Os03g0821300

Xb15 encodes a PP2C that negatively regulates the XA21-mediated innate immune response.

Annotated Information

Function

• The rice XA21 protein is representative of the large class of non-RD RKs predicted to be involved in plant innate immunity. XB15 encoded by Xb15 is an XA21 binding protein.

• XB15 negatively regulates cell death and the XA21- mediated innate immune response. This study suggests that XA21 is a substrate of XB15 and that the phosphatase activity of XB15 attenuates the XA21-mediated innate immune response.

Mutation

• To explore the role of Xb15 in resistance, the researchers examined whether constitutive overexpression of Xb15 would affect resistance to Xoo. The resulting transgenic plants (NTAP-XB15, pollen donor) were crossed with another Kitaake line possessing an N-terminal Myc-epitope-tagged XA21, under control of its native promoter (Myc-XA21, pollen recipient). The presence of the Myc-Xa21 and/or Ntap-Xb15 gene in the resulting F1 was confirmed by the PCR analysis (unpublished data). Next, they tested the expression of NTAP-XB15 and Myc-XA21 in the cross (XA21/XB15 17A-21 and 19A-72) using an anti-Myc antibody and peroxidase-anti-peroxidase (PAP, for detecting the TAP tag) (Figure 3A). Six-week-old progeny from self-pollinated XA21/XB15 17A-18, 17A-21, 19A-64, and 19A-72 were then inoculated with Xoo PR6 strain PXO99 expressing AvrXA21 and examined for cosegregation of genotype with phenotype by PCR analysis and measurement of the length of Xoo-induced lesions. All Myc-Xa21 plants overexpressing Ntap-Xb15 (Myc-Xa21/Ntap- Xb15, +/+) by PCR analysis showed accumulation of NTAPXB15 by protein gel blot analysis and displayed enhanced susceptibility to Xoo PR6 compared to transgenic plants carrying Myc-Xa21 alone (+/-) (Figure 3B). Figure 3C shows a picture of two typical leaves from each of the following inoculated rice plants: F2 segregants of XA21/ XB15 17A and 19A, and transgenic plants overexpressing NTAP only (NTAP) 16 d after Xoo PR6 inoculation.

Figure 3. XA21 Rice Plants Overexpressing Xb15 Are Susceptible to Xoo .

• To further investigate the in vivo function of Xb15, a knockout mutant line was identified from a collection of rice mutants generated by random insertion of the endogenous retrotransposon Tos17 (http://tos.nias.affrc.go.jp/;miyao/pub/tos17/). One mutant line (NF9014) carried a Tos17 insertion in the third exon of the Xb15 gene (Figure 5A). This insertion resulted in a lack of expression of the full-length Xb15 mRNA compared to the wild type Nipponbare plants when tested with appropriate primers by reverse transcription- PCR (RT-PCR) (Figure 5B). 18S ribosomal RNA (18S rRNA) was used as an internal control. The homozygous insertion mutant line (-/-) showed a severe cell death phenotype marked by the appearance of small necrotic lesions that were apparent at the vegetative stage (Figure 5C). We next tested if the cell death phenotype correlated with alterations in PR gene expression. Total RNA was extracted from the mutant line and the Nipponbare control. RT-PCR was performed with primers targeting PR genes as molecular markers. Figure 5D shows that the expression of full-length Xb15 was not detected in the Tos17 mutant line.

Figure 5. An Xb15 Tos17 Mutant Exhibits Cell Death Lesions .

Expression

• An XB15-smGFP2 fusion protein localizes to the plasma membrane.

Evolution

• The researchers next compared the PP2C domain of XB15 with other evolutionary related proteins from the reference plant species rice and Arabidopsis, such as Arabidopsis POL and POL-like proteins (PLLs) (Figure 2).

Figure 2. Phylogenetic Relationships among POL and PLL Genes from Rice and Arabidopsis .

Labs working on this gene

• Department of Plant Pathology, University of California Davis, Davis, California, United States of America
• Agricultural Research Service, Appalachian Fruit Research Station, United States Department of Agriculture, Kearneysville, West Virginia, United States of America