LeMPK3 Is a Mitogen-activated Protein Kinase with Dual Specificity Induced during Tomato Defense and Wounding Responses

Abstract

Mitogen-activated protein (MAP) kinase cascades are readily activated during the response of plants to avirulent pathogens or to pathogen-derived elicitors. Here we show that the tomato MAP kinase LeMPK3 is specifically induced at the mRNA level during elicitation of the hypersensitive response in resistant plants infected by avirulent strains of the phytopathogenic bacteria Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. tomato, as well as upon treatment with the fungal elicitor ethylene-inducing xylanase. LeMPK3 gene expression was also induced very rapidly by mechanical stress and wounding much earlier than upon pathogen infection, but not in response to the defenserelated plant hormones ethylene and jasmonic acid. Moreover, in resistant tomato plants infected by X. campestris pv. vesicatoria, transcript accumulation was followed by an increase in LeMPK3 kinase activity. Biochemical characterization of a glutathione S-transferase-LeMPK3 fusion protein revealed that the LeMPK3 MAP kinase autophosphorylates in vitro mainly on tyrosine and less so on threonine and serine, whereas it phosphorylates myelin basic protein on serine and threonine. In vitro phosphorylation of a poly-(Glu-Tyr) copolymer by LeMPK3 demonstrated its capability to phosphorylate tyrosine residues on substrates as well. By mutagenesis and phosphoamino acid analysis, Tyr-201 in the kinase activation domain was identified as the main LeMPK3 autophosphorylation site and as critical for kinase activity. Finally, LeMPK3 autophosphorylation showed a preference for Mn 2؉ cations and proceeded via an intramolecular mechanism with an estimated K m value for ATP of 9.5 M. These results define LeMPK3 as a MAP kinase with dual specificity and strongly suggest that it represents a convergence point for different signaling pathways inducing the activation of defense responses in tomato.

Identification of MAPKs and Their Possible MAPK Kinase Activators Involved in the Pto-mediated Defense Response of Tomato

“…Although we had not initially proposed that LeMPK3 acts downstream of LeMAPKKK␣ (34), its placement there is consistent with a previous report showing that VIGS of LeMPK3 compromised resistance to avirulent strains of Pst (33). More recently, LeMPK3 mRNA levels were shown to be induced in resistant plants by avirulent strains of Pst, further supporting its involvement downstream of Pto (25).…”

“…In tobacco, separate functions for SIPK and Ntf4 can be inferred from the temporal and tissue-specific expression of Ntf4 (24). LeMPK3 was also shown to be activated by ultraviolet-B radiation and has been shown to be regulated at the transcript level similar to WIPK (25).…”

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

“…Extracts from frozen leaf material were obtained and immunocomplex or in-gel kinase assays with myelin basic protein as an artificial MAPK substrate were carried out as described (14,15). To measure MPK3 activity, magnesium in the kinase reaction buffer was substituted by manganese (26).…”

“…We had shown earlier that mechanical wounding with a hemostat and systemin application results in the activation of two MAPKs in tomato (Lycopersicon esculentum), LeMPK1 and LeMPK2, which belong to the A2 subgroup of plant MAPKs (12,14,15). Wounding also activated LeMPK3, which belongs to the A1 subgroup (12,26). MPK1 and MPK2 are 95% identical at the amino acid level and coordinately activated by all stimuli we tested so far.…”

“…LeMPK1, -2, and -3 were also activated by the host-specific elicitors AvrPto and AvrPtoB from Pseudomonas syringae (28) and infection by Xanthomonas campestris (26). Loss-of-function studies had shown they are required for defense against P. syringae and Mi-1-mediated resistance to aphids (29).…”