Tomato MAPKs LeMPK1, LeMPK2, and LeMPK3 function in the systemin-mediated defense response against herbivorous insects

Abstract

Systemin is a wound-signaling peptide that mediates defenses of tomato plants against herbivorous insects. Perception of systemin by the membrane-bound receptor SR160 results in activation of MAPKs, synthesis of jasmonic acid (JA), and expression of defense genes. To test the function of MAPKs in the response to systemin, we used virus-induced gene silencing (VIGS) in plants that overexpress the systemin precursor prosystemin (35S::prosys plants). These transgenic plants accumulate high levels of defense proteins and exhibit increased resistance to herbivorous insects. Cosilencing of the MAPKs MPK1 and MPK2 reduced MPK1/2 kinase activity, JA biosynthesis, and expression of JA-dependent defense genes. Application of methyl-JA restored the full defense response. These data show that MPK1 and MPK2 are essential components of the systemin signaling pathway and most likely function upstream of JA biosynthesis. MPK1 and MPK2 are 95% identical at the amino acid level. Specific VIGS of only MPK1 or MPK2 resulted in the same reduction of defense gene expression as cosilencing of MPK1 and MPK2, indicating that gene dosage effects may be important for MPK signaling. In addition, VIGS of the closely related MPK3 also reduced systemin-induced defense responses. The function of MPK1/2 and orthologs in pathogen-induced defenses is well established. Here we show that cosilencing of MPK1 and MPK2 compromised prosystemin-mediated resistance to Manduca sexta (Lepidoptera) herbivory, demonstrating that MPK1 and MPK2 are also required for successful defenses against herbivorous insects.

Fig. 1.

Cosilencing of MPK1 and MPK2 attenuates wound-induced MPK1/2 activity and PI-II synthesis. (A) 35S::prosys plants were infiltrated with either pTRV-MPK1/2 or -GFP (Control). Six weeks later, leaves were wounded and analyzed by immunocomplex kinase assays 0 and 10 min after wounding by using specific antibodies against MPK1 and MPK2. Signals represent phosphorylated myelin basic protein, an artificial MAPK substrate. Representative plants (–3) are shown. (B) 35S::prosys plants from independent experiments were analyzed for MPK1 (n = 9), MPK2 (n = 9), and MPK3 (n = 5) mRNA levels by sqRT-PCR, for wound-induced MPK1 (n = 5), and MPK2 (n = 4) activity by immunocomplex kinase assays, and for PI-II protein levels by RIDA (n = 9). The levels in VIGS plants (mean ± SD) were expressed as percentages of the mean levels in control plants which were defined as 100%.

Fig. 2.

Cosilencing of MPK1 and MPK2 attenuates expression of late systemin-induced wound response genes. 35S::prosys plants were infiltrated with pTRV-MPK1/2 and -GFP (Control). Five weeks later, transcript levels of Actin (internal control), the late genes PI-I and PI-II and the early genes AOS2, LoxD, and AOC were assessed by sqRT-PCR in leaf tissue. Ethidium bromide-stained agarose gels containing RT-PCR products are shown (colors inverted). The experiments are representative of 10 plants from three independent experiments for the late genes and of six plants from three independent experiments for the early genes.

Fig. 3.

Cosilencing of MPK1 and MPK2 attenuates JA biosynthesis. (A) 35S::prosys plants were infiltrated with pTRV-MPK1/2 or -GFP (Control). Four weeks later, leaves were wounded, and JA levels were measured in unwounded and wounded leaves 1 hr later. The bars represent the mean ± SD in 10 plants from three independent experiments. (B) 35S::prosys plants were infiltrated with pTRV-MPK1/2 or -GFP (Control). Four weeks later, plants were exposed to MeJA vapor (open bars) or to the solvent ethanol (filled bars) in a closed environment for 12 hr. Twenty-four hours after the start of the experiment, PI-II protein levels in leaves were measured by RIDA. The bars represent mean ± SD (n ≥ 18; three independent experiments).

Fig. 4.

VIGS of individual MPKs attenuates systemin-induced late gene expression. 35S::prosys plants were infiltrated with pTRV-MPK1 (A and B), -MPK2 (C and D), -MPK3 (E–G), or -GFP (Control, A–G). Four weeks later, MPK, PI-I, and PI-II transcript levels and PI-II protein levels were determined in leaf tissue by sqRT-PCR and RIDAs, respectively. (A, C, and E) MPK, PI, and Actin transcript levels. Ethidium bromide-stained agarose gels of PCR products are shown (colors inverted). (B, D, and F) MPK transcript levels and PI-II protein levels. The levels in VIGS plants (mean ± SD) (open bars) were expressed as percentages of the mean levels in control plants (filled bars), which were defined as 100% (n ≥ 5; ≥ 2 independent experiments; mean PI-II in controls of B, D, and F: 78 ± 15, 61 ± 16, and 75 ± 14 μg/ml leaf juice, respectively). (G) Ethidium bromide-stained agarose gels of PCR products corresponding to the early genes AOS, AOC, and LoxD, and to the late genes PI-I and PI-II in pTRV-MPK3- and pTRV-GFP-infiltrated control plants (n = 8; four independent experiments).

Fig. 5.

M. sexta herbivory activates MPKs, and cosilencing of MPK1 and MPK2 reduces systemin-mediated resistance to M. sexta larvae. (A) M. sexta larvae were allowed to consume one-half of the terminal leaflet of the lower leaf of two-leaf stage WT tomato seedlings. Ten minutes after onset of feeding (M. sexta), the wounded (local) and unwounded (systemic) leaf and leaves of unwounded control plants (unt) were assayed for MPK1, MPK2, and MPK3 activity by an immunocomplex kinase assay. Phosphorylated myelin basic protein is shown. The experiment is representative of three similar experiments. (B) 35S::prosys plants were infiltrated with pTRV-MPK1/2 and -GFP (Control). Four weeks later, each plant was exposed to one M. sexta larva for 11 days. Leaf damage (Upper; 12 representative plants are shown) and larval size (Lower) were documented photographically. An additional experiment generated similar results (not shown).