MAPKKKalpha is a positive regulator of cell death associated with both plant immunity and disease

Abstract

Many plant pathogens cause disease symptoms that manifest over days as regions of localized cell death. Localized cell death (the hypersensitive response; HR) also occurs in disease-resistant plants, but this response appears within hours of attempted infection and may restrict further pathogen growth. We identified a MAP kinase kinase kinase gene (MAPKKKalpha) that is required for the HR and resistance against Pseudomonas syringae. Significantly, we found that MAPKKKalpha also regulates cell death in susceptible leaves undergoing P. syringae infection. Overexpression of MAPKKKalpha in leaves activated MAPKs and caused pathogen-independent cell death. By overexpressing MAPKKKalpha in leaves and suppressing expression of various MAPKK and MAPK genes by virus-induced gene silencing, we identified two distinct MAPK cascades that act downstream of MAPKKKalpha. These results demonstrate that signal transduction pathways associated with both plant immunity and disease susceptibility share a common molecular switch.

Figure 1

Cell death is suppressed in N. benthamiana plants silenced for MAPKKKα. (A) Inhibition of cell death on TRV∷NbMAPKKKα leaves compared with TRV-only leaves after Agro-infiltration with (1) Cf9/Avr9, (2) Pto, (3) AvrPto, (4) AvrPtoBΔ6, (5) Pto(Y207D), (6) Pto/AvrPto, (7) BaxΔC and (8) Bax. Cultures were mixed 1:1 (single transgenes were mixed with an Agrobacterium strain carrying an empty vector). Photographs were taken 5 days after Agro-infiltration. The blue circles indicate no cell death and the red circles indicate cell death. Similar results were obtained in five independent experiments, each consisting of 6–8 plants with 2–3 leaves infiltrated per plant. In these experiments, inhibition of Pto/AvrPto and Pto(Y207D) HR was scored based on the parameters described in Figure 3A and presented as % in the text. (B) NbMAPKKKα transcript abundance is reduced in TRV∷NbMAPKKKα-silenced plants compared to TRV-only plants as assessed by RT–PCR. First strand cDNA was prepared from leaf tissue surrounding the infiltrated areas and used in RT–PCR with primers specific for NbMAPKKKα (right panel) or actin (left panel). PCR products were sampled from each cycle indicated, separated on an agarose gel and stained with ethidium bromide. Lane M shows 1 kb DNA ladder (Promega Co., Madison, WI). Asterisks indicate NbMAPKKKα PCR product. (C) Electrolyte leakage is reduced in TRV∷NbMAPKKKα and TRV∷Prf plants compared to TRV-only plants. Leaves were Agro-infiltrated with Pto/AvrPto or Pto/empty vector as described in (A). Data presented are means of three plants/line and error bars represent standard deviations. The experiment was repeated three times with similar results.

Figure 2

Sequence of MAPKKKα and relationship to MAPKKKα from other plant species. (A) Alignment of NbMAPKKKα and LeMAPKKKα predicted protein sequences using Clustal W method. Numbers on the left indicate amino-acid positions. Identical residues are shaded in black. The serine-rich domain is marked with a dotted line. The deduced kinase domain is underlined with a solid black line. The catalytic ATP-binding lysine is marked with an asterisk (^*) and the signature motif for the plant MEKK family is indicated with crosses (+). (B) Neighbor-joining tree using MEGA.2.1 method (see Supplementary Methods) based on the deduced catalytic kinase domain of NbMAPKKKα (AY500155), LeMAPKKKα (AY500156) and orthologs (BnMAPKKKα [CAA08995], B. napus; AtMAPKKKα [At1g53570], A. thaliana; OsAAF34436, OsCAD40821, Oryza sativa), with MEKK A2 subgroup members (AtMAPKKKα-1 [At1g63700], AtMAPKKKγ [At5g66850]) and other plant MEKKs (AtMEKK1 [At4g08500]; NtNPK1 [A48084], Nicotiana tabacum) involved in disease signaling. Sequences were obtained from the NCBI database. Scale below represents amino-acid substitutions and numbers on the tree represent bootstrap scores.

Figure 3

MAPKKKα is required for the Pto-mediated HR. (A) N. benthamiana 35S∷Pto plants silenced for NbMAPKKKα or Prf showed less or no HR compared to TRV-only plants 18 h after inoculation with P. s. pv. tabaci (avrPto) at 10⁷ CFU/ml (photo panel on left). Results of HR assays are shown in the table on the right. HR occurred in >80% of the infiltrated area (+); HR cell death occurred in 20–80% of the infiltrated area (+/−); no cell death was observed (−). Number in each category is shown over total scored. Disease symptoms were not evident in TRV∷NbMAPKKKα or TRV-only plants but developed in TRV∷Prf plants 7 days after infection, at 10⁶ CFU/ml (photo panel on right). The blue circle indicates no cell death, the yellow circle indicates disease-associated cell death and the red circle indicates HR. (B) Detection of HR cell death by Trypan blue staining. RG-PtoR tomato leaves silenced for LeMAPKKKα or Prf were compared to TRV-only leaves 18 h after infiltration of 5 × 10⁷ CFU/ml of P. s. pv. tomato DC3000 (avrPto). The experiment was repeated three times with similar results. For each experiment, positive and negative controls were analyzed in parallel: TRV∷Prf (no HR observed), TRV-only (confluent blue area indicates HR-associated cell death). (C) Quantification of the HR cell death inhibition in tomato leaves shown in (B) by ion leakage of leaf discs following the procedure described in Figure 1C.

