April 2021

2021 has been a very exciting and productive year for our lab so far with one major manuscript from our lab and two other contributions together with the Hafrén (SLU Uppsala) about virus-proteasome interaction and Börnke Lab (IGZ Großbeeren) about bacterial effector XopS. We have finally finished one of our major stories about how a bacterial effector from Xanthomonas traps the autophagy machinery to cause disease:” “Self-ubiquitination of a pathogen type-III effector traps and blocks the autophagy machinery to promote disease”. You can find the updated link to the preprint here:

https://www.biorxiv.org/content/10.1101/2021.03.17.435853v2

The XopL journey:

Our journey on Xanthomonas effector XopL started in 2015 when we conducted Y2H screenings with various Xanthomonas effectors to identify new host targets. This approach was pretty successful as we identified many interesting putative host targets (including the proteasome). However, as usual, it takes a long time to verify these interactions and also its biological relevance (it took as almost 6 years!). In the same screen we also identified that another bacterial effector, XopS, interacted with WRKY40 transcription factor, which in the end resulted in the fantastic story “The Xanthomonas type-III effector protein XopS stabilizes CaWRKY40a to regulate defense hormone responses and preinvasion immunity in pepper” from the Börnke Lab, led by first author Margot Raffeiner. Read more about it here: https://www.biorxiv.org/content/10.1101/2021.03.31.437833v1

The Y2H screen yielded that XopL might interact with SH3P2, a component of the autophagy machinery. However, at this time, we were not really motivated to study these interactions, as the expression of XopL and also SH3P2 was pretty tricky in plants and things did not develop as quick as we thought. Meanwhile, when I started to do my postdoc in Sweden, SLU Uppsala, we found out that Pseudomonas, activated autophagy for proteasome degradation (and most likely other stuff) to promote its virulence (http://www.plantcell.org/content/30/3/668). These findings together with the interaction data of XopL and SH3P2 prompted us to investigate whether Xanthomonas utilizes autophagy in a similar or different manner for its own benefit (in the DFG funded Emmy Noether project).

My talented and super motivated PhD student Jia Xuan Leong joined our lab last year as a master student and was able to show that, in contrast to Pseudomonas, Xanthomonas blocks autophagy in an effector-dependent manner to enhance its pathogenicity. We then went on and screened a couple of effectors for their ability to block autophagy. We analysed XopJ, XopS and XopD, as they have known effects on proteolytic degradation pathways and of course included XopL, because of its ability to interact with SH3P2. Fortunately, it turned out that XopL is able to suppress autophagy which made us very confident that we have to further characterize its function. However, our major concern was that previous reports stated that XopL is not a major virulence factor of Xanthomonas.

Fortunately, with the help of Mary Beth Mudgett and her amazing lab, we were able to show that XopL is one of the major virulence factors required for the virulence of Xanthomonas in tomato and roq1 N. benthamiana. Interestingly, this effect is only present when plants are dip-inoculated and not when they are syringe-inoculated.Thanks again for the Mudgett Lab for this great contribution!

Meanwhile we got a bit confused, as we realized that XopL is also degraded by autophagy. XopL associates with selective autophagy receptor NBR1/Joka2 which mediates the degradation of XopL. So far NBR1/Joka2 was known to degrade aggregates (aggrephagy), viral proteins and intracellular pathogens (xenophagy) but not bacterial effector proteins. We further discovered that XopL undergoes self-ubiquitination in vitro and in planta, at Lysine 191. Although, mutation of this lysine stabilizes XopL, XopL is still ubiquitinated in plants. We assume (and have also indications) that other lysines within XopL are ubiquitinated.

But how does XopL suppress its own degradation to act as a virulence factor? We went back to our “old” interaction data and confirmed the interaction between XopL and SH3P2 using different techniques. Because XopL is a member of the IpaH3 E3 ligase family, we hypothesized that XopL might degrade SH3P2 and thereby block autophagy. Indeed, in vitro ubiquitination assays (performed by talented PhD Student Margot Raffeiner from the Börnke lab) revealed that XopL ubiquitinates SH3P2 and mediates its degradation by the proteasome. The role of SH3P2 in the literature was still a bit controversial, as it has multiple functions. However, we show that SH3P2 from Nicotiana benthamiana is partially required for autophagy. Autophagosome formation during autophagy induction seems impacted.

Finally, after huge efforts (I am really sorry that you had to perform so many bacterial growth assays ) Jia Xuan could show that loss of SH3P2 and Joka2/NBR1 is beneficial for Xanthomonas growth (also loss of general autophagy) and can even restore the reduced growth of a Xanthomonas strain lacking autophagy suppressor XopL. Taken together, we conclude that Xanthomonas effector XopL acts as a bait to trap and dampen the autophagy pathway resulting in enhance pathogenicity of Xanthomonas. We provide a unique mechanism how a bacterial effector undergoes self-ubiquitination to trick the autophagy machinery. We think that during this process XopL is partially sacrificed and degraded in order to access and dampen autophagy responses.

XopL belongs to the IpaH3 E3 ligase family with members in Salmonella & Shigella. Are these effectors also undergoing self-ubiquitination and do NBR1 homologue in animals recognize bacterial effectors? Superimposing XopL (brown) and IpaH3 (blue) structures reveal possible ubiquitination sites (green) in IpaH3 in the same region within the LRR domain. It might be interesting to look if this mechanism is conserved across kingdoms.

Kudos to first author Jia Xuan Leong who did an amazing job during the last year. She was so persistent, especially doing all the bacterial growths and silencing assays. I am so proud what you have achieved and sure that we‘ll discover more in the next years! We are thankful to everyone who contributed to this exciting project and grateful for generous funding by the DFG Emmy Noether Program.