Code-assisted discovery of TAL effector targets in bacterial leaf streak of rice reveals contrast with bacterial blight and a novel susceptibility gene

Citation: Cernadas RA, Doyle EL, Nin˜o-Liu DO, Wilkins KE, Bancroft T, et al. (2014) Code-Assisted Discovery of TAL Effector Targets in Bacterial Leaf Streak of Rice Reveals Contrast with Bacterial Blight and a Novel Susceptibility Gene. PLoS Pathog 10(2): e1003972. https://doi.org/10.1371/journal.ppat.1003972Bacterial leaf streak of rice, caused by Xanthomonas oryzae pv. oryzicola (Xoc) is an increasingly important yield constraint in this staple crop. A mesophyll colonizer, Xoc differs from X. oryzae pv. oryzae (Xoo), which invades xylem to cause bacterial blight of rice. Both produce multiple distinct TAL effectors, type III-delivered proteins that transactivate effector-specific host genes. A TAL effector finds its target(s) via a partially degenerate code whereby the modular effector amino acid sequence identifies nucleotide sequences to which the protein binds. Virulence contributions of some Xoo TAL effectors have been shown, and their relevant targets, susceptibility (S) genes, identified, but the role of TAL effectors in leaf streak is uncharacterized. We used host transcript profiling to compare leaf streak to blight and to probe functions of Xoc TAL effectors. We found that Xoc and Xoo induce almost completely different host transcriptional changes. Roughly one in three genes upregulated by the pathogens is preceded by a candidate TAL effector binding element. Experimental analysis of the 44 such genes predicted to be Xoc TAL effector targets verified nearly half, and identified most others as false predictions. None of the Xoc targets is a known bacterial blight S gene. Mutational analysis revealed that Tal2g, which activates two genes, contributes to lesion expansion and bacterial exudation. Use of designer TAL effectors discriminated a sulfate transporter gene as the S gene. Across all targets, basal expression tended to be higher than genome-average, and induction moderate. Finally, machine learning applied to real vs. falsely predicted targets yielded a classifier that recalled 92% of the real targets with 88% precision, providing a tool for better target prediction in the future. Our study expands the number of known TAL effector targets, identifies a new class of S gene, and improves our ability to predict functional targeting

The Xanthomonas citri effector protein PthA interacts with citrus proteins involved in nuclear transport, protein folding and ubiquitination associated with DNA repair

P>Xanthomonas axonopodis pv. citri utilizes the type III effector protein PthA to modulate host transcription to promote citrus canker. PthA proteins belong to the AvrBs3/PthA family and carry a domain comprising tandem repeats of 34 amino acids that mediates protein-protein and protein-DNA interactions. We show here that variants of PthAs from a single bacterial strain localize to the nucleus of plant cells and form homo- and heterodimers through the association of their repeat regions. We hypothesize that the PthA variants might also interact with distinct host targets. Here, in addition to the interaction with alpha-importin, known to mediate the nuclear import of AvrBs3, we describe new interactions of PthAs with citrus proteins involved in protein folding and K63-linked ubiquitination. PthAs 2 and 3 preferentially interact with a citrus cyclophilin (Cyp) and with TDX, a tetratricopeptide domain-containing thioredoxin. In addition, PthAs 2 and 3, but not 1 and 4, interact with the ubiquitin-conjugating enzyme complex formed by Ubc13 and ubiquitin-conjugating enzyme variant (Uev), required for K63-linked ubiquitination and DNA repair. We show that Cyp, TDX and Uev interact with each other, and that Cyp and Uev localize to the nucleus of plant cells. Furthermore, the citrus Ubc13 and Uev proteins complement the DNA repair phenotype of the yeast Delta ubc13 and Delta mms2/uev1a mutants, strongly indicating that they are also involved in K63-linked ubiquitination and DNA repair. Notably, PthA 2 affects the growth of yeast cells in the presence of a DNA damage agent, suggesting that it inhibits K63-linked ubiquitination required for DNA repair.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[98/14138-2]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[00/10266-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[03/08316-5]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[07/06686-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESPConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

Expression patterns of the most significantly differentially expressed rice genes.

<p>Normalized least square means of signal intensities (y-axis) at 2, 4, 8, 24, and 96 h after inoculation (x-axis) with <i>X. oryzae</i> pv. oryzicola BLS256 (Xoc), <i>X. oryzae</i> pv. oryzae strain PXO99^A (Xoo) or mock control are plotted for the genes most significantly differentially expressed relative to mock uniquely in response to Xoc (Xoc only), uniquely in response to Xoo (Xoo only), similarly in response to Xoc and Xoo (Xoc and Xoo similarly), and differently in response to Xoc and Xoo (Xoc and Xoo differently). Where two probe sets correspond to the same gene, the one with the lower <i>q</i>-value was selected for display. Locus IDs are given at right, omitting the prefix “LOC_Os”. Results were derived from a mixed linear model analysis with four replicates. Vertical bars represent standard error. Asterisks mark previously identified targets of Xoo TAL effectors, <i>TFIIaγ1</i>(<i>Os01g73890</i>) and <i>OsSWEET11</i> (<i>Os08g42350</i>), activated by PthXo7 and PthXo1, respectively. Daggers flag Xoc TAL effector targets discovered in this study.</p

Type III secretion system dependence of the most significant rice gene expression changes.

