A Strain of an Emerging Indian Xanthomonas oryzae pv. oryzae Pathotype Defeats the Rice Bacterial Blight Resistance Gene xa13 Without Inducing a Clade III SWEET Gene and Is Nearly Identical to a Recent Thai Isolate.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) injects transcription activator-like effectors (TALEs) that bind and activate host "susceptibility" (S) genes important for disease. Clade III SWEET genes are major S genes for bacterial blight. The resistance genes xa5, which reduces TALE activity generally, and xa13, a SWEET11 allele not recognized by the cognate TALE, have been effectively deployed. However, strains that defeat both resistance genes individually were recently reported in India and Thailand. To gain insight into the mechanism(s), we completely sequenced the genome of one such strain from each country and examined the encoded TALEs. Strikingly, the two strains are clones, sharing nearly identical TALE repertoires, including a TALE known to activate SWEET11 strongly enough to be effective even when diminished by xa5. We next investigated SWEET gene induction by the Indian strain. The Indian strain induced no clade III SWEET in plants harboring xa13, indicating a pathogen adaptation that relieves dependence on these genes for susceptibility. The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance.

Introduction

Xanthomonas oryzae pv. oryzae (Xoo), causes bacterial blight of rice, a yield-reducing disease widespread in Asia and Africa (Nino-Liu et al., 2006). Xoo relies on type III secreted, transcription activator-like effectors (TALEs) that directly activate specific host genes, called “susceptibility” (S) genes, which contribute to disease development (Hutin et al., 2015). A TALE finds its DNA target by virtue of a central repeat region (CRR) in the protein composed of nearly identical, direct repeats of 33–35 amino acid residues. Residues at the 12th and 13th positions in each repeat, together the “repeat-variable diresidue” (RVD), correspond to a single nucleotide in the effector binding element (EBE) in the DNA in a contiguous, code-like fashion such that the number and composition of RVDs predict the sequence of the EBE (Boch et al., 2009; Moscou and Bogdanove, 2009). The first residue of each RVD plays a stabilizing role and the second is the base-specifying residue. Characterized Xoo strains harbor 9 to nearly 20 different TALE-encoding (tal) genes, of which only one or two may encode a major virulence factor (Yang and White, 2004; Bogdanove et al., 2011; Hutin et al., 2015). All strains examined to date activate one of three members of clade III of the SWEET sucrose transporter gene family in rice (SWEET11, SWEET12, and SWEET14). These genes are major S genes, targeted by diverse TALEs from different strains (Hutin et al., 2015). In an experimental context, each of the other two members of SWEET clade III (SWEET12 and SWEET15), and no other SWEET genes tested, also functioned as a major S gene (Antony et al., 2010; Streubel et al., 2013). SWEET activation apparently leads to sucrose export into the xylem vessels, facilitating Xoo proliferation and symptom development by an as yet uncharacterized mechanism.

Host resistance is the most effective means of controlling rice bacterial blight. To date, 42 bacterial blight resistance genes, called Xa genes, have been identified from cultivated and wild rice species (Hutin et al., 2015; Kim et al., 2015; Busungu et al., 2016). The functions of most of the dozen or so that have been cloned and characterized relate to TALEs, and several are recessive. All but one of these recessive genes are alleles of a SWEET gene with a mutation at the EBE that prevents binding and activation by the cognate TALE, conferring resistance through reduced susceptibility. For example, xa13 is a variant of SWEET11 that lacks the PthXo1 EBE in its promoter and thereby confers resistance to strains that depend on PthXo1 (Chu et al., 2006). A strain can overcome xa13 if it expresses a TALE (such as PthXo2, PthXo3, AvrXa7, or TalC) that activates an alternate clade III SWEET gene (Zhou et al., 2015). The recessive bacterial blight resistance gene that is not a SWEET allele, xa5, acts more broadly. It is an allele of the general transcription factor subunit gene TFIIAγ5. The protein encoded by the dominant allele is an apparent contact point between TALEs and the transcriptional machinery. The product of xa5 harbors a single amino acid substitution that interferes with its interaction with TALEs and thereby reduces activation of their targets (Iyer-Pascuzzi et al., 2008; Huang et al., 2016). Interestingly, strains carrying PthXo1 are compatible with xa5. This compatibility is postulated to be due to the unusually strong activation of SWEET11 by PthXo1, which even diminished in the xa5 background is apparently high enough to render the plant susceptible (Huang et al., 2016).

