GENERAL CONTROL NONREPRESSIBLE4 Degrades 14-3-3 and the RIN4 Complex to Regulate Stomatal Aperture with Implications on Nonhost Disease Resistance and Drought Tolerance

Gene Constructs, Bacterial Strains, and Plant Materials

Agrobacterium tumefaciens strain GV3101 containing TRV2:4D7-2 (NbME04D07-2; http://vigs.noble.org) was grown at 28°C in Luria-Bertani medium supplemented with rifampicin (10 µg/mL) and kanamycin (50 µg/mL). VIGS was performed by mixing a 1:1 ratio of Agrobacterium strains containing TRV1 and TRV2 as described (Senthil-Kumar and Mysore, 2014). Pseudomonas syringae strains were grown in King’s B medium at 30°C supplemented with rifampicin (10 µg/mL), kanamycin (50 µg/mL), or streptomycin (50 µg/mL) when needed. The Arabidopsis thaliana GCN4 full-length CDS was expressed in pMDC32 vector (Curtis and Grossniklaus, 2003) with 2X35S promoter. Several putative gcn4 T-DNA insertion mutants (SAIL_589_H02.v2, SALK_080928, SALK_040028, SALK_040028.36.55.x, SALK_040028.56.00.x, SALK_040124.37.50.x, SALK_109597.47.30.x, SALK_017235.55.25.x.) are listed in the SALK database. We obtained these mutants and, after molecular characterization, realized that none of them are complete knockouts. Therefore, we decided to generate GCN4 RNAi lines. To generate AtGCN4 RNAi lines, pH7GWIW G2 (II) vector (https://gateway.psb.ugent.be/search/index/silencing/any) with two different ∼250-bp fragments of the AtGCN4 CDS from 395 to 620 bp (RNAi A4) or 1211 to 1460 bp (RNAi B1) was used. For co-IP studies, GCN4 was expressed in Gateway destination vector pMDC83 with GFP tag on the C terminus by Gateway cloning (http://www.botinst.uzh.ch/en/research/development/grossnik/vectors/MarkdGatewayVectors.html). Both 14-3-3 and RIN4 were expressed in the pDEST15 vector with the GST tag. For the immunoblot or protein degradation assay, GCN4-GFP was expressed in pMDC83, and 14-3-3-HA and RIN4-HA were expressed in the pEARLEYGATE 201 vector. To express GCN4-GFP under the control of its native promoter, GCN4 was cloned into the pMDC107 vector. Floral dip inoculation (Clough and Bent, 1998) was used to develop transgenic lines that were screened on hygromycin (25 μg/mL) and basta (25 μM/mL).

Phylogenetic Analysis

Amino acid sequences for the candidate GCN4 genes from different species were identified from the BLAST homology analysis. Sequence alignments were made using the geneious tool (http://www.geneious.com) and further refined. Bootstrapping was performed with stepwise addition of 1000 replicates. A phylogenetic tree was computed, annotated, and depicted using the geneious tool. The phylogenetic tree is also reconfirmed by the Molecular Evolutionary Genetic Analysis (MEGA 6.0) alignment tool (http://www.megasoftware.net).

Disease Development and HR Assay in Nicotiana benthamiana

Four-week-old N. benthamiana seedlings were inoculated with an Agrobacterium strain containing TRV:NbGCN4. Three weeks after TRV inoculation, the silenced and nonsilenced plants were vacuum infiltrated with host or nonhost pathogens. To study the role of NbGCN4 in HR to nonhost pathogens, the silenced and control plants were infiltrated with nonhost pathogens. To determine the bacterial titer, leaf discs at 0, 1, 2, 3, 5, and 7 dpi, from four biological replicates representing four individual plants, were collected using a 1-cm² core borer. Leaf samples were ground, subjected to serial dilution, plated on King’s B agar medium supplemented with appropriate antibiotics, and incubated at 28°C for 2 d for bacterial colony counting. For visualization of bacterial multiplication using GFPuv-expressing strains, plants were syringe infiltrated as described (Wang et al., 2007).

