Functional characterization of the arabidopsis hsp70-hsp90 organizing protein (hop) family in response to stress

Resumen

ABSTRACT HSP70-HSP90 organizing protein (HOP) is a conserved family of cytosolic cochaperones, whose role as scaffolding proteins of the chaperones HSP70 and HSP90 has been profusely studied in mammals and yeast. Although HOP family is also conserved in plants, their involvement in different protein complexes and their biological role in plant physiology has remained extremely elusive. Arabidopsis genome codes for three AtHOP proteins, AtHOP1, AtHOP2 and AtHOP3. In an attempt to determine the possible role of these proteins in response to different stresses, we started with the characterization of AtHOP3. HOP3 interacts in vivo with cytosolic HSP90 and HSP70, and, unexpectedly, with binding immunoglobulin protein (BiP), a member from the HSP70 family located in the endoplasmic reticulum (ER). Consistent with this interaction with BiP, HOP3 is partially localized at the ER. Moreover, HOP3 is induced both at transcript and protein levels by chemical agents inducing the unfolded protein response (UPR) through a mechanism dependent on inositol-requiring enzyme 1 (IRE1). Importantly, hop3 loss-of-function mutants show a reduction in pollen germination and a hypersensitive phenotype in the presence of ER stress inducer agents, a phenotype that is reverted by the addition of the chemical chaperone tauroursodeoxycholic acid (TUDCA). All these data demonstrate, for the first time in any eukaryote, a main role of HOP as an important regulator of the ER stress response. ER stress response is a process intimately associated with important specific developmental programs and to environmental stress sensing and response in plants. Since HOP3 plays a main role during ER stress and this process is highly induced during pathogen infection, we have studied the involvement of HOP3 in the plant defense response to different phytopathogens, specifically, to the necrotrophic fungus Botrytis cinerea, the vascular and hemibiotrophic fungus Fusarium oxysporum f. sp. conglutinans and the hemibiotrophic bacteria Pseudomonas syringae pv. tomato DC3000. This study demonstrates that HOP3 is required for the establishment of the defense response to these different pathogens. In addition, our data shows that HOP3 interacts with CORONATINE INSENSITIVE1 (COI1) in the yeast two-hybrid system and that the hop3-1 loss-of-function mutant shows a reduced sensitivity to MeJA. These data indicate that HOP3 is required for jasmonic acid (JA) signaling in plants. Finally, we have characterized the molecular role of the different members of the AtHOP family in thermotolerance. This analysis demonstrates that the three members of this family play a redundant role in the acquisition of long-term thermotolerance in Arabidopsis. Additionally, our data show that these proteins interact strongly with HSP90 and that part of the bulk of HOP shuttles from the cytoplasm to cytoplasmic foci and to the nucleus in response to heat stress. Consistent with this latter location and with the formation of a HOP complex with the heat shock factor HsfA1A, the heat shock response is altered in the hop1 hop2 hop3 triple mutant. This triple mutant also displays an unusual high accumulation of insoluble and ubiquitinated proteins under the heat challenge. These data reveal that HOPs are involved in two different aspects of the response to heat: the maintenance of protein homeostasis and the proper establishment of heat shock response, affecting the plant capacity to acclimate to heat stress for long periods. For all of it, the data presented in this thesis provide new evidences for the essential role of different members of the HOP family in response to different environmental stresses.