The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity

Abstract

Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.

Figure 1

N. benthamiana NbNOG1-silenced plants are compromised in nonhost resistance. (A,B) NbNOG1-silenced (TRV::NbNOG1) and non-silenced control (TRV::00) N. benthamiana plants were vacuum-infiltrated with nonhost pathogen P. syringae pv. tomato T1 (pDSK-GFP _uv) or host pathogen P. syringae pv. tabaci (pDSK-GFP _uv) to observe symptom development (left panels) or bacterial multiplication 3 days post-inoculation (dpi; right panels). An increase in GFP fluorescence associated with bacterial multiplication was observed in TRV::NbNOG1 plants but not in the TRV::00. To monitor bacterial multiplication in TRV::NbNOG1 and TRV::00, N. benthamiana plants were vacuum-infiltrated with P. syringae pv. tomato T1 (A) and P. syringae pv. tabaci (B) and bacterial multiplication was quantified at various dpi as indicated. Bars represent means and standard deviations for three independent experiments. Asterisks above bars indicate statistically significant difference between NbNOG1-silenced plants and control (Student’s t-test P < 0.05). (C) HR was observed between NbNOG1-silenced and control N. benthamiana plants. Plants were syringe-infiltrated with P. syringae pv. tomato T1 or X. campestris pv. vesicatoria (1 × 10⁶ CFU/ml) or Agrobacterium strains for transient expression of Pto and AvrPto, or Cf-9 and Avr-9, or INF1. Agrobacterium strain GV2260 with empty vector (EV) was used as a control. HR was observed at different hours post inoculation (hpi). This experiment was repeated at least three times and showed similar results. Each experiment had five replications.

Figure 2

Tomato SlNOG1–silenced plants are compromised in nonhost resistance. (A) SlNOG1-silenced tomato compromised nonhost resistance. TRV::NbNOG1 and TRV::00 inoculated tomato plants were sprayed with the nonhost pathogen P. syringae pv. tabaci (Pstab) and the host pathogen P. syringae pv. tomato DC3000 (Pst DC3K). Pictures were taken after 5 days after inoculation. (B) The bacterial growth of both pathogens was significantly higher in SlNOG1-silenced plants than TRV::00 plants. Bacterial growth was measured after 2 and 6 dpi. Bars represent means and standard deviation for three independent experiments. Asterisks represent statistically significant difference between treatments for equivalent time points using Student’s t- test (P < 0.05).

Figure 3

NOG1-1 and NOG1-2 are induced by ABA, PAMPs, host and nonhost bacterial pathogens. (A) Arabidopsis wild-type (Col-0) plants were individually syringe-infiltrated with ABA (10 µM), Flg22 (20 µM), or LPS (100ng), or flood-inoculated with the pathogens P. syringae pv. maculicola (Psm) and P. syringae pv. tabaci (Pstab) at 1 × 10⁴ CFU/ml. RNA was isolated from tissue samples harvested at 0 hr, 6 hr, 12 hr and 24 hr, and qRT-PCR was performed. Bars indicate relative gene expression in comparison with the housekeeping gene Ubiquitin (UBQ5) and in relation to 0 hr time that was considered as 1. Different letters above bars indicate a statistically significant difference within a treatment using two-way ANOVA (P < 0.01). Error bars represent the standard deviation of three biological replicates (three technical replicates for each biological replicate). (B) β-Glucuronidase (GUS) staining of pNOG1-1::GUS and pNOG1-2::GUS in response to ABA, PAMPs, bacterial pathogens. pNOG1-1::GUS (left panel) and pNOG1-2::GUS (right panel) expression was measured 12 hr after treatment with ABA, flg22, LPS, Psm and Pstab. Seedlings were flood-inoculated with both pathogens (1.4 × 10⁶ CFU/ml), ABA (10 µM) and PMAPs (flg22: 20 µM, and LPS: 100ng). After 2 hr of GUS straining, plants were washed with sterile water and images were obtained.

Figure 4

Arabidopsis NOG1-1-RNAi but not nog1-2 plants are compromised in nonhost resistance. (A,B) Arabidopsis wild type (Col-0), nog1-2 knockdown line, NOG1-1-RNAi, nog1-2 NOG1-1-RNAi double-gene knockdown lines, overexpression (NOG1-1-OE) and complementation lines (NOG1-2-comp) were flood-inoculated with Pstab (1.4 × 10⁶ CFU/ml) (A) or Psm (1 × 10⁴ CFU/ml) (B) to assess disease symptoms (upper panel) and bacterial growth (lower panel) at 1 and 3 days post inoculation (dpi). Different letters above bars indicate a statistically significant difference within a time point using two-way ANOVA (P < 0.01). Error bars represent the standard deviation of three biological replications (three technical replicates for each biological replication). All experiments were conducted using T2 lines.

