Rice XA21 binding protein 3 is a ubiquitin ligase required for full Xa21-mediated disease resistance.

XA21 is a receptor-like kinase protein in rice (Oryza sativa) that confers gene-for-gene resistance to specific races of the causal agent of bacterial blight disease, Xanthomonas oryzae pv oryzae. We identified XA21 binding protein 3 (XB3), an E3 ubiquitin ligase, as a substrate for the XA21 Ser and Thr kinase. The interaction between XB3 and the kinase domain of XA21 has been shown in yeast and in vitro, and the physical association between XB3 and XA21 in vivo has also been confirmed by coimmunoprecipitation assays. XB3 contains an ankyrin repeat domain and a RING finger motif that is sufficient for its interaction with the kinase domain of XA21 and for its E3 ubiquitin ligase activity, respectively. Transgenic plants with reduced expression of the Xb3 gene are compromised in resistance to the avirulent race of X. oryzae pv oryzae. Furthermore, reduced levels of Xb3 lead to decreased levels of the XA21 protein. These results indicate that Xb3 is necessary for full accumulation of the XA21 protein and for Xa21-mediated resistance.

The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase.

The rice gene Xa21 confers resistance against Xanthomonas oryzae pv. oryzae (Xoo). Xa21 encodes a receptor-like kinase (XA21). We demonstrate that XA21 autophosphorylates residues Ser686, Thr688 and Ser689 in vitro. Substitution of these residues with alanines did not affect the autophosphorylation function of this kinase, but specifically destabilized the resistance protein in vitro and in vivo. Plants carrying these same substitutions in XA21 were compromised in their resistance to the normally avirulent Xoo Philippine race 6. Additionally, we show that wild-type XA21 and the kinase-dead mutant with the invariable Lys736 residue mutated to glutamic acid were also proteolytically degraded in protein extracts. Finally, we show a correlation between the in vitro degradation and in vivo instability of the proteins. We propose that autophosphorylation of Ser686, Thr688 and Ser689 functions to stabilize XA21 against the developmentally controlled proteolytic activity.

The ubiquitin ligase XBAT32 regulates lateral root development in Arabidopsis.

Ubiquitin-mediated protein modification plays a key role in many cellular signal transduction pathways. The Arabidopsis gene XBAT32 encodes a protein containing an ankyrin repeat domain at the N-terminal half and a RING finger motif. The XBAT32 protein is capable of ubiquitinating itself. Mutation in XBAT32 causes a number of phenotypes including severe defects in lateral root production and in the expression of the cell division marker CYCB1;1::GUS. The XBAT32 gene is expressed abundantly in the vascular system of the primary root, but not in newly formed lateral root primordia. Treatment with auxin increases the expression of XBAT32 in the primary root and partially rescues the lateral root defect in xbat32-1 mutant plants. Thus, XBAT32 is a novel ubiquitin ligase required for lateral root initiation.

Biochemical characterization of the kinase domain of the rice disease resistance receptor-like kinase XA21.

The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn(2+) is at least 15 times higher than that with Mg(2+). The K(m) and V(max) of XA21 kinase for ATP are 0.3 microm and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.