A Comprehensive Overview of the Genes and Functions Required for Lettuce Infection by the Hemibiotrophic Phytopathogen Xanthomonas hortorum pv. vitians

Abstract

The successful infection of a host plant by a phytopathogenic bacterium depends on a finely tuned molecular cross talk between the two partners. Thanks to transposon insertion sequencing techniques (Tn-seq), whole genomes can now be assessed to determine which genes are important for the fitness of several plant-associated bacteria in planta. Despite its agricultural relevance, the dynamic molecular interaction established between the foliar hemibiotrophic phytopathogen Xanthomonas hortorum pv. vitians and its host, lettuce (Lactuca sativa), remains completely unknown. To decipher the genes and functions mobilized by the pathogen throughout the infection process, we conducted a Tn-seq experiment in lettuce leaves to mimic the selective pressure occurring during natural infection. This genome-wide screening identified 170 genes whose disruption caused serious fitness defects in lettuce. A thorough examination of these genes using comparative genomics and gene set enrichment analyses highlighted that several functions and pathways were highly critical for the pathogen's survival. Numerous genes involved in amino acid, nucleic acid, and exopolysaccharide biosynthesis were critical. The xps type II secretion system operon, a few TonB-dependent transporters involved in carbohydrate or siderophore scavenging, and multiple genes of the carbohydrate catabolism pathways were also critical, emphasizing the importance of nutrition systems in a nutrient-limited environment. Finally, several genes implied in camouflage from the plant immune system and resistance to immunity-induced oxidative stress were strongly involved in host colonization. As a whole, these results highlight some of the central metabolic pathways and cellular functions critical for Xanthomonas host adaptation and pathogenesis. IMPORTANCE Xanthomonas hortorum was recently the subject of renewed interest, as several studies highlighted that its members were responsible for diseases in a wide range of plant species, including crops of agricultural relevance (e.g., tomato and carrot). Among X. hortorum variants, X. hortorum pv. vitians is a reemerging foliar hemibiotrophic phytopathogen responsible for severe outbreaks of bacterial leaf spot of lettuce all around the world. Despite recent findings, sustainable and practical means of disease control remain to be developed. Understanding the host-pathogen interaction from a molecular perspective is crucial to support these efforts. The genes and functions mobilized by X. hortorum pv. vitians during its interaction with lettuce had never been investigated. Our study sheds light on these processes by screening the whole pathogen genome for genes critical for its fitness during the infection process, using transposon insertion sequencing and comparative genomics.

Keywords: Tn-seq; Xanthomonas hortorum; comparative genomics; lettuce; phytopathogen.

FIG 1

Validation of the experimental design and bottleneck assessment. (a) Evolution of the X. hortorum pv. vitians LM16734 population during lettuce infection, expressed as log₁₀-transformed CFU · cm⁻² of inoculated leaf. Red triangles, measured epiphytic populations following spray inoculation; green triangles, measured endophytic populations at each time point; gray bars, standard errors calculated on triplicates. (b and c) Read count correlation among Tn-seq duplicates for the X. hortorum pv. vitians LM16734 chromosome in (b) the inoculum and (c) lettuce leaves. (d and e) Volcano plots of the resampling results in (d) the inoculum versus the in vitro library condition and (e) lettuce leaves versus the inoculum condition. Plots represent the log₂ fold change of each genetic feature as a function of the negative log₁₀ of the resampling test P value. Genetic features that passed the FDR adjustment test (q value of ≤0.05) are indicated in red if they are conditionally essential and in blue if they provide a growth advantage. Nonsignificant genetic features are in gray.

FIG 2

Genome atlas of the critical genetic features required for lettuce infection by X. hortorum pv. vitians LM16734. From the inner to outer track are the mean read count over 1 kb in 1/10^th TSB and in lettuce, the gene state in the in vitro library, as described in reference , the log₂ FC score in lettuce leaves compared to the inoculum, clustering of critical genes, pathways, functions and structural components in lettuce, gene names or locus tag identifiers, and chromosomal tracks.

FIG 3

Schematic representation of (a) the critical genes in lettuce within the xps T2SS operon and (b) the LPS biosynthesis gene cluster of X. hortorum pv. vitians LM16734. Numbers in brackets indicate the width of the viewing window on the LM16734 genome. Green arrows, critical genes in lettuce; gold arrows, critical genes in 1/10th TSB; red arrows, essential genes in the in vitro library; gray arrows, nonsignificant genes. Gene names, log₂ FC values, and—for panel b—grouping in functional regions as described in reference are displayed below. Black bars, localizations of TA sites; bar plots, read counts at each TA site under the control and experimental conditions. (Values of >1,000 are not displayed.)

FIG 4

Schematic overview of the critical genes and processes required for lettuce infection by X. hortorum pv. vitians LM16734 as discussed in this work. Gray objects are known important virulence factors not screened as critical but discussed in the text. Abbreviations: EPS, exopolysaccharide; T2SS, type II secretion system; Glc, glucose; Fru, fructose; MeGX₃, methylglucuronoxylotriose; ROS, reactive oxygen species; T3SS, type III secretion system; T3Es, type III effectors; LPS, lipopolysaccharide; PRRs, pattern recognition receptors.