Workflow of the study.

<p>In grey, the procedure used to select candidate effector genes. Transcriptomic data from sunflower leaves infected by <i>Pl</i>. <i>halstedii</i> or isolated zoospores were analyzed by PSI-tBLASTn using the annotated sequences that were available in NCBI in March 2010 as models for RXLR and CRN effectors. In white, the procedure used to build the <i>Pl</i>. <i>halstedii</i> pathotype determination key. Identification of effector polymorphism was done by comparisons between genomic sequences obtained in 7 representative pathogen pathotypes (100, 300, 304, 334, 700, 703 and 710). Among the 22 <u>K</u>Bioscience <u>C</u>ompetitive <u>A</u>llele <u>S</u>pecific <u>P</u>CR (KASP) markers, eight were used in a determination key to discriminate <i>Pl</i>. <i>halstedii</i> pathotypes.</p

Polymorphism analysis of <i>Pl</i>. <i>halstedii</i> effector and non-effector genes.

<p>(A) Mean nucleotide diversity (π) calculated on non-effector genes (black bar) and effector genes (grey bar). Π values were calculated on DnaSP v5 software. Error bars represent two SEM. (B, C) Comparisons of polymorphism distributions (represented by SNP sparseness, i.e. minimum average distance between two polymorphisms) in <i>Pl</i>. <i>halstedii</i> effector (grey) and non-effector genes (black). (B) Frequency of genes in each class of index. (C) Frequency of predicted peptides in each class of SNP sparseness, among genes with nucleotide polymorphism.</p

Identification key for <i>Pl</i>. <i>halstedii</i> pathotypes using KASP markers designed on effector gene SNPs.

<p>First level (Key 1) separated the 14 <i>Pl</i>. <i>halstedii</i> pathotypes in 5 groups with 3 markers. Second level (Key 2) used five other markers to distinguish 12 subgroups of pathotypes, and especially 6 subgroups of multi-isolate pathotypes (Red boxes). 100 and 304, 314 and 714 pathotypes could not be distinguished. ¹<i>Pl</i>. <i>halstedii</i> pathotypes with only one isolate available.</p

<i>Plasmopara halstedii</i> geographical isolates used in this study.

<p><i>Plasmopara halstedii</i> geographical isolates used in this study.</p

Genotypes of 14 reference isolates (_ref) and 21 geographical isolates of <i>Pl</i>. <i>halstedii</i> for 22 KASP markers based on effector gene SNPs.

<p>The DNA base involved in polymorphism is indicated by a specific colour. Heterozygous DNA bases are separated with a slash. (–) sign corresponds to the absence of the indel (versus T in PhRXLR58_1). *Genomic sequences of effectors available for these pathotypes (S1_File). Not determined results: nd.</p

Data_Sheet_1_Investigating genetic diversity within the most abundant and prevalent non-pathogenic leaf-associated bacteria interacting with Arabidopsis thaliana in natural habitats.zip

Microbiota modulates plant health and appears as a promising lever to develop innovative, sustainable and eco-friendly agro-ecosystems. Key patterns of microbiota assemblages in plants have been revealed by an extensive number of studies based on taxonomic profiling by metabarcoding. However, understanding the functionality of microbiota is still in its infancy and relies on reductionist approaches primarily based on the establishment of representative microbial collections. In Arabidopsis thaliana, most of these microbial collections include one strain per OTU isolated from a limited number of habitats, thereby neglecting the ecological potential of genetic diversity within microbial species. With this study, we aimed at estimating the extent of genetic variation between strains within the most abundant and prevalent leaf-associated non-pathogenic bacterial species in A. thaliana located south-west of France. By combining a culture-based collection approach consisting of the isolation of more than 7,000 bacterial colonies with an informative-driven approach, we isolated 35 pure strains from eight non-pathogenic bacterial species. We detected significant intra-specific genetic variation at the genomic level and for growth rate in synthetic media. In addition, significant host genetic variation was detected in response to most bacterial strains in in vitro conditions, albeit dependent on the developmental stage at which plants were inoculated, with the presence of both negative and positive responses on plant growth. Our study provides new genetic and genomic resources for a better understanding of the plant-microbe ecological interactions at the microbiota level. We also highlight the need of considering genetic variation in both non-pathogenic bacterial species and A. thaliana to decipher the genetic and molecular mechanisms involved in the ecologically relevant dialog between hosts and leaf microbiota.</p