The xa5 and xa13 genes have been widely deployed, both singly and in combination (Jeung et al., 2006; Sundaram et al., 2008, 2009; Shanti et al., 2010). Their effectiveness, however, has varied in different rice growing countries. In India, which is the second largest producer of rice behind China and has a highly diverse Xoo population (Midha et al., 2017), xa13 has historically been effective, whereas xa5-compatible Xoo isolates can be found throughout the country (Figure 1A; Mishra et al., 2013; Yugander et al., 2017). In contrast, in Thailand, another major rice producer, xa13-breaking stains are common while xa5 has largely remained effective (Figure 1B) (Wonglom et al., 2015). Recently, strains compatible with either R gene have been reported in each country (Lore et al., 2011; Mishra et al., 2013; Wonglom et al., 2015; Yugander et al., 2017). To gain insight into the mechanism(s) by which such strains overcome xa5 and xa13, we completely sequenced and compared the genomes and encoded TALE repertoires of one such strain from each country, IX-280 from India (Yugander et al., 2017) and SK2-3 from Thailand (Wonglom et al., 2015), to each other and to those of other sequenced Xoo strains. Further, we examined the ability of the Indian strain to activate SWEET gene expression in rice genotypes harboring xa5, xa13, or both genes.

Figure 1

Distribution of Xoo strains compatible with rice varieties IRBB5 (xa5, white), IRBB13 (xa13, gray), or both IRBB5 and IRBB13 (xa5 and xa13 individually, black) in (A) India and (B) Thailand.

Materials and methods

Genomic DNA extraction and sequencing

DNA for complete-genome sequencing was isolated using the protocol described by Booher et al. (2015) with the following two modifications: after overnight culture and centrifugation, extracellular polysaccharide was removed by washing the bacterial pellet 7–8 times with NE buffer (0.15 M NaCl, 50 mM EDTA), and after cell lysis, DNA was extracted four times with phenol/chloroform and once with chloroform/isoamyl alcohol. For each strain, 4–7 μg of genomic DNA was used to prepare a 20 kb library and each library was sequenced by SMRT technology to >150X genome coverage using P6-C4 chemistry (Pacific Biosciences, Menlo Park, CA USA), as described (Booher et al., 2015).

Genome sequence assembly

De novo assembly of the sequence reads was performed using HGAP v.2.0 (HGAP2) and HGAP v. 3.0 (HGAP3) (Chin et al., 2013) as described (Booher et al., 2015). Since TALE encoding (tal) genes are often clustered and their repetitive sequences can lead to misassembly even using long-read technology, tal gene containing regions were separately assembled using the PBX toolkit, a pipeline that uses long, tal gene sequence-containing seed reads to assemble tal clusters with more accuracy (Booher et al., 2015). Length cutoff settings used for these seed reads were 16 kb (pbx16000), 12 kb (pbx12000), or 10 kb (pbx10000). After HGAP and PBX assemblies were completed, the HGAP assemblies with the fewest unitigs and the majority of the tal gene sequences found by PBX were chosen for manual closure and finishing.

Genome finishing, assembly verification, and annotation

To finish the genomes, the circular assemblies were polished twice more with Quiver and then checked for structural variants and misassemblies using PBHoney (English et al., 2014). The tal gene repertoires were verified by consensus with the local tal assemblies made with PBX and by Southern blots of genomic DNA digested with either BamHI or SphI, or with BamHI and EcoRI, and probed with the tal gene specific probe pZWavrXa7 (Yang and White, 2004). To confirm the absence of plasmids smaller than 20 kb that could have been excluded during library preparation, total DNA was prepared and examined by agarose gel electrophoresis as described, using Xanthomonas campestris pv. vesicatoria 85–10, which has four plasmids, as a positive control (Booher et al., 2015). After finishing and assembly verification, genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (Tatusova et al., 2016), and tal gene annotations were manually corrected.

