A local score approach improves GWAS resolution and detects minor QTL: application to Medicago truncatula quantitative disease resistance to multiple Aphanomyces euteiches isolates.

Quantitative trait loci (QTL) with small effects, which are pervasive in quantitative phenotypic variation, are difficult to detect in genome-wide association studies (GWAS). To improve their detection, we propose to use a local score approach that accounts for the surrounding signal due to linkage disequilibrium, by accumulating association signals from contiguous single markers. Simulations revealed that, in a GWAS context with high marker density, the local score approach outperforms single SNP p-value-based tests for detecting minor QTL (heritability of 5-10%) and is competitive with regard to alternative methods, which also aggregate p-values. Using more than five million SNPs, this approach was applied to identify loci involved in Quantitative Disease Resistance (QDR) to different isolates of the plant root rot pathogen Aphanomyces euteiches, from a GWAS performed on a collection of 174 accessions of the model legume Medicago truncatula. We refined the position of a previously reported major locus, underlying MYB/NB-ARC/tyrosine kinase candidate genes conferring resistance to two closely related A. euteiches isolates belonging to pea pathotype I. We also discovered a diversity of minor resistance QTL, not detected using p-value-based tests, some of which being putatively shared in response to pea (pathotype I and III) and/or alfalfa (race 1 and 2) isolates. Candidate genes underlying these QTL suggest pathogen effector recognition and plant proteasome as key functions associated with M. truncatula resistance to A. euteiches. GWAS on any organism can benefit from the local score approach to uncover many weak-effect QTL.

Two ancestral genes shaped the Xanthomonas campestris TAL effector gene repertoire.

Xanthomonas transcription activator-like effectors (TALEs) are injected inside plant cells to promote host susceptibility by enhancing transcription of host susceptibility genes. TALE-encoding (tal) genes were thought to be absent from Brassicaceae-infecting Xanthomonas campestris (Xc) genomes based on four reference genomic sequences. We discovered tal genes in 26 of 49 Xc strains isolated worldwide and used a combination of single molecule real time (SMRT) and tal amplicon sequencing to yield a near-complete description of the TALEs found in Xc (Xc TALome). The 53 sequenced tal genes encode 21 distinct DNA binding domains that sort into seven major DNA binding specificities. In silico analysis of the Brassica rapa promoterome identified a repertoire of predicted TALE targets, five of which were experimentally validated using quantitative reverse transcription polymerase chain reaction. The Xc TALome shows multiple signs of DNA rearrangements that probably drove its evolution from two ancestral tal genes. We discovered that Tal12a and Tal15a of Xcc strain Xca5 contribute together in the development of disease symptoms on susceptible B. oleracea var. botrytis cv Clovis. This large and polymorphic repertoire of TALEs opens novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease and for understanding the molecular processes underlying TALE evolution.

Genetic Characterization of 'Candidatus Liberibacter solanacearum' Haplotypes Associated with Apiaceous Crops in France.

'Candidatus Liberibacter solanacearum' (Lso) is an emerging phytopathogenic bacterium that causes significant crop losses worldwide. This bacterium has been identified in association with diseases of several solanaceous crops in the United States and New Zealand, and with carrot and celery crops in several European countries. Five Lso haplotypes (LsoA, LsoB, LsoC, LsoD, and LsoE) have now been described worldwide. In France, symptoms of Lso were observed on plants of the Apiaceae family in several regions. One hundred and ninety-two samples of apiaceous plants were collected from 2012 to 2016 in different geographical regions and were tested for the occurrence of Lso by real-time PCR assay. In addition to carrot and celery, Lso was detected in four other apiaceous crops: chervil, fennel, parsley, and parsnip. These new findings suggest that Lso has a wider natural host range within the Apiaceae family than expected. To identify the Lso haplotypes present in France, we sequenced and analyzed the 16S rRNA gene and the 50S ribosomal protein rpIJ-rpIL gene region from a representative bacterial collection of 44 Lso-positive samples. Our SNP analysis revealed the occurrence of two distinct bacterial lineages that correspond to haplotypes D and E. Then, we assessed the phylogenetic relationships between strains isolated from France and a worldwide collection of Lso isolates by using the rpIJ-rpIL gene region sequences. The neighbor-joining tree constructed delineated five clusters corresponding to the five Lso haplotypes, with LsoD and LsoE being closely related phylogenetically. Altogether, the data presented here constitute a first step toward a better understanding of the genetic diversity among Lso haplotypes in France, and provide new insights into the host range of this emerging bacterial species.

