The PTI-suppressing Avr2 effector from Fusarium oxysporum suppresses mono-ubiquitination and plasma membrane dissociation of BIK1

Plant pathogens use effector proteins to target host processes involved in pathogen perception, immune signalling, or defence outputs. Unlike foliar pathogens, it is poorly understood how root-invading pathogens suppress immunity. The Avr2 effector from the tomato root- and xylem-colonizing pathogen Fusarium oxysporum suppresses immune signalling induced by various pathogen-associated molecular patterns (PAMPs). It is unknown how Avr2 targets the immune system. Transgenic AVR2 Arabidopsis thaliana phenocopies mutants in which the pattern recognition receptor (PRR) co-receptor BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) or its downstream signalling kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) are knocked out. We therefore tested whether these kinases are Avr2 targets. Flg22-induced complex formation of the PRR FLAGELLIN SENSITIVE 2 and BAK1 occurred in the presence and absence of Avr2, indicating that Avr2 does not affect BAK1 function or PRR complex formation. Bimolecular fluorescence complementation assays showed that Avr2 and BIK1 co-localize in planta. Although Avr2 did not affect flg22-induced BIK1 phosphorylation, mono-ubiquitination was compromised. Furthermore, Avr2 affected BIK1 abundance and shifted its localization from nucleocytoplasmic to the cell periphery/plasma membrane. Together, these data imply that Avr2 may retain BIK1 at the plasma membrane, thereby suppressing its ability to activate immune signalling. Because mono-ubiquitination of BIK1 is required for its internalization, interference with this process by Avr2 could provide a mechanistic explanation for the compromised BIK1 mobility upon flg22 treatment. The identification of BIK1 as an effector target of a root-invading vascular pathogen identifies this kinase as a conserved signalling component for both root and shoot immunity

Conservation of the PBL-RBOH immune module in land plants

The rapid production of reactive oxygen species (ROS) is a key signaling output in plant immunity. In the angiosperm model species Arabidopsis thaliana (hereafter Arabidopsis), recognition of non- or altered-self elicitor patterns by cell-surface immune receptors activates the receptor-like cytoplasmic kinases (RLCKs) of the AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) family, particularly BOTRYTIS-INDUCED KINASE1 (BIK1).$^{1}$$^{,}$$^{2}$$^{,}$$^{3}$ BIK1/PBLs in turn phosphorylate the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to induce apoplastic ROS production.$^{4}$$^{,}$$^{5}$ PBL and RBOH functions in plant immunity have been extensively characterized in flowering plants. Much less is known about the conservation of pattern-triggered ROS signaling pathways in non-flowering plants. In this study, we show that in the liverwort Marchantia polymorpha (hereafter Marchantia), single members of the RBOH and PBL families, namely MpRBOH1 and MpPBLa, are required for chitin-induced ROS production. MpPBLa directly interacts with and phosphorylates MpRBOH1 at specific, conserved sites within its cytosolic N terminus, and this phosphorylation is essential for chitin-induced MpRBOH1-mediated ROS production. Collectively, our work reveals the functional conservation of the PBL-RBOH module that controls pattern-triggered ROS production in land plants

The phagocytosis oxidase/Bem1p domain-containing protein PB1CP negatively regulates the NADPH oxidase RBOHD in plant immunity

Perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors activates RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) through direct phosphorylation by BOTRYTIS-INDUCED KINASE 1 (BIK1) and induces the production of reactive oxygen species (ROS). RBOHD activity must be tightly controlled to avoid the detrimental effects of ROS, but little is known about RBOHD downregulation. To understand the regulation of RBOHD, we used co-immunoprecipitation of RBOHD with mass spectrometry analysis and identified PHAGOCYTOSIS OXIDASE/BEM1P (PB1) DOMAIN-CONTAINING PROTEIN (PB1CP). PB1CP negatively regulates RBOHD and the resistance against the fungal pathogen Colletotrichum higginsianum. PB1CP competes with BIK1 for binding to RBOHD in vitro. Furthermore, PAMP treatment enhances the PB1CP-RBOHD interaction, thereby leading to the dissociation of phosphorylated BIK1 from RBOHD in vivo. PB1CP localizes at the cell periphery and PAMP treatment induces relocalization of PB1CP and RBOHD to the same small endomembrane compartments. Additionally, overexpression of PB1CP in Arabidopsis leads to a reduction in the abundance of RBOHD protein, suggesting the possible involvement of PB1CP in RBOHD endocytosis. We found PB1CP, a novel negative regulator of RBOHD, and revealed its possible regulatory mechanisms involving the removal of phosphorylated BIK1 from RBOHD and the promotion of RBOHD endocytosis

