AvrE is an effector protein injected into the plant cell by the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 through the type three secretion system to promote disease. AvrE and its orthologs in other species of pathogenic bacteria, such as DspE in Erwinia amylovora and WtsE in Pantoea stewartii, are major virulence factors. Deletion of genes encoding AvrE-family effectors often greatly reduces the virulence of bacterial pathogens. Despite their importance in bacterial pathogenesis, the virulence functions of AvrE-family effectors remain enigmatic.This dissertation describes a detailed sequence-function study of the AvrE protein, which does not share any significant overall sequence similarities with other proteins, except for other members of the AvrE family. Several sequence motifs and conserved amino acid residues were identified and shown to be important for AvrE function. Most intriguing was the finding that the N-terminal and central regions of AvrE contain two WxxxE motifs, which are also found in some mammalian pathogen effectors that function as novel guanine nucleotide exchange factors (GEFs). Site-directed mutagenesis and pathogenesis assays were performed, yielding information about the essential amino acid features required for the function of the WxxxE motif in AvrE. In the C-terminus of AvrE, another conserved motif, LKKxGxE, was found. Results from site-directed mutagenesis and pathogenesis assays suggest that a pair of lysine residues in this motif is essential for the virulence function of AvrE.Transgenic expression of AvrE was found to be toxic to plant and yeast cells, suggesting that the host targets of AvrE may be conserved among eukaryotes. A large-scale screen was performed to identify Arabidopsis proteins that were capable of suppressing AvrE toxicity in yeast, by cotransforming avrE and an Arabidopsis cDNA library into yeast cells. In addition, non-toxic, truncated AvrE derivatives were used in yeast two-hybrid screens to identify Arabidopsis proteins that could interact with AvrE. Although several candidate host proteins were isolated, further studies to determine their relevance to the AvrE function during bacterial infection gave no definitive answers. Nonetheless, an AvrE fragment spanning the first 522 amino acids interacted with the Arabidopsis protein Rad23-A, a known interactor of another Pst DC3000 effector, HopM1. Previous studies have shown that AvrE and HopM1, although dissimilar in protein sequences, are functionally redundant, presumably because they affect the same host cellular processes. Rad23-A was found to be degraded during infection of Arabidopsis plants with Pst DC3000, but degradation was not observed when plants were infected with the CEL mutant bacteria, in which both avrE and hopM1 are deleted. The CEL mutant complemented with either the avrE or hopM1 gene induced degradation of Rad23-A during infection. Rad23-A may be used as a molecular marker for the virulence function of AvrE and HopM1 in Pst DC3000 infection of Arabidopsis.
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