RIN4 Functions with Plasma Membrane H ⁺-ATPases to Regulate Stomatal Apertures during Pathogen Attack

Abstract

In plants, the protein Rin4 acts with the plasma membrane H ⁺-ATPase to regulate pathogen entry and the innate immune response, in part, through the regulation of stomatal closure.

Abstract

Pathogen perception by the plant innate immune system is of central importance to plant survival and productivity. The Arabidopsis protein RIN4 is a negative regulator of plant immunity. In order to identify additional proteins involved in RIN4-mediated immune signal transduction, we purified components of the RIN4 protein complex. We identified six novel proteins that had not previously been implicated in RIN4 signaling, including the plasma membrane (PM) H ⁺-ATPases AHA1 and/or AHA2. RIN4 interacts with AHA1 and AHA2 both in vitro and in vivo. RIN4 overexpression and knockout lines exhibit differential PM H ⁺-ATPase activity. PM H ⁺-ATPase activation induces stomatal opening, enabling bacteria to gain entry into the plant leaf; inactivation induces stomatal closure thus restricting bacterial invasion. The rin4 knockout line exhibited reduced PM H ⁺-ATPase activity and, importantly, its stomata could not be re-opened by virulent Pseudomonas syringae. We also demonstrate that RIN4 is expressed in guard cells, highlighting the importance of this cell type in innate immunity. These results indicate that the Arabidopsis protein RIN4 functions with the PM H ⁺-ATPase to regulate stomatal apertures, inhibiting the entry of bacterial pathogens into the plant leaf during infection.

Author Summary

Plants are continuously exposed to microorganisms. In order to resist infection, plants rely on their innate immune system to inhibit both pathogen entry and multiplication. We investigated the function of the Arabidopsis protein RIN4, which acts as a negative regulator of plant innate immunity. We biochemically identified six novel RIN4-associated proteins and characterized the association between RIN4 and the plasma membrane H ⁺-ATPase pump. Our results indicate that RIN4 functions in concert with this pump to regulate leaf stomata during the innate immune response, when stomata close to block the entry of bacterial pathogens into the leaf interior.