Tree of the amino acid sequences of C-domains of strains GPE PC73, XaS3, X11-5A, BAI3, and BLS256 together with C-domains identified by Rausch et al. as starter C-domains or as dual C/E-domains (Additional file 2).

The tree was constructed using the maximum likelihood method and GTR as substitution model. Bootstrap percentages retrieved in 100 replications are shown at the main nodes. The scale bar (0.2) indicates the number of amino acid substitutions per site. C-domains belonging to the same clade as dual C/E-domains are in blue. C-domains belonging to the same clade as starter C-domains are in red. Putative starter C-domains of the loci META-A and META-C of strain GPE PC73, the contig G111 of strain XaS3 and the locus of strain BTAi similar to META-A and META-C are in green. C-domains Ax, Bx ad Cx correspond to C-domains of modules of the loci META-A, META-B and META-C of strain GPE PC73, respectively. C-domains Ox correspond to C-domains of modules of the locus META-B of strain BAI3. C-domains USxxx/x correspond to C-domains of modules of contigs of strain X11-5A. C-domains Gxxx/x correspond to C-domains of modules of contigs of strain XaS3. C-domains bradyx correspond to C-domains of the locus of Bradyrhizobium spp. strain BTAi similar to META-A and META-C (genes Bbta_6814, Bbta_6813, Bbta_6812). C-domains XOCx correspond to C-domains of the locus NRPS located in the same region as XaPPTase in strain BLS256. C-domains 0364 and 1145 correspond to C-domains of short NRPS genes XALc_0364 and XALc_1145 of strain GPE PC73, respectively. C-domain 0354XaS3 corresponds to the short NRPS gene of strain XaS3. C-domain Bbta4110 corresponds to the short NRPS gene of strain BTAi. C-domains identified by Rausch et al. [6] as starter C-domains were tagged “Starter1” to “Starter15” as follows: Starter1: Pseusyrin.NP_792633.1.m_1_leu Starter2: Pseusp.Q84BQ6.arfA_1_leu Starter3: Pseufluor.YP_259252.1.m_1_leu Starter4: Baciliche.YP_077640.1.lchAA_1_gln Starter5: Nocafarci.YP_117314.1.m_1_orn_lys_arg Starter6: Nocafarci.YP_119006.1.m_1_tyr Starter7: Nocafarci.YP_119328.1.m_1_ser Starter8: Nocafarci.YP_121279.1.m_1_ser Starter9: Strecoeli.NP_627443.1.m_1_ser Starter10: Strchrys.O68487.acmB_1_thr Starter11: Erwicarot.YP_049593.1.m_1_gln Starter12: Strprist.Q54959.snbC_1_thr Starter13: Bacisubti.NP_388230.1.srfAA_1_glu Starter14: Bacisubti.NP_389716.1.ppsA_1_glu Starter15: Baciliche.YP_090052.1.m_1_gln C-domains identified by Rausch et al. [6] as Dual C/E-domains were tagged “DualC/E1” to “DualC/E18” as follows: DualC/E1:Photlumin.NP_929905.1.m_9_thr_TO_val DualC/E2:Photlumin.NP_930489.1.m_2_val_TO_trp DualC/E3:Photlumin.NP_929905.1.m_6_bht_TO_trp DualC/E4:Bradjapon.NP_768748.1.m_3_ser_TO_phe DualC/E5:Chroviola.NP_902472.1.m_3_val_TO_ile_dual DualC/E6:Chroviola.NP_902472.1.m_1_thr_dual DualC/E7:Burkmalle.YP_106216.1.m_2_glu_TO_gly DualC/E8:Burkpseud.YP_111641.1.m_3_thr_TO_leu DualC/E9:Burkpseud.YP_111641.1.m_1_glu_gln DualC/E10:Pseusyrin.NP_792633.1.m_2_leu_TO_leu DualC/E11:Ralssolan.NP_522203.1.m_3_ser_TO_gly DualC/E12:Ralssolan.NP_522203.1.m_1_val DualC/E13:Pseufluor.YP_259253.1.m_4_leu_TO_ser DualC/E14:Pseufluor.YP_259253.1.m_2_thr_TO_ile DualC/E15:Pseusyrin.NP_792634.1.m_3_thr_TO_val DualC/E16:Pseusyrin.NP_792634.1.m_5_leu_TO_leu DualC/E17:Erwicarot.YP_049592.1.m_4_ser_TO_tyr_bht DualC/E18:Erwicarot.YP_049593.1.m_2_gln_TO_as

