Fig. 4

XopL mediates the ubiquitination and degradation of NbFd. a In vitro assay for XopL-mediated ubiquitination of NbFd. Anti-His antisera (ab-His) was used to detect His-tagged NbFd or its ubiquitinated products, anti-ubiquitin antibody (an-ub) was used to detect ubiquitin or polyubiquitin, and anti-GST antibody (ab-GST) was used to detect XopL. The experiment was repeated three times with similar results. b XopL mediates NbFd degradation in planta. NbFd was detected with anti-myc antibody at 2, 3 and 4 dpi. XopL was detected using anti-flag antisera, and total protein was monitored with anti-actin antibody. mRNA of transiently expressed NbFd-YFP and native EF1α were measured at each time interval by RT-PCR. c LRR domain of XopL fails to mediate NbFd degradation. NbFd:YFP and pHB-xopL-LRR (lacking E3 ligase domain) or empty pHB were agroinfiltrated into Nb. NbFd, XopL-LRR, and actin levels were measured by immunoblotting as described in panel b. d Effect of the proteasome inhibitor MG132 on the stability of ferredoxin. Anti-flag and actin antibodies were used to detect flag-tagged XopL and actin respectively. In panels b, c, and d, the values above each blotted band represent band intensity and were calculated using Image J software. In each panel, the first lane band intensity was normalized as 1.00. Experiments were repeated at least three times with similar results