High-density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance to Aphanomyces euteiches.

• The use of quantitative disease resistance (QDR) is a promising strategy for promoting durable resistance to plant pathogens, but genes involved in QDR are largely unknown. To identify genetic components and accelerate improvement of QDR in legumes to the root pathogen Aphanomyces euteiches, we took advantage of both the recently generated massive genomic data for Medicago truncatula and natural variation of this model legume. • A high-density (≈5.1 million single nucleotide polymorphisms (SNPs)) genome-wide association study (GWAS) was performed with both in vitro and glasshouse phenotyping data collected for 179 lines. • GWAS identified several candidate genes and pinpointed two independent major loci on the top of chromosome 3 that were detected in both phenotyping methods. Candidate SNPs in the most significant locus (σ(A)²= 23%) were in the promoter and coding regions of an F-box protein coding gene. Subsequent qRT-PCR and bioinformatic analyses performed on 20 lines demonstrated that resistance is associated with mutations directly affecting the interaction domain of the F-box protein rather than gene expression. • These results refine the position of previously identified QTL to specific candidate genes, suggest potential molecular mechanisms, and identify new loci explaining QDR against A. euteiches.

Type three effector gene distribution and sequence analysis provide new insights into the pathogenicity of plant-pathogenic Xanthomonas arboricola.

Xanthomonas arboricola is a complex bacterial species which mainly attacks fruit trees and is responsible for emerging diseases in Europe. It comprises seven pathovars (X. arboricola pv. pruni, X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. populi, X. arboricola pv. poinsettiicola, X. arboricola pv. celebensis, and X. arboricola pv. fragariae), each exhibiting characteristic disease symptoms and distinct host specificities. To better understand the factors underlying this ecological trait, we first assessed the phylogenetic relationships among a worldwide collection of X. arboricola strains by sequencing the housekeeping gene rpoD. This analysis revealed that strains of X. arboricola pathovar populi are divergent from the main X. arboricola cluster formed by all other strains. Then, we investigated the distribution of 53 type III effector (T3E) genes in a collection of 57 X. arboricola strains that are representative of the main X. arboricola cluster. Our results showed that T3E repertoires vary greatly between X. arboricola pathovars in terms of size. Indeed, X. arboricola pathovars pruni, corylina, and juglandis, which are responsible for economically important stone fruit and nut diseases in Europe, harbored the largest T3E repertoires, whereas pathovars poinsettiicola, celebensis, and fragariae harbored the smallest. We also identified several differences in T3E gene content between X. arboricola pathovars pruni, corylina, and juglandis which may account for their differing host specificities. Further, we examined the allelic diversity of eight T3E genes from X. arboricola pathovars. This analysis revealed very limited allelic variations at the different loci. Altogether, the data presented here provide new insights into the evolution of pathogenicity and host range of X. arboricola and are discussed in terms of emergence of new diseases within this bacterial species.

Reducing patient waiting time and length of stay in an Acute Care Pediatric Emergency Department.

Prolonged waiting times and length of stay in Pediatric Emergency Department, are the two of the most challenging patient and clinical outcomes of healthcare institution. These emerged due to various reasons, namely: the use of triaging process and patient flow criteria that eventually lead to bottlenecks and overcrowding in the ED. After realizing the root causes of the prolonged waiting times and length of stay, the KASCH ED instigated a team to study the factors and thereby arrive at a considerable conclusion that will result in an improvement. The quality improvement project was initiated and steps were undertaken to improve the flow, reduce the waiting times, and reduce the overcrowding in Pediatric Emergency Acute Care Unit. The primary cause identified was inadequate team awareness and lack of the ED process flow, thus creating confusion as to where the type of patients based on the triage level will be assessed, managed and treated. Using the Canadian Triage and Acuity Scale (CTAS) as guide in triaging patients, a theory called Pediatric Rapid Assessment and Management (PRAM) was introduced in the Acute Care Unit. This certain model is basically aimed to rapidly assess and managed the patients who were triaged as Level III and Level IV within a period of 30 minutes. Several PDSA cycles were tested and implemented in order to assure that the process fit the criteria and the process flow will be improved. Following the completion of each cycle, significant improvements were noted, such as patients being assessed in Initial Assessment Room on average time less than the target of 15 minutes. In like manner, patients' length of stay on average less than 15 minutes in PRAM bed. The total time for assessment and plan of management is with a target time of less than 30 minutes. The team continuously drive th process and monitored the key performance indicators of the PRAM during the study period and subsequent improvement strategies were likewise implemented.

