ser7 phosphorylation was diminished in the mpk3/MPK6RNAi plants compared to WT plants (Fig. 7E). Furthermore, the flg22-induced CDKC;1 mobility shift was blocked in the mpk3/MPK6RNAi plants (Fig. 7F), providing genetic evidence of MPK3 and MPK6 in flg22-induced CDKC;1 and CTD phosphorylation.

Discussion

We have demonstrated that the phosphorylation dynamics of the RNAPII core transcription machinery is regulated by the evolutionarily conserved kinases and phosphatases in response to pathogen attacks. Specifically, we revealed a MAMP-induced phosphorylation relay emanating from a MAPK cascade downstream of multiple PRRs to CDKCs and RNAPII CTD that activates the transcription machinery for immune gene expression. We also elucidated dephosphorylation of RNAPII CTD by CPL3 phosphatase as a counter-regulatory mechanism to fine tune transcriptional reprogramming for appropriate immune responses. Thus, both CDKCs and CPL3 target RNAPII CTD and oppositely regulate plant immune gene expression and disease resistance to bacterial and fungal pathogens (Fig. 7G). Considering the conservation of MAPKs and CTD kinases and phosphatases among eukaryotes, the immune signaling circuits identified in this study may represent an evolutionarily convergent regulatory mechanism that eukaryotic cells use to promptly respond to extracellular stimuli.

As an element of the core transcription machinery, RNAPII is subject to complex regulation that ensures proper processing of nascent mRNAs. The phosphorylation dynamics of RNAPII CTD has been extensively studied, in particular in yeast, and many CTD binding proteins and transcription events are associated with specific CTD phosphorylation patterns (Buratowski, 2009). A universal CTD cycle has been proposed to orchestrate the transcription of virtually all genes through complex interplay between different kinases, phosphatases and other modifying enzymes (Bataille et al., 2012). However, the manner in which specific cellular responses regulate CTD phosphorylation dynamics, which in turn controls gene expression, has been a mystery. We show here that MAMP perception markedly induces rapid and transient CTD phosphorylation on various Ser residues in its heptad repeats. This phosphorylation is achieved by a direct phosphorylation relay from a MAPK cascade to CDKCs and counter-regulated by the CPL3 phosphatase. The data suggest an important role of MAPK-mediated RNAPII CTD phosphorylation in transcriptional regulation of plant immune genes.

CPL3 belongs to a member of multigene family in Arabidopsis (Koiwa et al., 2002; Koiwa et al., 2006). Only CPL3 and CPL4 contain both FCPH and BRCT domains, whereas CPL1 and CPL2 contain FCPH and double-stranded RNA-binding domains. The cpl1 mutants display altered expression of cold-, salt-, ABA-, and osmotic stress-inducible genes, and cpl3 mutants show specific alternation to ABA responses (Jiang et al., 2013; Koiwa et al., 2002). Despite lacking the BRCT domain, CPL1 could specifically dephosphorylate Ser5 of Arabidopsis CTD (Koiwa et al., 2004). In addition, CPL1 dephosphorylates RNA binding protein HYPONASTIC LEAVES 1 (HYL1) to regulate processing and strand selection during plant miRNA biogenesis (Manavella et al., 2012). CPL3 is a prototype Fcp1 phosphatase containing both FCPH phosphatase catalytic domain and BRCT domain. Our results support that CPL3 is a genuine CTD Ser2 phosphatase and requires both FCPH and BRCT domains for its activity, which is similar to yeast Fcp1. However, distinct from Fcp1, CPL3 is not an essential gene. CPL4, the closest homolog of CPL3, is likely an essential gene involved in plant vegetative growth and development (Bang et al., 2006). Therefore, it appears that CPL4 is involved in the regulation of RNAPII activity for general transcription, whereas CPL3 specifically regulates RNAPII activity during plant stress responses. It remains unknown how CPL3 activity is regulated upon MAMP treatment. It is possible that CPL3 could be activated by upstream kinases or other regulators in MAMP signaling. Additionally, CPL3 may interact with specific transcription factors to regulate immune gene expression.

