, 2003), in P. syringae pv. phaseolicola 1448A annotated as PSPPH_A0122 (HopAW1) and PSPPH_A0129, and in Acidovorax avenae ssp. avenae annotated as Acav_0110 and Acav_4550. Such situation might be check details indicative of different substrates being targeted in the plant host or the same substrate cleaved at different positions (thus generating different

cleavage products) or function in different plant hosts. Other T3S effector genes are also present in duplicate in B. japonicum genome, such as NopE1 and NopE2 (Wenzel et al., 2010). The presence in a single strain of multiple members belonging to a T3S effector family also occurs in phytopathogenic bacteria. For example, multiple members of the YopJ family are present in P. syringae, Xanthomonas campestris, and Ralstonia solanacearum (Ma et al., 2006; Zhou et al., 2009; Szczesny et al., 2010; Lewis et al., 2011). The diversification of effector family

members is thought to have been evolved during the co-evolutionary arms race between plants and their attackers via both pathoadaptation and horizontal gene transfer (Ma et al., 2006). Further studies are needed to determine the driving forces in shaping the T3E repertoire of B. japonicum as well as the presence of multiple effector paralogs. Here, we present evidence that NopT1 and NopT2 are indeed cysteine proteases with autoproteolytic activity which is maximal at pH of 6.5, and it is completely abolished by the class-specific cysteine peptidase inhibitor, E-64. Moreover, single mutations disrupting the catalytic core residues Sorafenib supplier (C100, H213, and D228) of NopT1 diminished both the cysteine protease and the autoprocessing activities. These findings are consistent with previous reports indicating that some T3S effectors classified as clan CA proteases from plant pathogenic and symbiotic bacteria are autoproteolytically cleaved in E. coli (Puri et al., 1997; López-Solanilla Buspirone HCl et al., 2004; Dai et al., 2008; Dowen et al., 2009; Kambara

et al., 2009). It is interesting to note that two previous studies (Dai et al., 2008; Kambara et al., 2009) reached contradictory conclusions regarding the necessity of the catalytic residue H205 in the proteolytic activity of NopT from NGR234. Although these studies reached different conclusions, their primary data are not necessarily mutually exclusive because differences in methods applied might account for the seeming discrepancy. To gain insight into the role of the residues surrounding the autoproteolytic site in NopT1 autoprocessing, we mutated the amino acids immediately preceding the putative autocleavage sites at P3, P2, and P1 that are occupied by residues D47, K48, and M49, respectively. Triple substitution of P1–P3 sites with alanines completely abolished the autoproteolytic cleavage of NopT1. This finding is consistent with previous studies demonstrating that mutations of P1–P3 residues in AvrPphB, ORF4, NopT, RipT, and PBS1 prevent self-processing (Shao et al., 2003b; Zhu et al.