Variations in type III effector repertoires, pathological phenotypes and host range of Xanthomonas citri pv. citri pathotypes.

The mechanisms determining the host range of Xanthomonas are still undeciphered, despite much interest in their potential roles in the evolution and emergence of plant pathogenic bacteria. Xanthomonas citri pv. citri (Xci) is an interesting model of host specialization because of its pathogenic variants: pathotype A strains infect a wide range of Rutaceous species, whereas pathotype A*/A(W) strains have a host range restricted to Mexican lime (Citrus aurantifolia) and alemow (Citrus macrophylla). Based on a collection of 55 strains representative of Xci worldwide diversity assessed by amplified fragment length polymorphism (AFLP), we investigated the distribution of type III effectors (T3Es) in relation to host range. We examined the presence of 66 T3Es from xanthomonads in Xci and identified a repertoire of 28 effectors, 26 of which were shared by all Xci strains, whereas two (xopAG and xopC1) were present only in some A*/A(W) strains. We found that xopAG (=avrGf1) was present in all A(W) strains, but also in three A* strains genetically distant from A(W) , and that all xopAG-containing strains induced the hypersensitive response (HR) on grapefruit and sweet orange. The analysis of xopAD and xopAG suggested horizontal transfer between X. citri pv. bilvae, another citrus pathogen, and some Xci strains. A strains were genetically less diverse, induced identical phenotypic responses and possessed indistinguishable T3E repertoires. Conversely, A*/A(W) strains exhibited a wider genetic diversity in which clades correlated with geographical origin and T3E repertoire, but not with pathogenicity, according to T3E deletion experiments. Our data outline the importance of taking into account the heterogeneity of Xci A*/A(W) strains when analysing the mechanisms of host specialization.

Evolutionary history of the plant pathogenic bacterium Xanthomonas axonopodis.

Deciphering mechanisms shaping bacterial diversity should help to build tools to predict the emergence of infectious diseases. Xanthomonads are plant pathogenic bacteria found worldwide. Xanthomonas axonopodis is a genetically heterogeneous species clustering, into six groups, strains that are collectively pathogenic on a large number of plants. However, each strain displays a narrow host range. We address the question of the nature of the evolutionary processes--geographical and ecological speciation--that shaped this diversity. We assembled a large collection of X. axonopodis strains that were isolated over a long period, over continents, and from various hosts. Based on the sequence analysis of seven housekeeping genes, we found that recombination occurred as frequently as point mutation in the evolutionary history of X. axonopodis. However, the impact of recombination was about three times greater than the impact of mutation on the diversity observed in the whole dataset. We then reconstructed the clonal genealogy of the strains using coalescent and genealogy approaches and we studied the diversification of the pathogen using a model of divergence with migration. The suggested scenario involves a first step of generalist diversification that spanned over the last 25,000 years. A second step of ecology-driven specialization occurred during the past two centuries. Eventually, secondary contacts between host-specialized strains probably occurred as a result of agricultural development and intensification, allowing genetic exchanges of virulence-associated genes. These transfers may have favored the emergence of novel pathotypes. Finally, we argue that the largest ecological entity within X. axonopodis is the pathovar.

Natural genetic variation of Xanthomonas campestris pv. campestris pathogenicity on arabidopsis revealed by association and reverse genetics.

