Dissecting the causal polymorphism of the Lr67res multipathogen resistance gene.

Partial resistance to multiple biotrophic fungal pathogens in wheat (Triticum aestivum L.) is conferred by a variant of the Lr67 gene, which encodes a hexose-proton symporter. Two mutations (G144R, V387L) differentiate the resistant and susceptible protein variants (Lr67res and Lr67sus). Lr67res lacks sugar transport capability and was associated with anion transporter-like properties when expressed in Xenopus laevis oocytes. Here, we extended this functional characterization to include yeast and in planta studies. The Lr67res allele, but not Lr67sus, induced sensitivity to ions in yeast (including NaCl, LiCl, KI), which is consistent with our previous observations that Lr67res expression in oocytes induces novel ion fluxes. We demonstrate that another naturally occurring single amino acid variant in wheat, containing only the Lr67G144R mutation, confers rust resistance. Transgenic barley plants expressing the orthologous HvSTP13 gene carrying the G144R and V387L mutations were also more resistant to Puccinia hordei infection. NaCl treatment of pot-grown adult wheat plants with the Lr67res allele induced leaf tip necrosis and partial leaf rust resistance. An Lr67res-like function can be introduced into orthologous plant hexose transporters via single amino acid mutation, highlighting the strong possibility of generating disease resistance in other crops, especially with gene editing.

Expression of the wheat multipathogen resistance hexose transporter Lr67res is associated with anion fluxes.

Many disease resistance genes in wheat (Triticum aestivum L.) confer strong resistance to specific pathogen races or strains, and only a small number of genes confer multipathogen resistance. The Leaf rust resistance 67 (Lr67) gene fits into the latter category as it confers partial resistance to multiple biotrophic fungal pathogens in wheat and encodes a Sugar Transport Protein 13 (STP13) family hexose-proton symporter variant. Two mutations (G144R, V387L) in the resistant variant, Lr67res, differentiate it from the susceptible Lr67sus variant. The molecular function of the Lr67res protein is not understood, and this study aimed to broaden our knowledge on this topic. Biophysical analysis of the wheat Lr67sus and Lr67res protein variants was performed using Xenopus laevis oocytes as a heterologous expression system. Oocytes injected with Lr67sus displayed properties typically associated with proton-coupled sugar transport proteins-glucose-dependent inward currents, a Km of 110 ± 10 µM glucose, and a substrate selectivity permitting the transport of pentoses and hexoses. By contrast, Lr67res induced much larger sugar-independent inward currents in oocytes, implicating an alternative function. Since Lr67res is a mutated hexose-proton symporter, the possibility of protons underlying these currents was investigated but rejected. Instead, currents in Lr67res oocytes appeared to be dominated by anions. This conclusion was supported by electrophysiology and 36Cl- uptake studies and the similarities with oocytes expressing the known chloride channel from Torpedo marmorata, TmClC-0. This study provides insights into the function of an important disease resistance gene in wheat, which can be used to determine how this gene variant underpins disease resistance in planta.

Overgrowth mutants determine the causal role of gibberellin GA2oxidaseA13 in Rht12 dwarfism of wheat.

The induced dwarf mutant Rht12 was previously shown to have agronomic potential to replace the conventional DELLA mutants Rht-B1b/Rht-D1b in wheat. The Rht12 dwarfing gene is not associated with reduced coleoptile length (unlike the DELLA mutants) and it is dominant, characteristics which are shared with the previously characterized dwarfing genes Rht18 and Rht14. Using the Rht18/Rht14 model, a gibberellin (GA) 2-oxidase gene was identified in the Rht12 region on chromosome 5A. A screen for suppressor mutants in the Rht12 background identified tall overgrowth individuals that were shown to contain loss-of-function mutations in GA2oxidaseA13, demonstrating the role of this gene in the Rht12 dwarf phenotype. It was concluded that Rht12, Rht18, and Rht14 share the same height-reducing mechanism through the increased expression of GA 2-oxidase genes. Some of the overgrowth mutants generated in this study were semi-dwarf and taller than the original Rht12 dwarf, providing breeders with new sources of agronomically useful dwarfism.

