MAPK signaling regulates nitric oxide and NADPH oxidase-dependent oxidative bursts in Nicotiana benthamiana

Abstract

Nitric oxide (NO) and reactive oxygen species (ROS) act as signals in innate immunity in plants. The radical burst is induced by INF1 elicitin, produced by the oomycete pathogen Phytophthora infestans. NO ASSOCIATED1 (NOA1) and NADPH oxidase participate in the radical burst. Here, we show that mitogen-activated protein kinase (MAPK) cascades MEK2-SIPK/NTF4 and MEK1-NTF6 participate in the regulation of the radical burst. NO generation was induced by conditional activation of SIPK/NTF4, but not by NTF6, in Nicotiana benthamiana leaves. INF1- and SIPK/NTF4-mediated NO bursts were compromised by the knockdown of NOA1. However, ROS generation was induced by either SIPK/NTF4 or NTF6. INF1- and MAPK-mediated ROS generation was eliminated by silencing Respiratory Burst Oxidase Homolog B (RBOHB), an inducible form of the NADPH oxidase. INF1-induced expression of RBOHB was compromised in SIPK/NTF4/NTF6-silenced leaves. These results indicated that INF1 regulates NOA1-mediated NO and RBOHB-dependent ROS generation through MAPK cascades. NOA1 silencing induced high susceptibility to Colletotrichum orbiculare but not to P. infestans; conversely, RBOHB silencing decreased resistance to P. infestans but not to C. orbiculare. These results indicate that the effects of the radical burst on the defense response appear to be diverse in plant-pathogen interactions.

Figure 1.

NO Burst Induced by the MEK2-WIPK/SIPK Cascade but Not by the MEK1-NTF6 Cascade. (A) Effects of the expression of Nb MEK1^DD, St MEK2^DD, and inf1 on the activation of NTF6, WIPK, and SIPK. Protein was extracted from intact leaves (Cont) or from leaves 24 h after the inoculation with A. tumefaciens carrying the indicated gene constructs. Kinase activities of the extracts were assayed by IC kinase assay with anti-NTF6 antibody and WIPK- or SIPK-specific antiserum using myelin basic protein (MBP) as a substrate. Immunoblot analyses were done using anti-HA antibody. Protein loads were monitored by Coomassie blue (CBB) staining of the bands corresponding to ribulose-1,5-bisphosphate carboxylase (Rubisco) large subunit. The asterisk indicates a nonspecific protein. (B) Effects of the expression of inf1, St MEK2^DD, and Nb MEK1^DD on NO burst. N. benthamiana leaves were pretreated with A. tumefaciens containing the indicated gene constructs and an Agrobacterium infiltration buffer as a control for 36 h and then were infiltrated with 12.5 μM DAF-2 DA agent to detect NO. Sodium nitroprusside (SNP; 500 μM) was infiltrated into leaves with DAF-2 DA. The infiltrated leaf areas were monitored 1 h later under fluorescence stereomicroscopy. Signal intensities were quantified by color histogram analysis. Data are means ± se from three independent experiments. Bar = 1 mm. (C) Effects of silencing MEK1 and MEK2 on NO burst. After 3 weeks of inoculation with the gene silencing constructs TRV:MEK1, TRV:MEK2, and TRV as a control, silenced leaves inoculated with A. tumefaciens containing the indicated gene expression constructs for 36 h were analyzed as described in (B). Data are means ± se from four experiments. Bar = 1 mm.

Figure 2.

Effects of VIGS of NOA1 and NOS or NR Inhibitors on NO Burst Induced by INF1 and St MEK2^DD. NO production was estimated as described in Figure 1B. A. tumefaciens–inoculated leaves were infiltrated with cPTIO (500 μM), L-NAME (5 mM), or tungstate (100 μM) for 1 h before infiltration of DAF-2DA. Data are means ± se from four experiments. Bar = 1 mm.

Figure 3.

SIPK and NTF4 Participate in NOA1-Mediated NO Burst. (A) N. benthamiana leaves were inoculated with TRV:WIPK (W), TRV:SIPK (S), TRV:WIPK/SIPK (W/S), and TRV as a control. Total RNA was isolated from leaves 24 h after inoculation with A. tumefaciens containing inf1 and was used for RT-PCR with specific primers for WIPK, SIPK, and NTF4. The specificity of the primer pairs to SIPK or NTF4 was confirmed using cDNAs (pBin-Nb SIPK and pBin-Nb NTF4) as templates. (B) NO was measured as described in Figure 1B. Data are means ± se from 10 experiments. Bar = 1 mm. (C) IC kinase assay and immunoblot analysis were performed using anti-HA antibody as described in Figure 1A. (D) NO production was estimated as described in Figure 1B. Data are means ± se from four experiments. Bar = 1 mm.

Figure 4.

VIGS of SIPK Compromises the Expression of RBOHB Induced by St MEK2^DD but Not by INF1. N. benthamiana leaves were inoculated for gene silencing with TRV:WIPK (W), TRV:SIPK (S), TRV:WIPK/SIPK (W/S), and TRV as a control. Total RNA was isolated from the silenced leaves infiltrated with A. tumefaciens containing inf1 (top panel) or St MEK2^DD (bottom panel) at the indicated times (hours). Equal amounts of RNA (10 μg) were electrophoresed on a 1.2% agarose gel containing formaldehyde and were transferred to a membrane. Ethidium bromide staining of rRNA bands is shown as a loading control. Accumulation of transcripts for WIPK, SIPK, or RBOHB was confirmed by RNA gel blot hybridization.