ABSTRACT The pathogenic bacterium Xanthomonas campestris pv. campestris, the causal agent of black rot of Brassicaceae, manipulates the physiology and the innate immunity of its hosts. Association genetic and reverse-genetic analyses of a world panel of 45 X. campestris pv. campestris strains were used to gain understanding of the genetic basis of the bacterium's pathogenicity to Arabidopsis thaliana. We found that the compositions of the minimal predicted type III secretome varied extensively, with 18 to 28 proteins per strain. There were clear differences in aggressiveness of those X. campestris pv. campestris strains on two Arabidopsis natural accessions. We identified 3 effector genes (xopAC, xopJ5, and xopAL2) and 67 amplified fragment length polymorphism (AFLP) markers that were associated with variations in disease symptoms. The nature and distribution of the AFLP markers remain to be determined, but we observed a low linkage disequilibrium level between predicted effectors and other significant markers, suggesting that additional genetic factors make a meaningful contribution to pathogenicity. Mutagenesis of type III effectors in X. campestris pv. campestris confirmed that xopAC functions as both a virulence and an avirulence gene in Arabidopsis and that xopAM functions as a second avirulence gene on plants of the Col-0 ecotype. However, we did not detect the effect of any other effector in the X. campestris pv. campestris 8004 strain, likely due to other genetic background effects. These results highlight the complex genetic basis of pathogenicity at the pathovar level and encourage us to challenge the agronomical relevance of some virulence determinants identified solely in model strains. IMPORTANCE The identification and understanding of the genetic determinants of bacterial virulence are essential to be able to design efficient protection strategies for infected plants. The recent availability of genomic resources for a limited number of pathogen isolates and host genotypes has strongly biased our research toward genotype-specific approaches. Indeed, these do not consider the natural variation in both pathogens and hosts, so their applied relevance should be challenged. In our study, we exploited the genetic diversity of Xanthomonas campestris pv. campestris, the causal agent of black rot on Brassicaceae (e.g., cabbage), to mine for pathogenicity determinants. This work evidenced the contribution of known and unknown loci to pathogenicity relevant at the pathovar level and identified these virulence determinants as prime targets for breeding resistance to X. campestris pv. campestris in Brassicaceae.

Multilocus sequence analysis and type III effector repertoire mining provide new insights into the evolutionary history and virulence of Xanthomonas oryzae.

Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.

A "repertoire for repertoire" hypothesis: repertoires of type three effectors are candidate determinants of host specificity in Xanthomonas.

BACKGROUND: The genetic basis of host specificity for animal and plant pathogenic bacteria remains poorly understood. For plant pathogenic bacteria, host range is restricted to one or a few host plant species reflecting a tight adaptation to specific hosts. METHODOLOGY/PRINCIPAL FINDINGS: Two hypotheses can be formulated to explain host specificity: either it can be explained by the phylogenetic position of the strains, or by the association of virulence genes enabling a pathological convergence of phylogenically distant strains. In this latter hypothesis, host specificity would result from the interaction between repertoires of bacterial virulence genes and repertoires of genes involved in host defences. To challenge these two hypotheses, we selected 132 Xanthomonas axonopodis strains representative of 18 different pathovars which display different host range. First, the phylogenetic position of each strain was determined by sequencing the housekeeping gene rpoD. This study showed that many pathovars of Xanthomonas axonopodis are polyphyletic. Second, we investigated the distribution of 35 type III effector genes (T3Es) in these strains by both PCR and hybridization methods. Indeed, for pathogenic bacteria T3Es were shown to trigger and to subvert host defences. Our study revealed that T3E repertoires comprise core and variable gene suites that likely have distinct roles in pathogenicity and different evolutionary histories. Our results showed a correspondence between composition of T3E repertoires and pathovars of Xanthomonas axonopodis. For polyphyletic pathovars, this suggests that T3E genes might explain a pathological convergence of phylogenetically distant strains. We also identified several DNA rearrangements within T3E genes, some of which correlate with host specificity of strains. CONCLUSIONS/SIGNIFICANCE: These data provide insight into the potential role played by T3E genes for pathogenic bacteria and support a "repertoire for repertoire" hypothesis that may explain host specificity. Our work provides resources for functional and evolutionary studies aiming at understanding host specificity of pathogenic bacteria, functional redundancy between T3Es and the driving forces shaping T3E repertoires.