A β-glucuronidase (GUS) Based Bacterial Competition Assay to Assess Fine Differences in Fitness during Plant Infection

Luneau, Julien; Noël, Laurent; Lauber, Emmanuelle; Boulanger, Alice

doi:10.21769/bioprotoc.3776

Cited by 2 publications

(2 citation statements)

References 15 publications

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“…We mixed the Xcc strain 8004∷GUS*‐GFP* (hereafter WT*) and deletion mutants in the 8004∷GUS‐GFP background to assess the fitness of the latter as previously described (Luneau et al ., 2022b). Briefly, we inoculated cauliflower hydathodes with the same protocol as for the RB‐TnSeq assay except that we adjusted the inoculum suspensions to OD 600 = 0.05 for each strain (final OD 600 = 0.1) in 1 mM MgCl 2 + 0.5% (v/v) Tween 80.…”

Section: Methodsmentioning

confidence: 99%

“…The Xcc 8004∷GUS‐GFP strain (Cerutti et al ., 2017) was used as a recipient for the RB‐TnSeq library and all deletion mutants (Supporting Information Table S1). For competition assays, we used the Xcc 8004::GUS*‐GFP* variant, which contains point mutations inactivating the catalytic sites of both reporter proteins as described (Luneau et al ., 2022b). Xcc was cultivated in MOKA rich medium (Blanvillain et al ., 2007), MME glucose‐poor medium (Arlat et al ., 1991) or MME glucose complemented with dropout – leucine/tryptophan/histidine (LTH) supplements instead of casamino acids (10.5 g l −1 K 2 HPO 4 , 4.5 g l −1 KH 2 PO 4 , 1 g l −1 (NH 4 ) 2 SO 4 , 1 mM MgSO 4 , 0.15% (w/v) DropOut‐LTH, 20 mM glucose) at 28°C under agitation at 200 rpm or on MOKA‐agar plates.…”

Section: Methodsmentioning

confidence: 99%

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Genome‐wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection

et al. 2022

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Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages.Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media.Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation.This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genome‐wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection

et al. 2022

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Genome-wide identification of fitness determinants in theXanthomonas campestrisbacterial pathogen during early stages of plant infection

Luneau

Baudin

Quiroz-Monnens

et al. 2022

Preprint

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Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv. campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. 183 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism when only few genes known to be involved in virulence were found to be affected. The biological relevance of 13 genes was independently confirmed by phenotyping single mutants. Notably, we show that the XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study thus revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.

show abstract

A β-glucuronidase (GUS) Based Bacterial Competition Assay to Assess Fine Differences in Fitness during Plant Infection

Cited by 2 publications

References 15 publications

Genome‐wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection

Genome‐wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection

Genome-wide identification of fitness determinants in theXanthomonas campestrisbacterial pathogen during early stages of plant infection

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