Identification of two key genes involved in flavonoid catabolism and their different roles in apple resistance to biotic stresses.

Biosynthesis of flavonoid aglycones and glycosides is well established. However, key genes involved in their catabolism are poorly understood, even though the products of hydrolysis and oxidation play important roles in plant resistance to biotic stress. Here, we report on catabolism of dihydrochalcones (DHCs), the most abundant flavonoids in domesticated apple and wild Malus. Two key genes, BGLU13.1 and PPO05, were identified by activity-directed protein purification. BGLU13.1-A hydrolyzed phlorizin, (the most abundant DHC in domesticated apple) to produce phloretin which was then oxidized by PPO05. The process differed in some wild Malus, where trilobatin (a positional isomer of phlorizin) was mainly oxidized by PPO05. The effects of DHC catabolism on apple resistance to biotic stresses was investigated using transgenic plants. Either directly or indirectly, phlorizin hydrolysis affected resistance to the phytophagous pest two-spotted spider mite, while oxidation of trilobatin was involved in resistance to the biotrophic fungus Podosphaera leucotricha. DHC catabolism did not affect apple resistance to necrotrophic pathogens Valsa mali and Erwinia amylovara. These results suggest that different DHC catabolism pathways play different roles in apple resistance to biotic stresses. The role of DHC catabolism on apple resistance appeared closely related to the mode of invasion/damage used by pathogen/pest.

Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.

Biosynthesis of the Dihydrochalcone Sweetener Trilobatin Requires Phloretin Glycosyltransferase2.

Epidemics of obesity and type 2 diabetes drive strong consumer interest in plant-based low-calorie sweeteners. Trilobatin is a sweetener found at high concentrations in the leaves of a range of crabapple (Malus) species, but not in domesticated apple (Malus × domestica) leaves, which contain trilobatin's bitter positional isomer phloridzin. Variation in trilobatin content was mapped to the Trilobatin locus on LG 7 in a segregating population developed from a cross between domesticated apples and crabapples. Phloretin glycosyltransferase2 (PGT2) was identified by activity-directed protein purification and differential gene expression analysis in samples high in trilobatin but low in phloridzin. Markers developed for PGT2 cosegregated strictly with the Trilobatin locus. Biochemical analysis showed PGT2 efficiently catalyzed 4'-o-glycosylation of phloretin to trilobatin as well as 3-hydroxyphloretin to sieboldin. Transient expression of double bond reductase, chalcone synthase, and PGT2 genes reconstituted the apple pathway for trilobatin production in Nicotiana benthamiana Transgenic M. × domestica plants overexpressing PGT2 produced high concentrations of trilobatin in young leaves. Transgenic plants were phenotypically normal, and no differences in disease susceptibility were observed compared to wild-type plants grown under simulated field conditions. Sensory analysis indicated that apple leaf teas from PGT2 transgenics were readily discriminated from control leaf teas and were perceived as significantly sweeter. Identification of PGT2 allows marker-aided selection to be developed to breed apples containing trilobatin, and for high amounts of this natural low-calorie sweetener to be produced via biopharming and metabolic engineering in yeast.

Sensory-Directed Genetic and Biochemical Characterization of Volatile Terpene Production in Kiwifruit.