<p>RT-PCR results reflecting transcript abundance are shown for rice genes identified by GeneChip expression analysis as the ten (or fewer) most significantly differentially expressed in response to (A) <i>X. oryzae</i> pv. oryzicola BLS256 (Xoc), (B) <i>X. oryzae</i> pv. oryzae strain PXO99^A (Xoo), (C) Xoc and Xoo similarly, or (D) Xoc and Xoo to different extents. Leaf samples were harvested at 36 hours after inoculation with wild-type strains or with the type III secretion (T3S⁻) deficient derivatives. RT-PCR results for previously reported Xoo-induced genes, <i>OsSWEET11</i> and <i>TFIIAγ1</i> <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003972#ppat.1003972-Yang4" target="_blank">[9]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003972#ppat.1003972-Sugio1" target="_blank">[10]</a>, are omitted. An actin gene (<i>Os04g57210</i>) that is not differentially expressed was used as a reference for relative transcript abundance across samples. The experiment was repeated twice and yielded the same results.</p

Rice transcriptional responses to <i>Xanthomonas oryzae</i> pv. oryzicola BLS256 (Xoc) or <i>X. oryzae</i> pv. oryzae PXO99^A (Xoo).

<p>Distribution of genes differentially expressed over a 96(see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003972#s4" target="_blank">Materials and Methods</a>) in response to either strain relative to a mock inoculation is shown. Each circle of the Venn diagram represents a different pairwise comparison of treatments, as indicated in non-bold text. Results are based on mixed linear model analysis using four biological replicates for each time point of the study and an estimated false discovery rate of 0.3. The intersections represent the genes differentially expressed uniquely in response to the different treatments, indicated in bold text. Note that differentially expressed uniquely in response to mock means differentially expressed similarly in Xoc and Xoo relative to mock, and differentially expressed uniquely in response to all three treatments means differentially expressed both in Xoc and Xoo relative to mock, but also differentially between Xoc and Xoo. Also, since differential expression in a given pairwise comparison is determined using a statistical cutoff, transitive predictions, i.e., A = B and B = C, therefore A = C, may not hold.</p

Expression levels of probesets associated with <i>X. oryzae</i> pv. oryzicola BLS256 (Xoc) TAL effector targets relative to other probesets.

<p>Individual box plots show average normalized expression values over time for probesets associated with verified (real) Xoc TAL effector targets, probesets associated with genes predicted but shown not to be targeted by an Xoc TAL effector (falsely predicted targets), all probesets differentially expressed (DE) in the mock vs. Xoc comparison at <i>q</i>≤0.3, or all probesets on the chip. The top row of plots shows data from mock-inoculated plants and the bottom row data from plants inoculated with Xoc. For each plot, the central bar indicates the median value and the top and bottom of the box indicate the 75^th percentile and the 25^th percentile, respectively. Whiskers indicate the most extreme data points above and below the median that are not outliers, calculated as ≤1.5*(75^th percentile – 25^th percentile) above the 75^th percentile or below the 25^th percentile. Outliers are plotted individually. Boxplots were made using the ‘boxplot()’ function of the statistical software package R (<a href="http://www.r-project.org" target="_blank">www.r-project.org</a>).</p

Determination of <i>Os01g52130</i> as the relevant target of Tal2g using designer TAL effectors.

<p>(A) DNA sequence of the promoter regions of Tal2g induced genes <i>Os06g46500</i> and <i>Os01g52130</i> in rice cv. Nipponbare. The effector binding elements (EBEs) for Tal2g are in bold. The EBEs for designer TAL effectors dT434 targeting <i>Os06g46500</i> and dT436 and dT437 targeting <i>Os01g52130</i> are underlined and labeled above. Periods indicate transcriptional start sites and italics indicate translational start sites, per the Rice Genome Annotation Project (Release 7, <a href="http://rice.plantbiology.msu.edu" target="_blank">http://rice.plantbiology.msu.edu</a>). (B) Activation of <i>Os06g46500</i> and <i>Os1g52130</i> by Tal2g, and specific activation respectively of <i>Os06g46500</i> and <i>Os01g52130</i> by dT434, and dT436 or dT437. Shown are the results of RT-PCR amplification from leaf RNA isolated 48 h after inoculation by infiltration with <i>X. oryzae</i> pv. oryzicola BLS256 (WT), the <i>tal2g</i> knockout derivative M27 carrying an empty plasmid vector (ev), M27 carrying the vector with the cloned <i>tal2g</i> gene, or M27 carrying the vector with coding sequences for dT436, dT436, or dT437 as indicated. The actin gene <i>Os04g57210</i> was used as a reference for relative transcript abundance across samples. (C) Rescue of the virulence defect of M27 by dT436 or dT437 but not dT434 in the lesion length assay. Lesion lengths were measured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003972#ppat-1003972-g004" target="_blank">Figure 4</a>, 10 days after inoculation with the indicated strains. Values labeled with the same letter are not significantly different and those labeled with different letters are (Student's <i>t</i>-test, p<0.01). Error bars represent standard deviation (N≥10). Experiments were repeated twice with consistent results. (D) A rice (cv. Nipponbare) leaf showing bacterial leaf streak symptoms two days after inoculation with a suspension of WT cells at an OD₆₀₀ of 0.5 (approximately 1×10⁸ CFU/ml) by infiltration using a needleless syringe over a 4 mm diameter leaf area, and labeled to indicate the site of inoculation, at which surface bacterial populations were quantified, and the three 12 mm long leaf sections in which total bacterial populations were quantified, as presented in panel E. (E) Restoration of the surface population and the total population spread of M27 to wild-type levels by dTAL437 but not dTAL434. Populations were quantified at 2, 5, 8 and 11 days after inoculation. Results are the means and standard deviations of samples from three leaves; nd, not detected. At each time point (not across time points), values labeled with the same letter are not significantly different, and those labeled with different letters are (Student's <i>t</i>-test <i>p</i><0.0001).</p