Genomic comparisons

For structural comparison, complete genomes were aligned using progressiveMauve (Darling et al., 2010) in the MegAlign Pro module of the DNAStar Suite (Lasergene 13.0.0.357) with default settings. For phylogenetic analysis, complete and draft genomes were aligned using Mauve v2.3.1 (Darling et al., 2004), and core alignment was used to infer phylogeny using PhyML v3.1 (Guindon et al., 2010). The core alignment and maximum likelihood tree was further subjected to ClonalFrameML (Didelot and Wilson, 2015) analysis with 100 bootstrap replicates to refine the phylogeny considering the impact of recombination. The ClonalFrameML tree was visualized using iTOL v3 (Letunic and Bork, 2016).

TALE analysis and target prediction

All tal gene sequences were extracted using the PBX exporter (Booher et al., 2015) or AnnoTALE (Grau et al., 2016). Orthology of IX-280 and SK2-3 TALEs to previously sequenced TALEs was determined using FuncTAL (Pérez-Quintero et al., 2015) and AnnoTALE (Grau et al., 2016). RVD or amino acid sequence was used as input for FuncTAL, and DNA sequence for AnnoTALE. AnnoTALE class builder files used to assign TALEs to families were downloaded on July 1, 2017. The results from the two tools were consistent. Targets of IX-280 and SK2-3 TALEs of interest were predicted using the TALE-NT 2.0 Target Finder tool (Doyle et al., 2012) and TalGetter (Grau et al., 2013). Predictions were made for both forward and reverse strands of promoter sequences, defined as the 1,000 bp upstream of a transcriptional start site to the translational start site for TALgetter, and 1,000 bp upstream of the translational start site for TALE-NT 2.0, and using MSU Rice Genome Annotation Project Release 7 (http://rice.plantbiology.msu.edu/). Default settings were used for Target Finder (upstream base of binding site = T, score cutoff = 3.0, Doyle et al. scoring matrix) and for TALgetter (standard model, p = 0.000001, upstream/downstream offset = 0).

Bacterial and plant growth conditions and disease and gene expression assays

Plants were grown in a growth chamber maintained at 28°C and 85% relative humidity with a photoperiod of 12 h. The bacterium was cultured at 28°C on modified Wakimoto agar medium. For the disease assay, bacterial cells were resuspended in sterile water at an OD600 of 0.2 and clip-inoculated (Kauffman, 1973) to fully expanded leaves of 40–45 day-old plants. Lesions were photographed 14 days later. For gene expression assays, bacterial cells were resuspended in sterile water at an OD600 of 0.5 and infiltrated into leaves of 3-week-old plants using a needleless syringe. Water was used for mock inoculation as a control. The inoculated portions of leaves were harvested 24 h later, and total RNA was extracted using the PureLink™ RNA Mini kit (Invitrogen, Carlsbad, California, USA) following the manufacturer's instructions. RNA was further treated with DNase (Invitrogen) to remove genomic DNA contamination. Quality and quantity of RNA were analyzed by 1.0% agarose gel electrophoresis and spectrophotometry using a Nanodrop (Thermo Scientific, Waltham, Massachusetts, USA). cDNA was generated from 1 μg purified RNA using the Superscript™ Vilo™ cDNA synthesis kit (Invitrogen) with random primers. Quantitative real time PCR (qPCR) was performed on a Light cycler® 480 Instrument II (Roche Molecular Diagnostics, Santa Barbara, California, USA). About 250 ng of cDNA was used for each qPCR reaction with gene specific primers (Supplementary Table S1). Each gene was tested with three biological replicates, with three technical replicates each. The average threshold cycle (Ct) was used to determine the fold change of gene expression. The expression of each gene was normalized to the expression of the 18S rRNA gene. The 2−ΔΔCt method was used for relative quantification (Livak and Schmittgen, 2001).

Results

Assembly of the complete IX-280 and SK2-3 genomes

Single Molecule Real-Time (SMRT) DNA sequence data for IX-280 assembled using either HGAP2 or HGAP3 (see Methods) resulted into two contigs, corresponding to a chromosome and a 43 kb plasmid. We named the plasmid pXOO43. The HGAP2 assembly, though it yielded an intact, self-complementary chromosomal contig, collapsed one cluster of four tal genes into three, indicated by a coverage spike in that cluster. A comparison of the ends of the misassembled cluster to pbx12000 and pbx16000 assemblies generated using the PBX toolkit (Booher et al., 2015) showed overlap with several that included an intact cluster of four tal genes. We chose a pbx16000 contig assembled using settings of 3,000 kb read overlap and 97% read identity to replace the misassembled cluster in the HGAP2 assembly. We also verified the presence of the cluster of four tal genes in the raw sequence of IX-280. To further confirm our final assembly, we obtained additional long reads from a separate DNA preparation of the same isolate and reassembled with HGAP3 using all available reads; the resulting HGAP3 assembly was consistent with the manually corrected HGAP2 assembly.

