Resistance and susceptibility QTL identified in a rice MAGIC population by screening with a minor-effect virulence factor from Xanthomonas oryzae pv. oryzae

Abstract

Effective and durable disease resistance for bacterial blight (BB) of rice is a continuous challenge due to the evolution and adaptation of the pathogen, Xanthomonas oryzae pv. oryzae (Xoo), on cultivated rice varieties. Fundamental to this pathogens' virulence is transcription activator-like (TAL) effectors that activate transcription of host genes and contribute differently to pathogen virulence, fitness or both. Host plant resistance is predicted to be more durable if directed at strategic virulence factors that impact both pathogen virulence and fitness. We characterized Tal7b, a minor-effect virulence factor that contributes incrementally to pathogen virulence in rice, is a fitness factor to the pathogen and is widely present in geographically diverse strains of Xoo. To identify sources of resistance to this conserved effector, we used a highly virulent strain carrying a plasmid borne copy of Tal7b to screen an indica multi-parent advanced generation inter-cross (MAGIC) population. Of 18 QTL revealed by genome-wide association studies and interval mapping analysis, six were specific to Tal7b (qBB-tal7b). Overall, 150 predicted Tal7b gene targets overlapped with qBB-tal7b QTL. Of these, 21 showed polymorphisms in the predicted effector binding element (EBE) site and 23 lost the EBE sequence altogether. Inoculation and bioinformatics studies suggest that the Tal7b target in one of the Tal7b-specific QTL, qBB-tal7b-8, is a disease susceptibility gene and that the resistance mechanism for this locus may be through loss of susceptibility. Our work demonstrates that minor-effect virulence factors significantly contribute to disease and provide a potential new approach to identify effective disease resistance.

Keywords: MAGIC; TAL effector; bacterial blight; loss of susceptibility; quantitative resistance; quantitative susceptibility.

Figure 1

Tal7b is conserved among Xanthomonas oryzae pv. oryzae (Xoo) strains of Asian origin. (a) TAL effector profiles from 24 Asian strains of Xoo and one representative strain of African Xoo (BAI3), and an Asian X. o. pv. oryzicola (Xoc) strain (BLS256) were classified into groups according to DisTAL tree (Pérez‐Quintero et al., 2015). TAL groups are listed in order from left to right and conserved TAL effector groups are outlined in red; major TAL effector groups containing PthXo1 and AvrXa7 are boxed in blue; grey/white shading indicates the presence or absence of TAL effector in a strain; black represents multiple copies of that TAL effector or a homologue in that strain. The tree at the top shows the phylogenetic distances between TAL effector groups, and the parsimony tree on the right is based on whole genome sequences. (b) Variation and phylogenetic relationships of Tal7b homologues in Asian strains of Xoo are shown for each TAL effector along with their RVD sequence, TAL ID, strain name and specific geographic origin. The heatmap indicates nucleotide identity for each repeat, where blue represents sequence mismatch as indicated in identity key (top left) and white shading represents 100% match among sequences. Red letters indicate the two RVD where TAL effector family (TEF) 7 and 26 members differ and missing repeats are represented as dashed red lines inside grey boxes.

Figure 2

Distribution of the phenotypic response (lesion length in cm) of MAGIC AILs to strain PXO99^A with tal7b (pHMI::tal7b) or the empty vector control (pHMI::EV). (a) Violin plots represent the distribution of the least square means (LS‐Means) of bacterial blight disease lesion lengths on 330 MAGIC AILs and the eight founders inoculated with PXO99^A pHMI::EV (left) or pHMI::tal7b (right). Embedded box and whisker plots represent 50% of the values around the median (bold). The asterisk (*) represents Tukey adjusted P‐value < 0.001. (b) Each point on the scatter plot represents the LS‐Means of lesion length for each of the 330 AIL screened. The black lines connecting each point highlight the 63 AIL with significantly longer lesion lengths in response to PXO99^A pHMI::EV (left) compared to PXO99^A pHMI::tal7b (right) (Tukey adjusted P‐value < 0.05). Histograms represent the frequency distribution of the lesion length (LS‐Means) in the MAGIC AILs after inoculation with (c) PXO99^A pHMI::EV and (d) PXO99^A pHMI::tal7b. Histograms are overlaid on LS‐Means of the lesion lengths for the eight founder parents of the indica MAGIC population. PSBRc82 (F) was not included in the PXO99^A pHMI::EV screen in panel c because of poor germination.

Figure 3

Detection of QTL conferring resistance to PXO99^A carrying pHMI::EV or pHMI::tal7b. Manhattan plots and simple interval mapping show the genomic location on the x‐axis and the negative logarithm of the P‐values on the y‐axis. Solid blue line indicates a significance threshold of P ≤ 0.001. (a, b) GWAS results show significant associations to (a) PXO99^A pHMI::EV on chromosomes 5 and 11 (cyan; Table S7) and (b) PXO99^A pHMI::tal7b on chromosomes 5, 8, 11 and 12 (magenta; Table S8). (c, d) Simple interval mapping featuring unique QTL to Tal7b on chromosome 8, qBB‐PXO99^A‐8 and qBB‐tal7b‐8. Shaded regions indicate 1‐LOD supporting intervals for (c) qBB‐PXO99^A‐8 to PXO99^A pHMI::EV, which is distinct from the peak for (d) qBB‐tal7b‐8 to PXO99^A pHMI::tal7b.

Figure 4

Predicted Tal7b gene targets overlap with QTL conferring resistance to PXO99^A pHMI::tal7b. (a) Integrated physical map of rice chromosomes 1, 3, 5, 8, 10, 11 and 12 showing QTL identified in this study for PXO99^A pHMI::EV (cyan), PXO99^A pHMI::tal7b (magenta), predicted Tal7b targets (horizontal lines) and previously described resistance loci for bacterial blight (vertical lines). Detailed physical location for mapped loci is listed in Table S8. (b) Sequence logo showing predicted EBE of Tal7b using predicted targets in PXO99^A pHMI::tal7b‐specific QTL only. (c) Sequence logo showing predicted EBE of Tal7b using predicted targets from a genome‐wide scan. (d) Predicted Tal7b gene targets per QTL unique for PXO99^A pHMI::tal7b. (e) Top ten predicted Tal7b gene targets that overlap with PXO99^A pHMI::tal7b‐specific QTL.

Figure 5

Allelic variation in predicted Tal7b rice targets in MAGIC founders identify putative susceptibility genes for Tal7b. Tal7b predicted gene target with (a) polymorphic events in the EBE site itself; (b) downstream or upstream shifts in the promoter region between the EBE and the start codon or the region 1 kb upstream the EBE site (or both); (c) indels; and (d) complete absence of the EBE site in at least one or more of the eight founders (Table S9). (e) Lesion lengths on AIL and Nipponbare (Nipp) inoculated with PXO99^A pHMI::EV or PXO99^A pHM1::tal7b. The AIL ID number is listed above each pairwise comparison box. Asterisks indicate significant differences between treatments, as determined by one‐way ANOVA (P‐value ≤ 0.05, Table S11). NS represents no significant differences between treatments. (f‐h) Model proposing how resistance in q‐BBtal7b‐8 may contribute to resistance through loss of susceptibility by harbouring polymorphisms in the (g) Tal7b EBE or (h) S‐gene coding sequence, disabling activation or function of the putative S‐gene.