CDKs are often activated by phosphorylation of conserved Thr residues within the T-loops by CDK-activating kinases (CAKs). A plant specific CDK, CDKF;1 phosphorylates the T-loops of CDKDs and activates their kinase activity towards CTD (Hajheidari et al., 2012). Here, we identified the activation of CDKCs by flg22-induced MAPK cascade. In vivo and in vitro data provide evidence that MPK3 directly phosphorylates CDKC;1 and CDKC;2, and activates CDKC kinase activity towards CTD. In fission yeast, CTD Ser2 phosphorylation is elevated under nitrogen starvation condition, which depends on a stress-responsive MAPK pathway (Sukegawa et al., 2011). In human T-cell receptor and phorbal ester signaling, MAPKs are involved in phosphorylation of CDK9 to enhance HIV transcription (Mbonye et al., 2013). Apparently, activation of CDKs by MAPK cascades might be a conserved mechanism for the regulation of RNAPII CTD phosphorylation.

Experimental Procedures

Plant growth, generation of pFRK1∷LUC transgenic plants and mutant screens

Arabidopsis accession Col-0, Ler, pFRK1∷LUC wild-type (WT) and mutant (aggie) transgenic plants, cpl2-2 (SALK_059753), cpl3-3 (SALK_094720C), cpl3-4 (SALK_051322C), cdkc;2-1 (SALK_149280C) and fls2 were grown in pots containing soil (Metro Mix 360) in a growth room at 23°C, 60% relative humidity and 75μE m-2s-1 light with a 12-hr photoperiod. To detect CTD phosphorylation and gene induction, one-week-old or 12-day-old seedlings grown on ½ MS medium were transferred to water for overnight and then treated with 100 nM flg22 for indicated time points.

The pFRK1∷LUC construct in protoplast transient expression vector (He et al., 2006) was sub-cloned into a binary vector pCB302 and introduced into Arabidopsis Col-0 plants. The transgenic plants were selected with Basta resistance and analyzed with flg22-induced pFRK1∷LUC expression. The seeds of homozygous pFRK1∷LUC transgenic plants were mutagenized by 0.4% EMS. Approximately 6,000 M2 seedlings were grown on liquid ½ MS medium for 14 days, transferred to water for overnight and then treated with 10 nM flg22. The individual seedlings were transferred to each well of 96-well plate 12 hr after flg22 treatment and sprayed with 0.2 mM luciferin. The plate was put in the dark for 20 min, and the bioluminescence signal was read by a luminometer (Perkin Elmer). The putative mutants were transferred to solid ½ MS medium for additional 10 days and then transferred to soil to set seeds.

Plasmid construction

The CTD, CDKC;1, CDKC;2, CYCT1;3, CPL3, CPL3N, CPL3C and CPL3FCPH genes were amplified from Col-0 cDNA library with primers containing BamHI or NcoI at N terminus and StuI at C terminus, and introduced into a plant expression vector pHBT with an HA or FLAG epitope-tag at C terminus. The point mutations of CDKC;1S94A, CDKC;1S259A, CDKC;2S94A, CDKC;2S259A and different CPL3C mutants were generated by site-directed mutagenesis. A 400-bp fragment of CDKC;1 was amplified from Col-0 cDNA with primers containing EcoRI at N terminus and KpnI at C terminus,, and inserted into VIGS pYL156 (pTRV-RNA2) vector by EcoRI and KpnI digestion. The CDKC;1 RNAi construct was obtained from Dr. Z. Chen (Cui et al., 2007). The primers for cloning and point mutations were listed in the Supplemental Experimental Procedures. The CDKC;1 and CDKC;2 were sub-cloned into a modified GST fusion protein expression vector pGEX4T-1 (Pharmacia) or a modified pMAL-c2 vector (New England BioLabs) with BamHI and StuI digestion and different CPL3 constructs were sub-cloned into a modified pMAL-c2 vector with SpeI or BamHI and StuI digestion. The recombinant CDKC and CPL3 fusion protein expression vectors were introduced into E. coli strain BL21 (DE3). Expression of fusion proteins and affinity purification were performed with standard protocol. The CPL3N, CPL3C and FCPH genes were sub-cloned into a modified pGBKT7 vector (Clontech) with SpeI or BamHI and StuI digestion, and the CTD gene was sub-cloned into a modified pGADT7 vector (Clontech) with BamHI and StuI digestion for yeast two-hybrid assay. The constructs of GST-CTD, pCPL3∷CPL3, MAPK fusion protein and protoplast expression vectors were reported previously (He et al., 2006; Koiwa et al., 2002; Koiwa et al., 2004). The MAPK-specific phosphatase (MKP) was cloned from mouse (Kovtun et al., 1998).