Structural and functional insights into the modulation of the activity of a flax cytokinin oxidase by flax rust effector AvrL567-A.

During infection, plant pathogens secrete effector proteins to facilitate colonization. In comparison with our knowledge of bacterial effectors, the current understanding of how fungal effectors function is limited. In this study, we show that the effector AvrL567-A from the flax rust fungus Melampsora lini interacts with a flax cytosolic cytokinin oxidase, LuCKX1.1, using both yeast two-hybrid and in planta bimolecular fluorescence assays. Purified LuCKX1.1 protein shows catalytic activity against both N6-(Δ2-isopentenyl)-adenine (2iP) and trans-zeatin (tZ) substrates. Incubation of LuCKX1.1 with AvrL567-A results in increased catalytic activity against both substrates. The crystal structure of LuCKX1.1 and docking studies with AvrL567-A indicate that the AvrL567 binding site involves a flexible surface-exposed region that surrounds the cytokinin substrate access site, which may explain its effect in modulating LuCKX1.1 activity. Expression of AvrL567-A in transgenic flax plants gave rise to an epinastic leaf phenotype consistent with hormonal effects, although no difference in overall cytokinin levels was observed. We propose that, during infection, plant pathogens may differentially modify the levels of extracellular and intracellular cytokinins.

The Wheat Lr67 Gene from the Sugar Transport Protein 13 Family Confers Multipathogen Resistance in Barley.

Fungal pathogens are a major constraint to global crop production; hence, plant genes encoding pathogen resistance are important tools for combating disease. A few resistance genes identified to date provide partial, durable resistance to multiple pathogens and the wheat (Triticum aestivum) Lr67 hexose transporter variant (Lr67res) fits into this category. Two amino acids differ between the wild-type and resistant alleles - G144R and V387L. Exome sequence data from 267 barley (Hordeum vulgare) landraces and wild accessions was screened and neither of the Lr67res mutations was detected. The barley ortholog of Lr67, HvSTP13, was functionally characterized in yeast as a high affinity hexose transporter. The G144R mutation was introduced into HvSTP13 and abolished Glc uptake, whereas the V387L mutation reduced Glc uptake by ∼ 50%. Glc transport by HvSTP13 heterologously expressed in yeast was reduced when coexpressed with Lr67res Stable transgenic Lr67res barley lines exhibited seedling resistance to the barley-specific pathogens Puccinia hordei and Blumeria graminis f. sp. hordei, which cause leaf rust and powdery mildew, respectively. Barley plants expressing Lr67res exhibited early senescence and higher pathogenesis-related (PR) gene expression. Unlike previous observations implicating flavonoids in the resistance of transgenic sorghum (Sorghum bicolor) expressing Lr34res, another wheat multipathogen resistance gene, barley flavonoids are unlikely to have a role in Lr67res-mediated resistance. Similar to observations made in yeast, Lr67res reduced Glc uptake in planta These results confirm that the pathway by which Lr67res confers resistance to fungal pathogens is conserved between wheat and barley.

Developmentally regulated HEART STOPPER, a mitochondrially targeted L18 ribosomal protein gene, is required for cell division, differentiation, and seed development in Arabidopsis.

Evidence is presented for the role of a mitochondrial ribosomal (mitoribosomal) L18 protein in cell division, differentiation, and seed development after the characterization of a recessive mutant, heart stopper (hes). The hes mutant produced uncellularized endosperm and embryos arrested at the late globular stage. The mutant embryos differentiated partially on rescue medium with some forming callus. HES (At1g08845) encodes a mitochondrially targeted member of a highly diverged L18 ribosomal protein family. The substitution of a conserved amino residue in the hes mutant potentially perturbs mitoribosomal function via altered binding of 5S rRNA and/or influences the stability of the 50S ribosomal subunit, affecting mRNA binding and translation. Consistent with this, marker genes for mitochondrial dysfunction were up-regulated in the mutant. The slow growth of the endosperm and embryo indicates a defect in cell cycle progression, which is evidenced by the down-regulation of cell cycle genes. The down-regulation of other genes such as EMBRYO DEFECTIVE genes links the mitochondria to the regulation of many aspects of seed development. HES expression is developmentally regulated, being preferentially expressed in tissues with active cell division and differentiation, including developing embryos and the root tips. The divergence of the L18 family, the tissue type restricted expression of HES, and the failure of other L18 members to complement the hes phenotype suggest that the L18 proteins are involved in modulating development. This is likely via heterogeneous mitoribosomes containing different L18 members, which may result in differential mitochondrial functions in response to different physiological situations during development.

