Richard K Hughes

John Innes Centre · Department of Biological Chemistry

Plant diseases caused by pathogens and pests are a constant threat to global food security. Direct crop losses, and the measures used to control disease (e.g. application of pesticides), have significant agricultural, economic and societal impacts. Therefore, it is essential we understand the molecular mechanisms of the plant immune system, a syste...

Plant NLRs are modular immune receptors that trigger rapid cell death in response to attempted infection by pathogens. A highly conserved nucleotide-binding domain shared with APAF-1, various R-proteins and CED-4 (NB-ARC domain) is proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies of plant...

Autophagy-related protein 8 (ATG8) is a highly conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and a number of proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. Howev...

Plant NLRs are modular immune receptors that trigger rapid cell death in response to attempted infection by pathogens. A highly conserved nucleotide-binding domain shared with APAF-1, various R-proteins and CED-4 (NB-ARC domain) is proposed to act as a molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies of plant...

The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern‐triggered immunity (PTI) following perception of Microbe‐Associated Molecular Patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 was previously shown to supp...

ATG8 is a highly-conserved ubiquitin-like protein that modulates autophagy pathways by binding autophagic membranes and numerous proteins, including cargo receptors and core autophagy components. Throughout plant evolution, ATG8 has expanded from a single protein in algae to multiple isoforms in higher plants. However, the degree to which ATG8 isof...

Fig. S1 ΔspAvr2 R45H complements the virulence defect of a FolΔAvr2 strain. Fig. S2 Side‐by‐side representations of the structures of Avr2 and its structural homologs: human Speckle‐type POZ protein, human TRAF6 and human SIAH1. Fig. S3 Overlays of the structure of Avr2 with structural homologs; Ptr‐ToxA, human Speckle‐type POZ protein, human TRA...

Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I‐2 carrying tomato (Solanum lycopersicum). Fol strains that evade I‐2 recognition carry point mutations...

Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8-family interacting motif (AIM) and perturbs host selective autophagy. However, the structural basis of this interaction remains unknown. Here we define the crystal structure o...

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid...

Plant proteins that associate with PexRD54 as identified by mass spectrometry after immunoprecipitation (IP). FLAG:PexRD54 was transiently expressed in N. benthamiana leaves and proteins were extracted two days after infiltration and used in IP experiments. Unique spectral counts are given for each control and PexRD54 sample. Proteins related to ve...

Unique peptides obtained from liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of PexRD54 immunoprecipitation experiments suggest specific association with ATG8CL. Although ATG8 protein family is highly conserved, and there are eight ATG8 variants in N. benthamiana, unique peptides obtained from LC-MS/MS analysis enabled specific...

Primers used in this study. DOI: http://dx.doi.org/10.7554/eLife.10856.035

Plants use autophagy to safeguard against infectious diseases. However, how plant pathogens interfere with autophagy-related processes is unknown. Here, we show that PexRD54, an effector from the Irish potato famine pathogen Phytophthora infestans, binds host autophagy protein ATG8CL to stimulate autophagosome formation. PexRD54 depletes the autoph...

An estimated 15% of global crop production is lost to pre-harvest disease every year. New ways to manage plant diseases are required. A mechanistic understanding of how plant pathogens re-program their hosts to enable colonisation may provide novel genetic or chemical opportunities to interfere with disease. One notorious plant parasite is the Iris...

Structural analysis of RXLR effector proteins from oomycete plant pathogens is an emerging area of research. These studies are aimed at understanding the molecular basis of how these proteins manipulate plant cells to promote infection and also to help define how they can lead to activation of the plant innate immune system. Here, we describe a med...

Sulfonucleotide reductases catalyse the first reductive step of sulfate assimilation. Their substrate specificities generally correlate with the requirement for a [Fe4S4] cluster, where adenosine 5'-phosphosulfate (APS) reductases possess a cluster and 3'-phosphoadenosine 5'-phosphosulfate reductases do not. The exception is the APR-B isoform of AP...

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for ess...

Background: Glycosyltransferases (GTs) have important functions in plant secondary metabolism. Results: A gene encoding an N-methylanthranilic acid O-glucosyltransferase forms part of a biosynthetic cluster for the synthesis of acylated defense compounds in oat. Conclusion: This GT synthesizes the activated acyl donor required for triterpene acylat...

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The abili...

Gram-negative phytopathogenic bacteria translocate effector proteins into plant cells to subvert host defenses. These effectors can be recognized by plant nucleotide-binding-leucine-rich repeat immune receptors, triggering defense responses that restrict pathogen growth. AvrRps4, an effector protein from Pseudomonas syringae pv. pisi, triggers RPS4...

The cycle inhibiting factors (Cifs) are a family of translocated effector proteins, found in diverse pathogenic bacteria, that interfere with the host cell cycle by catalyzing the deamidation of a specific glutamine residue (Gln40) in NEDD8 and the related protein ubiquitin. This modification prevents recycling of neddylated cullin-RING ligases, le...

