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Dynamic interplay between phosphorylation and ubiquitination during plant receptor kinase-mediated immunity

Periodic Reporting for period 1 - PHOSPH-UBIQ-IMMUN (Dynamic interplay between phosphorylation and ubiquitination during plant receptor kinase-mediated immunity)

Reporting period: 2017-07-01 to 2019-06-30

The growing population and changing climate challenge the quest for food security. Plant pathogens lead to annual global food production yield losses up to 16-20%. Our travel systems enable pathogens to rapidly spread globally, allowing new diseases to emerge, as shown by the 2016 wheat blast epidemy in Bangladesh, a global food security threat. Thus, plant biologists urgently need to find solutions for existing and emerging diseases.
To help plants better protect themselves, we need to know the nature of plant-pathogen interactions and underlying mechanisms of plant immunity by studying how plants respond to pathogen attack, from the moment of recognition of pathogen-associated molecular patterns (PAMPs) to well-orchestrated defense responses. Many PAMPs and their corresponding pattern recognition receptors (PRRs) have been identified, and common principles in activation of PTI have arisen. PAMP-driven interaction of receptor and co-receptor leads to transphosphorylation events resulting in complex activation and phosphorylation of downstream cytoplasmic kinases. The last ones emerged as integrative points of signal transduction, with BIK1 being a key convergent point in signaling triggered by different PAMPs.
BIK1 is genetically involved in most PTI outputs, however, many of its downstream targets are still unknown. Prof. Zipfel’s lab recently identified a potential BIK1 phosphorylation motif. A search for this motif in proteins known from the literature to be phosphorylated in response to PAMP treatment, led to the identification of candidate BIK1 substrates. These candidates were verified in a preliminary screen based on the ability of BIK1 to phosphorylate its targets in in vitro kinase assays and to interact with them in planta. Among them, an E3 ubiquitin ligase, which we named BIK1 PHOSPHORYLATION SUBSTRATE 1 (BPS1), was found; as well as two ubiquitin-specific proteases, which we called BPS2 and BPS3. PHOSPH-UBIQ-IMMUN aimed to understand how BIK1 regulates PTI via the characterization of its substrates, thus revealing a dynamic interplay between phosphorylation and ubiquitination events in plant immune signalling. Characterization of key components of immune signalling will ultimately lead to our ability to manipulate them to strengthen plant defense.
Firstly, I confirmed that BPS1/BPS2/BPS3 are indeed BIK1 substrates: they associate with BIK1, when co-expressed in N. benthamiana, and are phosphorylated by BIK1 in vitro. Due to time / technical constraints, most of my subsequent work focused on BPS1. Thus, I found that in planta BPS1 is a highly phosphorylated protein. To find BIK1-dependent phosphosites on BPS1, I performed in vitro kinase assays and identified phosphosites by mass spectrometry. I also demonstrated that BIK1 and BPS1 associate within the same complex in Arabidopsis; the interaction being independent of PAMP treatment.
Secondly, I verified whether BPS1 is genetically involved in PTI. I analysed several PTI responses in bsp1 mutants. I found that reactive oxygen species (ROS) production was reduced in bsp1 compared to wild-type. bsp1 mutants demonstrated reduced seedling growth inhibition and impaired stomata closure in response to PAMP treatment. Together, these data suggest that BPS1 acts genetically as a positive regulator of PTI.
Then, I asked which proteins BPS1 associates with in planta? I purified FLAG-tagged BPS1 and identified its associated proteins with or without PAMP treatment. I found BPS1 interacted with proteins of the FLS2/BAK1 PRR complex after PAMP treatment, thus confirming its involvement in very early PTI signalling. I found that BPS1 is phosphorylated in vitro by the co-receptor protein BAK1, and identified the corresponding phosphosites using mass spectrometry. Thus, the above data place BPS1 at the level of the PRR complex and associated cytoplasmic kinases.
The fact that PTI responses triggered by different PAMPs are affected in bps1 mutant suggests that BPS1 acts on a common regulator of different pathways. Thus, BIK1 itself becomes a good candidate for being a BPS1 target. BIK1 is known to be tightly regulated at the protein level; with a balance between its “inactive” and “activated” form being important for the amplitude of immune signalling. Therefore, I checked BIK1 protein levels in bps1 and BPS1 overexpression lines. Before PAMP treatment, BIK1 protein level is reduced in BPS1 overexpression line, while it is increased in bps1 mutant line. This result is consistent with the observed reduced ROS production in BPS1 overexpression line, as ROS production is BIK1-dependent. However, after PAMP treatment, in bps1 mutant there is a clear reduction in the accumulation of activated BIK1 (phosphorylated form). Moreover, inhibition of the proteasome leads to the restoration of activated BIK1 levels in bps1. Thus, in bps1, there is a lack of activated, signalling-competent form of BIK1. This fits with the observed reduced PTI outputs in bps1 line. Together, these results demonstrate that BPS1 acts on a convergent point of PTI signalling, BIK1, thus affecting defence responses triggered by different PAMPs. Moreover, BPS1 protein level is important for correct accumulation of both “inactive” and “activated” forms of BIK1.

I presented my results at the SEB Cell Symposium 2017 “From proteome to phenotype: role of post-translational modifications”, Edinburgh and at the International Conference on Arabidopsis Research (ICAR) 2018 in Turku, Finland. I regularly presented my project at Prof. Zipfel’s lab meetings as well as at TSL seminars.
I have identified BPS1 as a new key player in plant immune signalling. BPS1 affects protein levels of the cytoplasmic kinase BIK1 known to be a convergent point of signalling triggered by various PAMPs. BPS1 protein level itself is important for the balance of “inactive” and “activated” forms of BIK1, and thus contributes to the amplitude of defence responses. My results advance our understanding of function and regulation of BIK1 and demonstrate the importance of the interplay between phosphorylation and ubiquitination during PTI. PHOSPH-UBIQ-IMMUN has implications in fundamental science as it increases our knowledge about signalling networks involved in plant immunity. It also opens new directions of research to be continued in Prof. Zipfel’s lab. A manuscript describing my results is currently in preparation.
I have also illustrated that BPS1 could be used as a new tool for fine-tuning plant immune signalling, which is of interest to applied science. Together, my results have helped us to better understand plant defence responses leading to an ultimate goal of making plants more resistant to pathogen attacks.

I discussed the broad idea of my project and importance of plant immunity with members of the general public during the 2018 Norwich Science Festival. To promote science and especially women in science, I participated in Soap Box Science 2018, talking to the general public about my work as a woman in science. I also talked about my scientific career and my work with STEM students in a “meet a professional” session at the John Innes Centre in 2018, encouraging school pupils to consider developing scientific careers.
Fig. 1. Janus-faced BPS1: its effect on BIK1 stability before and after PAMP treatment.