Circular representation of the genome sequence of <i>Xam</i> CIO151.

<p>From outside to inside: first circle in blue indicates CDS predicted in the positive strands for the scaffolds classified as probable chromosomal regions. Second circle in red indicates the CDS predicted in the negative strand. Red spots in the black third circle indicate the region identified with atypical nucleotide composition. The fourth circle indicates the deviation pattern from the average G+C content. Inner circle shows GC skew values, positive values are shown in purple and negative values are shown in orange. Numbers correspond to scaffold IDs.</p

Phylogeny of conserved effectors in the genus <i>Xanthomonas</i>.

<p>Phylogenetic tree of concatenated conserved effector protein sequences of AvrBs2, XopK, XopL, XopN, XopQ XopR families and the Hpa1 protein, obtained with a Bayesian approach. Numbers on branches indicate Bayesian support values. Length of branches indicates the number of amino acid substitutions per site.</p

Organization of pathogenicity-related gene clusters in the <i>Xam</i> CIO151 genome.

<p>Open arrows with labels indicate genes with assigned functions, black arrows indicate genes with early stop codons, open arrows without labels indicate conserved hypothetical proteins and grey arrows indicate non-conserved hypothetical proteins. Graphs above clusters show the G+C content and deviations from the average value. <b>A</b>. Xanthomonadin gene cluster; * indicates genes related to <i>pigB</i> genomic region and ** indicates genes reported as important for cluster functionality. Abbreviations used are: <b>H</b> = halogenase (xanmn_chr15_0075), <b>BP</b> = xanthomonadin biosynthesis protein (xanmn_chr15_0079), <b>E = </b>xanthomonadin exporter (xanmn_chr15_0373), <b>PSP</b> = putative secreted protein (xanmn_chr15_0080), <b>BACPD</b> = xanthomonadin biosynthesis acyl carrier protein dehydratase (xanmn_chr15_0082), <b>BA</b> = putative xanthomonadin biosynthesis acyltransferase (xanmn_chr15_0081 and xanmn_chr15_0083), <b>BMP</b> = putative xanthomonadin biosynthesis membrane protein (xanmn_chr15_0084), <b>ACP</b> = acyl carrier protein (xanmn_chr15_0085), <b>XanB1</b> = putative reductase/halogenase (xanmn_chr15_0086), <b>XanB2</b> = putative pteridine-dependent deoxygenase like protein (xanmn_chr15_0087), <b>AMP-l</b> = AMP-ligase (xanmn_chr15_0088), <b>DP</b> = dipeptidyl peptidase (xanmn_chr15_0090). <b>B</b>. Cluster implicated in xanthan production (<i>gum</i>). <b>C</b>. Regulation of pathogenicity factors (<i>rpf</i>) cluster. <b>D</b>. Type III secretion system (<b>T3SS</b>) cluster.</p