Pathogen infections, NGS, RNA-seq, kinase assay, CTD phosphorylation and dephosphorylation, VIGS, Co-IP and MS assays are listed in Supplemental Experimental Procedures.

Supplementary Material

1

Figure S1, related to Figure 1. Enhanced pFRK1∷LUC expression in aggie1 mutant. (A) The pFRK1∷LUC activity was enhanced in aggie1 mutant upon chitin treatments. Plants were treated with 50 ug/ml chitin, and luciferase activity was measured 12 hr after infiltration. (B) The aggie1 mutant is more resistant to Pst infection. The plants were dip inoculated with Pst at OD600 = 2×10-1 and bacterial growth assays were performed 3 dpi. (C) Disease symptom of WT and aggie1 mutant. The picture was taken 4 days after dip inoculation. (D) flg22-induced BIK1 phosphorylation in WT and aggie1 mutant. BIK1-HA was expressed in WT or aggie1 mutant protoplasts, treated with 100 nM flg22 for 10 min, and detected with an α-HA WB. The BIK1 phosphorylation is shown as a mobility-shift with WB.

The above experiments were repeated 4 times with similar results.

Figure S2, related to Figure 2. Aggie1 and Aggie3 encode CPL3. (A) Mapping of aggie1 on chromosome 2 between marker F4P9-3 and T1B8-2. (B) The alignment of genomic DNA, cDNA (CDS) and proteins of CPL3, aggie1 and aggie3. The red stars indicate G to A mutations in aggie1 and aggie3. Mutations of G1294A in aggie1 and G962A in aggie3 lead to alternative splicing of CPL3.

Figure S3, related to Figure 3. Comparison of CPL3-regulated genes with SA, ACC, MeJA and ABA-regulated genes. The expression data of cpl3-3 regulated genes without flg22 treatment (A) and flg22-induced genes (B) are cross-referenced with public database for their relative expression profile (in log2 ratio) with SA, ABA, ACC and MeJA using Genevestigator v3. Group I, II, III and IV contain genes in the 4 clusters highlighted on Fig. 3C. The color red or green indicates gene induction or repression as illustrated by the fold change color bars.

Figure S4, related to Figure 4. CPL3 localizes to the nucleus. (A) CPL3 localizes in nucleus. GFP-tagged CPL3, CPL3C and CPL3N were expressed in protoplasts and GFP signal was observed 12 hr after transfection with a confocal microscope. Nuclear-localized RFP (NLS-RFP) was co-transfected as a nuclear localization control. The schematic structure and domains of CPL3 are shown on the bottom. * indicates the position of a putative nuclear localization signal: DGPAWKRQKSD. (B) The flg22-treatment induces mobility shift of NLS-CTD. Protoplasts were expressed with NLS-CTD-FLAG, and stimulated with 100 nM flg22 for 10 min. (C) Phosphorylation of endogenous RNAPII could be removed by CIP. The seedlings were treated with 100 nM flg22 for 30 or 60 min, and the sample with 30 min flg22-treatment was further treated with 10 units of CIP (Biolabs) at 37°C for 1 hr. The CTD phosphorylation was detected with an α-pSer2 antibody WB. The WB of α-H3 antibody was used as a loading control.

The above experiments were repeated 2 times (A & C) and three time (B) with similar results.

