A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.

Induced proximity of a TIR signaling domain on a plant-mammalian NLR chimera activates defense in plants.

Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.

Distinct modes of derepression of an Arabidopsis immune receptor complex by two different bacterial effectors.

Plant intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors often function in pairs to detect pathogen effectors and activate defense. The Arabidopsis RRS1-R-RPS4 NLR pair recognizes the bacterial effectors AvrRps4 and PopP2 via an integrated WRKY transcription factor domain in RRS1-R that mimics the effector's authentic targets. How the complex activates defense upon effector recognition is unknown. Deletion of the WRKY domain results in an RRS1 allele that triggers constitutive RPS4-dependent defense activation, suggesting that in the absence of effector, the WRKY domain contributes to maintaining the complex in an inactive state. We show the WRKY domain interacts with the adjacent domain 4, and that the inactive state of RRS1 is maintained by WRKY-domain 4 interactions before ligand detection. AvrRps4 interaction with the WRKY domain disrupts WRKY-domain 4 association, thus derepressing the complex. PopP2-triggered activation is less easily explained by such disruption and involves the longer C-terminal extension of RRS1-R. Furthermore, some mutations in RPS4 and RRS1 compromise PopP2 but not AvrRps4 recognition, suggesting that AvrRps4 and PopP2 derepress the complex differently. Consistent with this, a "reversibly closed" conformation of RRS1-R, engineered in a method exploiting the high affinity of colicin E9 and Im9 domains, reversibly loses AvrRps4, but not PopP2 responsiveness. Following RRS1 derepression, interactions between domain 4 and the RPS4 C-terminal domain likely contribute to activation. Simultaneous relief of autoinhibition and activation may contribute to defense activation in many immune receptors.

Protein-protein interactions in the RPS4/RRS1 immune receptor complex.

Plant NLR (Nucleotide-binding domain and Leucine-rich Repeat) immune receptor proteins are encoded by Resistance (R) genes and confer specific resistance to pathogen races that carry the corresponding recognized effectors. Some NLR proteins function in pairs, forming receptor complexes for the perception of specific effectors. We show here that the Arabidopsis RPS4 and RRS1 NLR proteins are both required to make an authentic immune complex. Over-expression of RPS4 in tobacco or in Arabidopsis results in constitutive defense activation; this phenotype is suppressed in the presence of RRS1. RRS1 protein co-immunoprecipitates (co-IPs) with itself in the presence or absence of RPS4, but in contrast, RPS4 does not associate with itself in the absence of RRS1. In the presence of RRS1, RPS4 associates with defense signaling regulator EDS1 solely in the nucleus, in contrast to the extra-nuclear location found in the absence of RRS1. The AvrRps4 effector does not disrupt RPS4-EDS1 association in the presence of RRS1. In the absence of RRS1, AvrRps4 interacts with EDS1, forming nucleocytoplasmic aggregates, the formation of which is disturbed by the co-expression of PAD4 but not by SAG101. These data indicate that the study of an immune receptor protein complex in the absence of all components can result in misleading inferences, and reveals an NLR complex that dynamically interacts with the immune regulators EDS1/PAD4 or EDS1/SAG101, and with effectors, during the process by which effector recognition is converted to defense activation.

Pathogen perception by NLRs in plants and animals: Parallel worlds.

Intracellular NLR (Nucleotide-binding domain and Leucine-rich Repeat-containing) receptors are sensitive monitors that detect pathogen invasion of both plant and animal cells. NLRs confer recognition of diverse molecules associated with pathogen invasion. NLRs must exhibit strict intramolecular controls to avoid harmful ectopic activation in the absence of pathogens. Recent discoveries have elucidated the assembly and structure of oligomeric NLR signalling complexes in animals, and provided insights into how these complexes act as scaffolds for signal transduction. In plants, recent advances have provided novel insights into signalling-competent NLRs, and into the myriad strategies that diverse plant NLRs use to recognise pathogens. Here, we review recent insights into the NLR biology of both animals and plants. By assessing commonalities and differences between kingdoms, we are able to develop a more complete understanding of NLR function.

Glucan, Water Dikinase Exerts Little Control over Starch Degradation in Arabidopsis Leaves at Night.

The first step on the pathway of starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night is the phosphorylation of starch polymers, catalyzed by glucan, water dikinase (GWD). It has been suggested that GWD is important for the control of starch degradation, because its transcript levels undergo strong diel fluctuations, its activity is subject to redox regulation in vitro, and starch degradation is strongly decreased in gwd mutant plants. To test this suggestion, we analyzed changes in GWD protein abundance in relation to starch levels in wild-type plants, in transgenic plants in which GWD transcripts were strongly reduced by induction of RNA interference, and in transgenic plants overexpressing GWD. We found that GWD protein levels do not vary over the diel cycle and that the protein has a half-life of 2 d. Overexpression of GWD does not accelerate starch degradation in leaves, and starch degradation is not inhibited until GWD levels are reduced by 70%. Surprisingly, this degree of reduction also inhibits starch synthesis in the light. To discover the importance of redox regulation, we generated transgenic plants expressing constitutively active GWD. These plants retained normal control of degradation. We conclude that GWD exerts only a low level of control over starch degradation in Arabidopsis leaves.

A Comparative Overview of the Intracellular Guardians of Plants and Animals: NLRs in Innate Immunity and Beyond.

