A method for quantitation of apoplast hydration in Arabidopsis leaves reveals water-soaking activity of effectors of Pseudomonas syringae during biotrophy

Abstract

The plant apoplast has a crucial role in photosynthesis and respiration due to its vital function in gas exchange and transpiration. The apoplast is also a dynamic environment that participates in many ion and nutrient transport processes via plasma membrane-localized proteins. Furthermore, diverse microbes colonize the plant apoplast, including the hemibiotrophic bacterial pathogen, Pseudomonas syringae pv. tomato (Pto) strain DC3000. Pto DC3000 initiates pathogenesis upon moving through stomata into the apoplast and then proliferating to high levels. Here we developed a centrifugation-based method to isolate and quantify the apoplast fluid in Arabidopsis leaves, without significantly damaging the tissue. We applied the simple apoplast extraction method to demonstrate that the Pto DC3000 type III bacterial effectors AvrE1 and HopM1 induce hydration of the Arabidopsis apoplast in advance of macroscopic water-soaking, disruption of host cell integrity, and disease progression. Finally, we demonstrate the utility of the apoplast extraction method for isolation of bacteria proliferating in the apoplast.