Figure S5, related to Figure 5. MAPKs phosphorylate CDKCs. (A) CYCT1;3-induced CDKC;1 mobility shift. Protoplasts were expressed with CDKC;1-HA or CDKC;1-HA and CYCT1;3-FLAG and treated with 7.5 μM PD184161 for 1 hr before 100 nM flg22 treatment for 15 min. The proteins were detected with an α-HA WB in a 10% SDS-PAGE gel supplemented with 50 μM phos-tag (Wako chemicals USA, Inc.). The WB of α-H3 is shown as a loading control. (B) PD184161 and MKP suppress MAPK activation. The MAPK activation was detected by α-ERK antibody. (C) Expression of MAPK proteins used in Fig. 5E. The proteins were detected with α-FLAG WB. (D) Side-by-side control reactions using myelin basic protein as a substrate validated the activities of MPK3 and MPK6 for Fig. 5F. (E) MPK6 interacts with CDKCs. FLAG epitope-tagged CDKC and HA epitope-tagged MPK6 were co-expressed in protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immuno-blotted with an α-HA or α-FLAG antibody. The input of MPK6 and CDKCs is shown by WB. (F) MPK3/6 do not interact with CYCT1;3. HA epitope-tagged CYCT1;3 and FLAG epitope-tagged MPK3/6 were co-expressed in protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immuno-blotted with α-HA or α-FLAG antibody. The input of MPK3/6 and CYCT is shown by WB. (G) CDKC;1S94A and CDKC;2S94A block mobility shift. Protoplasts were expressed with CDKCs or their S94A or S259A variants, and treated with 100 nM flg22 for 15 min. The proteins were separated in a regular SDS-PAGE gel (top panel) or 50 μM phos-tag gel (middle panel). The WB of α-H3 was used as a loading control (bottom panel). (H) CDKC;2 S94 is phosphorylated by MPK3 as evidenced by sequencing of a doubly charged peptide ion at m/z 469.72 that matches to EIVTpSPGR of CDKC;2. (I) CDKC;1 S94 is phosphorylated by MPK6 as shown by similar MS analysis. (J) CDKC;2 S94 is phosphorylated by MPK6 as shown by similar MS analysis. The above experiments (except MS assays) were repeated 3 times with similar results.

Figure S6, related to Figure 6. CPL3 dephosphorylates CTD. (A) MPK3-activated CDKC;1 is responsible for GST-CTD phosphorylation in vitro. MPK3-HA was expressed in protoplasts treated with 100 nM flg22. MPK3 was immunoprecipitated with α-HA agarose beads, incubated with MBP-CDKC;1 or MBP proteins. The phosphorylated MBP proteins were collected by centrifugation as supernatant (MPK3-HA conjugated beads were in pellets) and used to phosphorylate GST-CTD. MBP supernatant did not phosphorylate Ser2, Ser5 and Ser7 of CTD, which was used as a control for Fig. 6A to show the activity of MBP-CDKC;1. (B) CPL3 and CPL3C dephosphorylate GST-CTD. CDKC;2-HA and CYCT1;3-HA were co-expressed in protoplasts, immunoprecipitated with α-HA agarose beads and used to phosphorylate GST-CTD. The phosphorylated GST-CTD was dephosphorylated by MBP-CPL3 or MBP-CPL3C. CTD phosphorylation was detected by WB with an α-pSer2, α-pSer5, or α-pSer7 antibody. The proteins of MBP-CPL3 and MBP-CPL3C are shown on the right. (C) The schematic domain organization of CPL3 used in this study. (D) CPL3C interacts with CTD. Pull-down assay was performed by incubating MBP-CPL3C, MBP-CPL3N or MBP-FCPH with glutathione beads containing CTD or phosphorylated CTD (pCTD). The HA-tagged CPL3 proteins were detected with an α-HA WB after glutathione bead pull-down (PD). The input control for GST-CTD, GST and CPL3-HA is shown with WB. (E) The CPL3 mutations did not affect the interaction with CTD. The sequence alignment of FCPH domain in CPL1, CPL2, CPL3, CPL4, ScFcp1 and HsFcp1 is shown on top panel and the conserved D933, K1046 and D1064D1065 of CPL3 residues are in red. In vitro pull-down assay was performed using GST, GST-pCTD (phosphorylated CTD by CDKC;2 as shown in Fig. S6B) or GST-CTD beads against fusion proteins of MBP-CPL3C or mutant variants tagged with an HA epitope. (F) CPL3D933A mutant lost function in protoplasts. Protoplasts from cpl3-4 mutants were co-expressed with CPL3C or its D933A mutant and pFRK1∷LUC reporter, and treated with 100 nM flg22 for 4 hr. The luciferase activity was normalized with internal GUS control. The protein expression of WT and mutant CPL3 is shown with WB on the bottom.