In Planta Bacterial Number and Disease Development in Arabidopsis

For flood inoculation, 4-week-old plants grown on Murashige and Skoog plates were incubated for 1 min with 40 mL of bacterial suspension as described (Ishiga et al., 2011). Symptoms were observed after 5 d. To examine bacterial growth, the entire rosette was harvested, ground, and serially diluted as described (Ishiga et al., 2011). For syringe inoculation, 6-week-old plants were infiltrated on the abaxial side of the leaves with the pathogen. Leaf samples from three biological replicates from three individual plants were collected, and the bacteria were quantified as described above for N. benthamiana.

Gene Expression Analyses

To test the downregulation of GCN4 transcripts in N. benthamiana silenced plants, tissue was collected 3 weeks after TRV inoculation. To determine the expression of GCN4 in the Arabidopsis wild type (Col-0), plants were treated with the host pathogen P. syringae pv maculicola (1 × 10⁴ cfu/mL) and the nonhost pathogen P. syringae pv tabaci (1 × 10⁴ cfu/mL) by flood inoculation, and leaf tissue was collected at 12 and 24 hpi. Total RNA was extracted according to the manufacturer’s instructions using a Qiagen Total RNA Extraction Kit. The cDNA was synthesized by oligo(dT) primers using moloney murine leukemia virus reverse transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. RT-qPCR was performed with the Sigma-Aldrich KicQStart SYBR Green Kit. The conditions for the PCR were as follows: 95°C for 2 min, 25 cycles of denaturation at 94°C for 45 s, annealing for 30 s at 58°C, and polymerization for 45 s at 72°C, followed by plate reading at 72°C for 5 min, estimation of melting curve from 50 to 95°C, and incubation at 72°C for 4 min. The Arabidopsis Actin gene was used as a reference to normalize RT-qPCR, and expression was quantified as described (Ruijter et al., 2009). The primers used in the study are given in Supplemental Table 1.

Localization of GCN4

For the subcellular localization assay, native promoter-driven AtGCN4 was fused to the N-terminal part of GFP and cloned into the binary vector pMDC107. Stable Arabidopsis transgenic lines were developed by floral dip transformation, and GFP fluorescence was captured using a Perkin-Elmer ultraview spinning disk confocal microscope. For extraction of the PM fraction and cytosolic fraction proteins, 13-d-old stable AtGCN4-GFP Arabidopsis plants were harvested and then homogenized in homogenization buffer (50 mM MOPS, pH 7.0, 5 mM EDTA, 0.33 M sucrose, 1.5 mM ascorbate, 0.2% [w/v] insoluble polyvinylpolypyrrolidone, 2 mM DTT, 1 mM PMSF, 1 µg/mL leupeptin, and 1 µg/mL pepstatin A) on ice. The homogenate was filtered through a 240-µm nylon net and centrifuged at 700g for 10 min at 4°C. The PM Protein Extraction Kit (ab65400; Abcam) was used to extract PM and cytosol proteins. GFP antibody (lot number 120-002-105, catalog number 130-091-833; Miltenyl Biotec) was used to detect GCN4, and H⁺-ATPase antibody (lot number 1510, catalog number ABIN1720784; Abcam) was used as a PM marker protein.

To test the tissue-specific expression of GCN4, the pGCN4:GCN4-GUS construct was cloned into the binary vector pMDC162. The stable transgenic lines expressing AtGCN4-GUS were developed by floral dip transformation (Mara et al., 2010). After selecting the stable lines on hygromycin, the whole plants were stained with X-gluc substrate (Jefferson et al., 1987). To see the expression of GCN4 in guard cells, the leaf samples were analyzed under a microscope.

Drought Stress Imposition and Measurement of Water Loss

The moisture stress was imposed on Col-0, RNAi, and overexpression lines by gradual reduction of water by bringing the field capacity from 100 to 40% in 9 d (Babitha et al., 2013). The pots were filled with a known quantity of soil and completely saturated with water to arrive at the amount of water required to maintain 100% field capacity. Gradual moisture stress was imposed by weighing the pots, and the loss of water in the pots was replenished with the required amount of water to arrive at the desired field capacity of the soil. At the end of the stress period, the pots reached 35% field capacity. The duration of stress imposition was for a period of 9 d. The plants were allowed to recover by rewatering for 10 d. To measure relative water loss, the detached leaves from 4-week-old plants were air dried at room temperature, the weight of leaves was determined every 1 h, and relative water loss percentage was calculated over the initial leaf weight.