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The...

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The...

Cereals contain xylanase inhibitor proteins (XIPs) which inhibit microbial xylanases from glycoside hydrolase families 10 and 11. Here, we report for the first time the isolation and characterisation of a genomic clone containing a xylanase inhibitor gene. This gene, Xip-II, isolated from a durum wheat genomic library (Triticum durum Desf.) encodes...

Serine carboxypeptidase-like (SCPL) proteins have recently emerged as a new group of plant acyltransferases. These enzymes share homology with peptidases but lack protease activity and instead are able to acylate natural products. Several SCPL acyltransferases have been characterized to date from dicots, including an enzyme required for the synthes...

Not just another P450: Shown here is a model of the overall structure of CYP74C3 with the putative membrane-binding region that is required for enzyme activation. Members of the CYP74 family of cytochrome P450 enzymes are specialised in the metabolism of hydroperoxides and play an important role in oxylipin metabolism, which is one of the main defe...

Sortases are a family of Gram-positive bacterial transpeptidases that anchor secreted proteins to bacterial cell surfaces. These include many proteins that play critical roles in the virulence of Gram-positive bacterial pathogens such that sortases are attractive targets for development of novel antimicrobial agents. All Gram-positive pathogens exp...

In silico structural analysis of CYP74C3, a membrane-associated P450 enzyme from the plant Medicago truncatula (barrel medic) with hydroperoxide lyase (HPL) specificity, showed that it had strong similarities to the structural folds of the classical microsomal P450 enzyme from rabbits (CYP2C5). It was not only the secondary structure predictions th...

Hydroperoxide lyase (HPL) is a key enzyme in plant oxylipin metabolism that catalyses the cleavage of polyunsaturated fatty acid hydroperoxides produced by the action of lipoxygenase (LOX) to volatile aldehydes and oxo acids. The synthesis of these volatile aldehydes is rapidly induced in plant tissues upon mechanical wounding and insect or pathoge...

The attB1 site in the Gateway (Invitrogen) bacterial expression vector pDEST17, necessary for in vitro site-specific recombination, contains the sequence AAA-AAA. The sequence A-AAA-AAG within the Escherichia coli dnaX gene is recognized as ‘slippery’ and promotes −1 translational frameshifting. We show here, by expressing in E. coli several plant...

CYP74C3 (cytochrome P450 subfamily 74C3), an HPL (hydroperoxide lyase) from Medicago truncatula (barrel medic), and CYP74A1, an AOS (allene oxide synthase) from Arabidopsis thaliana, are key membrane-associated P450 enzymes in plant oxylipin metabolism. Both recombinant detergent-free enzymes are monomeric proteins with dual specificity and very lo...

We investigate the effects of detergent on the kinetics and oligomeric state of allene oxide synthase (AOS) from Arabidopsis thaliana (CYP74A1). We show that detergent-free CYP74A1 is monomeric and highly water soluble with dual specificity, but has relatively low activity. Detergent micelles promote a 48-fold increase in k(cat)/K(m) (to 5.9 x 10(7...

We describe the detailed biochemical characterization of CYP74C3 (cytochrome P450 subfamily 74C3), a recombinant plant cytochrome P450 enzyme with HPL (hydroperoxide lyase) activity from Medicago truncatula (barrel medic). Steady-state kinetic parameters, substrate and product specificities, RZ (Reinheitszahl or purity index), molar absorption coef...

The oligomeric state of fatty acid hydroperoxide lyase (HPL), of molar mass ∼55 kDa is uncertain and it has been reported as a trimer or tetramer in vivo. The enzyme has been found to be bi-functional and is active even in the absence of detergent. The association with detergent is known to stabilise the binding of the enzyme to its substrate and t...

Oxylipin metabolism represents one of many defence mechanisms employed by plants. It begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases to form fatty acid hydroperoxides that are substrates for several enzymes, including specialized cytochrome P450s known as CYP74s. The targeting of a new CYP74, a 9-hydroperoxide lyase (HPL...

The xylanase inhibitor protein I (XIP-I), recently identified in wheat, inhibits xylanases belonging to glycoside hydrolase families 10 (GH10) and 11 (GH11). Sequence and structural similarities indicate that XIP-I is related to chitinases of family GH18, despite its lack of enzymatic activity. Here we report the identification and biochemical char...

Lipoxygenases (LOXs) are a class of non-heme iron containing dioxygenases which catalyse the hydroperoxidation of polyunsaturated fatty acids such as linoleic and linolenic acids. Plant LOXs are commonly believed to play an important role in fundamental plant processes such as defence, development and senescence. However, the physiological role of...

