Os8N3 is a host disease-susceptibility gene for bacterial blight of rice

Abstract

Many bacterial diseases of plants depend on the interaction of type III effector genes of the pathogen and disease-susceptibility genes of the host. The host susceptibility genes are largely unknown. Here, we show that expression of the rice gene Os8N3, a member of the MtN3 gene family from plants and animals, is elevated upon infection by Xanthomonas oryzae pv. oryzae strain PXO99(A) and depends on the type III effector gene pthXo1. Os8N3 resides near xa13, and PXO99(A) failed to induce Os8N3 in rice lines with xa13. Silencing of Os8N3 by inhibitory RNA produced plants that were resistant to infection by strain PXO99(A) yet remained susceptible to other strains of the pathogen. The effector gene avrXa7 from strain PXO86 enabled PXO99(A) compatibility on either xa13- or Os8N3-silenced plants. The findings indicate that Os8N3 is a host susceptibility gene for bacterial blight targeted by the type III effector PthXo1. The results support the hypothesis that X. oryzae pv. oryzae commandeers the regulation of otherwise developmentally regulated host genes to induce a state of disease susceptibility. Furthermore, the results support a model in which the pathogen induces disease susceptibility in a gene-for-gene manner.

Fig. 1.

Northern hybridization analysis of Os8N3 expression. (A) Os8N3 is induced in a TAL effector-dependent manner upon infection. Rice leaves were inoculated with water (mock), type III secretion mutant PXO99^AME7 (TTSS⁻), wild-type parent (PXO99^A), pthXo1⁻ (ME2), and ME2 with pthXo1 (ME2/pthXo1) reintroduced on pZWpthXo1 in pHM1, as indicated above each lane. Total RNA was extracted 24 h after inoculation and probed with a ³²P-labeled gene-specific 3′ probe of Os8N3. (B) Os8N3 is induced within 6 h after bacterial infection. Rice leaves were inoculated with PXO99^A, and RNA was extracted at the indicated time points (in hours). Filter was probed as in A. (C) Expression of Os8N3 is not elevated after infection of rice leaves with other strains of the pathogen. Rice leaves were inoculated with strains of X. oryzae pv. oryzae as indicated above each lane, and RNA was extracted 24 h after inoculation. Filter was probed as in A. (D) Os8N3 is expressed in developing inflorescence. Total RNA was extracted from the uninoculated plant tissues, as indicated above each lane. Filter was probed as in A. Total RNA loading is shown below the lanes (tlRNA) for each blot.

Fig. 2.

Os8N3 is a member of the MtN3 family. (A) Os8N3 is expressed as a 1.6-kb messenger RNA. Os8N3 is shown as represented by the full-length cDNA AK070510. Exons are represented by the solid black bars, and introns are indicated above the bars. Numbers indicate the number of base pairs of nucleotides. The first and last numbers for exons indicate the predicted 5′ and 3′ untranslated regions, respectively. 5′ and 3′ probes used throughout the study for hybridization (see Results) are indicated below the diagram. (B) Os8N3 product is predicted to encode a 307-aa integral membrane protein. The underlined region is a predicted uncleaved leader peptide. Eight predicted transmembrane regions are in bold. (C) Alignment of representative related proteins of the MtN3 family. Sequences were aligned by using clustalw (38) and displayed by using boxshade (www.ch.embnet.org/software/BOX_form.html). Protein sequence files: Medicago truncatula nodulin protein CAA69976 (MtN3), Arabidopsis MtN3-like protein AAL47380 (Arab); recombination activating gene 1 gene activation protein AAH14292 (Mouse), Drosophila melanogaster saliva protein AAD03390 (Fly), and Caenorhabditis elegans protein AAC02609 (Nema).

Fig. 3.

Os8N3 is not induced in xa13 plants. (A) Os8N3 is located on rice chromosome 8 in the region of the recessive resistance gene xa13. xa13 has been mapped to within a 14-kb region between two sequence-tagged markers, ST9 and RP7 (19). The position of Os8N3 within the interval is indicated by the horizontal arrow above the map. (B) Os8N3 is not induced in rice cultivars homozygous for xa13. Leaf RNA was isolated from the indicated rice cultivars 24 h after challenge with PXO99^A and probed with the 3′ probe for Os8N3. (C) Sequence polymophisms are present between IR24 and IRBB13 in the Os8N3 promoter. The Os8N3 promoter is shown, including 60 bp of coding sequence. The start of transcription is shown by the horizontal arrow, and the start of the coding sequence is indicated by “ATG.” A 243-bp insertion of DNA found in the IRBB13 sequence is indicated by the box on the promoter region. Other arrows indicate one to three nucleotide insertions present in either IRBB13 or IR24 in comparison with each sequence.

