Xanthomonas albilineans is able to move outside of the sugarcane xylem despite its reduced genome and the absence of a Hrp type III secretion system.

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, is a pathogen that experienced genome reduction during its speciation. Additionally, this xanthomonad is notably missing the Hrp type III secretion system and the xanthan gene cluster that are commonly found in pathogenic Xanthomonas species. X. albilineans was up to now considered as limited to the xylem of sugarcane. However, recently published studies suggested that X. albilineans was able to invade tissues other than the xylem of sugarcane leaves but the occurrence of X. albilineans outside the xylem has not been clearly proven. In this study, we used confocal microscopy and transmission electron microscopy to investigate the localization of this pathogen in diseased leaves and stalks of sugarcane. Three sugarcane cultivars with different levels of resistance to leaf scald were inoculated with the green fluorescent protein labelled X. albilineans strains XaFL07-1 (from Florida) and GPE PC73 (from Guadeloupe). Sections of sugarcane leaves and stalks were examined 8-60 days after inoculation in order to localize X. albilineans in the different plant tissues. Confocal microscopy observation of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and the metaxylem, however, X. albilineans was also observed in the phloem, the parenchyma and the bulliform cells of the leaves. Similarly, the protoxylem and the metaxylem of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in the intercellular spaces between these cells. Several of these observations made by confocal microscopy have been confirmed by transmission electron microscopy. X. albilineans can therefore no longer be considered as a xylem-limited pathogen. To our knowledge, this is the first description of a plant pathogenic bacterium invading apparently intact non-vascular plant tissue and multiplying in parenchyma cells. The mechanisms and virulence factors used by X. albilineans to enter and invade different tissues of sugarcane remain to be identified. (Résumé d'auteur

In planta localization of Xanthomonas albilineans and identification of molecular factors involved in epiphytic colonization of its host plant, the sugarcane.

Xanthomonas albilineans est l'agent causal de l'échaudure des feuilles, une des principales maladies bactériennes de la canne à sucre dont l'impact peut être très important lorsque des variétés sensibles sont infectées au champ. Les mécanismes qui régissent les interactions entre cet agent pathogène et la canne à sucre sont encore très peu connus. Les objectifs de ce travail étaient (i) d'identifier des déterminants moléculaires impliqués dans la survie épiphyte de X. albilineans et (ii) de préciser la localisation de la bactérie dans les tissus de la canne à sucre. Parmi les facteurs étudiés, les polysaccharides de surface et une protéine de la membrane externe (XaOmpA1) de X. albilineans s'avèrent indispensables pour la survie épiphyte de cet agent pathogène. En revanche, la molécule signal diffusible DSF et les métabolites secondaires codés par les gènes NRPS (« Non Ribosomal Peptide Synthetases ») ne sont pas requis pour l'installation de la bactérie en surface des feuilles, au moins en l'absence d'autres microorganismes compétiteurs. Toutefois, la colonisation optimale de la phyllosphère de la canne à sucre nécessite la présence d'un système DSF/RpfGC intact. Dans la deuxième partie de ce travail, nous avons vérifié la localisation in planta de X. albilineans par microscopie confocale, immunocytochimie, et microscopie électronique à transmission. Les observations microscopiques réalisées ont permis de montrer que X. albilineans n'est pas limitée au xylème de la canne à sucre, comme on le considérait jusqu'à présent. Bien au contraire, cette bactérie est capable de quitter le système vasculaire de sa plante hôte et de pénétrer dans d'autres types cellulaires, notamment les cellules du parenchyme non vasculaire. Il s'agit là, à notre connaissance, d'un nouveau mécanisme de colonisation d'une plante par une bactérie phytopathogène qui reste à décrypter.Xanthomonas albilineans is the causal agent of leaf scald, a lethal disease of sugarcane that can significantly impact infected susceptible varieties in the field. The mechanisms that govern the interactions between this bacterial pathogen and its host plant are not well known. The objectives of this study were (i) to identify molecular factors involved in epiphytic survival of X. albilineans and (ii) to verify the localization of X. albilineans in sugarcane tissues. Among the studied factors, surface polysaccharides and an outer-membrane protein (XaOmpA1) of X. albilineans were crucial for epiphytic survival of this pathogen. Secondary metabolites synthesized by non-ribosomal peptide synthetases and the diffusible signal factor DSF were not critical for survival of X. albilineans on the sugarcane leaf surface, at least in absence of competing microorganisms. However, an intact DSF/RpfGC system was necessary for optimal colonization of the phyllosphere. In the second part of this study, we verified in planta localization of X. albilineans by confocal microscopy, immunochemistry and transmission electron microscopy. Microscopic observations allowed us to show that X. albilineans is not a xylem limited bacterium as it was believed until now. This pathogen is able to invade numerous cellular types including vascular and non-vascular parenchyma cells. To our knowledge, this is a novel invasion strategy of a plant pathogenic bacterium that has not previously been described, and that remains to be deciphered

Breaking dogmas: the plant vascular pathogen Xanthomonas albilineans is able to invade non-vascular tissues despite its reduced genome

BGPI : équipe 3Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is missing the Hrp type III secretion system that is used by many Gram-negative bacteria to colonize their host. Until now, this pathogen was considered as strictly limited to the xylem of sugarcane. We used confocal laser scanning microscopy, immunocytochemistry and transmission electron microscopy (TEM) to investigate the localization of X. albilineans in diseased sugarcane. Sugarcane plants were inoculated with strains of the pathogen labelled with a green fluorescent protein. Confocal microscopy observations of symptomatic leaves confirmed the presence of the pathogen in the protoxylem and metaxylem; however, X. albilineans was also observed in phloem, parenchyma and bulliform cells of the infected leaves. Similarly, vascular bundles of infected sugarcane stalks were invaded by X. albilineans. Surprisingly, the pathogen was also observed in apparently intact storage cells of the stalk and in intercellular spaces between these cells. Most of these observations made by confocal microscopy were confirmed by TEM. The pathogen exits the xylem following cell wall and middle lamellae degradation, thus creating openings to reach parenchyma cells. This is the first description of a plant pathogenic vascular bacterium invading apparently intact non-vascular plant tissues and multiplying in parenchyma cells