The above experiments were repeated 3 times with similar results.

Figure S7, related to Figure 7. The mutational and VIGS analyses of CDKCs. (A) PCR analysis of cdkc;2 genomic DNA. The specific amplification of PCR product with primers of LBb1 and RP, but not LP and RP, indicates that it is a homozygous T-DNA insertion. The Ctrl was PCR reaction without DNA. The scheme of CDKC;2 gene, T-DNA insertion and primers is indicated on the bottom. The black boxes indicate exon of CDKC;2. (B) Expression of CDKC;2 in Col-0 WT and cdkc;2 mutant. The RT-PCR was performed for 35 cycles, and the UBQ10 is the control. (C) Expression of CDKC;1 in Col-0 and cdkc;1RNAi plants. The RT-PCR was performed for 35 cycles, and the UBQ10 is the control. (D) flg22-induced CTD phosphorylation in seedlings. The band intensity of pSer2, pSer5 and pSer7 in Fig. 7A was quantified by the Image J software and presented with mean ± s.e. (n=3) from three independent biological replicates. (E) The cdkc;2 plants are more susceptible to Psm infections. The WT and cdkc;2 plants were inoculated with Psm at OD600 = 2×10-4 and the bacterial counting was performed 2 dpi. The data are shown as mean ± se from three independent repeats with Student's t-test. * indicates p<0.05 when compared to WT. (F) The phenotype of VIGS plants. WT or cdkc;2 plants were inoculated with pYL156-EV (empty vector) or pYL156-CDKC;1. The pictures were taken 16 and 28 days after VIGS inoculation. The plants silenced with Cla1 were used as a visual marker for VIGS efficiency. (G) The flg22-induced gene expression in CDKC-silenced plants. VIGS-CDKC;1 or control plants were treated with 100 nM flg22 for 3 hr, and RNA was isolated for qRT-PCR analysis. The data are shown as mean ± se from three independent repeats. (H) Protein expression of MPK3 and MPK6 in WT and mpk3/6 mutant. WT and mpk3/6 seedlings were grown on ½ MS medium and treated with 5 μM estradiol for 2 days. MPK3 and MPK6 proteins were detected by WB with α-MPK3 (Sigma, M8318) and α-MPK6 (Sigma, #A7104) antibodies. Ponceau staining for RBC was used as a loading control.

The above experiments were repeated 3 times with similar results.

Table S1, related to Figure 3. The list of CPL3-regulated genes without treatment (see Excel file Table S1).

Table S2, related to Figure 3. The list of flg22-induced genes in WT and/or cpl3-3 (see Excel file Table S2).

Table S3, related to Figure 3. The list of CPL3-dependent flg22-induced genes (see Excel file Table S3).

Table S4, related to Figure 3. Enrichment analysis of GO terms for CPL3-dependent flg22-induced genes (see Excel file Table S4).

Table S5, related to Figure 3. The list of flg22-reduced genes in WT and/or cpl3-3 (see Excel file Table S5).

Table S6. Primers used in this study, Related to Experimental Procedures 

Table S7: AGI number and predicted MW of genes/proteins used in this study, Related to Experimental Procedures 

2
3
4
5
6
7

Highlights.

  • RNA polymerase II CTD phosphatase (CPL3) mutants show enhanced immune gene activation

  • MAMPs induce cyclin-dependent kinases CDKCs and RNA pol II CTD phosphorylation

  • Direct CDKC phosphorylation by MAP kinase (MAPK) activates immune gene expression

  • CPL3 counteracts MAPK-CDKC regulation via RNA pol II CTD dephosphorylation

Acknowledgments

We thank Salk Institute and ABRC for the Arabidopsis T-DNA insertion lines, Dr. Zhixiang Chen for CDKC and CYCT fusion protein and RNAi constructs, Dr. Shuqun Zhang for MPK fusion protein constructs, Dr. Keiko Torii for pTK103 vector, Dr. Ning Zhu for technical assistance of MS analysis, Drs. Greg Martin and Paul de Figueiredo for critical reading of the manuscript. The work was supported by NIH (R01GM092893) and NSF (IOS-1252539) to P.H and NIH (R01GM097247) and the Robert A. Welch foundation (A-1795) to L.S. The NGS was supported by Texas AgriLife Genomics Seed Grant. F. L. and F. C. were partially supported by China Scholarship Council. M.V.V.O and A.C.I. were partially supported by Rio de Janeiro State Research Foundation (FAPERJ), Brazil. K.B. was supported by NSF REU program.

