Identification of MPK4 interacting proteins in guard cells

Plants as sessile organisms are challenged by numerous biotic and abiotic stresses. Stomatal guard cells on the leaf surface are at the frontline of biotic and abiotic stress responses. Mitogen-activated protein kinase 4 (MPK4) has higher expression levels in guard cells than in mesophyll cells. The specific functions of MPK4 in guard cells are unknown. In this study, when MPK4 was overexpressed in Arabidopsis, bacterial entry of Pseudomonas syringae (Pst) into the plants was significantly decreased. The MPK4 overexpression plants had a similar trend of stomatal movement as wild-type Col-0, but had a smaller stomatal aperture than the Col-0, highlighting MPK4 plays a role in stomatal immune response. This function of the MPK4 requires its kinase activity because the MPK4 kinase-dead mutant did not have a significant difference in stomatal aperture compared to the Col-0. To understand MPK4 functions in guard cells, we investigated MPK4-associated protein complexes in guard cells using affinity purification mass spectrometry. A total of 145 proteins were identified to be in the MPK4-complex. Ten potential MPK4-interacting proteins were cloned and tested for physical interactions with the MPK4 using a yeast two hybrid (Y2H) system. Four proteins were newly identified to interact directly with the MPK4. MPK4 is highly abundant in stomatal guard cells, but its specific functions in guard cells are largely unknown. Through a bacterial entry assay of MPK4 overexpression plants, we found that MPK4 may play an important role in stomatal immune response. To understand the molecular mechanisms underlying the MPK4 functions in guard cells, we characterized the MPK4-associated protein complex in guard cells. Many of the 145 identified proteins were involved in plant immunity and development. Four of the proteins were newly identified to interact directly with the MPK4. This work has provided additional evidence for the MPK4 function as a positive regulator for stomatal immunity. The guard cell MPK4 protein complex and the four new interacting proteins were revealed. Whether MPK4 directly phosphorylates these interacting proteins deserves further investigation. These newly discovered proteins have chartered exciting directions toward understanding new functions of the MPK4 kinase.