Lipoxygenases catalyze the conversion of polyunsaturated fatty acids into hydroperoxides, that are in turn converted to oxylipins, which play important roles in defence reactions in plants and animals. This review describes the distribution of lipoxygenases in Nature, their diversity in terms of structure and catalytic activity, and their significa...

Lipoxygenases catalyze the conversion of polyunsaturated fatty acids into hydroperoxides, that are in turn converted to oxylipins, which play important roles in defence reactions in plants and animals. This review describes the distribution of lipoxygenases in Nature, their diversity in terms of structure and catalytic activity, and their significa...

We previously reported on the xylanase-inhibiting protein I (XIP-I) from wheat [McLauchlan, Garcia-Conesa, Williamson, Roza, Ravestein and Maat (1999), Biochem. J. 338, 441-446]. In the present study, we show that XIP-I inhibits family-10 and -11 fungal xylanases. The K(i) values for fungal xylanases ranged from 3.4 to 610 nM, but bacterial family-...

Using expressed sequence tag data, we obtained a full-length cDNA encoding a wheat protein inhibitor of xylanases (XIP-I). The 822 bp open reading frame encoded a protein of 274 amino acids with a molecular mass of 30.2 kDa, in excellent agreement with the native protein. Expression in Escherichia coli confirmed that the cDNA encoded a functional e...

Using expressed sequence tag data, we obtained a full-length cDNA encoding a wheat protein inhibitor of xylanases (XIP-I). The 822 bp open reading frame encoded a protein of 274 amino acids with a molecular mass of 30.2 kDa, in excellent agreement with the native protein. Expression in Escherichia coli confirmed that the cDNA encoded a functional e...

We have used a series of near-isogenic mutant pea lines defective in starch biosynthesis to identify genes that exert pleiotropic effects on the protein content and composition of the pea seed. The total protein contents of the flours obtained from the dry seed of these lines, determined using a new method based on amino acid analysis and validated...

We have produced a model to define the linoleate-binding pocket of pea 9/13-lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were asso...

A new potato tuber lipoxygenase full-length cDNA sequence (lox1:St:2) has been isolated from potato tubers and used to express in Escherichia coli and characterize a novel recombinant lipoxygenase (potato 13/9-lipoxygenase). Like most plant lipoxygenases it produced carbonyl compounds from linoleate (the preferred substrate) and was purified in the...

A number of products including apocarotenal, epoxycarotenal, apocarotenone, and epoxycarotenone generated by lipoxygenase (LOX) catalyzed co-oxidation of beta-carotene have been tentatively identified through the use of GC/MS and HPLC combined with photodiode array detection. Because of the large number of high molecular weight products detected an...

Mature pea seeds contain two major lipoxygenases (LOX) isoenzymes designated LOX-2 and LOX-3. The thermal inactivation of crude pea LOX and the recombinant LOX (rLOX) were studied. Heat-inactivation plots for crude extracts of pea LOX were linear from which thermodynamic activation parameters, ΔH#, ΔS# sand ΔG# have been estimated. The enzymatic ac...

We can produce sufficient amounts of specific pea seed and potato tuber lipoxygenase (LOX) by fermentation in Escherichia coil to assess their use in a number of specific applications. This has also allowed us to gain a more fundamental understanding of their differences in behaviour from a comparative analysis of the recombinant enzymes and from t...

The two major isoforms of lipoxygenase (LOX-2 and LOX-3) from pea (Pisum sativum L. cv. Birte) seeds have been cloned and expressed from full-length cDNAs as soluble, active, non-fusion proteins in Escherichia coli. A comparison of both isoforms purified to apparent homogeneity from E. coli and pea seeds has confirmed the authenticity of the recomb...

The two major forms of lipoxygenase from pea seeds (LOX 2 and 3) have been cloned and expressed as soluble, active, non-fusion proteins in Escherichia coli. Unambiguous measurements to date comparing the homogeneously purified LOX 3 from E. coli and pea seeds have confirmed the authenticity of this recombinant product. Preliminary comparisons of LO...

Two ecotypes of Arabidopsis thaliana were transformed with the gene encoding tobacco mosaic virus (TMV) movement protein (P30). P30 accumulated largely in a subcellular fraction containing cell wall components and as a soluble protein. The protein migrated in denaturing gels with an M(r) of 30K, significantly faster than P30 (M(r) approximately 34K...

The usefulness in structure/function studies of molybdenum-containing hydroxylases in work with rosy mutant strains of Drosophila melanogaster has been investigated. At least 23 such strains are available, each corresponding to a single known amino acid change in the xanthine dehydrogenase sequence. Sequence comparisons permit identification, with...

Xanthine dehydrogenase has been purified to homogeneity by conventional procedures from the wild-type strain of the fruit fly Drosophila melanogaster, as well as from a rosy mutant strain (E89 --> K, ry5231) known to carry a point mutation in the iron-sulfur domain of the enzyme. The wild-type enzyme had all the specific properties that are peculia...