Terpene volatiles are found in many important fruit crops, but their relationship to flavor is poorly understood. Here, we demonstrate using sensory descriptive and discriminant analysis that 1,8-cineole contributes a key floral/eucalyptus note to the aroma of ripe 'Hort16A' kiwifruit (Actinidia chinensis). Two quantitative trait loci (QTLs) for 1,8-cineole production were identified on linkage groups 27 and 29a in a segregating A. chinensis population, with the QTL on LG29a colocating with a complex cluster of putative terpene synthase (TPS)-encoding genes. Transient expression in Nicotiana benthamiana and analysis of recombinant proteins expressed in Escherichia coli showed four genes in the cluster (AcTPS1a-AcTPS1d) encoded functional TPS enzymes, which produced predominantly sabinene, 1,8-cineole, geraniol, and springene, respectively. The terpene profile produced by AcTPS1b closely resembled the terpenes detected in red-fleshed A chinensis AcTPS1b expression correlated with 1,8-cineole content in developing/ripening fruit and also showed a positive correlation with 1,8-cineole content in the mapping population, indicating the basis for segregation is an expression QTL. Transient overexpression of AcTPS1b in Actinidia eriantha fruit confirmed this gene produced 1,8-cineole in Actinidia Structure-function analysis showed AcTPS1a and AcTPS1b are natural variants at key TPS catalytic site residues previously shown to change enzyme specificity in vitro. Together, our results indicate that AcTPS1b is a key gene for production of the signature flavor terpene 1,8-cineole in ripe kiwifruit. Using a sensory-directed strategy for compound identification provides a rational approach for applying marker-aided selection to improving flavor in kiwifruit as well as other fruits.

Chemical synthesis and characterization of a new quinazolinedione competitive antagonist for strigolactone receptors with an unexpected binding mode.

Strigolactones (SLs) are multifunctional plant hormones regulating essential physiological processes affecting growth and development. In vascular plants, SLs are recognized by α/ß hydrolase-fold proteins from the D14/DAD2 (Dwarf14/Decreased Apical Dominance 2) family in the initial step of the signaling pathway. We have previously discovered that N-phenylanthranilic acid derivatives (e.g. tolfenamic acid) are potent antagonists of SL receptors, prompting us to design quinazolinone and quinazolinedione derivatives (QADs and QADDs, respectively) as second-generation antagonists. Initial in silico docking studies suggested that these compounds would bind to DAD2, the petunia SL receptor, with higher affinity than the first-generation compounds. However, only one of the QADs/QADDs tested in in vitro assays acted as a competitive antagonist of SL receptors, with reduced affinity and potency compared with its N-phenylanthranilic acid 'parent'. X-ray crystal structure analysis revealed that the binding mode of the active QADD inside DAD2's cavity was not that predicted in silico, highlighting a novel inhibition mechanism for SL receptors. Despite a ∼10-fold difference in potency in vitro, the QADD and tolfenamic acid had comparable activity in planta, suggesting that the QADD compensates for lower potency with increased bioavailability. Altogether, our results establish this QADD as a novel lead compound towards the development of potent and bioavailable antagonists of SL receptors.

Inhibition of strigolactone receptors by N-phenylanthranilic acid derivatives: Structural and functional insights.

The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified N-phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and Arabidopsis (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and in silico modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets.

The AAT1 locus is critical for the biosynthesis of esters contributing to 'ripe apple' flavour in 'Royal Gala' and 'Granny Smith' apples.

The 'fruity' attributes of ripe apples (Malus × domestica) arise from our perception of a combination of volatile ester compounds. Phenotypic variability in ester production was investigated using a segregating population from a 'Royal Gala' (RG; high ester production) × 'Granny Smith' (GS; low ester production) cross, as well as in transgenic RG plants in which expression of the alcohol acyl transferase 1 (AAT1) gene was reduced. In the RG × GS population, 46 quantitative trait loci (QTLs) for the production of esters and alcohols were identified on 15 linkage groups (LGs). The major QTL for 35 individual compounds was positioned on LG2 and co-located with AAT1. Multiple AAT1 gene variants were identified in RG and GS, but only two (AAT1-RGa and AAT1-GSa) were functional. AAT1-RGa and AAT1-GSa were both highly expressed in the cortex and skin of ripe fruit, but AAT1 protein was observed mainly in the skin. Transgenic RG specifically reduced in AAT1 expression showed reduced levels of most key esters in ripe fruit. Differences in the ripe fruit aroma could be perceived by sensory analysis. The transgenic lines also showed altered ratios of biosynthetic precursor alcohols and aldehydes, and expression of a number of ester biosynthetic genes increased, presumably in response to the increased substrate pool. These results indicate that the AAT1 locus is critical for the biosynthesis of esters contributing to a 'ripe apple' flavour.

