ABSTRACTTHE ROLE OF ARABIDOPSIS ACTIN DEPOLYMERIZING FACTOR 4 IN IMMUNE SIGNALING AND GENE EXPRESSIONByKatie Porter Arabidopsis thaliana actin-depolymerizing factor 4 (AtADF4) is a member of the over 75 characterized actin binding proteins (ABPs) including the 11 ADFs in Arabidopsis. As an ADF, AtADF4 has been shown to possess many of the biochemical and cellular functions associated with its role in the modification and regulation of the actin cytoskeleton. The collective works of numerous studies over the past few decades have demonstrated that ADFs from both plants and animals bind, sever, and/or depolymerize the aging pointed ends of filamentous actin. In the present study, I demonstrated that AtADF4 also contributes to a cellular function that has not previously been shown. In brief, the current body of work shows that AtADF4 specifically is required for the resistance of Arabidopsis when infected with Pseudomonas syringae pv. tomato expressing the bacterial effector AvrPphB, a cysteine protease known to target components of the immune-signaling responsible for recognition of non-self. While it is apparent that AtADF4 is required for resistance to Pst AvrPphB, the exact mechanism by which loss of AtADF4 (adf4) results in enhanced susceptibility remains largely unknown. Plant immunity is often achieved through recognition of bacterial effectors by a cognate resistance gene (R-gene). The R-gene of Arabidopsis that confers resistance to Pst AvrPphB is resistance to Pseudomonas syringae-5 (RPS5). Analysis of the expression of known R-genes of Arabidopsis in adf4 revealed a significant reduction in the expression of RPS5 while expression of other R-genes was not affected. Mitogen-activated protein kinase (MAPK) activation was examined in adf4 for the ability to recognize non-self through the pattern recognition receptor flagellin-sensitive 2 (FLS2) in the presence of AvrPphB. It was found that MAPK activation was reduced specifically in the adf4, while MAPK-signaling was not affected in the wild-type Col-0 or the rps5 mutant. The reduction of MAPK activation in adf4 but not rps5 suggests that in addition to regulating the expression of RPS5, AtADF4 also plays a role in FLS2-MAPK signaling in the presence of AvrPphB. The loss of resistance to Pst AvrPphB could be alleviated in adf4 when complemented with the serine-6 phosphorylation mimic AtADF4S6D. Although phosphorylation of serine-6 of plant ADFs is often associated with a reduced affinity for the actin cytoskeleton and is considered the inactive form of ADF, phosphorylation of serine-6 is in fact required for the immune-related functions of AtADF4. Establishment of the correlation of AtADF4 phosphorylation at serine-6 and resistance led to the examination of AtADF4 for additional unique biochemical features that may be related to immunity. Comparison of AtADF4 with its closest homologue AtADF1 revealed potential motifs of AtADF4 that may contribute to immune signaling, including phosphorylation of an additional residue, tyrosine-53. Interestingly, other recent examples of ADFs being required for immunity support these predictions. Taken together, the data presented herein identify the components of the immune response to which AtADF4 is associated, including the regulation of gene expression and recognition of non-self. These results provide a foundation for further defining the biochemical properties of AtADF4's role(s) in immune signaling.
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