Tomato SlMKK2 and SlMKK4 contribute to disease resistance against Botrytis cinerea

Background: Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that mediate the transduction of extracellular stimuli via receptors/sensors into intracellular responses and play key roles in plant immunity against pathogen attack. However, the function of tomato MAPK kinases, SlMKKs, in resistance against Botrytis cinerea remains unclear yet. Results: A total of five SlMKK genes with one new member, SlMKK5, were identified in tomato. qRT-PCR analyses revealed that expression of SlMKK2 and SlMKK4 was strongly induced by B. cinerea and by jasmonic acid and ethylene precursor 1-amino cyclopropane-1-carboxylic acid. Virus-induced gene silencing (VIGS)-based knockdown of individual SlMKKs and disease assays identified that SlMKK2 and SlMKK4 but not other three SlMKKs (SlMKK1, SlMKK3 and SlMKK5) are involved in resistance against B. cinerea. Silencing of SlMKK2 or SlMKK4 resulted in reduced resistance to B. cinerea, increased accumulation of reactive oxygen species and attenuated expression of defense genes after infection of B. cinerea in tomato plants. Furthermore, transient expression of constitutively active phosphomimicking forms SlMKK2(DD) and SlMKK4(DD) in leaves of Nicotiana benthamiana plants led to enhanced resistance to B. cinerea and elevated expression of defense genes. Conclusions: VIGS-based knockdown of SlMKK2 and SlMKK4 expression in tomato and gain-of-function transient expression of constitutively active phosphomimicking forms SlMKK2(DD) and SlMKK2(DD) in N. benthamiana demonstrate that both SlMKK2 and SlMKK4 function as positive regulators of defense response against B. cinerea.