Figure 5

ABA, PAMPs, and nonhost bacterial pathogens induce stomatal closure in NOG1-2-dependent manner. (A,B) The nog1-2 line impairs ABA-, PAMPs- and nonhost-bacterial-pathogen-induced stomatal closure. To observe stomatal behavior, epidermal peels of Col-0, nog1-2, NOG1-1-RNAi2, and NOG1-2 complemented lines were treated with stomata-opening buffer (KCl-MES), ABA (10 µM or 50 µM), flg22 (20 µM), Pstab and Psm at 1 × 10⁴ CFU/ml. Microscopic images were taken 3 hr after inoculation. The aperture size of stomata was measured after 30 min for ABA, 1 hr for flg22 and LPS, and 3 hr for Pstab and Psm. Asterisks indicate significant difference by Student’s t-test (P < 0.05). Error bars indicate standard error for counting 50 stomata/each epidermal peel. Three samples were examined for each treatment, and the experiment was repeated at least three times with similar results. (C) Bacterial entry through stomata in nog1-2 and NOG1-1-RNAi2 lines. To quantify bacterial entry, detached Arabidopsis leaves from wild-type Col-0 and nog1-2 and NOG1-1-RNAi2 were floated in bacterial suspensions (1 × 10⁴ CFU/ml) of the nonhost pathogen (Pstab) or host pathogen (Psm). After 1 hpi and 3 hpi, leaves were surface-sterilized with 10% bleach, ground, serially diluted and plated on KB media (B). After 2 days, the number of bacterial colony was counted. This experiment was repeated three times and showed similar results: five replications in each experiment. Asterisks indicate significant difference by Student’s t-test (P < 0.05).

Figure 6

NOG1-2 has GTPase activity and is functionally involved in JA- and ABA-mediated signaling pathway. (A) Left Panel: Rate of Pi release due to the GTPase activity of NOG1-2 protein (1 µM) in the presence of varying concentrations of GTP. Experiments were repeated three times, and data were averaged. Error bars represent the mean ± S.E. Right panel: The transgenic Arabidopsis or N. benthamiana plants expressing AtNOG1-2-GFP under native promoter of AtNOG1-2 or NbNOG1-GFP under 35 S promoter, respectively. Arrows represent nuclei in guard cells. One week of seedlings were observed for the localization of AtNOG1-2 under confocal laser microscopy. Scale bar is 10 µM. Atnog1-2 is less sensitive to JA than Col-0. (B) Atnog1-2 line, compared to wild-type Col-0, is less sensitive to JA. Seeds of different Arabidopsis lines were grown in ½ MS medium plates with or without 30 and 50 µM of MeJA, and 7 days later root lengths were measured. Three independent experiments were done, with at least 10 seedlings for each line. Bars represent means ± SD. Asterisks indicate significant difference from Col-0 by Student’s t-test (P < 0.05). (C) The mutation of AtNOG1-2 increases sensitivity to drought stress and ABA. Wild-type (Col-0) and nog1-2 plants were grown for four weeks (21 °C/14 hr day and 18 °C/10 hr night), then plants were dehydrated until drought symptom appeared. After leaves were completely collapsed, plants were re-watered to revive. nog1-2 seedlings are less sensitive to ABA. Seedlings of Col-0 and nog1-2 were grown in MS or MS with ABA (1uM) for 2-weeks.

Figure 7

MAPMAN visualization of Arabidopsis Affymetrix data describing the differentially expressed genes involved in plant defense responses in NOG1-1 RNAi and nog1-2 lines. The Affymetrix microarray analysis showed a number of up- and down-regulated genes in NOG1-1 RNAi and nog1-2 lines compared to wild-type (Col-0) without treatment. MAPMAN was used to analyze the gene function and biological pathways of NOG1-1 and NOG1-2. Four-week old seedlings grown on half MS media were collected for RNA extraction. Three biological replicates were used for each NOG1-1 RNAi and nog1-2 without any treatments. Color patterns from red (upregulation) to green (downregulation) indicate the change of gene expression.