Molecular dissection of the pseudokinase ZED1 expands effector recognition to the tomato immune receptor ZAR1.

The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) is a bacterial pathogen recognition hub that mediates resistance by guarding host kinases for modification by pathogen effectors. The pseudokinase HOPZ-ETI DEFICIENT 1 (ZED1) is the only known ZAR1-guarded protein that interacts directly with a pathogen effector, HopZ1a, from the bacterial pathogen Pseudomonas syringae, making it a promising system for rational design of effector recognition for plant immunity. Here, we conducted an in-depth molecular analysis of ZED1. We generated a library of 164 random ZED1 mutants and identified 50 mutants that could not recognize the effector HopZ1a when transiently expressed in Nicotiana benthamiana. Based on our random mutants, we generated a library of 27 point mutants and found evidence of minor functional divergence between Arabidopsis (Arabidopsis thaliana) and N. benthamiana ZAR1 orthologs. We leveraged our point mutant library to identify regions in ZED1 critical for ZAR1 and HopZ1a interactions and identified two likely ZED1-HopZ1a binding conformations. We explored ZED1 nucleotide and cation binding activity and showed that ZED1 is a catalytically dead pseudokinase, functioning solely as an allosteric regulator upon effector recognition. We used our library of ZED1 point mutants to identify the ZED1 activation loop regions as the most likely cause of interspecies ZAR1-ZED1 incompatibility. Finally, we identified a mutation that abolished ZAR1-ZED1 interspecies incompatibility while retaining the ability to mediate HopZ1a recognition, which enabled recognition of HopZ1a through tomato (Solanum lycopersicum) ZAR1. This provides an example of expanded effector recognition through a ZAR1 ortholog from a non-model species.

Utilising natural diversity of kinases to rationally engineer interactions with the angiosperm immune receptor ZAR1.

The highly conserved angiosperm immune receptor HOPZ-ACTIVATED RESISTANCE1 (ZAR1) recognises the activity of diverse pathogen effector proteins by monitoring the ZED1-related kinase (ZRK) family. Understanding how ZAR1 achieves interaction specificity for ZRKs may allow for the expansion of the ZAR1-kinase recognition repertoire to achieve novel pathogen recognition outside of model species. We took advantage of the natural diversity of Arabidopsis thaliana kinases to probe the ZAR1-kinase interaction interface and found that A. thaliana ZAR1 (AtZAR1) can interact with most ZRKs, except ZRK7. We found evidence of alternative splicing of ZRK7, resulting in a protein that can interact with AtZAR1. Despite high sequence conservation of ZAR1, interspecific ZAR1-ZRK pairings resulted in the autoactivation of cell death. We showed that ZAR1 interacts with a greater diversity of kinases than previously thought, while still possessing the capacity for specificity in kinase interactions. Finally, using AtZAR1-ZRK interaction data, we rationally increased ZRK10 interaction strength with AtZAR1, demonstrating the feasibility of the rational design of a ZAR1-interacting kinase. Overall, our findings advance our understanding of the rules governing ZAR1 interaction specificity, with promising future directions for expanding ZAR1 immunodiversity.

Genome-wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection.

Plant diseases are an important threat to food production. While major pathogenicity determinants required for disease have been extensively studied, less is known on how pathogens thrive during host colonization, especially at early infection stages. Here, we used randomly barcoded-transposon insertion site sequencing (RB-TnSeq) to perform a genome-wide screen and identify key bacterial fitness determinants of the vascular pathogen Xanthomonas campestris pv campestris (Xcc) during infection of the cauliflower host plant (Brassica oleracea). This high-throughput analysis was conducted in hydathodes, the natural entry site of Xcc, in xylem sap and in synthetic media. Xcc did not face a strong bottleneck during hydathode infection. In total, 181 genes important for fitness were identified in plant-associated environments with functional enrichment in genes involved in metabolism but only few genes previously known to be involved in virulence. The biological relevance of 12 genes was independently confirmed by phenotyping single mutants. Notably, we show that XC_3388, a protein with no known function (DUF1631), plays a key role in the adaptation and virulence of Xcc possibly through c-di-GMP-mediated regulation. This study revealed yet unsuspected social behaviors adopted by Xcc individuals when confined inside hydathodes at early infection stages.