Lr34 multi-pathogen resistance ABC transporter: molecular analysis of homoeologous and orthologous genes in hexaploid wheat and other grass species.

The Triticum aestivum (bread wheat) disease resistance gene Lr34 confers durable, race non-specific protection against three fungal pathogens, and has been a highly relevant gene for wheat breeding since the green revolution. Lr34, located on chromosome 7D, encodes an ATP-binding cassette (ABC) transporter. Both wheat cultivars with and without Lr34-based resistance encode a putatively functional protein that differ by only two amino acid polymorphisms. In this study, we focused on the identification and characterization of homoeologous and orthologous Lr34 genes in hexaploid wheat and other grasses. In hexaploid wheat we found an expressed and putatively functional Lr34 homoeolog located on chromosome 4A, designated Lr34-B. Another homoeologous Lr34 copy, located on chromosome 7A, was disrupted by the insertion of repetitive elements. Protein sequences of LR34-B and LR34 were 97% identical. Orthologous Lr34 genes were detected in the genomes of Oryza sativa (rice) and Sorghum bicolor (sorghum). Zea mays (maize), Brachypodium distachyon and Hordeum vulgare (barley) lacked Lr34 orthologs, indicating independent deletion of this particular ABC transporter. Lr34 was part of a gene-rich island on the wheat D genome. We found gene colinearity on the homoeologous A and B genomes of hexaploid wheat, but little microcolinearity in other grasses. The homoeologous LR34-B protein and the orthologs from rice and sorghum have the susceptible haplotype for the two critical polymorphisms distinguishing the LR34 proteins from susceptible and resistant wheat cultivars. We conclude that the particular Lr34-haplotype found in resistant wheat cultivars is unique. It probably resulted from functional gene diversification that occurred after the polyploidization event that was at the origin of cultivated bread wheat.

Characterization of gibberellin receptor mutants of barley (Hordeum vulgare L.).

The sequence of Gid1 (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthologue from barley. This was expressed in E. coli, and produced a protein that was able to bind GA in vitro with both structural specificity and saturability. Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gse1 mutants). Sixteen different gse1 mutants each carried a unique nucleotide substitution in this sequence. In all but one case, these changes resulted in single amino acid substitutions, and, for the remaining mutant, a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation. There was perfect linkage in segregating populations between new mutant alleles and the gse1 phenotype, leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gse1 locus. Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA(1), and a deficit of C(20) GA precursors. All of the gse1 mutants had reduced sensitivity to exogenous GA(3), and to AC94377 (a GA analogue) at concentrations that are normally 'saturating', but, at much higher concentrations, there was often a considerable response. The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

Isolation and expression analysis of multiple isoforms of putative farnesoic acid O-methyltransferase in several crustacean species.

