13Plants have many, highly variable resistance (R) gene loci, which provide resistance to a variety of 14 pathogens. The first R gene to be cloned, maize (Zea mays) Hm1, was published over 25 years ago, R genes are only understood for a small proportion of known R genes, a clearer understanding of 26 mechanisms is emerging and will be crucial for rational engineering and deployment of novel R 27 genes. 28 29

Reference datasets are critical in computational biology. They help define canonical biological features and are essential for benchmarking studies. Here, we describe a comprehensive reference dataset of experimentally validated plant nucleotide-binding leucine-rich repeat (NLR) immune receptors. RefPlantNLR consists of 481 NLRs from 31 genera belonging to 11 orders of flowering plants. This reference dataset has several applications. We used RefPlantNLR to determine the canonical features of functionally validated plant NLRs and to benchmark 5 NLR annotation tools. This revealed that although NLR annotation tools tend to retrieve the majority of NLRs, they frequently produce domain architectures that are inconsistent with the RefPlantNLR annotation. Guided by this analysis, we developed a new pipeline, NLRtracker, which extracts and annotates NLRs from protein or transcript files based on the core features found in the RefPlantNLR dataset. The RefPlantNLR dataset should also prove useful for guiding comparative analyses of NLRs across the wide spectrum of plant diversity and identifying understudied taxa. We hope that the RefPlantNLR resource will contribute to moving the field beyond a uniform view of NLR structure and function.

RNA-binding proteins (RBPs) play a crucial role in regulating RNA function and fate. However, the full complement of RBPs has only recently begun to be uncovered through proteome-wide approaches such as RNA interactome capture (RIC). RIC has been applied to various cell lines and organisms, including plants, greatly expanding the repertoire of RBPs. However, several technical challenges have limited the efficacy of RIC when applied to plant tissues. Here, we report an improved version of RIC that overcomes the difficulties imposed by leaf tissue. Using this improved RIC method in Arabidopsis leaves, we identified 717 RBPs, generating a deep RNA-binding proteome for leaf tissues. While 75% of these RBPs can be linked to RNA biology, the remaining 25% were previously not known to interact with RNA. Interestingly, we observed that a large number of proteins related to photosynthesis associate with RNA in vivo, including proteins from the four major photosynthetic supercomplexes. As has previously been reported for mammals, a large proportion of leaf RBPs lack known RNA-binding domains, suggesting unconventional modes of RNA binding. We anticipate that this improved RIC method will provide critical insights into RNA metabolism in plants, including how cellular RBPs respond to environmental, physiological and pathological cues.

ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.

Proteolytic cascades regulate immunity and development in animals, but these cascades in plants have not yet been reported. Here we report that the extracellular immune protease Rcr3 of tomato is activated by P69B and other subtilases (SBTs), revealing a proteolytic cascade regulating extracellular immunity in solanaceous plants. Rcr3 is a secreted papain-like Cys protease (PLCP) of tomato that acts both in basal resistance against late blight disease (Phytophthora infestans) and in gene-for-gene resistance against the fungal pathogen Cladosporium fulvum (syn. Passalora fulva). Despite the prevalent model that Rcr3-like proteases can activate themselves at low pH, we found that catalytically inactive proRcr3 mutant precursors are still processed into mature mRcr3 isoforms. ProRcr3 is processed by secreted P69B and other Asp-selective SBTs in solanaceous plants, providing robust immunity through SBT redundancy. The apoplastic effector EPI1 of P. infestans can block Rcr3 activation by inhibiting SBTs, suggesting that this effector promotes virulence indirectly by preventing the activation of Rcr3(-like) immune proteases. Rcr3 activation in Nicotiana benthamiana requires a SBT from a different subfamily, indicating that extracellular proteolytic cascades have evolved convergently in solanaceous plants or are very ancient in the plant kingdom. The frequent incidence of Asp residues in the cleavage region of Rcr3-like proteases in solanaceous plants indicates that activation of immune proteases by SBTs is a general mechanism, illuminating a proteolytic cascade that provides robust apoplastic immunity.

