The virulence factor AvrXa7 of Xanthomonas oryzae pv. oryzae is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein

Abstract

AvrXa7 is a member of the avrBs3 avirulence gene family, which encodes proteins targeted to plant cells by a type III secretion apparatus. AvrXa7, the product of avrXa7, is also a virulence factor in strain PXO86 of Xanthomonas oryzae pv. oryzae. Avirulence and virulence specificities are associated with the central repeat domain, which, in avrXa7, consists of 25.5 direct repeat units. Mutations in three C-terminal nuclear localization signal motifs eliminated avirulence and virulence activities in rice and severely reduced nuclear localization in a yeast assay system. Both pathogenicity functions and nuclear localization were restored on the addition of the sequence for the nuclear localization signal motif from SV40 T-antigen. The loss of avirulence activity because of mutations in the acidic transcriptional activation domain was restored by addition of the activation domain from the herpes simplex viral protein VP16. The activation domain was also required for virulence activity. However, the VP16 domain could not substitute for the endogenous domain in virulence assays. In gel shift assays, AvrXa7 bound double-stranded DNA with a preference for dA/dT rich sequences. The results indicate that products of the avrBs3-related genes are virulence factors targeted to host cell nuclei and have the potential to interact with the host DNA and transcriptional machinery as part of their mode of action. The results also suggest that the host defensive recognition mechanisms are targeted to the virulence factor site of action.

Figure 1

Map of avrXa7. The repeat domain is shown as a series of boxes in the middle of the gene. nls 1 2 3, NLS sequences 1, 2, and 3. AD, acidic transcriptional activation domain. E, EcoRI; P, PstI; S, SphI; H, HindIII. Arrow indicates position of Tn5-B20∷53 insertion used to generate PXO86mx53 (see Results).

Figure 2

Sequence analysis of avrXa7. (A) Schematic representation of the repeat domain of the avrXa7 gene product by using the single amino acid code for the twelfth and thirteenth codons of the repeat units. GenBank accession nos. for proteins are as follows: AvrXa7, AF275317; AvrXa10, AAA92974; AvrBs3, CAA34257; AvrBs3–2, S34809; Avrb6, AAB00675; PthA, AAC43587; PthN, AAB69865; PthB, AAD01494. (B) Unusual repeat thirteen of avrXa7. Duplicated region is underlined. Asterisks indicate positions of missing nucleotides or amino acid residues of the prototypic repeat unit.

Figure 3

Repeat domain of avrXa7 controls virulence specificity. (A) Leaves were inoculated with wild-type PXO86 (leaf 1); PXO86mx53 (pHM1) with the mutated copy of avrXa7 and the cloning vector alone (leaf 2); and PXO86mx53 (pZWavrXa7) (leaf 3). Leaves were photographed 3 days after inoculation. Dark areas represent water-soaked tissue. (B) Lesion length measurements (cm) 11 days after leaf-clip inoculation.

Figure 4

Virulence activity of AvrXa7 requires C-terminal AD. Rice leaves were syringe inoculated with PXO86mx53 (leaves 1–3) or PXO99 (leaves 4–8) containing the following genes: 1, avrXa7; 2, avrXa7^TGA; 3, avrXa7^VP16; 4, vector control; 5, avrXa7; 6, avrXa7^TGA; 7, avrXa7^VP16; 8, avrXa7^VP16. Leaves 1–3 and 8 are from IR24. Leaves 4–7 are from IRBB7 (Xa7).

Figure 5

NLS mutations and yeast nuclear localization assay. (A) Amino acid replacements at NLS 1, 2, and 3 are underlined. Only regions of NLS motifs are shown. KKKRK was introduced into AvrXa7 M123 to create AvrXa7SV40. (B) Each gene was fused to the LexA coding DNA-binding domain in pNIA vector and expressed in yeast. Strain L40 contains LexA-binding sites (UAS_LexA) immediately upstream of lacZ and His 3 genes. Induction of lacZ and His3 depends on the nuclear localization of the LexA fusion product. Induction leads to increased β-galactosidase activity and the ability of the yeast strain to grow in histidine-deficient media.

Figure 6

Effects of NLS mutation on the avirulence and virulence activities of avrXa7. (A) Leaves of IRBB7 rice containing the resistance gene Xa7 (Upper) and the susceptible variety IR24 (Lower) were syringe inoculated with PXO99 or PXO86mx53, respectively, containing the following genes: (1) pHM1, vector alone; (2) pZWavrXa7; (3) pZWavrXa7M123; (4) pZWavrXa7SV40. (B) Lesion length measurements 11 days after leaf-clip inoculations of IRBB7 with PXO99 (hatched bars) or IR24 with PXO86mx53 (solid bars). Numbering is as for A.

Figure 7

AvrXa7 is a DNA-binding protein. (A) Single-stranded and double-stranded oligonucleotides were end labeled with ³²P, mixed with AvrXa7 or GST (glutathione S-transferase), and subjected to polyacrylamide electrophoresis. Arrow indicates position of well. (B) DNA-binding competition to AvrXa7. Random oligonucleotides were labeled with ³²P and mixed with AvrXa7 and the indicated unlabeled DNA in 100-fold excess. A, T, C, G, indicate unlabeled single-stranded homopolymers; A/T and C/G indicate unlabeled double-stranded polymers. +/− indicate presence or absence of component.