From the Academy: Colloquium review. Unique characteristics of Xanthomonas oryzae pv. oryzae AvrXa21 and implications for plant innate immunity

Abstract

This article provides a brief overview of some of the major concepts and molecular features of plant and animal innate immune systems. The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the AvrXa21 elicitor. Xa21 codes for a receptor-like kinase consisting of an extracellular leucine-rich repeat domain, a transmembrane domain, and a cytoplasmic kinase domain. We show that AvrXa21 activity requires the presence of rax (required for AvrXa21) A, raxB, and raxC genes that encode components of a type one secretion system. In contrast, an hrpC(-) strain deficient in type three secretion maintains AvrXa21 activity. Xanthomonas campestris pv. campestris can express AvrXa21 activity if raxST, encoding a putative sulfotransferase, and raxA are provided in trans. Expression of rax genes depends on population density and other functioning rax genes. This and other data suggest that the AvrXa21 pathogen-associated molecule is involved in quorum sensing. Together these data suggest that AvrXa21 represents a previously uncharacterized class of Gram-negative bacterial signaling molecules. These results from our studies of the XA21/AvrXa21 interaction call for some modifications in the way we think about innate immunity strategies.

Fig. 1.

Working model for the synthesis, regulation, and function of AvrXa21. Functions assigned to each of the rax gene products based on sequence homology and/or functional studies are as follows (–45): RaxH, histidine kinase; RaxR, response regulator; RaxP, ATP sulfurylase; RaxQ, adenosine-5′-phosphosulfate kinase; RaxST, sulfotransferase; RaxA, membrane fusion protein, spanning the inner membrane and the periplasmic space; RaxB, ATP-binding cassette transporter; and RaxC, outer-membrane protein. See text for elaboration.

Fig. 2.

Bioassay showing that AvrXa21 activity is present in medium of PXO99 wild-type and TTSS-deficient mutant (hrpC⁻) strains but not in a TOSS-deficient mutant (raxA⁻) strain or a sulfuryltransferase-deficient (raxST⁻) strain. Leaves from 6-week-old TP309 and TP309 transgenic for XA21 (TP309-XA21) rice were inoculated with PXO99 wild-type (first and third leaves from left) or raxST⁻ (second and fourth leaves from left) Xoo strains by using the standard clipping method (46). To measure AvrXa21 activity, TP309-XA21 leaves were pretreated for 5 h with cell-free supernatant of PXO99 wild-type, raxST⁻, raxA⁻, and hrpC⁻ strains followed by inoculation with the raxST⁻ strain. Shown are representative leaves 2–3 weeks after inoculation from one of three independent experiments.

Fig. 3.

Cell density and AvrXa21-dependent expression of the raxST gene (filled symbols) and rRNA (open symbols). Xoo bacteria were cultured for 72 h and then diluted with fresh media to ≈10⁵ cfu/ml. The diluted cultures were returned to the incubator, and, as they grew, aliquots were removed for RNA isolation at different cell population densities. Equal amounts (1 μg) of bacterial RNA extracted with TRIzol reagent (Invitrogen, Carlsbad, CA) were used for real-time quantitative PCR. Three primer sets for each rax gene were designed to amplify ≈300-bp segments corresponding to three different parts of each gene. Primer sequences are provided in Table 3, which is published as supporting information on the PNAS web site. Expression levels are reported on a log scale and normalized to the copy number in the samples immediately after dilution. Data are from one of set of primers and two independent experiments. Similar trends were seen with all of the primer pairs. Expression is high at 10⁵ cfu/ml because the bacteria in this sample behave as if they are still at high density. (A) Wild-type PXO99 (circles) and raxR⁻ (squares) strains. (B) Diluted PXO99 Xoo cultures were recultured without treatment (circles), with AvrXa21-active HPLC fractions (no. 2, triangles; no. 17, diamonds) or an inactive HPLC fraction (no. 24, squares).