- Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. - The upstream regulators of the T3SS were exclusively up- regulated in the apoplast, preceding the induction of their downstream targets. - Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - For example, flagellar motility and chemotaxis-related genes were transcribed in the epi- phytic phase, while genes controlling metabolism were expressed in the apoplast [43]. - solanacearum plant infection through roots is highly variable due to stochastic changes in the physiological state of the plant, the initial inoculum and available root entry sites. - Comparison of the in planta tran- scriptomes with that obtained in axenic growth in rich medium identified 418 differentially expressed genes (DEGs) in the apoplast, 531 in the early xylem and 922 in the late xylem (log 2 fold change ≥ |1.5| and adjusted p-value ≤ 0.01). - Of these genes, 226 and 192 were up- and down-regulated, respectively, in the apoplast, 290 and 241 in the early xylem, and 378 and 544 in the late xylem (Fig. - The differentially expressed genes in the xylem genetic programme (both time points analysed) included a total of 162 and 156 up- and down-regulated genes. - Finally, 100 and 80 genes were, respectively, up- or down-regulated solely in the plant apoplast and 96 and 278 only in the late xylem condition, when plants are mostly dead. - Each vertical bar plot represents the number of shared DE between the conditions indicated by the lines and dots in the schematic below. - However, a substantial fraction of genes was only differentially expressed in the late xylem (40%. - Thus, we investigated the enrichment of KEGG pathways and GO terms in the genes that appeared DE in all in planta conditions. - As expected, the created “virulence and parasitic fitness” category was clearly enriched in the up-regulated genes in the in planta genetic programme (p-value . - Similarly, the polygalacturonase gene pglA, encoding one out of the six cell-wall degrading enzymes in the genome was also up-regulated in the plant. - which can be explained by the high expres- sion of the exopolysaccharide synthesis operon in the reference rich medium [38].. - Flagellar genes and the upstream regulators of the T3SS are exclusively up-regulated in the apoplast. - The KEGG flagellar assembly pathway was enriched in the genes exclusively up-regulated in the apoplast (Fig. - A closer perusal of the list of up-regulated genes in the apoplast genetic programme also revealed that the “virulence and parasitic fitness” category was enriched (p-value . - None of the KEGG pathways nor GO terms were enriched amongst the genes down-regulated in the apoplast.. - solana- cearum genes was DE in the xylem genetic programme, both at early and late conditions (Fig. - Almost one third (12 out of 38) of the genes with associated KEGG pathways differentially up-regulated in the xylem irre- spective of the condition belonged to the enriched cat- egory two-component system (Fig. - Three other categories were enriched in the genes up-regulated. - in the xylem: oxidative phosphorylation (six genes), bac- terial chemotaxis (five genes) and nitrogen metabolism (five genes). - The up-regulated nitrogen metabolism genes included nitrate transporters (nark1/2), enzymes involved in the denitrification pathway (aniA, norB) and in the dissimilatory nitrate reduction pathway (narG/H/. - The “virulence and parasitic fitness” category was also enriched in the xylem genetic programme up-regulated genes (p-value . - Other overexpressed genes in the category included 10 motility genes and the cytokinin biosynthesis gene tzs. - Finally, amongst the 102 KEGG tagged down-regulated genes in the xylem, the enriched categories were: ribosome, oxi- dative phosphorylation and citrate (TCA) cycle (Fig. - solanacearum genes was found DE in the xylem throughout infection, including up-regulation of nitro- gen utilisation and virulence genes, such as T3Es and down-regulation of genes encoding the citrate cycle en- zymes and the electron transport chain.. - Besides the DE genes in the xylem throughout infection, a large set of R. - solanacearum genes was exclusively DE in the Late xylem genetic programme, at late stages of infection when plants are already wilted (Fig. - Sur- prisingly, no KEGG category was enriched in this abun- dant set of up-regulated genes, but our “virulence and parasitic fitness” category was enriched in the up- regulated genes (p-value = 5·10 − 3. - In the genes differentially down- regulated in the late xylem condition, five KEGG cat- egories were enriched: carbon metabolism (18 out of 108 genes tagged), ribosome (17 genes), TCA cycle (9 genes), RNA degradation (six genes) and protein export (six genes) (Fig. - solanacearum exclusively downregulates at late infection stages in the xylem a large subset of genes involved in the central metabolism and its derived metabolic pathways.. - Expression profiles reinforce the