HGAP2 and HGAP3 assemblies of SK2-3 yielded a single chromosomal contig, but each terminated at a partial cluster of four tal genes. The intact cluster was present in pbx10000 assemblies. We selected a contig assembled using settings of 3,000 kb read overlap and 97% read identity to replace the broken cluster in the HGAP2 assembly and manually closed the genome.

The quality-control tool PBHoney (English et al., 2014) indicated no major inversions, deletions, or duplications in the assemblies. The proportion of mapped reads to post-filtered reads was 94.9% for IX-280 and 92% for SK2-3. Coverage graphs for the final assemblies showed no unusual peaks or dips that might indicate collapsed or expanded genomic repeats. PBX results were consistent with tal gene sequences extracted from the genomes, as were Southern blots hybridized with a tal gene-specific probe (Supplementary Figure S1). Separate DNA extraction and gel electrophoresis for both strains confirmed the absence of any small plasmids that might have been missed by SMRT sequencing (not shown).

Comparison of the IX-280 and SK2-3 genomes

The IX-280 plasmid pXOO43 has not been found in other Xanthomonas genomes, but some regions have a high degree of nucleotide identity with regions of pXAC64 from Xanthomonas citri ssp. citri (Da Silva et al., 2002). There are no predicted type III effector genes on the plasmid, but it harbors a cluster of genes annotated as type VI secretion genes. Associated with this cluster is an apparent operon containing pemK, encoding a toxin in a toxin/antitoxin system (Agarwal et al., 2007), and a gene encoding a protein of the XF1863 family, hypothesized to function as its antitoxin (Makarova et al., 2009). None of the pXOO43 content is found in the SK2-3 genome.

The IX-280 and SK2-3 chromosomes are entirely syntenous (Figure 2A, Supplementary Figure S2), including the tal genes, which show no duplications, deletions, or rearrangements in one genome relative to the other (Figure 2B). To determine how the genome structure of IX-280 and SK2-3 compares with that of other Xoo strains, we aligned the genomes with those of select strains for which complete genome sequences have been published. The complete Xoo genomes published to date sort into three East Asian lineages and a more distant African lineage (Quibod et al., 2016). We included representatives of each: Philippines strain PXO71 and Japanese strain MAFF311018 representing lineage PX-A, Philippines strain PXO86 representing lineage PX-B, Philippines strain PXO99A representing lineage PX-C, and the African strain AXO1947. The alignment shows no relationship between geographic area of isolation and genome arrangement (Figure 2A). Like IX-280 and SK2-3, the genome structures of PXO71 (Philippines) and MAFF311018 (Japan) are similar to one another, despite the strains being from different countries. In contrast, PXO86, PXO71, and PXO99A, all from the Philippines, have undergone genomic rearrangements relative to one another. The genome structure of the African strain, AXO1947, is distinct from those of the other Xoo strains, showing some of the genomic variability encompassed by the species. Though there are areas of similarity, the genomic arrangement of IX-280 and SK2-3 is not shared by any of the other strains.

Figure 2

Synteny between IX-280 and SK2-3 genomes and comparison of their tal genes. (A) Progressive Mauve alignment of the chromosomes of IX-280 and SK2-3 and other representative Xoo strains. (B) Map of the tal genes in IX-280 and SK2-3. Black arrows represent full-length tal genes, gray arrows truncTALE genes, and white arrows tal pseudogenes. Solid lines connect tal genes with >99% nucleotide identity and identical RVD sequence, and dotted lines connect less similar but clearly orthologous genes.