The Phloem as an Arena for Plant Pathogens.

Although the phloem is a highly specialized tissue, certain pathogens, including phytoplasmas, spiroplasmas, and viruses, have evolved to access and live in this sequestered and protected environment, causing substantial economic harm. In particular, Candidatus Liberibacter spp. are devastating citrus in many parts of the world. Given that most phloem pathogens are vectored, they are not exposed to applied chemicals and are therefore difficult to control. Furthermore, pathogens use the phloem network to escape mounted defenses. Our review summarizes the current knowledge of phloem anatomy, physiology, and biochemistry relevant to phloem/pathogen interactions. We focus on aspects of anatomy specific to pathogen movement, including sieve plate structure and phloem-specific proteins. Phloem sampling techniques are discussed. Finally, pathogens that cause particular harm to the phloem of crop species are considered in detail.

What the Wild Things Do: Mechanisms of Plant Host Manipulation by Bacterial Type III-Secreted Effector Proteins.

Phytopathogenic bacteria possess an arsenal of effector proteins that enable them to subvert host recognition and manipulate the host to promote pathogen fitness. The type III secretion system (T3SS) delivers type III-secreted effector proteins (T3SEs) from bacterial pathogens such as Pseudomonas syringae, Ralstonia solanacearum, and various Xanthomonas species. These T3SEs interact with and modify a range of intracellular host targets to alter their activity and thereby attenuate host immune signaling. Pathogens have evolved T3SEs with diverse biochemical activities, which can be difficult to predict in the absence of structural data. Interestingly, several T3SEs are activated following injection into the host cell. Here, we review T3SEs with documented enzymatic activities, as well as T3SEs that facilitate virulence-promoting processes either indirectly or through non-enzymatic mechanisms. We discuss the mechanisms by which T3SEs are activated in the cell, as well as how T3SEs modify host targets to promote virulence or trigger immunity. These mechanisms may suggest common enzymatic activities and convergent targets that could be manipulated to protect crop plants from infection.

Identification of a Putative DNA-Binding Protein in Arabidopsis That Acts as a Susceptibility Hub and Interacts With Multiple Pseudomonas syringae Effectors.

Phytopathogens use secreted effector proteins to suppress host immunity and promote pathogen virulence, and there is increasing evidence that the host-pathogen interactome comprises a complex network. To identify novel interactors of the Pseudomonas syringae effector HopZ1a, we performed a yeast two-hybrid screen that identified a previously uncharacterized Arabidopsis protein that we designate HopZ1a interactor 1 (ZIN1). Additional analyses in yeast and in planta revealed that ZIN1 also interacts with several other P. syringae effectors. We show that an Arabidopsis loss-of-function zin1 mutant is less susceptible to infection by certain strains of P. syringae, while overexpression of ZIN1 results in enhanced susceptibility. Functionally, ZIN1 exhibits topoisomerase-like activity in vitro. Transcriptional profiling of wild-type and zin1 Arabidopsis plants inoculated with P. syringae indicated that while ZIN1 regulates a wide range of pathogen-responsive biological processes, the list of genes more highly expressed in zin1 versus wild-type plants is particularly enriched for ribosomal protein genes. Altogether, these data illuminate ZIN1 as a potential susceptibility hub that interacts with multiple effectors to influence the outcome of plant-microbe interactions.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Arabidopsis Abscisic Acid Repressor 1 is a susceptibility hub that interacts with multiple Pseudomonas syringae effectors.

Pathogens secrete effector proteins into host cells to suppress host immunity and promote pathogen virulence, although many features at the molecular interface of host-pathogen interactions remain to be characterized. In a yeast two-hybrid assay, we found that the Pseudomonas syringae effector HopZ1a interacts with the Arabidopsis transcriptional regulator Abscisic Acid Repressor 1 (ABR1). Further analysis revealed that ABR1 interacts with multiple P. syringae effectors, suggesting that it may be targeted as a susceptibility hub. Indeed, loss-of-function abr1 mutants exhibit reduced susceptibility to a number of P. syringae strains. The ABR1 protein comprises a conserved APETALA2 (AP2) domain flanked by long regions of predicted structural disorder. We verified the DNA-binding activity of the AP2 domain and demonstrated that the disordered domains act redundantly to enhance DNA binding and to facilitate transcriptional activation by ABR1. Finally, we compared gene expression profiles from wild-type and abr1 plants following inoculation with P. syringae, which suggested that the reduced susceptibility of abr1 mutants is due to the loss of a virulence target rather than an enhanced immune response. These data highlight ABR1 as a functionally important component at the host-pathogen interface.