Footnotes

The authors have declared no conflict of interests.

Author contributions: F.L. performed biochemical assays of CPL3, CTD, CDKC and MAPK activities, and analyzed CDKC plants; C.C performed phenotypic analysis and cloning of aggie1, RNA-seq analysis, and VIGS assays; F.C. performed screen, phenotypic analysis, cloning and NGS of aggie3; M.O. sequenced candidate genes of Aggie1, and performed RNA-seq analysis and CDKC plants; X.Y. generated mpk6RNAi plants and performed CDKC and CTD phosphorylation in these plants; X. Meng performed MAPK phosphorylation on CDKCs; A.I. sequenced candidate genes of Aggie1; K.B. screened aggie1; M.L. performed Y2H assay; B.L. generated mpk6RNAi plants and validated MAPK inhibitor and MKP; S.C. performed MS assay; X. Ma and S.X. performed powdery mildew assay; Y.Z. and Z.F. analyzed RNA-seq data; R.M. and C.J. performed NGS; H.K. provided reagents; W.S., Z.L., and G.S.F. analyzed data; L.S and P.H. generated mutant population, designed experiments, analyzed data, and wrote the paper with inputs from other authors.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Figure S1, related to Figure 1. Enhanced pFRK1∷LUC expression in aggie1 mutant. (A) The pFRK1∷LUC activity was enhanced in aggie1 mutant upon chitin treatments. Plants were treated with 50 ug/ml chitin, and luciferase activity was measured 12 hr after infiltration. (B) The aggie1 mutant is more resistant to Pst infection. The plants were dip inoculated with Pst at OD600 = 2×10-1 and bacterial growth assays were performed 3 dpi. (C) Disease symptom of WT and aggie1 mutant. The picture was taken 4 days after dip inoculation. (D) flg22-induced BIK1 phosphorylation in WT and aggie1 mutant. BIK1-HA was expressed in WT or aggie1 mutant protoplasts, treated with 100 nM flg22 for 10 min, and detected with an α-HA WB. The BIK1 phosphorylation is shown as a mobility-shift with WB.

The above experiments were repeated 4 times with similar results.

Figure S2, related to Figure 2. Aggie1 and Aggie3 encode CPL3. (A) Mapping of aggie1 on chromosome 2 between marker F4P9-3 and T1B8-2. (B) The alignment of genomic DNA, cDNA (CDS) and proteins of CPL3, aggie1 and aggie3. The red stars indicate G to A mutations in aggie1 and aggie3. Mutations of G1294A in aggie1 and G962A in aggie3 lead to alternative splicing of CPL3.

Figure S3, related to Figure 3. Comparison of CPL3-regulated genes with SA, ACC, MeJA and ABA-regulated genes. The expression data of cpl3-3 regulated genes without flg22 treatment (A) and flg22-induced genes (B) are cross-referenced with public database for their relative expression profile (in log2 ratio) with SA, ABA, ACC and MeJA using Genevestigator v3. Group I, II, III and IV contain genes in the 4 clusters highlighted on Fig. 3C. The color red or green indicates gene induction or repression as illustrated by the fold change color bars.

Figure S4, related to Figure 4. CPL3 localizes to the nucleus. (A) CPL3 localizes in nucleus. GFP-tagged CPL3, CPL3C and CPL3N were expressed in protoplasts and GFP signal was observed 12 hr after transfection with a confocal microscope. Nuclear-localized RFP (NLS-RFP) was co-transfected as a nuclear localization control. The schematic structure and domains of CPL3 are shown on the bottom. * indicates the position of a putative nuclear localization signal: DGPAWKRQKSD. (B) The flg22-treatment induces mobility shift of NLS-CTD. Protoplasts were expressed with NLS-CTD-FLAG, and stimulated with 100 nM flg22 for 10 min. (C) Phosphorylation of endogenous RNAPII could be removed by CIP. The seedlings were treated with 100 nM flg22 for 30 or 60 min, and the sample with 30 min flg22-treatment was further treated with 10 units of CIP (Biolabs) at 37°C for 1 hr. The CTD phosphorylation was detected with an α-pSer2 antibody WB. The WB of α-H3 antibody was used as a loading control.