existence of specific genetic programmes in the apoplast and the xylem The findings described so far strongly suggest that R.. - solanacearum UY031 genes in the three in planta conditions: apoplast, early and late xylem. - contained 807 genes up-regulated in the apoplast but down-regulated in early and late xylem (Fig. - 2a), and the profile “specific xylem” contained 1286 genes down- regulated in the apoplast but up-regulated in the other conditions (Fig. - 2f) in the early xylem that showed the opposite trend in the apoplast and late xylem were 105 and 107, respectively.. - Enriched KEGG pathways in the “specific apoplast”. - In the “specific xylem” profile, the KEGG enrichment analysis yielded terms related with metabolism adaptation such as microbial metabolism in diverse environments (106 out of 411 tagged genes), ABC transporters (63 genes), and nitrogen metabolism (19 genes) among others (Fig. - This pro- vided an unbiased view on the gene expression data avoiding the effect of the reference condition in the DESeq analysis. - Both the rip T3Es and the hrp/hrc genes displayed a very homogeneous ex- pression pattern with high expression levels in the xylem genetic programme (early and late) and low expression levels in the apoplast. - Heatmap visualisation of the nor- malised transcriptomic data also indicated that flagellar genes —essential for swimming motility— were highly expressed in all in planta conditions, but to a higher ex- tent in the apoplast (Fig. - accordance with the enrichment of this category in planta and in the late xylem genetic programmes up- regulated genes, as well as in the specific apoplast pro- file. - The pil twitching motility genes encoding type IV pili followed a similar trend, although their expression was more similar in the apoplast and the xylem (Fig. - pilE1, pilY1, pilW, pilV, pilX), which were down-regulated in the apoplast compared to the xylem. - solanacearum UY031 in the reference condition and in planta apoplast, early and late conditions. - The genes encoding chemotactic sensors and chemotaxis signal transduction proteins showed low expression levels in the apoplast and progressive induction in the early and late xylem condi- tions (Additional File 10) in accordance with the enrich- ment of these specific genes in the late xylem genetic programme. - This stable expression in all in planta conditions was also observed in the differential expression analysis (Additional File 12).. - solanacearum during the infection are grouped in four biologically relevant genetic programmes: genes commonly DE in all in planta conditions, genes exclusively DE in the apo- plast, genes expressed in the xylem at any stage of the disease and genes exclusively DE in the xylem when plants are already wilted (Fig. - solanacearum in different genetic programmes, the largest number of DEGs appeared exclusively up-regulated in the xylem genetic programme (1286 genes) and the apoplast (807 genes). - T3Es expression is prevalent throughout the in planta infection process, especially in the xylem. - Our gene expression dataset also shows that most of the 60 T3Es are highly induced in the xylem genetic programme, confirming our previous results [34] that challenged the view of T3Es as key only early after infec- tion [32, 40]. - In agreement our finding that almost all T3Es are simultaneously expressed in the xylem, a re- cently published study showed that deletion of 42 R.. - 2/3/5/7, HLK1/2/3 and GALA were clearly induced in the xylem throughout infection (Fig. - Their expression pattern suggests that these biochemical activities are likely carried out in the. - Similarly, The T3E ripAY, which has been proven to impair the redox status of the plant cell degrading glutathione through its gamma-glutamyl cyclotransferase activity [57–60], was clearly induced in the xylem (Fig. - For in- stance, ripE2 can be clearly classified as an “early effector” since it was highly induced in the apoplast compared to the other conditions, while ripD and ripAD were highly induced in all in planta conditions (Fig. - This fact, linked with its high ex- pression in the apoplast and the activation of flagellar genes in this condition, suggests that R. - GABA in plant cells in the absence of sufficient bac- teria to consume it has been shown to induce cell death [62]. - 4), most of the flagellar-encoding genes were highly induced in the. - Log 2 fold-change expression profile in the Apoplast, Early and Late Xylem of the genes involved in the T3SS regulatory cascade and downstream activated genes. - apoplast and, to a lower extent, in the early and late xylem, supporting the previously mentioned hypothesis.. - A small subset of flagellar genes including the motor (motA, motB), the flagellin subunit (fliC) and the fila- ment cap (fliD) among others showed low expression in the apoplast, for which we have no plausible explanation.. - In our transcriptomic data, twitching motility genes showed a similar