IX-280 and SK2-3 belong to a highly clonal lineage

The striking genomic similarity of IX-280 and SK2-3 despite their geographic separation led us to explore their relatedness with other Xoo strains more broadly. Using all published complete Asian Xoo genomes and draft (short-read derived) genome sequences of 100 Indian Xoo strains previously subjected to phylogenetic analysis (Midha et al., 2017), we generated a phylogenetic tree using regions not affected by recombination. The previous phylogenetic analysis of the 100 Indian strains had revealed five lineages (Midha et al., 2017). Both IX-280 and SK2-3 map to the youngest and a highly clonal lineage, L-I (Figure 3). Of the strains examined, SK2-3 is the only non-Indian strain in this lineage.

Figure 3

Positions of IX-280 and SK2-3 on a clonal lineage tree derived from genomic sequences of 100 Indian Xoo strains and other Xoo strains from Asia. Lineages are block shaded in different colors. IX-280 and SK2-3 (blue font) are in lineage L-I.

The TALE repertoires suggest possible mechanisms of xa5 and xa13 defeat

The TALE repertoires of IX-280 and SK2-3 each consist of 15 TALEs and two truncTALEs, which are TALE variants with shortened N- and C-termini that can function as suppressors of resistance mediated by certain non-executor R genes (Ji et al., 2016; Read et al., 2016); each strain also harbors a tal pseudogene (Figure 4). The RVD sequence of each IX-280 TALE and truncTALE is identical to that of its counterpart in SK2-3, except for the truncTALE Tal2b, of which repeats 10-15 are missing in SK2-3. Since truncTALEs do not bind DNA and a specific RVD sequence is not critical to their function (Read et al., 2016), this difference in Tal2b between the two strains is likely functionally irrelevant.

Figure 4

RVD sequences of IX-280 and SK2-3 TALEs. An asterisk indicates that the second amino acid in the RVD is absent, resulting in a 33 aa repeat. RVDs in bold are different in PXO99A orthologs. A dagger indicates a truncTALE. The underlined RVD of Tal2a resides in a truncated (28 aa) repeat. Lower case italicized RVDs are untranslated following a frameshift. Blue font highlights TALEs for which EBEs in rice were predicted.

Tal1c of both strains is an ortholog of PthXo1 (Figure 4), which likely explains the ability of each strain to overcome xa5. PthXo1 in IX-280 and SK2-3 differs from PthXo1 in PXO99A at one RVD, but the base-specifying residue of that RVD is the same (Supplementary Figure S3). Notably, an ortholog of PthXo7, the PXO99A TALE that induces TFIIAγ1, is also present in both strains (Tal7). Compatibility with xa5 had been postulated to be due to activation of the paralog TFIIAγ1 by PthXo7 (Sugio et al., 2007), but it was recently shown that only TFIIAγ5, and not TFIIAγ1, interacts in planta with tested TALEs (Yuan et al., 2016).

TALEs that could enable defeat of xa13 are less apparent. IX-280 and SK2-3 have no ortholog of known, major virulence factors such as PthXo2, which drives expression of SWEET13 (also called Os12N3 or Xa25), or PthXo3, TalC, Tal5, or AvrXa7, which activate SWEET14 (Yang et al., 2006; Antony et al., 2010; Liu et al., 2011; Streubel et al., 2013; Wang et al., 2015). In all, eleven of the 15 TALEs of IX-280 and SK2-3 (including Tal1c and Tal7) are apparent orthologs of TALEs found in PXO99A, which does not overcome xa13 (Figure 4, Supplementary Figure S3). Of these, six are identical to their PXO99A counterpart and the others have from one to several differences in RVD sequence. The gene encoding Tal4b of IX-280 and SK2-3 has an ortholog in PXO99A that is pseudogenized by a frameshift early in the coding sequence. We reason that the xa13-compatibility of IX-280 and SK2-3 is conferred by one or more of the TALEs with no apparent, intact ortholog in PXO99A (i.e., Tal1b, Tal1a, Tal4b, and Tal5c) or with a difference in RVD sequence relative to the PXO99A counterpart (i.e., Tal1c, Tal5b, Tal6b, Tal6c, Tal6d, and Tal7).