Farnesoic acid O-methyltransferase (FaMeT) is the enzyme responsible for the conversion of farnesoic acid (FA) to methyl farnesoate (MF) in the final step of MF synthesis. Multiple isoforms of putative FaMeT were isolated from six crustacean species belonging to the families Portunidae, Penaeidae, Scyllaridae and Parastacidae. The portunid crabs Portunus pelagicus and Scylla serrata code for three forms: short, intermediate and long. Two isoforms (short and long) were isolated from the penaeid prawns Penaeus monodon and Fenneropenaeus merguiensis. Two isoforms were also identified in the scyllarid Thenus orientalis and parastacid Cherax quadricarinatus. Putative FaMeT sequences were also amplified from the genomic DNA of P. pelagicus and compared to the putative FaMeT transcripts expressed. Each putative FaMeT cDNA isoform was represented in the genomic DNA, indicative of a multi-gene family. Various tissues from P. pelagicus were individually screened for putative FaMeT expression using PCR and fragment analysis. Each tissue type expressed all three isoforms of putative FaMeT irrespective of sex or moult stage. Protein domain analysis revealed the presence of a deduced casein kinase II phosphorylation site present only in the long isoform of putative FaMeT.

Proanthocyanidin synthesis and expression of genes encoding leucoanthocyanidin reductase and anthocyanidin reductase in developing grape berries and grapevine leaves.

Proanthocyanidins (PAs), also called condensed tannins, can protect plants against herbivores and are important quality components of many fruits. Two enzymes, leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), can produce the flavan-3-ol monomers required for formation of PA polymers. We isolated and functionally characterized genes encoding both enzymes from grapevine (Vitis vinifera L. cv Shiraz). ANR was encoded by a single gene, but we found two highly related genes encoding LAR. We measured PA content and expression of genes encoding ANR, LAR, and leucoanthocyanidin dioxygenase in grape berries during development and in grapevine leaves, which accumulated PA throughout leaf expansion. Grape flowers had high levels of PA, and accumulation continued in skin and seeds from fruit set until the onset of ripening. VvANR was expressed throughout early flower and berry development, with expression increasing after fertilization. It was expressed in berry skin and seeds until the onset of ripening, and in expanding leaves. The genes encoding LAR were expressed in developing fruit, particularly in seeds, but had low expression in leaves. The two LAR genes had different patterns of expression in skin and seeds. During grape ripening, PA levels decreased in both skin and seeds, and expression of genes encoding ANR and LAR were no longer detected. The results indicate that PA accumulation occurs early in grape development and is completed when ripening starts. Both ANR and LAR contribute to PA synthesis in fruit, and the tissue and temporal-specific regulation of the genes encoding ANR and LAR determines PA accumulation and composition during grape berry development.

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development.

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.

Proanthocyanidin biosynthesis in plants. Purification of legume leucoanthocyanidin reductase and molecular cloning of its cDNA.

Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of approximately 10 micromol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known. Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome. This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.

Identification and biochemical characterization of mutants in the proanthocyanidin pathway in Arabidopsis.

Proanthocyanidin (PA), or condensed tannin, is a polymeric flavanol that accumulates in a number of tissues in a wide variety of plants. In Arabidopsis, we found that PA precursors (detected histochemically using OsO(4)) accumulate in the endothelial cell layer of the seed coat from the two-terminal cell stage of embryo development onwards. To understand how PA is made, we screened mature seed pools of T-DNA-tagged Arabidopsis lines to identify mutants defective in the synthesis of PA and found six tds (tannin-deficient seed) complementation groups defective in PA synthesis. Mutations in these loci disrupt the amount (tds1, tds2, tds3, tds5, and tds6) or location and amount of PA (tds4) in the endothelial cell layer. The PA intermediate epicatechin has been identified in wild type and mutants tds1, tds2, tds3, and tds5 (which do not produce PA) and tds6 (6% of wild-type PA), whereas tds4 (2% of wild-type PA) produces an unidentified dimethylaminocinnamaldehyde-reacting compound, indicating that the mutations may be acting on genes beyond leucoanthocyanidin reductase, the first enzymatic reduction step dedicated to PA synthesis. Two other mutants were identified, an allele of tt7, which has a spotted pattern of PA deposition and produces only 8% of the wild-type level of type PA as propelargonidin, and an allele of tt8 producing no PA. Spotted patterns of PA deposition observed in seed of mutants tds4 and tt7-3 result from altered PA composition and distribution in the cell. Our mutant screen, which was not exhaustive, suggests that the cooperation of many genes is required for successful PA accumulation.