Figure 4

MAPKKKα plays a role in development of bacterial disease symptoms. (A) Symptoms in N. benthamiana plants silenced for NbMAPKKKα or TRV-only inoculated with 5 × 10⁵ CFU/ml of P. s. pv. tabaci 5 days after infection (left panel). Disease severity (at 4 or 5 days after infection) is shown in the table. Disease symptoms occurred in >80% of the infiltrated area (+); disease symptoms occurred in 20–80% of the infiltrated area (+/−); no disease symptoms were observed (−). The experiment was performed with three plants per line in which two leaves were inoculated per plant. The blue circle indicates no cell death and the yellow circle indicates disease-related cell death. (B) Tomato leaves susceptible to bacterial speck disease (RG-prf3) were silenced for TRV∷LeMAPKKKα or TRV-only were inoculated with 10⁴ CFU/ml DC3000. After 4 days, leaves were destained and photographed. TRV-only leaves developed more lesions than TRV∷LeMAPKKKα-silenced leaves. (C) Growth of DC3000 on TRV∷LeMAPKKKα tomato leaves (RG-prf3) is reduced ∼80-fold compared to TRV-only leaves 4 days after bacterial inoculation. A total of three TRV-only and seven TRV∷LeMAPKKKα-infected RG-prf3 tomato plants were vacuum-infiltrated with 10⁴ CFU/ml DC3000. For bacterial growth measurements, 3 leaf discs/plant (1 cm²) were obtained from silenced leaves. Tissue was collected from the same areas for RT–PCR to monitor silencing efficiency (see Supplementary Figure S3) and bacterial counts from plants that did not show satisfactory silencing were excluded. Data presented are the means of three plants and error bars represent the standard deviation.

Figure 5

Overexpression of LeMAPKKKα in leaves causes cell death and stimulates MAPK activity. (A) Assessment of cell death in N. benthamiana leaves after infiltration of Agrobacterium strains carrying (1) empty vector, (2) LeMAPKKKα, (3) LeMAPKKKα^KD (LeMAPKKKα^202–458) or (4) LeMAPKKKα^KD− (LeMAPKKKα^{202–458/K231M}). All genes were tagged with the double hemagglutinin (HA) epitope and expressed from an estradiol-inducible promoter. The blue circle indicates no cell death and the red circle indicates cell death. Photograph was taken 36 h after application of estradiol. (B) Expression of LeMAPKKKα activates MAPK activity in leaf tissue. Protein extracts from N. benthamiana leaves transformed with the constructs described in (A) were assayed for MAPK activity using the in-gel kinase assay (top panel) 6 h after application of estradiol. Expression of the transiently expressed proteins was monitored by immunoblot analysis with anti-HA antibody (bottom panel). Lanes are numbered corresponding to (A).

Figure 6

Identification of two MAPK cascades acting downstream of MAPKKKα. (A) Overexpression of LeMAPKKKα^KD and LeMAPKKKα^KD− using an estradiol-inducible promoter in N. benthamiana leaves silenced for the MAPKK and MAPK genes shown, and for Prf and in TRV-only leaves. The blue circle indicates no cell death and the red circle indicates cell death. The photographs were taken 48 h after induction with estradiol. (B) Overexpression of LeMAPKKKα^KD, LeMEK2^DD, Pto/AvrPto or Bax (and their respective controls; LeMAPKKKα^KD−; LeMEK2; Pto/empty vector; BaxΔC) via Agro-infiltration (as described in Figure 1A) in N. benthamiana leaves silenced for the genes indicated or in TRV-only leaves. Expression of the transgenes was induced by estradiol 48 h after Agro-infiltration. Cell death was recorded 48 h after estradiol application (i.e., 4 days after bacteria infiltration) by using the following scoring system: 2 for full HR; 1 for partial HR; and 0 for complete inhibition of HR. Shown are the means of four independent experiments for LeMAPKKKα^KD, Pto/AvrPto and Bax and three independent experiments for LeMEK2^DD. Four to six silenced plants per construct (with 2–3 leaves infiltrated in each) were used in each experiment. Error bars represent the standard deviation. ^*Not performed.

Figure 7

Model for MAPKKKα-mediated cell death signaling. Model based on epistasis analysis combining cell death assays with N. benthamiana plants silenced for various MAPKK and MAPK genes. Two MAPK cascades are shown acting downstream of Pto in a MAPKKKα-mediated cell death signaling pathway. A MEK2/WIPK cascade might also contribute to cell death via a MAPKKKα-independent pathway. Depiction of a role for MAPKKKα in disease-associated cell death is based on reduced bacterial speck disease symptoms in N. benthamiana or in tomato silenced for MAPKKKα. ‘X' indicates a possible upstream activator of MAPKKKα lying between Pto and MAPKKKα. Dotted lines indicate the possibility of the two MAPK cascades acting either sequentially or in a parallel co-dependent manner. ‘Y' indicates a possible integrator of signals if the pathways act in parallel.