Ichinose and Renier A. L. van der Hoorn INTRODUCTION: Immunogenic flagellin fragments are a signature of bacterial invasion in both plants and animals. Plants recognize flagellin fragments via FLS2, a model receptor kinase that is highly conserved amongst angiosperms. However, little is known about events upstream of flagellin perception by FLS2. The flagellin fragments recognized by FLS2 are buried in the flagellin polymer structure and require hydrolytic release before recognition can occur, yet the hydrolases releasing these elicitors remain to be identified. Uncovering their identity is a daunting task because the extracellular space of plants (the apoplast) is known to contain hundreds of uncharacterized glycosidases and proteases. RATIONALE:We reasoned that pathogenic bacteria would suppress plant hydrolases that are important for immunity. We therefore monitored the activity of apoplastic hydrolases using activitybased protein profiling (ABPP) using chemical probes that irreversibly label the active site of hydrolases in an activity-dependent manner. We applied this strategy to study the infection of the tobacco relative Nicotiana benthamiana with the model bacterial pathogens Pseudomonas syringae pathovars tomato DC3000 (PtoDC3000); tabaci (Pta6605); and syringae (PsyB728a). RESULTS:Glycosidase activity profiling of apoplastic fluids isolated from PtoDC3000-infected plants revealed that the activity of the β-galactosidase BGAL1 is suppressed in the apoplast during infection.BGAL1 suppression is caused by a heat-stable, basic, small inhibitor molecule that is produced by the bacteria under the control of the hrpR/S/L virulence regulators. Null mutants of N. benthamiana lacking BGAL1 generated by genome editing are more susceptible, demonstrating that BGAL1 contributes to immunity. When studying why BGAL1 is suppressed, we discovered that treatment of PtoDC3000 and Pta6605 with apoplastic fluids containing BGAL1 releases an elicitor that triggers the release of reactive oxygen species (ROS) in leaf discs, a signature immune response in plants. The released elicitor is flagellin-derived because the ROS burst requires both the FLS2 receptor in the plant and flagellinencoding fliC in the bacteria. The flagellin polymer of both PtoDC3000 and Pta6605 is O-glycosylated with a glycan consisting of several rhamnose residues and a terminal modified viosamine (mVio).Treatment of mutant Pta6605 bacteria carrying nonglycosylated flagellin triggers the ROS burst when treated with apoplastic fluids, even in the absence of BGAL1, demonstrating that BGAL1 requires

SummaryAgroinfiltrated Nicotiana benthamiana is a flexible and scalable platform for recombinant protein (RP) production, but its great potential is hampered by plant proteases that degrade RPs. Here, we tested 29 candidate protease inhibitors (PIs) in agroinfiltrated N. benthamiana leaves for enhancing accumulation of three unrelated RPs: glycoenzyme α‐Galactosidase; glycohormone erythropoietin (EPO); and IgG antibody VRC01. Of the previously described PIs enhancing RP accumulation, we found only cystatin SlCYS8 to be effective. We identified three additional new, unrelated PIs that enhance RP accumulation: N. benthamiana NbPR4, NbPot1 and human HsTIMP, which have been reported to inhibit cysteine, serine and metalloproteases, respectively. Remarkably, accumulation of all three RPs is enhanced by each PI similarly, suggesting that the mechanism of degradation of unrelated RPs follows a common pathway. Inhibitory functions HsTIMP and SlCYS8 are required to enhance RP accumulation, suggesting that their target proteases may degrade RPs. Different PIs additively enhance RP accumulation, but the effect of each PI is dose‐dependent. Activity‐based protein profiling (ABPP) revealed that the activities of papain‐like Cys proteases (PLCPs), Ser hydrolases (SHs) or vacuolar processing enzymes (VPEs) in leaves are unaffected upon expression of the new PIs, whereas SlCYS8 expression specifically suppresses PLCP activity only. Quantitative proteomics indicates that the three new PIs affect agroinfiltrated tissues similarly and that they all increase immune responses. NbPR4, NbPot1 and HsTIMP can be used to study plant proteases and improve RP accumulation in molecular farming.

Reference datasets are critical in computational biology. They help define canonical biological features and are essential for benchmarking studies. Here, we describe a comprehensive reference dataset of experimentally validated plant NLR immune receptors. RefPlantNLR consists of 415 NLRs from 31 genera belonging to 11 orders of flowering plants. We used RefPlantNLR to determine the canonical features of functionally validated plant NLRs. This reference dataset should prove useful for benchmarking NLR annotation tools, guiding comparative analyses of NLRs across the wide spectrum of plant diversity and identifying under-studied taxa. We hope that the RefPlantNLR resource will contribute to moving the field beyond a uniform view of NLR structure and function.