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Publications - Cell and Metabolic Biology
Publications
Pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. Effector protein delivery is controlled by the T3S chaperone HpaB, which presumably escorts effector proteins to the secretion apparatus. One intensively studied effector is the transcription activator-like (TAL) effector AvrBs3, which binds to promoter sequences of plant target genes and activates plant gene expression. It was previously reported that type III-dependent delivery of AvrBs3 depends on the N-terminal protein region. The signals that control T3S and translocation of AvrBs3, however, have not yet been characterized. In the present study, we show that T3S and translocation of AvrBs3 depend on the N-terminal 10 and 50 amino acids, respectively. Furthermore, we provide experimental evidence that additional signals in the N-terminal 30 amino acids and the region between amino acids 64 and 152 promote translocation of AvrBs3 in the absence of HpaB. Unexpectedly, in vivo translocation assays revealed that AvrBs3 is delivered into plant cells even in the absence of HrpF, which is the predicted channel-forming component of the T3S translocon in the plant plasma membrane. The presence of HpaB- and HrpF-independent transport routes suggests that the delivery of AvrBs3 is initiated during early stages of the infection process, presumably before the activation of HpaB or the insertion of the translocon into the plant plasma membrane.
Publications
In plant secondary metabolism, β‐acetal ester‐dependent acyltransferases, such as the 1‐O ‐sinapoyl‐β‐glucose:l ‐malate sinapoyltransferase (SMT; EC 2.3.1.92), are homologous to serine carboxypeptidases. Mutant analyses and modeling of Arabidopsis SMT (AtSMT) have predicted amino acid residues involved in substrate recognition and catalysis, confirming the main functional elements conserved within the serine carboxypeptidase protein family. However, the functional shift from hydrolytic to acyltransferase activity and structure–function relationship of AtSMT remain obscure. To address these questions, a heterologous expression system for AtSMT has been developed that relies on Saccharomyces cerevisiae and an episomal leu2‐d vector. Codon usage adaptation of AtSMT cDNA raised the produced SMT activity by a factor of approximately three. N‐terminal fusion to the leader peptide from yeast proteinase A and transfer of this expression cassette to a high copy vector led to further increase in SMT expression by factors of 12 and 42, respectively. Finally, upscaling the biomass production by fermenter cultivation lead to another 90‐fold increase, resulting in an overall 3900‐fold activity compared to the AtSMT cDNA of plant origin. Detailed kinetic analyses of the recombinant protein indicated a random sequential bi‐bi mechanism for the SMT‐catalyzed transacylation, in contrast to a double displacement (ping‐pong) mechanism, characteristic of serine carboxypeptidases.
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