Crystal structure of kiwellin, a major cell-wall protein from kiwifruit.

Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4 kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05 Å resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi ß-barrel fold hooked to an N-terminal ß hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.

Ligand promiscuity within the internal cavity of Epiphyas postvittana Takeout 1 protein.

Takeout proteins are found across a diverse range of insect species and are thought to be involved in important aspects of insect physiology and behavior. These proteins act as ligand carriers, but the nature of their endogenous ligands remains unknown. The crystal structure of Epiphyas postvittana Takeout 1 (EpTo1), the only structure for any Takeout protein to date, revealed an α/ß-wrap fold with a purely hydrophobic internal cavity of tubular shape. When recombinantly expressed in Escherichia coli, we previously showed that a surrogate ubiquinone-8 ligand binds within the internal cavity of EpTo1 with excellent shape complementarity. We have now expressed EpTo1 in an insect cell expression system devoid of ubiquinone-8, and solved its crystal structure at 2.2Å resolution. Using combined information from crystallography and mass spectrometry, we identify a mixture of fatty acid moieties, mostly myristic and palmitic acid, bound inside the EpTo1 cavity, mimicking the structure of the longer ubiquinone-8 compound. No significant alteration of the internal cavity was observed regardless of the bound ligands, ubiquinone-8 or fatty acids, suggesting that the internal cavity of EpTo1 forms a rigid scaffold that imposes strict structural constraints for selectivity and specificity of ligand(s) in vivo.

Structure of an antennally-expressed carboxylesterase suggests lepidopteran odorant degrading enzymes are broadly tuned.

Insects rely on the detection of chemical cues present in the environment to guide their foraging and reproductive behaviour. As such, insects have evolved a sophisticated chemical processing system in their antennae comprised of several types of olfactory proteins. Of these proteins, odorant degrading enzymes are responsible for metabolising the chemical cues within the antennae, thereby maintaining olfactory system function. Members of the carboxyl/cholinesterase gene family are known to degrade odorant molecules with acetate-ester moieties that function as host recognition cues or sex pheromones, however, their specificity for these compounds remains unclear. Here, we evaluate expression levels of this gene family in the light-brown apple moth, Epiphyas postvittana, via RNAseq and identify putative odorant degrading enzymes. We then solve the apo-structure for EposCCE24 by X-ray crystallography to a resolution of 2.43 Å and infer substrate specificity based on structural characteristics of the enzyme's binding pocket. The specificity of EposCCE24 was validated by testing its ability to degrade biologically relevant and non-relevant sex pheromone components and plant volatiles using GC-MS. We found that EposCCE24 is neither capable of discriminating between linear acetate-ester odorant molecules of varying chain length, nor between molecules with varying double bond positions. EposCCE24 efficiently degraded both plant volatiles and sex pheromone components containing acetate-ester functional groups, confirming its role as a broadly-tuned odorant degrading enzyme in the moth olfactory organ.

Expression, purification and characterisation of the recombinant possum lipocalin vulpeculin.

BACKGROUND: Lipocalins are a large family of proteins, which possess a highly conserved eight-stranded antiparallel beta-barrel structure as distinctive trait. This family includes Major Urinary Proteins (MUPs) from rats and mouse, studied for their role in urinary protein-mediated chemosignalling. Vulpeculin has been identified as the most abundant protein in the urine of the common brushtail possum, Trichosurus vulpecula. On the basis of high similarity with other MUPS, we hypothesised that vulpeculin might have a role in possum chemosignalling and investigated its stability and binding ability. METHODS: We expressed and purified vulpeculin using an E.coli-based system and confirmed correct folding by circular dichroism (CD) spectroscopy. Thermal stability was studied by CD and binding properties were investigated using two optical probes N-phenyl-naphthylamine (NPN) and 8-anilino-1-naphthalene sulphonic acid (ANS). RESULTS: CD revealed a secondary structure typical of a predominantly ß-sheet protein, consistent with the beta barrel structure of the lipocalin family. Vulpeculin showed a high level of thermostability, as assessed by CD, exhibiting a small shift in the secondary structure even at 95 °C. Binding assays indicated that vulpeculin cannot accommodate the NPN ligand but can bind ANS. CONCLUSION: The urinary secretion, high degree of sequence similarity with other lipocalins, its beta sheet structure assessed by CD and potential to bind hydrophobic ligands in the hydrophobic cavity or an external hydrophobic pocket, suggest vulpeculin may be involved in possum chemosignalling. GENERAL SIGNIFICANCE: This work represents a first step towards the further investigation of the newly discovered lipocalin and its role in possum chemosignalling.