The above experiments were repeated 2 times (A & C) and three time (B) with similar results.

Figure S5, related to Figure 5. MAPKs phosphorylate CDKCs. (A) CYCT1;3-induced CDKC;1 mobility shift. Protoplasts were expressed with CDKC;1-HA or CDKC;1-HA and CYCT1;3-FLAG and treated with 7.5 μM PD184161 for 1 hr before 100 nM flg22 treatment for 15 min. The proteins were detected with an α-HA WB in a 10% SDS-PAGE gel supplemented with 50 μM phos-tag (Wako chemicals USA, Inc.). The WB of α-H3 is shown as a loading control. (B) PD184161 and MKP suppress MAPK activation. The MAPK activation was detected by α-ERK antibody. (C) Expression of MAPK proteins used in Fig. 5E. The proteins were detected with α-FLAG WB. (D) Side-by-side control reactions using myelin basic protein as a substrate validated the activities of MPK3 and MPK6 for Fig. 5F. (E) MPK6 interacts with CDKCs. FLAG epitope-tagged CDKC and HA epitope-tagged MPK6 were co-expressed in protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immuno-blotted with an α-HA or α-FLAG antibody. The input of MPK6 and CDKCs is shown by WB. (F) MPK3/6 do not interact with CYCT1;3. HA epitope-tagged CYCT1;3 and FLAG epitope-tagged MPK3/6 were co-expressed in protoplasts. The proteins were immunoprecipitated with α-FLAG agarose beads, immuno-blotted with α-HA or α-FLAG antibody. The input of MPK3/6 and CYCT is shown by WB. (G) CDKC;1S94A and CDKC;2S94A block mobility shift. Protoplasts were expressed with CDKCs or their S94A or S259A variants, and treated with 100 nM flg22 for 15 min. The proteins were separated in a regular SDS-PAGE gel (top panel) or 50 μM phos-tag gel (middle panel). The WB of α-H3 was used as a loading control (bottom panel). (H) CDKC;2 S94 is phosphorylated by MPK3 as evidenced by sequencing of a doubly charged peptide ion at m/z 469.72 that matches to EIVTpSPGR of CDKC;2. (I) CDKC;1 S94 is phosphorylated by MPK6 as shown by similar MS analysis. (J) CDKC;2 S94 is phosphorylated by MPK6 as shown by similar MS analysis. The above experiments (except MS assays) were repeated 3 times with similar results.

Figure S6, related to Figure 6. CPL3 dephosphorylates CTD. (A) MPK3-activated CDKC;1 is responsible for GST-CTD phosphorylation in vitro. MPK3-HA was expressed in protoplasts treated with 100 nM flg22. MPK3 was immunoprecipitated with α-HA agarose beads, incubated with MBP-CDKC;1 or MBP proteins. The phosphorylated MBP proteins were collected by centrifugation as supernatant (MPK3-HA conjugated beads were in pellets) and used to phosphorylate GST-CTD. MBP supernatant did not phosphorylate Ser2, Ser5 and Ser7 of CTD, which was used as a control for Fig. 6A to show the activity of MBP-CDKC;1. (B) CPL3 and CPL3C dephosphorylate GST-CTD. CDKC;2-HA and CYCT1;3-HA were co-expressed in protoplasts, immunoprecipitated with α-HA agarose beads and used to phosphorylate GST-CTD. The phosphorylated GST-CTD was dephosphorylated by MBP-CPL3 or MBP-CPL3C. CTD phosphorylation was detected by WB with an α-pSer2, α-pSer5, or α-pSer7 antibody. The proteins of MBP-CPL3 and MBP-CPL3C are shown on the right. (C) The schematic domain organization of CPL3 used in this study. (D) CPL3C interacts with CTD. Pull-down assay was performed by incubating MBP-CPL3C, MBP-CPL3N or MBP-FCPH with glutathione beads containing CTD or phosphorylated CTD (pCTD). The HA-tagged CPL3 proteins were detected with an α-HA WB after glutathione bead pull-down (PD). The input control for GST-CTD, GST and CPL3-HA is shown with WB. (E) The CPL3 mutations did not affect the interaction with CTD. The sequence alignment of FCPH domain in CPL1, CPL2, CPL3, CPL4, ScFcp1 and HsFcp1 is shown on top panel and the conserved D933, K1046 and D1064D1065 of CPL3 residues are in red. In vitro pull-down assay was performed using GST, GST-pCTD (phosphorylated CTD by CDKC;2 as shown in Fig. S6B) or GST-CTD beads against fusion proteins of MBP-CPL3C or mutant variants tagged with an HA epitope. (F) CPL3D933A mutant lost function in protoplasts. Protoplasts from cpl3-4 mutants were co-expressed with CPL3C or its D933A mutant and pFRK1∷LUC reporter, and treated with 100 nM flg22 for 4 hr. The luciferase activity was normalized with internal GUS control. The protein expression of WT and mutant CPL3 is shown with WB on the bottom.