Nucleotide-binding domain leucine-rich repeat receptors (NLRs) play important roles in the innate immune systems of both plants and animals. Recent breakthroughs in NLR biochemistry and biophysics have revolutionized our understanding of how NLR proteins function in plant immunity. In this review, we summarize the latest findings in plant NLR biology and draw direct comparisons to NLRs of animals. We discuss different mechanisms by which NLRs recognize their ligands in plants and animals. The discovery of plant NLR resistosomes that assemble in a comparable way to animal inflammasomes reinforces the striking similarities between the formation of plant and animal NLR complexes. Furthermore, we discuss the mechanisms by which plant NLRs mediate immune responses and draw comparisons to similar mechanisms identified in animals. Finally, we summarize the current knowledge of the complex genetic architecture formed by NLRs in plants and animals and the roles of NLRs beyond pathogen detection.

Chromatic photoacclimation extends utilisable photosynthetically active radiation in the chlorophyll d-containing cyanobacterium, Acaryochloris marina.

Chromatic photoacclimation and photosynthesis were examined in two strains of Acaryochloris marina (MBIC11017 and CCMEE5410) and in Synechococcus PCC7942. Acaryochloris contains Chl d, which has an absorption peak at ca 710 nm in vivo. Cultures were grown in one of the three wavelengths (525 nm, 625 nm and 720 nm) of light from narrow-band photodiodes to determine the effects on pigment composition, growth rate and photosynthesis: no growth occurred in 525 nm light. Synechococcus did not grow in 720 nm light because Chl a does not absorb effectively at this long wavelength. Acaryochloris did grow in 720 nm light, although strain MBIC11017 showed a decrease in phycobilins over time. Both Synechococcus and Acaryochloris MBIC11017 showed a dramatic increase in phycobilin content when grown in 625 nm light. Acaryochloris CCMEE5410, which lacks phycobilins, would not grow satisfactorily under 625 nm light. The cells adjusted their pigment composition in response to the light spectral conditions under which they were grown. Photoacclimation and the Q (y) peak of Chl d could be understood in terms of the ecological niche of Acaryochloris, i.e. habitats enriched in near infrared radiation.

Spectral properties of bacteriophytochrome AM1_5894 in the chlorophyll d-containing cyanobacterium Acaryochloris marina.

Acaryochloris marina, a unicellular oxygenic photosynthetic cyanobacterium, has uniquely adapted to far-red light-enriched environments using red-shifted chlorophyll d. To understand red-light use in Acaryochloris, the genome of this cyanobacterium was searched for red/far-red light photoreceptors from the phytochrome family, resulting in identification of a putative bacteriophytochrome AM1_5894. AM1_5894 contains three standard domains of photosensory components as well as a putative C-terminal signal transduction component consisting of a histidine kinase and receiver domain. The photosensory domains of AM1_5894 autocatalytically assemble with biliverdin in a covalent fashion. This assembled AM1_5894 shows the typical photoreversible conversion of bacterial phytochromes with a ground-state red-light absorbing (Pr) form with λBV max[Pr] 705 nm, and a red-light inducible far-red light absorbing (Pfr) form with λBV max[Pfr] 758 nm. Surprisingly, AM1_5894 also autocatalytically assembles with phycocyanobilin, involving photoreversible conversion of λPCB max[Pr] 682 nm and λPCB max[Pfr] 734 nm, respectively. Our results suggest phycocyanobilin is also covalently bound to AM1_5894, while mutation of a cysteine residue (Cys11Ser) abolishes this covalent binding. The physiological function of AM1_5894 in cyanobacteria containing red-shifted chlorophylls is discussed.

Comparative genomic analysis of multiple strains of two unusual plant pathogens: Pseudomonas corrugata and Pseudomonas mediterranea.

The non-fluorescent pseudomonads, Pseudomonas corrugata (Pcor) and P. mediterranea (Pmed), are closely related species that cause pith necrosis, a disease of tomato that causes severe crop losses. However, they also show strong antagonistic effects against economically important pathogens, demonstrating their potential for utilization as biological control agents. In addition, their metabolic versatility makes them attractive for the production of commercial biomolecules and bioremediation. An extensive comparative genomics study is required to dissect the mechanisms that Pcor and Pmed employ to cause disease, prevent disease caused by other pathogens, and to mine their genomes for genes that encode proteins involved in commercially important chemical pathways. Here, we present the draft genomes of nine Pcor and Pmed strains from different geographical locations. This analysis covered significant genetic heterogeneity and allowed in-depth genomic comparison. All examined strains were able to trigger symptoms in tomato plants but not all induced a hypersensitive-like response in Nicotiana benthamiana. Genome-mining revealed the absence of type III secretion system and known type III effector-encoding genes from all examined Pcor and Pmed strains. The lack of a type III secretion system appears to be unique among the plant pathogenic pseudomonads. Several gene clusters coding for type VI secretion system were detected in all genomes. Genome-mining also revealed the presence of gene clusters for biosynthesis of siderophores, polyketides, non-ribosomal peptides, and hydrogen cyanide. A highly conserved quorum sensing system was detected in all strains, although species specific differences were observed. Our study provides the basis for in-depth investigations regarding the molecular mechanisms underlying virulence strategies in the battle between plants and microbes.