In this report we show that the T3SS of H. rubrisubalbicans is important for establishing pathogenic

interactions with sugarcane, lesion formation in V. unguiculata leaves as well as endophytic colonization of a rice cultivar and maize. The gene organization of the H. rubrisubalbicans hrp/hrc cluster is identical to that of H. seropedicae [25]. The T3SS gene cluster of phytopathogenic bacteria can be divided into two groups based on DNA homology, genetic organization, and regulation pattern [35]. The structural organization of hrcUhrpXhrcShrcRhrcQ and hrpBhrcJhrpDhrpE genes in the H. rubrisubalbicans hrp cluster resembles that of bacteria such as Pseudomonas syringae, Erwinia amylovora, and Pantoea stewartii. H. rubrisubalbicans also possesses a hrpL gene, a characteristic of bacteria from group I. The HrpL protein, a member of the ECF family of alternative sigma factors, regulates the expression INK 128 clinical trial of hrp genes in group I [27, 50, 51]. Interestingly, H. rubrisubalbicans hrpL has no σ54 promoter sequence, a feature conserved in group I organisms, but contains a gene highly similar to hrpG. The HrpG protein is involved in the expression of group II hrp genes [52, 53]. Upstream from orf1, orf6, hrpO, orf8, hrpB and orf10 are conserved sequences that are similar to the hrp box sequences which are recognized by

HrpL of P. syringae [27–29] suggesting PCI-32765 research buy the presence of at least six HrpL dependent operons. This is consistent with the observation that hrp genes are commonly organized in large gene clusters, consisting of multiple transcriptional units. For instance, P. syringae pv. syringae and E. amylovora contain a 25 Kb cluster with eight transcriptional units [54]. Blast search using the available sequence allowed to identify five candidates for H. rubrisubalbicans effector proteins: Hrop1, Hrop2, HropAV1, HropAN1 and HropF1. Only HropAN1 has a counterpart in Etoposide mouse H. seropedicae, the other effector proteins are unique to H. rubrisubalbicans and could be involved in the

pathogenic phenotype of H. rubrisubalbicans. To determine if the T3SS of H. rubrisubalbicans is functional we constructed and characterized hrcN and hrpE mutants. T3SS-associated ATPases (HrcN proteins) have long been predicted to be the key energizers of the T3SS. The H. rubrisubalbicans hrcN mutant failed to cause the mottled stripe disease in sugarcane variety B-4362, demonstrating that the HrcN of H. rubrisubalbicans is important for bacterial pathogenicity. Similar results were observed in other plant pathogens, such as Xanthomonas oryzae pathovar oryzae KACC10859, whose hrcN mutant completely lost virulence [55]. X. campestris pv. vesicatoria strain 85, whose hrcN mutant failed to induce plant reactions in susceptible and resistant pepper plants [56], and a R. solanacearum hrcN mutant lost virulence on tomato [57]. The H. rubrisubalbicans hrpE mutant also lost the ability to cause disease.