2d). As shown in Fig. 4h, the triple mutant NopT1-GCC was not capable of causing cell death in tobacco following transient expression by Agrobacterium as the wild-type protein did. This result suggests that the putative palmitoylation sites may be more important than myristoylation for plant plasma membrane association and the subsequent cell death in tobacco. To investigate whether the NopT1 autoprocessing is required to reveal the embedded acylation sites, we created another mutant (NopT1-DKM)
by substituting residues D47, K48, and M49 with alanines (Fig. 2d). In this mutant, both acylation sites were intact, while Akt inhibitor the amino acids immediately preceding the putative check details NopT1 autocleavage site were modified. This mutant was inactive in eliciting cell death in tobacco (Fig. 4i). To further test whether the mutant proteins NopT1-GCC and NopT1-DKM are autoprocessed, we expressed them in E. coli and analyzed the purified proteins by SDS-PAGE and Western blotting. The NopT1-DKM was completely resistant to autocleavage (Fig. 2c), suggesting that the residues D47, K48, and M49 are required for autoprocessing of the N-terminal region. In contrast, the protein mutated in residues G50, C52, and C53 (NopT1-GCC) still shows autocleavage (Fig. 2c). It is interesting
to note that the wild-type NopT1 was very rapidly processed in E. coli, and we were able to detect the full-length protein only when short times of induction (e.g. 2–4 h) were chosen. In contrast, the full-length protein of the GCC mutant was still detectable in substantial amounts upon induction of protein expression for 12 h in E. coli. Although these results indicate that mutation in the G50, C52, and
C53 residues partially affects the autoproteolytic activity of NopT1, significant autocleavage activity is observed for NopT1-GCC protein. Together, the results suggest that autoprocessing of NopT1 is required to unmask its putative acylation sites. In this study, we Forskolin in vitro demonstrated for the first time that NopT1, but not NopT2, of B. japonicum elicits cell death in plants tested. Both proteins possess cysteine protease activity that is essential for the cell death–eliciting activity in the case of NopT1. Many members of the YopT/AvrPphB effector family have been shown to possess cysteine protease activity (Shao et al., 2002; López-Solanilla et al., 2004), although some of them are not autoprocessed or acylated (Dowen et al., 2009). In plant symbiotic bacteria, three genes encoding YopT family members have been found: one in Rhizobium NGR234, named nopT (Dai et al., 2008), and two in B. japonicum (nopT1 and nopT2). Multiple proteases of the YopT family can be found in a single strain, for example, in Pseudomonas syringae pv.