초록
<P>Many plant-pathogenic bacteria utilize type II secretion (T2S) systems to secrete degradative enzymes into the extracellular milieu. T2S substrates presumably mediate the degradation of plant cell wall components during the host-pathogen interaction and thus promote bacterial virulence. Previously, the Xps-T2S system from <I>Xanthomonas campestris</I> pv. vesicatoria was shown to contribute to extracellular protease activity and the secretion of a virulence-associated xylanase. The identities and functions of additional T2S substrates from <I>X. campestris</I> pv. vesicatoria, however, are still unknown. In the present study, the analysis of 25 candidate proteins from <I>X. campestris</I> pv. vesicatoria led to the identification of two type II secreted predicted xylanases, a putative protease and a lipase which was previously identified as a virulence factor of <I>X. campestris</I> pv. vesicatoria. Studies with mutant strains revealed that the identified xylanases and the protease contribute to virulence and <I>in planta</I> growth of <I>X. campestris</I> pv. vesicatoria. When analyzed in the related pathogen <I>X. campestris</I> pv. campestris, several T2S substrates from <I>X. campestris</I> pv. vesicatoria were secreted independently of the T2S systems, presumably because of differences in the T2S substrate specificities of the two pathogens. Furthermore, in <I>X. campestris</I> pv. vesicatoria T2S mutants, secretion of T2S substrates was not completely absent, suggesting the contribution of additional transport systems to protein secretion. In line with this hypothesis, T2S substrates were detected in outer membrane vesicles, which were frequently observed for <I>X. campestris</I> pv. vesicatoria. We, therefore, propose that extracellular virulence-associated enzymes from <I>X. campestris</I> pv. vesicatoria are targeted to the Xps-T2S system and to outer membrane vesicles.</P><P><B>IMPORTANCE</B> The virulence of plant-pathogenic bacteria often depends on TS2 systems, which secrete degradative enzymes into the extracellular milieu. T2S substrates are being studied in several plant-pathogenic bacteria, including <I>Xanthomonas campestris</I> pv. vesicatoria, which causes bacterial spot disease in tomato and pepper. Here, we show that the T2S system from <I>X. campestris</I> pv. vesicatoria secretes virulence-associated xylanases, a predicted protease, and a lipase. Secretion assays with the related pathogen <I>X. campestris</I> pv. campestris revealed important differences in the T2S substrate specificities of the two pathogens. Furthermore, electron microscopy showed that T2S substrates from <I>X. campestris</I> pv. vesicatoria are targeted to outer membrane vesicles (OMVs). Our results, therefore, suggest that OMVs provide an alternative transport route for type II secreted extracellular enzymes.</P>