A natural diversity screen in Arabidopsis thaliana reveals determinants for HopZ1a recognition in the ZAR1-ZED1 immune complex.

Pathogen pressure on hosts can lead to the evolution of genes regulating the innate immune response. By characterizing naturally occurring polymorphisms in immune receptors, we can better understand the molecular determinants of pathogen recognition. ZAR1 is an ancient Arabidopsis thaliana NLR (Nucleotide-binding [NB] Leucine-rich-repeat [LRR] Receptor) that recognizes multiple secreted effector proteins from the pathogenic bacteria Pseudomonas syringae and Xanthomonas campestris through its interaction with receptor-like cytoplasmic kinases (RLCKs). ZAR1 was first identified for its role in recognizing P. syringae effector HopZ1a, through its interaction with the RLCK ZED1. To identify additional determinants of HopZ1a recognition, we performed a computational screen for ecotypes from the 1001 Genomes project that were likely to lack HopZ1a recognition, and tested ~300 ecotypes. We identified ecotypes containing polymorphisms in ZAR1 and ZED1. Using our previously established Nicotiana benthamiana transient assay and Arabidopsis ecotypes, we tested for the effect of naturally occurring polymorphisms on ZAR1 interactions and the immune response. We identified key residues in the NB or LRR domain of ZAR1 that impact the interaction with ZED1. We demonstrate that natural diversity combined with functional assays can help define the molecular determinants and interactions necessary to regulate immune induction in response to pathogens.

A compact Cascade-Cas3 system for targeted genome engineering.

CRISPR-Cas technologies have enabled programmable gene editing in eukaryotes and prokaryotes. However, the leading Cas9 and Cas12a enzymes are limited in their ability to make large deletions. Here, we used the processive nuclease Cas3, together with a minimal Type I-C Cascade-based system for targeted genome engineering in bacteria. DNA cleavage guided by a single CRISPR RNA generated large deletions (7-424 kilobases) in Pseudomonas aeruginosa with near-100% efficiency, while Cas9 yielded small deletions and point mutations. Cas3 generated bidirectional deletions originating from the programmed site, which was exploited to reduce the P. aeruginosa genome by 837 kb (13.5%). Large deletion boundaries were efficiently specified by a homology-directed repair template during editing with Cascade-Cas3, but not Cas9. A transferable 'all-in-one' vector was functional in Escherichia coli, Pseudomonas syringae and Klebsiella pneumoniae, and endogenous CRISPR-Cas use was enhanced with an 'anti-anti-CRISPR' strategy. P. aeruginosa Type I-C Cascade-Cas3 (PaeCas3c) facilitates rapid strain manipulation with applications in synthetic biology, genome minimization and the removal of large genomic regions.

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay.

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide. Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for activity.

NLR (nucleotide-binding [NB] leucine-rich repeat [LRR] receptor) proteins are critical for inducing immune responses in response to pathogen proteins, and must be tightly modulated to prevent spurious activation in the absence of a pathogen. The ZAR1 NLR recognizes diverse effector proteins from Pseudomonas syringae, including HopZ1a, and Xanthomonas species. Receptor-like cytoplasmic kinases (RLCKs) such as ZED1, interact with ZAR1 and provide specificity for different effector proteins, such as HopZ1a. We previously developed a transient expression system in Nicotiana benthamiana that allowed us to demonstrate that ZAR1 function is conserved from the Brassicaceae to the Solanaceae. Here, we combined structural modelling of ZAR1, with molecular and functional assays in our transient system, to show that multiple intramolecular and intermolecular interactions modulate ZAR1 activity. We identified determinants required for the formation of the ZARCC oligomer and its activity. Lastly, we characterized intramolecular interactions between ZAR1 subdomains that participate in keeping ZAR1 immune complexes inactive. This work identifies molecular constraints on immune receptor function and activation.