Induction of vacuolar invertase inhibitor mRNA in potato tubers contributes to cold-induced sweetening resistance and includes spliced hybrid mRNA variants.

Cold storage of tubers of potato (Solanum tuberosum L.) compromises tuber quality in many cultivars by the accumulation of hexose sugars in a process called cold-induced sweetening. This is caused by the breakdown of starch to sucrose, which is cleaved to glucose and fructose by vacuolar acid invertase. During processing of affected tubers, the high temperatures involved in baking and frying cause the Maillard reaction between reducing sugars and free amino acids, resulting in the accumulation of acrylamide. cDNA clones with deduced proteins homologous to known invertase inhibitors were isolated and the two most abundant forms, termed INH1 and INH2, were shown to possess apoplastic and vacuolar localization, respectively. The INH2 gene showed developmentally regulated alternative splicing, so, in addition to the INH2α transcript encoding the full-length protein, two hybrid mRNAs (INH2ß*A and INH2ß*B) that encoded deduced vacuolar invertase inhibitors with divergent C-termini were detected, the result of mRNA splicing of an upstream region of INH2 to a downstream region of INH1. Hybrid RNAs are common in animals, where they may add to the diversity of the proteome, but are rarely described in plants. During cold storage, INH2α and the hybrid INH2ß mRNAs accumulated to higher abundance in cultivars resistant to cold-induced sweetening than in susceptible cultivars. Increased amounts of invertase inhibitor may contribute to the suppression of acid invertase activity and prevent cleavage of sucrose. Evidence for increased RNA splicing activity was detected in several resistant lines, a mechanism that in some circumstances may generate a range of proteins with additional functional capacity to aid adaptability.

The Use of Differential Scanning Fluorimetry to Assess Strigolactone Receptor Function.

Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (Tm) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the Tm tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.

Crystal structure of Epiphyas postvittana pheromone binding protein 3.

The insect olfactory system operates as a well-choreographed ensemble of molecules which functions to selectively translate volatile chemical messages present in the environment into neuronal impulses that guide insect behaviour. Of these molecules, binding proteins are believed to transport hydrophobic odorant molecules across the aqueous lymph present in antennal sensilla to receptors present in olfactory sensory neurons. Though the exact mechanism through which these proteins operate is still under investigation, these carriers clearly play a critical role in determining what an insect can smell. Binding proteins that transport important sex pheromones are colloquially named pheromone binding proteins (PBPs). Here, we have produced a functional recombinant PBP from the horticultural pest, Epiphyas postvittana (EposPBP3), and experimentally solved its apo-structure through X-ray crystallography to a resolution of 2.60 Å. Structural comparisons with related lepidopteran PBPs further allowed us to propose models for the binding of pheromone components to EposPBP3. The data presented here represent the first structure of an olfactory-related protein from the tortricid family of moths, whose members cause billions of dollars in losses to agricultural producers each year. Knowledge of the structure of these important proteins will allow for subsequent studies in which novel, olfactory molecule-specific insecticides can be developed.

Synergistic improvement in the performance of insect odorant receptor based biosensors in the presence of Orco.