Measurement of Stomatal Aperture

Fresh leaves from 3-week-old plants at similar developmental stages were harvested. The epidermal peels from leaves were then incubated in stomata opening buffer (5 mM KCl, 50 mM CaCl₂, and 10 mM MES-Tris, pH 6.1) or 0.5 ng/μL COR (purchased from C. Bender, Oklahoma State University), or 1 ng/μL fusicoccin (Sigma-Aldrich) or 10 μM ABA (Sigma-Aldrich), or pathogens or various combinations at room temperature under high light for 3 h. Photographs were taken using a Nikon Optishot-2 camera at 40×, and stomatal apertures were measured using ImageJ software (Chitrakar and Melotto, 2010).

PM H⁺-ATPase Activity Assay

Various Arabidopsis lines were grown for 5 weeks in soil at pH 7.5. RIN4-expressing lines were sprayed with 20 mM dexamethasone in 0.025% Silwet, while the remaining lines were sprayed with water and 0.025% Silwet. Leaf tissue was harvested after 48 h. One set of plants was used as a control without pathogen treatment and another set of plants was sprayed with P. syringae pv tomato (DC3000), and tissue was harvested after 4 h. For all experiments, PM was immediately purified after harvesting leaf tissue (Liu et al., 2009). H⁺-pump activity was detected by a decrease of acridine orange absorbance at 495 nm as described (Liu et al., 2009). The assay buffer contained 20 mM MES-KOH, pH 7.0, 140 mM KCl, 3 mM ATP-Na₂, 30 mM acridine orange, 0.05% Brij 58, and 50 mg of PM protein in a total volume of 1 mL. Membranes were preincubated at 25°C for 5 min in assay buffer. The assay was initiated by the addition of 3 mM MgSO₄. Each experiment was repeated two times with independent PM isolations.

Isolation of Guard Cells and RNA Extraction

Arabidopsis Col-0 plants were grown under 8-h-light (300 µmol m⁻¹ s⁻¹ with LED bulbs)/16-h-dark conditions for 5 weeks. Plants were sprayed with either 4.0 × 10⁷ cfu/mL P. syringae pv tabaci or P. syringae pv tomato (DC3000) in water containing 0.025% Silwet L-77 or with water containing 0.025% Silwet L-77 as mock. Guard cell enrichment was done as described with slight modification (Misra et al., 2015). Ten grams of leaves with main veins removed was harvested and blended four times for 20 s each in 100 mL of cold sterile distilled water with the transcription inhibitors cordycepin (0.01%) and actinomycin D (0.0033%) using a Waring Laboratory Blender (http://www.waringlab.com/). Transcription inhibitors were used to inhibit the potential induction of transcript during guard cell enrichment (Obulareddy et al., 2013). The blended mixture was filtered through a 100-μm nylon mesh (sefar.com), and remnant was washed with cold distilled water until the flow-through was clear of mesophyll cells, debris, and plastids. These epidermal peels were digested with a mixture of 0.7% cellulysin and 0.025% macerozyme R10 with 0.1% polyvinylpyrrolidone 40 and 0.25% BSA for 1 h in darkness with shaking at 140 rpm. The digest was washed on a 100-μm nylon mesh using 750 mL of cold basic solution (560 mM sorbitol, 5 mM MES, 0.5 mM CaCl₂, 0.5 mM MgCl₂, and 10 μM KH₂PO₄, pH 5.5) to remove broken epidermal cells. Guard cell enrichment was verified by observing a small portion of tissue under a Nikon Optiphot-2 microscope (Nikon Instruments). RNA was isolated from guard cell-enriched tissue using TRIzol reagent (Life Technologies), and cDNA synthesis and RT-qPCR analysis were performed as described previously (Pant et al., 2015). In brief, RNA was treated with DNase I using the TURBO DNA-free Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, and cDNA was synthesized from 1 µg of RNA using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. The expression profiling of genes was performed using a 7900HT Real-Time PCR System (Thermo Fisher Scientific). Enrichment of guard cells was also verified at the molecular level by checking the expression of At5g46240 (KAT1) and At1g62400 (HT1) transcripts known to be induced in guard cells (Bates et al., 2012). The experiment was repeated using independently grown plant material.