Comparative Genomics Screen Identifies Microbe-Associated Molecular Patterns from 'Candidatus Liberibacter' spp. That Elicit Immune Responses in Plants.

Citrus huanglongbing (HLB), caused by phloem-limited 'Candidatus Liberibacter' bacteria, is a destructive disease threatening the worldwide citrus industry. The mechanisms of pathogenesis are poorly understood and no efficient strategy is available to control HLB. Here, we used a comparative genomics screen to identify candidate microbe-associated molecular patterns (MAMPs) from 'Ca. Liberibacter' spp. We identified the core genome from multiple 'Ca. Liberibacter' pathogens, and searched for core genes with signatures of positive selection. We hypothesized that genes encoding putative MAMPs would evolve to reduce recognition by the plant immune system, while retaining their essential functions. To efficiently screen candidate MAMP peptides, we established a high-throughput microtiter plate-based screening assay, particularly for citrus, that measured reactive oxygen species (ROS) production, which is a common immune response in plants. We found that two peptides could elicit ROS production in Arabidopsis and Nicotiana benthamiana. One of these peptides elicited ROS production and defense gene expression in HLB-tolerant citrus genotypes, and induced MAMP-triggered immunity against the bacterial pathogen Pseudomonas syringae. Our findings identify MAMPs that boost immunity in citrus and could help prevent or reduce HLB infection.

Protein Acetylation in Pathogen Virulence and Host Defense: In Vitro Detection of Protein Acetylation by Radiolabeled Acetyl Coenzyme A.

Protein acetylation has emerged as a common modification that modulates multiple aspects of protein function, including localization, stability, and protein-protein interactions. It is increasingly evident that protein acetylation significantly impacts the outcome of host-microbe interactions. In order to characterize novel putative acetyltransferase enzymes and their substrates, we describe a simple protocol for the detection of acetyltransferase activity in vitro. Purified proteins are incubated with 14C-acetyl CoA and separated electrophoretically, and acetylated proteins are detected by phosphorimaging or autoradiography.

The enemy within: phloem-limited pathogens.

The growing impact of phloem-limited pathogens on high-value crops has led to a renewed interest in understanding how they cause disease. Although these pathogens cause substantial crop losses, many are poorly characterized. In this review, we present examples of phloem-limited pathogens that include intracellular bacteria with and without cell walls, and viruses. Phloem-limited pathogens have small genomes and lack many genes required for core metabolic processes, which is, in part, an adaptation to the unique phloem environment. For each pathogen class, we present multiple case studies to highlight aspects of disease caused by phloem-limited pathogens. The pathogens presented include Candidatus Liberibacter asiaticus (citrus greening), Arsenophonus bacteria, Serratia marcescens (cucurbit yellow vine disease), Candidatus Phytoplasma asteris (Aster Yellows Witches' Broom), Spiroplasma kunkelii, Potato leafroll virus and Citrus tristeza virus. We focus on commonalities in the virulence strategies of these pathogens, and aim to stimulate new discussions in the hope that widely applicable disease management strategies can be found.

Soil mixture composition alters Arabidopsis susceptibility to Pseudomonas syringae infection.

Pseudomonas syringae is a gram-negative bacterial pathogen that causes disease on more than 100 different plant species, including the model plant Arabidopsis thaliana. Dissection of the Arabidopsis thaliana-Pseudomonas syringae pathosystem has identified many factors that contribute to successful infection or immunity, including the genetics of the host, the genetics of the pathogen, and the environment. Environmental factors that contribute to a successful interaction can include temperature, light, and the circadian clock, as well as the soil environment. As silicon-amended Resilience soil is advertised to enhance plant health, we sought to examine the extent to which this soil might affect the behavior of the A. thaliana-P. syringae model pathosystem and to characterize the mechanisms through which these effects may occur. We found that plants grown in Si-amended Resilience soil displayed enhanced resistance to bacteria compared to plants grown in non-Si-amended Sunshine soil, and salicylic acid biosynthesis and signaling were not required for resistance. Although silicon has been shown to contribute to broad-spectrum resistance, our data indicate that silicon is not the direct cause of enhanced resistance and that the Si-amended Resilience soil has additional properties that modulate plant resistance. Our work demonstrates the importance of environmental factors, such as soil in modulating interactions between the plant and foliar pathogens, and highlights the significance of careful annotation of the environmental conditions under which plant-pathogen interactions are studied.