Figure 5.

Nb MEK1^DD Induces RBOHB-Dependent Oxidative Burst. (A) Effects of the expression of inf1, St MEK2^DD, and Nb MEK1^DD on oxidative burst. N. benthamiana leaves were inoculated with A. tumefaciens containing inf1, St MEK2^DD, Nb MEK1^DD, or GUS as a control. These different inoculation sites on the same leaf were infiltrated with 0.5 mM L-012 solution (a reagent to detect ROS) 24 h after the inoculation and were monitored using a CCD camera. White circles, whose diameters are ∼1.5 cm, indicate areas infiltrated with L-012. Chemiluminescence intensities were quantified by a program equipped with a photon image processor. Data are means ± se from eight experiments. (B) Profile of transcript accumulation of RBOHB induced by INF1, St MEK2^DD, and Nb MEK1^DD. Total RNA was isolated from leaves infiltrated with A. tumefaciens carrying the indicated gene expression constructs or infiltration buffer for infiltration control at the indicated times. RBOHB transcript accumulation was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (C) VIGS of NTF6 compromised the expression of RBOHB induced by Nb MEK1^DD. Total RNA was isolated from NTF6-silenced leaves infiltrated with A. tumefaciens containing Nb MEK1^DD at the indicated times. Transcript accumulation was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (D) Effects of VIGS of RBOHA and RBOHB on oxidative burst induced by INF1, St MEK2^DD, or Nb MEK1^DD. Silenced leaves inoculated with A. tumefaciens for 24 h were analyzed as described in (A). Data are means ± se from four experiments.

Figure 6.

SIPK, NTF4, and NTF6 Participate in the Oxidative Burst and the Expression of RBOHB Induced by INF1. (A) N. benthamiana leaves were inoculated with the gene silencing constructs TRV:NTF6 (NTF6), TRV:SIPK/NTF6 (S/NTF6), or TRV alone as a control. Total RNA was isolated from silenced leaves infiltrated with A. tumefaciens containing inf1 at the indicated times. Accumulation of RBOHB mRNA was monitored by RNA gel blot hybridization; ethidium bromide staining of rRNA is shown as a loading control. (B) Effects of VIGS of SIPK (S), NTF6 (NTF6), or SIPK/NTF6 (S/NTF6) on the oxidative burst induced by INF1 or GUS as a control. Silenced leaves inoculated with A. tumefaciens for 24 h were analyzed as described in Figure 5A. Data are means ± se from twelve experiments. (C) Transcript accumulation of RBOHB induced by St MEK2^DD, SIPK, and NTF4. Total RNA was isolated from leaves 24 h after inoculation with A. tumefaciens carrying expression constructs for the indicated proteins and was used for RT-PCR with specific primers for RBOHB. (D) TRV control- and RBOHB-silenced leaves were inoculated with A. tumefaciens containing the indicated gene expression constructs. ROS were measured 24 h after the inoculation as described in Figure 5A. Data are means ± se from four experiments.

Figure 7.

Effects of VIGS of NOA1 and RBOHB on Susceptibility to P. infestans and C. orbiculare. (A) Effects of silencing of NOA1, RBOHB, or NOA1/RBOHB (N/B) on the phenotype of the leaves. Silenced leaves were photographed 3 weeks after initiation of VIGS by TRV infection with no additional challenge. (B) Susceptibility to P. infestans in the silenced plants. Silenced leaves were inoculated with drops of a P. infestans zoospore suspension (2 × 10⁵ zoospores/mL). Inoculated leaves were photographed 4 d after the inoculation. Red boxes: close-up shown in lower photographs. (C) Susceptibility to C. orbiculare in the silenced plants. Silenced leaves were inoculated with C. orbiculare conidial suspension (1 × 10⁶ conidia/mL) using a vaporizer. Inoculated leaves were photographed 6 d after the inoculation. Red boxes: close-up shown in lower photographs. (D) Evaluation of disease symptom and biomass of P. infestans. Disease symptoms were evaluated by the following scoring system: disease symptom as percentage of whole leaf: 0, <20%; 1, 20 to 50%; 2, >50%. Data are means ± se from seven experiments. To determine the biomass of P. infestans, real-time PCR was performed with P. infestans–specific primers using DNA isolated from inoculated leaves in (B). Data are means ± se from four experiments. (E) Number and size of lesion spots counted 6 d after inoculation with C. orbiculare. Diameter of lesion spots was recorded using the following scoring system: 0, <1 mm; 1, 1 to 2 mm; 2, >2 mm. Data are means ± se from eight experiments.

Figure 8.

Scheme of Proposed Model for MAPK Cascade Signaling Pathway Leading to NO and Oxidative Bursts. INF1 induces NO burst by means of MEK2-SIPK/NTF4 cascade and oxidative burst by means of MEK2-SIPK/NTF4 and (NPK1)-MEK1-NTF6 cascades. Based on loss-of-function and gain-of-function analyses, SIPK and NTF4 play important roles in NOA1-mediated NO burst and Nb RBOHB–dependent oxidative burst. NTF6 is also responsible for Nb RBOHB–dependent oxidative burst. The question mark indicates unidentified MAPKKK(s).