The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria uses the type III secretion system (T3SS) to inject effector proteins into cells of its Solanaceous host plants. It is generally assumed that these effectors manipulate host pathways to favor bacterial replication and survival. However, the molecular mechanisms by which type III effectors suppress host defense responses are far from being understood. Based on sequence similarity, Xanthomonas outer protein J (XopJ) is a member of the YopJ/AvrRxv family of SUMO peptidases and acetyltranferases, although its biochemical activity has not yet been demonstrated. Confocal laser scanning microscopy revealed that green fluorescent protein (GFP) fusions of XopJ are targeted to the plasma membrane when expressed in plant cells, which most likely involves N-myristoylation. In contrast to a XopJ(C235A) mutant disrupted in the catalytic triad sequence, the wild-type effector GFP fusion protein was also localized in vesicle-like structures colocalizing together with a Golgi marker protein, suggesting an effect of XopJ on vesicle trafficking. To explore an effect of XopJ on protein secretion, we used a GFP-based secretion assay. When a secreted (sec)GFP marker was coexpressed with XopJ in leaves of Nicotiana benthamiana, GFP fluorescence was retained in reticulate structures. In contrast, in plant cells expressing secGFP alone or along with the XopJ(C235A) mutant, no GFP fluorescence accumulated within the cells. Moreover, coexpressing secGFP together with XopJ led to a reduced accumulation of secGFP within the apoplastic fluid of N. benthamiana leaves, further showing that XopJ affects protein secretion. Transgenic expression of XopJ in Arabidopsis suppressed callose deposition elicited by a T3SS-negative mutant of Pseudomonas syringae pv. tomato DC3000. A role of XopJ in the inhibition of cell wall-based defense responses is discussed.

2The plasma membrane is at the interface of plant-pathogen interactions and thus many bacterial type-3

The fine-tuned balance of protein level, conformation and location within the cell is vital for the dynamic changes required for a cell to respond to a given stimulus. This requires the regulated turnover of damaged or short-lived proteins through the ubiquitin proteasome system (UPS). Thus, the protease activity of the proteasome is adjusted to meet the current demands of protein degradation via the UPS within the cell. We describe the adaptation of an intramolecular quenched fluorescence assay utilizing substrate-mimic peptides for the measurement of proteasome activity in total plant extracts.The peptide substrates contain donor-quencher pairs that flank the scissile bond. Following cleavage, the increase in dequenched donor emission of the product is subsequently measured over time and used to calculate the relative proteasome activity.

Beyond its role in cellular homeostasis, autophagy is considered to play anti- and pro-microbial roles in host-microbe interactions, both in animals and plants. One of the prominent roles of anti-microbial autophagy in animals is to degrade intracellular pathogens or microbial molecules, in a process termed “xenophagy”. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. However, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that NBR1/Joka2-dependent selective autophagy functions in plant defence by degrading the bacterial type-III effector (T3E) XopL from Xanthomonas campestris pv. vesicatoria (Xcv). We show how XopL associates with the autophagy machinery and undergoes self-ubiquitination, subsequently triggering its own degradation by the NBR1/Joka2-mediated selective autophagy. Intriguingly, Xcv is also able to suppress autophagy in a T3E-dependent manner by utilizing the same T3E XopL that interacts and degrades the autophagy component SH3P2 via its E3 ligase activity. Thus, XopL is able to escape its own degradation and promote pathogenicity of Xcv by inhibiting autophagy through SH3P depletion. Together, we reveal a novel phenomenon how NBR1/Joka2 contributes to anti-microbial autophagy that we termed “effectorphagy”. We provide a unique mechanism how a T3E undergoes self-modification to act as a bait to trap host cellular degradation machineries.Significant statementAutophagy has anti- and pro-microbial roles in host-microbe interactions. Its anti-microbial role is derived from its ability to degrade intracellular pathogens, termed “xenophagy”. The contribution of xenophagy to host-bacteria interactions in plants and its substrates remains elusive. Here, we reveal that NEIGHBOR OF BRCA1 (NBR1)-mediated autophagy has an anti-microbial role towards bacteria by degrading the type-III effector (T3E) XopL from Xanthomonas campestris pv. vesicatoria (Xcv), a process we termed “effectorphagy”. We unveil that the same T3E is able to perturb autophagy to escape its own degradation and to boost bacterial virulence. These findings highlight a novel role of xenophagy that is conserved across kingdoms and we offer new perspectives on how T3Es undergo self-modification to trap host cellular degradation pathways.