The PTI-suppressing Avr2 effector from Fusarium oxysporum suppresses mono-ubiquitination and plasma membrane dissociation of BIK1

Plant pathogens use effector proteins to target host processes involved in pathogen perception, immune signalling, or defence outputs. Unlike foliar pathogens, it is poorly understood how root-invading pathogens suppress immunity. The Avr2 effector from the tomato root- and xylem-colonizing pathogen Fusarium oxysporum suppresses immune signalling induced by various pathogen-associated molecular patterns (PAMPs). It is unknown how Avr2 targets the immune system. Transgenic AVR2 Arabidopsis thaliana phenocopies mutants in which the pattern recognition receptor (PRR) co-receptor BRI1-ASSOCIATED RECEPTOR KINASE (BAK1) or its downstream signalling kinase BOTRYTIS-INDUCED KINASE 1 (BIK1) are knocked out. We therefore tested whether these kinases are Avr2 targets. Flg22-induced complex formation of the PRR FLAGELLIN SENSITIVE 2 and BAK1 occurred in the presence and absence of Avr2, indicating that Avr2 does not affect BAK1 function or PRR complex formation. Bimolecular fluorescence complementation assays showed that Avr2 and BIK1 co-localize in planta. Although Avr2 did not affect flg22-induced BIK1 phosphorylation, mono-ubiquitination was compromised. Furthermore, Avr2 affected BIK1 abundance and shifted its localization from nucleocytoplasmic to the cell periphery/plasma membrane. Together, these data imply that Avr2 may retain BIK1 at the plasma membrane, thereby suppressing its ability to activate immune signalling. Because mono-ubiquitination of BIK1 is required for its internalization, interference with this process by Avr2 could provide a mechanistic explanation for the compromised BIK1 mobility upon flg22 treatment. The identification of BIK1 as an effector target of a root-invading vascular pathogen identifies this kinase as a conserved signalling component for both root and shoot immunity

Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in<i>Arabidopsis thaliana</i>

Summar The calcium-dependent protein kinase CPK28 is a regulator of immune homeostasis in multiple plant species. Here, we used a proteomics approach to uncover CPK28-associated proteins. We found that CPK28 associates with subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39, and trans-phosphorylates RAF26 and RAF39. Metazoan Raf kinases function in mitogen-activated protein kinase (MAPK) cascades as MAPK kinase kinases (MKKKs). Although Raf-like kinases share some features with MKKKs, we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis MKKs. We show that MRK1, RAF26, and RAF39 localize to the cytosol and endomembranes, and we define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal immunity, and possess substrate specificities distinct from canonical MKKKs

Conservation of the PBL-RBOH immune module in land plants

The rapid production of reactive oxygen species (ROS) is a key signaling output in plant immunity. In the angiosperm model species Arabidopsis thaliana (hereafter Arabidopsis), recognition of non- or altered-self elicitor patterns by cell-surface immune receptors activates the receptor-like cytoplasmic kinases (RLCKs) of the AVRPPHB SUSCEPTIBLE 1 (PBS1)-like (PBL) family, particularly BOTRYTIS-INDUCED KINASE1 (BIK1).1,2,3 BIK1/PBLs in turn phosphorylate the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) to induce apoplastic ROS production.4,5 PBL and RBOH functions in plant immunity have been extensively characterized in flowering plants. Much less is known about the conservation of pattern-triggered ROS signaling pathways in non-flowering plants. In this study, we show that in the liverwort Marchantia polymorpha (hereafter Marchantia), single members of the RBOH and PBL families, namely MpRBOH1 and MpPBLa, are required for chitin-induced ROS production. MpPBLa directly interacts with and phosphorylates MpRBOH1 at specific, conserved sites within its cytosolic N terminus, and this phosphorylation is essential for chitin-induced MpRBOH1-mediated ROS production. Collectively, our work reveals the functional conservation of the PBL-RBOH module that controls pattern-triggered ROS production in land plants