Recombinant Protein Expression and ATPase Activity of GCN4

To determine if purified GCN4 protein has ATPase activity, the GCN4-His-tagged protein was overexpressed in Escherichia coli BL21 expression cells using pDEST17 vector and induced with IPTG. The protein was purified using Ni-NTA agarose (Qiagen). Protein was confirmed by His tag-specific antiserum (catalog number H1029, batch number 033M4785; Sigma-Aldrich) by protein blotting. To determine ATPase activity, an EnzChek Phosphate Assay Kit was used (Thermo Fisher Scientific). Three milligrams of purified recombinant GCN4 protein was added to the assay medium and preincubated at 25°C for 10 min before the addition of MgSO₄. The Bradford assay was used to calculate total protein content (Bradford, 1976).

Yeast Two-Hybrid Assays

Yeast two-hybrid assays were performed following the manufacturer’s protocol using the ProQuest Two-Hybrid System (Thermo Fisher Scientific). AtGCN4 was fused to the GAL4 DNA binding domain in pDEST32 as the bait construct. AtRIN4, At14-3-3, AtAHA1 (At2g18960), and AtAHA2 (At4g30190.2) proteins were used as prey proteins and fused to the GAL4 activation domain in pDEST22. The AAAD1 and AAAD2 domains of GCN4 were expressed separately in pDEST32 vector. Bait and prey constructs were cotransformed into yeast MaV203 competent cells. Positive clones were identified by their ability to grow on synthetic defined medium minus Leu/Trp/His (triple dropout medium) or Leu/Trp/His/Ura (quadruple dropout medium) containing 20 mM 3-aminotriazole. Liquid medium contained X-Gal to detect interaction by the development of blue color.

BiFC Assay

AtGCN4 was fused to the N-terminal EYFP in the pSITE-nEYFP vector. AtRIN4 and At14-3-3 were fused with the C-terminal part of EYFP in pSITE-cEYFP (Kudla and Bock, 2016). The AAAD1 and AAAD2 domains of GCN4 were expressed separately in the N-terminal pSITE-nEYFP vector. These vectors were transiently expressed in N. benthamiana. Leaves were collected 3 d after infiltration. Fluorescence was detected using either a Leica TCS SP2 or a Bio-Rad MRC 1024 ES confocal laser-scanning microscope.

Co-IP Assay

For immunoprecipitation and co-IP assays, N. benthamiana leaves were harvested 2 d after infiltration with an Agrobacterium strain containing the Pro35S:GCN4 construct within the T-DNA and were homogenized in protein extraction buffer (50 mM Tris-HCl, pH 7.5, 75 mM NaCl, 0.2% Triton X-100, 5 mM EDTA, 5 mM EGTA, 1 mM DTT, 100 µM MG132, 10 mM NaF, 2 mM Na₂VO₄, and 1% protease inhibitor cocktail [P9599; Sigma-Aldrich]). The interacting proteins RIN4 and 14-3-3 were individually expressed with C-terminal GST tag in pDEST15-expressing rosette cells. The proteins were purified with GST resin and confirmed by GST-specific antisera (lot number GR 9, 3884-10, catalog number ab58626; Abcam). The protein content was quantified using a Pierce 660-nm protein assay reagent (Thermo Fisher Scientific). After protein extraction, 5 µg of purified RIN4/14-3-3 from E. coli was added to 100 µg of total protein and incubated overnight at 4°C. The mixture was incubated for 2 h at 4°C with GFP Trap-A (Chromotek). The precipitated samples were washed, released by 2× SDS protein loading buffer, resolved by SDS-PAGE, and then transferred to nitrocellulose membranes. These membranes were incubated in blocking buffer (1× Tris-buffered saline buffer including 0.1% Tween 20 and 5% dried nonfat milk) for 1 h at room temperature, then with horseradish peroxidase-conjugated anti-GFP (Miltenyl Biotec) and anti-GST (Abcam) antibodies overnight at 4°C. An enhanced chemiluminescence system (GE Healthcare) was used for detection (Golemis, 2002).