Fig. 4.

Silencing of Os8N3 expression by using iRNA. (A) Schematic of silencing vector for inhibition of Os8N3 mRNA. A 598-bp fragment from the 3′ region of Os8N3 (see Fig. 2A, 3′ probe) was cloned in two orientations in pANDA with a fragment of the uidA gene as an internal linker (GUS linker) behind the maize Ubi1 promoter. (B) Northern analysis of Os8N3-silenced rice plants. RNA was isolated from leaves of control plants containing only transformation vector sequences (Vector) and four independent transformed plants expressing the dsRNA sequence as indicated by the 3′ probe (Top, untreated, lanes 1–4). Expression was then analyzed with a 5′ probe to determine the level of expression for the endogenous Os8N3 before (Middle, untreated) and after challenge by strain PXO99^A (Bottom, PXO99^A).

Fig. 5.

Virulence assay on Os8N3-silenced plants. (A) Silenced plants have reduced bacterial populations. Bacterial populations at 8 days were measured from 10 plants of the control plant line with vector-only sequences (V) and three silenced plant lines (columns 1–3). Bacterial populations were measured as cell-forming units (cfu) per leaf. (B) Silenced plants display reduced lesion lengths. Lesion measurements in cm were taken at 8 days after inoculation from 10 leaves of the control line (V) and 10 leaves of each of four silenced lines (columns 1–4). (C) Os8N3-silenced plants have low pollen viability. Low pollen viability was assessed by the presence of starch (which stains dark blue) accumulation in pollen. Mature pollen grains from control plant (Ca) and silenced plant (Cb) were stained with 1% I-KI solution.

Fig. 6.

Strain-specific susceptibility mediated by Os8N3 and PthXo1. (A) avrXa7 enables PXO99^A compatibility on IRBB13. Leaves of 60-day-old IRBB13 plants containing xa13 and the near-isogenic parent IR24 were inoculated with the bacterial strain as indicated below each column. The average lesion length for 10 plants after 16 days was determined. (B) avrXa7 enables PXO99^A compatibility on Os8N3-silenced plants. Leaves of 40-day-old Os8N3-silenced plants were inoculated with the bacterial strain as indicated below each column. The average lesion length for six plants was determined after 8 days. (C) avrXa7 does not direct elevated expression of Os8N3 in IRBB13 or IR24. IRBB13 and IR24 plants were inoculated with the strain indicated above each lane. Total RNA was extracted at 24 h after inoculation and analyzed with the 3′ probe of Os8N3 (as in Fig. 1A). Total RNA loading is shown below the blots (tlRNA). Lanes are numbered for reference in the text. Plant variety is indicated below the lane numbers.

Fig. 7.

PXO99^A incites an atypical resistance reaction on IRBB13. (A) PX099^A does not elicit a typical hypersensitive reaction on leaves of IRBB13. Each leaf was inoculated with PXO99^A containing the gene indicated below the leaf. The number below each leaf photograph is included for reference in the text. The HR (brown tissue in the region of inoculation of leaf 3) is due to the presence of a dominant R gene present in IRBB13 and the avirulence gene avrXa7-sacB50. The avirulence gene was derived from a deletion analysis of avrXa7 and directs the elicitation of an HR in IR24 and related near-isogenic lines such as IRBB13 (22). The leaves were photographed 4 days after inoculation. (B) Rice peroxidase gene POX22.3 is not induced during challenge of IRBB13 with PXO99^A. Three sets of IRBB13 plants were inoculated as in A with PXO99^A (avrXa7), PXO99^A, and PXO99^A (avrXa7-SacB50), and total RNA was isolated at 0, 6, 12, and 24 h after inoculation. cDNA prepared from the samples was subjected to quantitative RT-PCR using POX22.3-specific primers. Gene-specific primers for the rice gene OsTFIIAγ5 were used for control.