Predicted targets of possible xa13-breaking TALEs

Toward identifying the basis for IX-280 and SK2-3 compatibility with xa13, we generated lists of candidate target genes in rice (cv. Nipponbare) for their Tal1a, Tal1b, Tal4b, and Tal5c, which are either not found in PXO99A or differ by more than 6 RVDs from the most similar TALE in PXO99A (see Materials and Methods). Tal1a contains several instances of RVDs NN and NS, which have dual and lax specificity, respectively, so EBEs were predicted in most promoters. Among the candidates for Tal1b was a SWEET gene, SWEET2b, but SWEET2b was shown previously not to function as an S gene (Streubel et al., 2013). Another was a putative sulfate transporter gene, OsSULTR3;3 (Os04g55800.1). The distinct putative sulfate transporter gene OsSULTR3;6 is an S gene for bacterial leaf streak caused by X. oryzae pv. oryzicola (Cernadas et al., 2014), but whether sulfate transporters might confer susceptibility in bacterial blight is unknown; heterologous expression of an OsSULTR3;6-inducing TALE in the TALE-deficient strain X11-5A did not increase the extent of bacterial blight caused by this strain (Verdier et al., 2012). For Tal4b, EBEs were predicted in the promoters of three SWEET genes, SWEET1b (clade I and shown not to function as an S gene by Streubel et al., 2013), SWEET7e (clade II and not tested in that study), and SWEET14, within 350 bp of the transcriptional start sites (TSS). For Tal5c, EBEs were predicted in the promoters of SWEET15 within 100 bp of the TSS, SWEET13 within 250 bp, and SWEET12 within 50 bp.

IX-280 compatibility with xa5 is associated with induction of SWEET11 but IX-280 induces no clade III SWEET in compatible xa13 plants

To determine the mechanism by which these strains overcome xa13, we focused on the clade III SWEET genes. We inoculated IX-280 to rice cultivar IR24, which harbors neither xa5 nor xa13, and near isogenic cultivars IRBB5 (xa5), IRBB13 (xa13), and IRBB53 (xa5 and xa13). Each of these cultivars except IRBB53 is susceptible to IX-280 (Figure 5A; Yugander et al., 2017). We hypothesized that IX-280, by virtue of its PthXo1 ortholog Tal1c, induces SWEET11 strongly in IR24 and sufficiently in IRBB5, and that for compatibility in IRBB13 it induces another SWEET gene or the xa13 allele of SWEET11 by virtue of some other TALE. Further, we hypothesized that induction of the alternate SWEET gene is not as strong as that of SWEET11, such that when diminished by xa5 it is insufficient for susceptibility, explaining incompatibility with the combined xa5 and xa13 rice genotype IRBB53. We first compared expression of SWEET11 across each of the cultivars, using quantitative RT-PCR of RNA harvested from leaf tissue 24 h after inoculation. It was induced to 799-fold in IR24, to 553-fold in IRBB5, and not at all in IRBB13 or IRBB53, relative to mock (water) inoculation (Figure 5B). For reference we also examined expression of the bZIP transcription factor gene TFX1 and the TFIIAγ5 paralog TFIIAγ1. These are targets of PXO99A TALEs PthXo6 and PthXo7; these TALEs contribute moderately to virulence (Sugio et al., 2007) and an ortholog of each (Tal3c and Tal7, respectively) is present in IX-280 and SK2-3. In IR24 and IRBB13 each of the transcription factor genes was moderately induced (20 to 35-fold) in IX-280-inoculated leaves relative to mock (Figure 5B). This induction provides evidence that Tal3c and Tal7 are delivered and functional, and that the single RVD difference between PthXo7 and Tal7 does not impact targeting of TFIIAγ1. In IRBB5 and IRBB53, TFX1 and TFIIAγ1 induction was reduced to just 3 to 5-fold relative to mock (Figure 5B). This result is consistent with the observation that the xa5 allele reduces generally the ability of TALEs to induce their targets (Yuan et al., 2016). Next, we assayed the ability of IX-280 inoculated to IRBB13 plants to induce any of the other clade III SWEET genes. Contrary to our hypothesis, it induced none (Figure 5C). Finally, we tested each of the additional (non-clade III) SWEET genes that were identified as candidate targets of the IX-280 and SK2-3 TALEs that differ from TALEs of PXO99A, noted in the previous section: SWEET2b, SWEET1b, and SWEET7e. We also tested OsSULTR3;3. None of these was induced either (Supplementary Table S2).

