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. 2015 Nov 18:16:975.
doi: 10.1186/s12864-015-2190-0.

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome

Brice Roux  1   2 , Stéphanie Bolot  1   2 , Endrick Guy  1   2   3 , Nicolas Denancé  1   2   4 , Martine Lautier  1   2   5 , Marie-Françoise Jardinaud  1   2   6 , Marion Fischer-Le Saux  7 , Perrine Portier  7 , Marie-Agnès Jacques  7 , Lionel Gagnevin  8 , Olivier Pruvost  8 , Emmanuelle Lauber  1   2 , Matthieu Arlat  1   2   5 , Sébastien Carrère  1   2 , Ralf Koebnik  9 , Laurent D Noël  10   11
Affiliations

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome

Brice Roux et al. BMC Genomics. .

Abstract

Background: The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani).

Results: In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins.

Conclusions: This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris.

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Figures

Fig. 1
Fig. 1
Comparison of 13 publically available and newly sequenced Xanthomonas campestris proteomes. Orthologous proteins were determined using OrthoMCL software using homogenously re-annotated genomes. a A phylogenetic tree of X. campestris core proteomes (3481 orthologous coding sequences, CDS) was generated using the PhyML software (Default parameters). Bootstrap values are indicated in grey for each branch. b Size of X. campestris core ORFeome was determined considering only CDS with a single ortholog per genome. c Numbers above black bars indicate the size of the pan ORFeome. Only one CDS per orthology group was considered. The number of annotated CDS per genome is indicated (open bar). The number of isolate-specific CDS is given (green bars). d Venn diagram illustrating the number of coding sequences shared among the core ORFeomes (as defined in (b)) in the four X. campestris pathovars. Numbers in brackets indicate the number of genes in the core genome of the pathovars. Xcc (blue): X. campestris pv. campestris, Xci (red): X. campestris pv. incanae, Xcr (black): X. campestris pv. raphani, XcNP (green): X. campestris non-pathogenic
Fig. 2
Fig. 2
Conservation in Xanthomonas campestris of flg22 (a) and elf18 (b) peptides from FliC and EF-Tu PAMP proteins, respectively. Peptides with/without predicted elicitor activity are in black/grey, respectively. Residues which are essential for elicitor activity and polymorphic in X. campestris are underlined in black
Fig. 3
Fig. 3
Diversity of type III secretome in Xanthomonas campestris. a Presence (blue)/absence (white) matrix of genes encoding putative T3SP including T3E (black font) and other accessory proteins (others; grey font) was searched in genomic sequences by tblastn using known T3SP protein sequences (http://www.xanthomonas.org/t3e.html). X. campestris core T3E are indicated in red. Grey and blue boxes correspond to protein identities ranging from 40-60 % and 60-100 % over the entire length of the reference protein. Numbers indicate copy numbers of hax genes. The minimal size of the secretome is indicated below the table. b Venn diagram illustrating the overlap between the core secretomes of X. campestris pathovars: campestris (Xcc), non-pathogenic (XcNP), incanae (Xci) and raphani (Xcr). Numbers in brackets indicate the number of the core and accessory type III secreted proteins as inferred from published genome sequences in those pathovars. Genes orthologous to known type III secreted proteins (www.xanthomonas.org) were identified by Blast. Asterisk indicates that all predicted type III secreted proteins of strain CFBP 5825R were used in this analysis. Xcc: X. campestris pv. campestris, Xci: X. campestris pv. incanae, Xcr: X. campestris pv. raphani, XcNP: X. campestris non-pathogenic
Fig. 4
Fig. 4
Identification of the hrpG regulon of strain CFBP 5828R of X. campestris pv. raphani by RNA sequencing. a Dot plot of the normalized expression levels of all genes in presence of hrpG* versus the empty vector (EV). The diagonal indicates genes which expression level is identical in both conditions. Red lines indicate boundaries where gene expression is induced five-fold relative to the other condition. Red dots indicate genes which expression is significantly induced at least five fold relative to the other condition. The hrpG gene is indicated in green. b Dot plot of the expression ratios (hrpG* versus EV) of 13 genes determined either by RNA sequencing (RNAseq) or quantitative RT-PCR (RT-qPCR). The diagonal indicates genes which expression ratios are identical with both techniques. Bars indicate standard deviations. c Box plot of the normalized expression ratios of hrpG-induced genes encoding the T3S system (T3SS) and type III secreted proteins (T3SP) versus the other genes (−). Box plot of the normalized expression ratios of hrpG-induced genes with a PIP box (TTCGB-N15-TTCGB) in their promoter region versus the other genes (−). Red dots indicate the mean values. Significant mean difference was detected using a Welch-test (***, P-value < 0.001)
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