Bootstrap values are shown as percentage (>50%) from 1,000 replicates for each node. The tree is unrooted tree. Scale bar represents number of nucleotide substitutions per site. GenBank accession numbers are in parenthesis. Sequences similar to the HrpL-dependent promoter consensus (GGAACC-N15-CCACTCAAT) [26–29] were detected upstream from orf1, orf6, hrpO, orf8, hrpB and orf10 (Figure 3a, b). The ORFs from orf8 to orf9, from hrpB to hrpE and from orf10 to hrcC overlap or are spaced by less than 94 nucleotides apart, suggesting that these three groups of genes are part of three distinct operons. The ORFs

from orf6 to hrcN appear to belong to the same operon, although a 114 bp gap is found between orf6 and orf7, but no promoter was found upstream from orf7. Likewise, the intergenic regions orf1 orf2 and orf3 orf4 contain 336 bp and 249 bp, respectively, but no promoter sequence selleck was identified. This HSP inhibitor analysis suggests that H. rubrisubalbicans hrp/hrc genes are probably organized in six HrpL-dependent operons. Figure 3 (a) Putative promoter sequences of the orf1,orf6, orf8, hrpB

and orf10 operons and hrpO gene of H. rubrisubalbicans. (b) Schematic conserved nucleotide bases found in the promoter regions – H. rubrisubalbicans Hrp-box. H. rubrisubalbicans hrp associated genes Two Hrp associated genes called hpaB (JN256204) and hpaB1 (JN256205) encode general T3SS chaperones, which promote secretion and translocation of multiple effectors proteins [30]. The hpaB and hpaB1 genes are predicted to belong to the TIR chaperone protein family. The hpaB1 gene was found

approximately 12 kb downstream from the hrcC gene and it encodes a small acidic chaperone. H. rubrisubalbicans T3SS effector proteins Type III secretion systems have been characterized in a variety of plant pathogenic bacteria. The structural proteins of these systems are highly conserved, but the T3SS effector proteins, that play a central role in virulence, are less conserved and difficult to identify. A BlastX search of the H. rubrisubalbicans partial genome sequence (30%) against NCBI-nr database allowed identification of five candidates of H. rubrisubalbicans effector proteins HropAN1 (H. rubrisubalbicans outer protein) (JN256208), HropAV1 (JN256209), HropF1 (JN256210), Hrop1 (JN256206) and Hrop2 (JN256207) (Table 1). Hrop1 and Hrop2 were Carteolol HCl also identified as T3SS effectors by the program EffectiveT3 (http://​www.​effectors.​org/​) [31]. The genes encoding these proteins are located apart from the hrp/hrc genes cluster. Table 1 Type III-effector proteins of H. rubrisubalbicans Putative Effector Protein Homology (Gene Bank accession number) Identity/Similarity % Predicted size aa HropAV1 type III effector, HopAV1 family [Ralstonia solanacearum] (CBJ40351.1) 56/70 784 HropAN1 type III effector Hrp-dependent outer protein [Burkholderia sp. Ch1-1] (ZP_06844144.1) 78/86 428 HropF1 XopF1 effector [Xanthomonas oryzae pv. oryzae PXO99A] (YP_001911267.