Protein Degradation and Gel Blot Analyses

GCN4-GFP, HA-RIN4, and 14-3-3-GFP recombinant proteins were transiently expressed either individually or coexpressed in various combinations in N. benthamiana leaves using Agrobacterium. The bacterial infiltrations were performed with the same Agrobacterium concentration (0.6 OD₆₀₀) for all the constructs. Total protein was quantified using the Bradford method, and equal known concentrations were taken for the protein degradation assay. After 48 hpi with Agrobacterium, the host pathogen P. syringae pv tabaci was infiltrated at the same spot where Agrobacterium was infiltrated, and tissue was frozen after 0, 8, 12, 24, and 48 hpi. Total protein was extracted from leaf samples with extraction buffer (50 mM Tris-MES, pH 7.5, 80 mM NaCl, 10 mM MgCl₂, 10% glycerol, 0.2% Nonidet P-40, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail [Sigma-Aldrich]). Samples were incubated at room temperature. MG132 (1.6 μM; Sigma-Aldrich) was added to part of the samples to inhibit the proteasome-mediated protein degradation. Later, proteins were blotted on PVDF membrane, and GFP antiserum (Miltenyl Biotec) or HA antiserum (Sigma-Aldrich) was used to detect the GCN4, RIN4, and 14-3-3 protein levels. Actin antiserum (catalog number A0480, lot number 054M4805V; Sigma-Aldrich) was used to detect endogenous Actin protein that served as a loading control. The primary horseradish peroxidase-conjugated GFP, HA, and Actin antisera were diluted to 1:10,000 followed by secondary goat anti-rabbit antibody (lot number GR 1, 46572-6, catalog number ab9110; Abcam) conjugated to horseradish peroxidase and visualized using enhanced chemiluminescence solution (GE Healthcare Bio-Sciences), and protein gel blots were imaged.

Accession Numbers

Sequence data from this article can be found in the GenBank data library under accession numbers At3g54540 (AtGCN4), NbME04D07-2 (NbGCN4), At3g25070 (AtRIN4), At1g34760 (At14-3-3), At2g18960 (AtAHA1), and At4g30190.2 (AtAHA2).

Supplemental Data

• Supplemental Figure 1. Silencing of 4D7-2 in N. benthamiana Compromises Nonhost Disease Resistance.

• Supplemental Figure 2. AtGCN4 Overexpression and RNAi Lines.

• Supplemental Figure 3. Arabidopsis Plants Overexpressing AtGCN4 Are Resistant to Host Pathogen P. syringae pv maculicola upon Flood Inoculation.

• Supplemental Figure 4. AtGCN4 Localizes to PM and Guard Cells.

• Supplemental Figure 5. Scanning Electron Micrographs of the Adaxial Leaf Surface of Col-0, AtGCN4-RNAiA4, AtGCN4-RNAiB1, AtGCN4-OE6 and AtGCN4-OE16 at Magnification of 100 μm and 20 μm.

• Supplemental Figure 6. Expression of Recombinant AtGCN4 Protein in Escherichia coli to Determine ATPase Activity.

• Supplemental Figure 7. Stomatal Closure on AtGCN4 RNAi Lines Is Insensitive to Fusicoccin, Coronatine, and Nonhost Pathogen P. syringae pv tabaci.

• Supplemental Figure 8. Open Stomata Phenotype in Nb4D7-2 (NbGCN4) Silenced N. benthamiana Plants upon Nonhost Pathogen Infection.

• Supplemental Figure 9. A Negative Control for Y2H Assay and Bimolecular Fluorescence Complementation Assay with Split Enhanced Yellow Fluorescent Protein.

• Supplemental Table 1. List of Primers Used in the Study.

• Supplemental Data Set 1. GCN4 Sequence and Its Alignment with Its Orthologs.