The above experiments were repeated 3 times with similar results.

Figure S7, related to Figure 7. The mutational and VIGS analyses of CDKCs. (A) PCR analysis of cdkc;2 genomic DNA. The specific amplification of PCR product with primers of LBb1 and RP, but not LP and RP, indicates that it is a homozygous T-DNA insertion. The Ctrl was PCR reaction without DNA. The scheme of CDKC;2 gene, T-DNA insertion and primers is indicated on the bottom. The black boxes indicate exon of CDKC;2. (B) Expression of CDKC;2 in Col-0 WT and cdkc;2 mutant. The RT-PCR was performed for 35 cycles, and the UBQ10 is the control. (C) Expression of CDKC;1 in Col-0 and cdkc;1RNAi plants. The RT-PCR was performed for 35 cycles, and the UBQ10 is the control. (D) flg22-induced CTD phosphorylation in seedlings. The band intensity of pSer2, pSer5 and pSer7 in Fig. 7A was quantified by the Image J software and presented with mean ± s.e. (n=3) from three independent biological replicates. (E) The cdkc;2 plants are more susceptible to Psm infections. The WT and cdkc;2 plants were inoculated with Psm at OD600 = 2×10-4 and the bacterial counting was performed 2 dpi. The data are shown as mean ± se from three independent repeats with Student's t-test. * indicates p<0.05 when compared to WT. (F) The phenotype of VIGS plants. WT or cdkc;2 plants were inoculated with pYL156-EV (empty vector) or pYL156-CDKC;1. The pictures were taken 16 and 28 days after VIGS inoculation. The plants silenced with Cla1 were used as a visual marker for VIGS efficiency. (G) The flg22-induced gene expression in CDKC-silenced plants. VIGS-CDKC;1 or control plants were treated with 100 nM flg22 for 3 hr, and RNA was isolated for qRT-PCR analysis. The data are shown as mean ± se from three independent repeats. (H) Protein expression of MPK3 and MPK6 in WT and mpk3/6 mutant. WT and mpk3/6 seedlings were grown on ½ MS medium and treated with 5 μM estradiol for 2 days. MPK3 and MPK6 proteins were detected by WB with α-MPK3 (Sigma, M8318) and α-MPK6 (Sigma, #A7104) antibodies. Ponceau staining for RBC was used as a loading control.

The above experiments were repeated 3 times with similar results.

Table S1, related to Figure 3. The list of CPL3-regulated genes without treatment (see Excel file Table S1).

Table S2, related to Figure 3. The list of flg22-induced genes in WT and/or cpl3-3 (see Excel file Table S2).

Table S3, related to Figure 3. The list of CPL3-dependent flg22-induced genes (see Excel file Table S3).

Table S4, related to Figure 3. Enrichment analysis of GO terms for CPL3-dependent flg22-induced genes (see Excel file Table S4).

Table S5, related to Figure 3. The list of flg22-reduced genes in WT and/or cpl3-3 (see Excel file Table S5).

Table S6. Primers used in this study, Related to Experimental Procedures 

Table S7: AGI number and predicted MW of genes/proteins used in this study, Related to Experimental Procedures 

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