Insect odorant receptors (ORs) are believed to be a complex of an odorant binding subunit, OrX, and an ion channel forming subunit, Orco. In our previous study, we showed that the OrX subunit on its own in liposomes could detect volatile organic compounds (VOCs) ultrasensitively using Electrochemical Impedance Spectroscopy (EIS). In this study, we investigated the effect of the presence of Orco on the response of the OrX subunit to detect the VOCs. The OrXs - Or10a, Or22a, Or35a and Or71a, together with Orco, were recombinantly expressed, purified and integrated into liposomes. These OrX/Orco liposomes were covalently attached to a gold surface modified with N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide) (NHS/EDC)-activated self-assembled monolayers (SAMs) of 6-mercaptohexanoic acid (MHA). It was demonstrated that the OrX/Orco liposomes could sensitively and selectively detect their ligands by monitoring a change in frequency and impedance signal upon binding with both Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) and EIS. Using EIS, three OrXs (Or10a, Or22a and Or35a) showed a shift in their dose-response curves when Orco was co-integrated, reflecting an increase in ligand sensitivity and a decrease in limit of detection (LOD). Or71a in the presence of Orco did not show any improvement in ligand sensitivity as this is a highly tuned receptor which may be already at the sensitivity limit for EIS. The observed enhancement in sensor performance is believed to be an effect of Orco which is stabilizing the OrX in a more active conformation and amplifying charge transfer to result in a greater reduction in impedance.

An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants.

Herein, we present that insect odorant receptors reconstituted into the lipid bilayers of liposomes can be successfully immobilized onto a gold surface and selectively and sensitively detect odorant molecules. The odorant receptors (OrXs) Or10a, Or22a, and Or71a from the common fruit fly, Drosophila melanogaster, were recombinantly expressed, purified and integrated into nano-liposomes (100-200 nm). These liposomes were covalently attached to the self-assembled monolayers (SAMs) of a 6-mercaptohexanoic acid (MHA)-modified gold surface. X-ray Photo Electron Spectroscopy (XPS) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements confirmed the successful modification of the gold surface and immobilization of liposomes. Atomic Force Microscopy (AFM) revealed that the liposomes were covalently attached to the surface without any disruption of vesicles. The liposomes tethered to the gold sensor surface were then treated with a range of known ligands of various concentrations. We demonstrated by Electrochemical Impedance Spectroscopy (EIS) that an OrX/liposome EIS sensor can sensitively and selectively detect its known ligand to femtomolar concentrations by detecting a change in electrical signal upon binding. Our study is the first step towards using purified insect odorant receptors alone in biosensors to enable the development of novel ultrasensitive volatile sensors for medical diagnostic, air quality, food safety and border security applications.

Data on preparation and characterization of an insect odorant receptor based biosensor.

Insect Odorant receptors (OrXs) can be used as the recognition element in a biosensor as they demonstrate high levels of sensitivity and selectivity towards volatile organic compounds. Herein, we describe a method to express and purify insect odorant receptors and reconstitute them into artificial lipid bilayers (liposomes). These OrX/liposomes were covalently attached to a gold surface and characterized using quartz crystal microbalance with dissipation monitoring (QCM-D). The interaction of OrX/liposomes immobilized on a gold surface to positive and negative odorants were studied by means of electrochemical impedance spectroscopy (EIS) and QCM-D. The data presented in this article are related to the research article titled "An ultrasensitive electrochemical impedance-based biosensor using insect odorant receptors to detect odorants" [1].

The various and varying roles of specific chaperones in type III secretion systems.

The type III secretion pathway is used by numerous Gram-negative pathogenic bacteria to deliver proteins within the membrane or the cytoplasm of eukaryotic cells with which these bacteria interact. Secretion is regulated by external signals. This requires that, before being secreted, proteins are stored in the cytoplasm where they need to be stabilised, separated from other interaction partners, and maintained in a secretion-competent state. Specialised, energy-independent chaperones play various roles in these functions by associating in the cytoplasm with proteins before their secretion. Some chaperones are also directly involved in modulating transcription in response to secretion.

Repeat-containing protein effectors of plant-associated organisms.

Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs) that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.