The Xanthomonas Ax21 protein is processed by the general secretory system and is secreted in association with outer membrane vesicles

Abstract

Pattern recognition receptors (PRRs) play an important role in detecting invading pathogens and mounting a robust defense response to restrict infection. In rice, one of the best characterized PRRs is XA21, a leucine rich repeat receptor-like kinase that confers broad-spectrum resistance to multiple strains of the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In 2009 we reported that an Xoo protein, called Ax21, is secreted by a type I-secretion system and that it serves to activate XA21-mediated immunity. This report has recently been retracted. Here we present data that corrects our previous model. We first show that Ax21 secretion does not depend on the predicted type I secretion system and that it is processed by the general secretion (Sec) system. We further show that Ax21 is an outer membrane protein, secreted in association with outer membrane vesicles. Finally, we provide data showing that ax21 knockout strains do not overcome XA21-mediated immunity.

Keywords: Outer membrane vesicles; PAMPs; Plant immunity; Rice; Secretion; XA21; Xanthomonas.

Figure 1. Ax21 secretion does not depend on RaxABC.

Western blot analysis, using an anti-Ax21 antibody (upper panel), was carried out on total cells and cell-free supernatants of wild type strain PXO99 and the PXO99ΔraxA, PXO99ΔraxB and PXO99ΔraxC knockout strains grown under Ax21-enriching conditions (see material and methods). Similar levels of Ax21 are detected in supernatants of all tested strains. In all strains Ax21 is of similar size (predicted size of a mature, processed Ax21 protein is ∼19 kDa). Loading control with Coomassie brilliant blue staining (CBB) is presented in the lower panel.

Figure 2. Ax21 is targeted to the Sec pathway.

(A) The first 20 amino acids of Ax21 are predicted to constitute a Sec signal peptide containing a positively charged polar n-region, a hydrophobic central region, and a c-region containing the leader peptidase motif AXA. (B) Recombinant Ax21 was expressed in BL21.DE3 E. coli with a C-terminal His tag. Samples from total cultures, cell-free supernatants, and resuspended cell pellets were analyzed by SDS-PAGE followed by Western blot analysis. An anti-His antibody was used to detect the C-terminal His tag. Processed Ax21-His (21 kDa) is present in E. coli cells, but is not detected in the supernatant. When the leader peptidase motif AXA is mutated to DDD (Ax21-sp-mut), the protein is not processed (23 kDa).

Figure 3. Ax21 is an outer membrane protein secreted in association with outer membrane vesicles (OMVs).

Xoo cell-free supernatants were centrifugation at 180,000 g for 2 h to separate OMVs from soluble proteins. Fractions were then subjected to Western blot analysis with an anti-Ax21 antibody. Input: cell-free supernatant before centrifugation, ultra supernatant: supernatant after ultracentrifugation, ultra pellet: OMVs pellet after ultracentrifugation that was resuspended in 200 µL of water, diluted pellet: pellet sample diluted back to the original volume of the input sample. This figure shows that Ax21 is present in cell-free supernatants exclusively in an insoluble form in membrane vesicles. This experiment was repeated twice with similar results.

Figure 4. Ax21 insertion mutant strains retain the ability to activate XA21-mediated immunity.

To re-examine whether the ax21 mutant strain can evade XA21-mediated immunity, we used a validated PXO99Δax21 (see Fig. S5) to inoculate rice using a standard leaf-clipping assay. We have also added two newly generated ax21 insertion mutant strains labeled ax21 mut1-1 and ax21 mut1-4. PXO99 and PXO99ΔraxST were used as control strains on XA21-expressing plants. Disease lesions were scored 13–14 days post inoculation. Bars represent averages of at least 15 leaves ± SE. Statistical analysis was done using the Tukey-Kramer honestly significant difference test for mean comparison using the JMP software. Different letters within plant genotype, represent significant differences (P < 0.01) within each plant genotype.