Crystal Structure of Xanthomonas AvrRxo1-ORF1, a Type III Effector with a Polynucleotide Kinase Domain, and Its Interactor AvrRxo1-ORF2

Abstract

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak (BLS) disease on rice plants. Xoc delivers a type III effector AvrRxo1-ORF1 into rice plant cells that can be recognized by disease resistance (R) protein Rxo1, and triggers resistance to BLS disease. However, the mechanism and virulence role of AvrRxo1 is not known. In the genome of Xoc, AvrRxo1-ORF1 is adjacent to another gene AvrRxo1-ORF2, which was predicted to encode a molecular chaperone of AvrRxo1-ORF1. We report the co-purification and crystallization of the AvrRxo1-ORF1:AvrRxo1-ORF2 tetramer complex at 1.64 Å resolution. AvrRxo1-ORF1 has a T4 polynucleotide kinase domain, and expression of AvrRxo1-ORF1 suppresses bacterial growth in a manner dependent on the kinase motif. Although AvrRxo1-ORF2 binds AvrRxo1-ORF1, it is structurally different from typical effector-binding chaperones, in that it has a distinct fold containing a novel kinase-binding domain. AvrRxo1-ORF2 functions to suppress the bacteriostatic activity of AvrRxo1-ORF1 in bacterial cells.

3′-NADP and 3′-NAADP, Two Metabolites Formed by the Bacterial Type III Effector AvrRxo1

“…1). In fact, neither we nor others (19) could show that AvrRxo1 catalyzes the transfer of the γ-phosphate group of ATP onto UNAG to a significant extent because only minor traces of a potential UNAG-3P species were detected when a reaction mixture of AvrRxo1 incubated with UNAG and ATP for 5 h was analyzed using anion exchange chromatography (Fig. 2 a ).…”

“…Apart from the structural homology of this central AvrRxo1 domain with polynucleotide kinases, the recently reported 3D structure of AvrRxo1 revealed a strong homology of AvrRxo1 with UDP- N -acetylglucosamine (UNAG) kinases of the ϵ-ζ toxin-antitoxin family (19). This raised the question whether AvrRxo1 might be an authentic UNAG kinase or not as the previously identified, characteristic UNAG binding motif of ζ kinases (25) cannot be identified in the effector's primary structure (Fig.…”

“…By a similar approach, the structural homology of the effector protein AvrRxo1 from the pathogen Xanthomonas oryzae pv. oryzicola to nucleotide kinases led to the recent proposal that AvrRxo1 contains a polynucleotide kinase domain with an unknown toxic mechanism in plants (19). …”

“…The role of AvrRxo1 as a virulence factor is further supported by the finding that it is toxic when expressed in tobacco and yeast cells (20, 21, 23). Furthermore, when ectopically expressed in Escherichia coli , AvrRxo1 was shown to inhibit cell growth, a phenotype that could be suppressed by co-expression of AvrRxo2 (19). …”

“…oryzicola (20). Inhibition of the bacteriostatic phenotype is most likely accomplished by complex formation as inferred from the extensive interaction interface between AvrRxo1 and AvrRxo2 observed in the crystal structure (19). Given that X. oryzae pv.…”