Figure 5

Compatibility and ability of IX-280 to induce known or potential bacterial blight S genes in near-isogenic rice lines IR24, IRBB5 (xa5), IRBB13 (xa13), and IRBB53 (xa5 and xa13). (A) Representative lesions at 14 days after clip inoculation. Arrows indicate the distance the lesion progressed. (B) Fold induction of SWEET11, TFX1, and TFIIAγ1 24-27 h after inoculation by syringe infiltration of IX-280 relative to mock (water)-inoculated leaves, measured by qRT-PCR. Each bar represents the mean of three replicates. Error bars represent standard deviation. (C) Fold induction, as in (B), of the other clade III SWEET genes by IX-280 and selected positive control strains. ME2 is a pthXo1 knockout derivative of PXO99A (Yang and White, 2004) used here to deliver artificial TALEs ArtTAL12-2 and ArtTAL15-1, which are targeted to the SWEET12 and SWEET15 promoters, respectively (Streubel et al., 2013). PXO339 is a Philippines race 9 Xoo strain that induces SWEET13 (Liu et al., 2011). PXO86 is a Philippines race 1 Xoo strain that induces SWEET14 (Bai et al., 2000; Antony et al., 2010).

Discussion

This study presents the first completely assembled genome sequence of an Indian Xoo strain and the first genome sequence of a Thai strain. The genome comparisons we carried out (Figure 2A) and comparisons published elsewhere (Salzberg et al., 2008; Quibod et al., 2016) demonstrate the high level of variability in genome structure across different strains of Xoo and a general lack of relationship between genome structure and the geographical location at which a strain was isolated. Like other Xoo strains, both IX-280 and SK2-3 contain hundreds of IS elements and other transposons in their genomes (Table 1) that likely contribute to genome plasticity (Salzberg et al., 2008; Booher et al., 2015). Despite the overall genome structure variability in the species and the geographic separation of IX-280 and SK2-3, strikingly these two strains are part of a young and highly clonal lineage prevalent in India, L-I (Midha et al., 2017), in which no other characterized, non-Indian strains cluster. This observation and the relative rarity of xa5 compatibility in Thailand (Figure 1B) suggest introduction of SK2-3 or a recent progenitor in lineage L-I to Thailand directly, or indirectly, from India. Since we cannot rule out L-I having originated outside of India, however, it is alternatively possible that members of the lineage were introduced separately to Thailand and to India.

Table 1

The IX-280 and SK2-3 genome assemblies.

	IX-280	SK2-3
Chromosome	4,963,593 bp	4,934,446 bp
Plasmid	42,975 bp	–
Final coverage	164.0x	156.4x
% Mapped reads	94.6%	92.0%
Annotated genes	5,041	4,926
Annotated IS elements	411	407
Annotated transposases	730	698
tal genes	17	17

The strains IX-280 and SK2-3 are of special interest because of their compatibility with multiple single R genes (Wonglom et al., 2015; Yugander et al., 2017), in particular xa5 and xa13. Previously, strains compatible with xa5 and with xa13 were only found in the genomically and geographically diverse lineage L-III (Midha et al., 2017); IX-280 and SK2-3 represent the first example of strains compatible with xa5 and with xa13 in the much more genetically homogeneous lineage L-I. In light of the expanding R gene compatibility and geographic spread of strains in L-I, the fact that IX-280 and SK2-3 are incompatible with the xa5 and xa13 stacked line IRBB53 underscores the potential benefit of deploying such R gene stacks. The results also illustrate the importance of complete genome sequencing in monitoring Xoo populations to develop and deploy varieties with effective disease resistance.

The basis for the compatibility of IX-280 and SK2-3 with xa5 is almost certainly their ability to sufficiently activate SWEET11 even under the dampening effect of xa5 (Figure 5B). Their PthXo1 ortholog, Tal1c, is presumably responsible for this; the single difference in RVD sequence between Tal1c and PthXo1 does not affect the base specifying residue (Supplementary Figure S3). Induction of TFX1 by Tal3c (the PthXo6 ortholog), and of TFIIAγ1 by Tal7 (ortholog of PthXo7), though reduced by xa5, may also contribute. As noted, PthXo6 is a demonstrated virulence factor and TFX1 is a verified S gene (Sugio et al., 2007). PthXo7 is also a demonstrated virulence factor, and although activation of TFIIAγ1 was observed only by the xa5-compatible strain PXO99A (Sugio et al., 2007), silencing it decreased susceptibility to PXO99A even in an xa5 background (Yuan et al., 2016). We also observed that despite induction of TFIIAγ1 by Tal7, activation of SWEET11, TFX1, and TFIIAγ1 itself remain dampened in IRBB5 relative to IR24 and IRBB13 (Figure 5B). Thus, activation of TFIIAγ1 by Tal7 appears to contribute to susceptibility in some way other than providing a substitute for TFIIAγ5.