A Practical Guide to Quantitative Interactor Screening with Next-Generation Sequencing (QIS-Seq).

Yeast two-hybrid screens are a powerful approach to identify protein-protein interactions; however, they are typically limited in the number of interactions identified, and lack quantitative values to ascribe confidence scores to the interactions that are obtained. We have developed a high-throughput, quantitative, yeast two-hybrid screening approach coupled with next-generation sequencing. This strategy allows the identification of interacting proteins that are preferentially associated with a bait of interest, and helps eliminate nonspecific interacting proteins. The method is high-throughput, allowing many more baits to be tested and many more candidate interacting proteins to be identified. Quantitative data allows the interactors to be ascribed confidence scores based on their enrichment with particular baits, and can identify both common and rare interacting proteins.

Analysis of the ZAR1 Immune Complex Reveals Determinants for Immunity and Molecular Interactions.

Plants depend on innate immunity to prevent disease. Plant pathogenic bacteria, like Pseudomonas syringae and Xanthomonas campestris, use the type III secretion system as a molecular syringe to inject type III secreted effector (T3SE) proteins in plants. The primary function of most T3SEs is to suppress immunity; however, the plant can evolve nucleotide-binding domain-leucine-rich repeat domain-containing proteins to recognize specific T3SEs. The AtZAR1 NLR induces strong defense responses against P. syringae and X. campestris The P. syringae T3SE HopZ1a is an acetyltransferase that acetylates the pseudokinase AtZED1 and triggers recognition by AtZAR1. However, little is known about the molecular mechanisms that lead to AtZAR1-induced immunity in response to HopZ1a. We established a transient expression system in Nicotiana benthamiana to study detailed interactions among HopZ1a, AtZED1, and AtZAR1. We show that the AtZAR1 immune pathway is conserved in N. benthamiana and identify AtZAR1 domains, and residues in AtZAR1 and AtZED1, that are important for immunity and protein-protein interactions in planta and in yeast (Saccharomyces cerevisiae). We show that the coiled-coil domain of AtZAR1 oligomerizes, and this domain acts as a signal to induce immunity. This detailed analysis of the AtZAR1-AtZED1 protein complex provides a better understanding of the immune signaling hub controlled by AtZAR1.

Die another day: Molecular mechanisms of effector-triggered immunity elicited by type III secreted effector proteins.

Bacterial pathogens inject type III secreted effector (T3SE) proteins into their hosts where they display dual roles depending on the host genotype. T3SEs promote bacterial virulence in susceptible hosts, and elicit immunity in resistant hosts. T3SEs are typically recognized when they modify a host target that is associated with a NOD-like receptor protein. We focus on the molecular mechanisms of T3SE recognition in plants. Plants guard multiple nodes of the immune signaling pathway, from recognition at the cell surface by receptor-like kinases to nuclear signaling. Some nodes are bacterial virulence targets, while other nodes are decoys that resemble true virulence targets.

Lewis Acid Zeolites for Biomass Conversion: Perspectives and Challenges on Reactivity, Synthesis, and Stability.

Zeolites containing Sn, Ti, Zr, Hf, Nb, or Ta heteroatoms are versatile catalysts for the activation and conversion of oxygenated molecules owing to the unique Lewis acid character of their tetrahedral metal sites. Through fluoride-mediated synthesis, hydrophobic Lewis acid zeolites can behave as water-tolerant catalysts, which has resulted in a recent surge of experimental and computational studies in the field of biomass conversion. However, many open questions still surround these materials, especially relating to the nature of their active sites. This lack of fundamental understanding is exemplified by the many dissonant results that have been described in recent literature reports. In this review, we use a molecular-based approach to provide insight into the relationship between the structure of the metal center and its reactivity toward different substrates, with the ultimate goal of providing a robust framework to understand the properties that have the strongest influence on catalytic performance for the conversion of oxygenates.