expression pattern than swimming motility, but they were less in- duced in the apoplast and their expression was often maintained in early and late xylem (Fig. - Finally, pilI, which encodes the type IV pili chemosensor protein, was especially induced in the apoplast (Fig. - solanacearum specifically activates different nitrogen metabolism genes to thrive in the xylem. - Our gene expression dataset shows a faint induction of the nitrogen metabolism in the apoplast, reaching its expression peak in the xylem (Fig. - When nitrate is available in the extracellular space, it diffuses the outer membrane and is imported to the cytoplasm by NarK1/2. - We found both the transporters- and reductase-encoding genes induced in the xylem (Fig. - Expression of these denitrification path- way genes is also induced in the xylem (Fig. - Log 2 fold-change expression profile in the Apoplast, Early and Late Xylem of the genes involved in the denitrification ( aniA, norB. - reactive nitrogen species produced during nitrate dissimi- latory pathway in the anaerobic xylem vessels [29].. - The nitrate present in the cytoplasm is reduced to nitrite by NasA/B. - The fact that nasA is induced in the xylem and not in the apoplast is in dis- agreement with these results and may indicate strain- or condition-specific roles of N genes in R. - Finally, the nitric oxide anion in the cytoplasm can be detoxified using HmpX, whose expression is also highly induced in the xylem genetic programme (Fig. - solanacearum contains several genes that code for ROS scavenging enzymes, helping the bacterium survive in the plant apoplast and xylem [72].. - solanacearum has the highest expression of motility genes in the apoplast, while the majority of T3Es and nitrogen metabolism genes are highly induced in the xylem environment. - To avoid bias of quorum sensing signals in the xylem stages and not in the apoplast, similar bacterial yields were infiltrated in potato leaves for the initial stage. - As shown in Additional File 2 A, bacterial densities recovered from the three in planta conditions were in the same order of magnitude (be- tween 10 7 and 10 8 CFUs/ml). - The aerial part of the plants was vacuum-infiltrated for 30 s to 1 min and the leaves were dried in paper towel before incubating the plants in the inoculation chamber (27 °C, 12 h / 12 h). - In all other cases, RNA-sequencing was performed in the Shanghai PSC Genomics facility. - Raw sequencing data will be available upon publication in the Sequence Read Archive under Bio Project: PRJNA660623 (accession codes SAMN15955133 to SAMN15955144).. - The UpSetR [84] R package was used to visualise the intersection of DE genes in the different in planta conditions. - the gene was allocated in the same cluster in 30 out of 40 iterations with the membership value set to μ ≥ 0.75.. - (A) Experimental set-up for the three in planta conditions, corre- sponding to an early (leaf apoplast), mid (xylem from asymptomatic plants) and late stages (xylem from dead plants) of the disease. - (C) Two-dimensional Principal Compo- nent Analysis representation of the expression data of the conditions ’ biological replicates used in the study.. - Additional file 3: DEGs in the three in planta conditions. - Each ver- tical bar plot represents the number of shared DE between the condi- tions indicated by the lines and dots in the schematic below. - Heatmap showing the normalised transcripts per million (TPM) of the genes involved in the T3SS regulatory cascade and the T3SS apparatus in the reference and in the in planta conditions.. - Heatmap showing the normalised transcripts per million (TPM) of the genes involved in chemosensing and signal transduction in the reference and in the in planta conditions.. - Heatmap showing the normalised transcripts per million (TPM) of the genes involved in phytohormones biosynthesis in the reference and in the in planta conditions.. - Heatmap showing the normalised transcripts per million (TPM) of the genes coding for ROS scavenging enzymes in the reference condition and in planta apoplast, early and late condition.. - Ralstonia solanacearum, a widespread bacterial plant pathogen in the post-genomic era. - Repertoire, unified nomenclature and evolution of the Type III effector gene set in the Ralstonia solanacearum species complex. - Ralstonia solanacearum uses inorganic nitrogen metabolism for virulence, ATP production, and detoxification in the oxygen-limited host xylem environment. - A single regulator mediates strategic switching between attachment/spread and growth/virulence in the plant pathogen Ralstonia solanacearum. - Role of alkyl hydroperoxide reductase (AhpC) in the biofilm formation of Campylobacter jejuni. - Differential roles of OxyR-controlled antioxidant enzymes alkyl hydroperoxide reductase (AhpCF) and catalase (KatB) in the protection of Pseudomonas aeruginosa against hydrogen peroxide in biofilm vs
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