The basis for the compatibility of IX-280 and SK2-3 with xa13 is yet to be determined. Despite some of their TALEs being predicted to target clade III SWEET genes, no clade III SWEET gene was induced by IX-280 in IRBB13 plants. Nor were any of a handful of other candidate targets of interest, including SWEET genes of other clades and a paralog of a putative sulfate transporter gene that confers susceptibility to bacterial leaf streak. Possible reasons for such false positive predictions include competing endogenous DNA-binding proteins or DNA methylation at the target, or binding that does not lead to gene activation due to position in the promoter. Compatibility with xa13, which harbors a promoter deletion that eliminates the binding site of PthXo1 (and of the IX-280 and SK2-3 ortholog Tal1c), but not with xa5 and xa13 together, thus points to a second major TALE in IX-280 and SK2-3 that activates an alternative, novel S gene. Because of the incompatibility with stacked xa5 and xa13, such as in IRBB53, one would predict that the induction of this alternative S gene by the TALE is not strong enough to remain effective when dampened by xa5. Though we predicted targets only for IX-280 (and SK2-3) TALEs most dissimilar to those of the xa13-incompatible strain PXO99A, it is possible that one of the IX-280 TALEs more closely related to a PXO99A TALE is responsible: even a single RVD difference could confer the ability to target a new gene. It is also possible that the promoter sequence of the alternative S gene is different in IRBB13 and not represented in our predictions using the Nipponbare reference. Thus, future work should begin with loss- and gain-of-function experiments for each of the IX-280 and SK2-3 TALEs that are not precisely conserved in PXO99A, followed by transcript profiling of the IRBB13 and IR24 responses for any unique TALE revealed to be important for compatibility in IRBB13 plants.

Studies of diverse strains have suggested that induction of a clade III SWEET gene is a fundamental requirement for Xoo to cause bacterial blight of rice, but the African Xoo strain BAI3 was recently reported to be compatible on a rice line from which the binding site for its major TALE, TalC, in the promoter of SWEET14, was removed by genome editing, and the strain induced no clade III SWEET in that line (Blanvillain-Baufume et al., 2017). We have shown for the first time compatibility of an Asian Xoo strain without clade III SWEET gene induction. The emerging picture suggests some degree of selection on Xoo populations to evolve to target alternative S genes, perhaps due to the extensive deployment of R genes like xa13 and xa25 (a recessive allele of the SWEET13/Xa25 S gene widely used in China; Chen et al., 2002; Liu et al., 2011; Zhou et al., 2015). It will be of interest to determine whether the evolutionarily divergent IX-280 (and SK2-3) and BAI3 lineages have converged on the same new S gene, or if they target distinct ones. Further, determining the biochemical function(s) of the new S gene(s), which ostensibly can substitute for sucrose export in rendering the plant susceptible, promises to shed light on the mechanism by which clade III SWEET genes contribute to disease development.

Data availability statement

The IX-280 genome assembly has been deposited in GenBank under accessions CP019226 (chromosome) and CP019227 (plasmid pXOO43) and the SK2-3 assembly under accession CP019515. Raw data and associated metadata are available from the Sequence Read Archive (SRA) under accessions SRR5989134 (IX-280) and SRR5990719, SRR5990720, and SRR5990721 (SK2-3).

Author contributions

GL, RO, SP, and RR conceived the study. SC, AB, and RR designed the experiments. SC, PM, CG, PD, LW, SM, WK, JL, and RR performed the experiments and/or generated data. SC, LW, PP, NS, KS, AB, and RR analyzed and interpreted the data. SC, AB, and RR wrote the manuscript with input from RO.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.