The rice CYP78A gene BSR2 confers resistance to Rhizoctonia solani and affects seed size and growth in Arabidopsis and rice.

The fungal pathogen Rhizoctonia solani causes devastating diseases in hundreds of plant species. Among these, R. solani causes sheath blight, one of the three major diseases in rice. To date, few genes have been reported that confer resistance to R. solani. Here, rice-FOX Arabidopsis lines identified as having resistance to a bacterial pathogen, Pseudomonas syringae pv. tomato DC3000, and a fungal pathogen, Colletotrichum higginsianum were screened for disease resistance to R. solani. BROAD-SPECTRUM RESISTANCE2 (BSR2), a gene encoding an uncharacterized cytochrome P450 protein belonging to the CYP78A family, conferred resistance to R. solani in Arabidopsis. When overexpressed in rice, BSR2 also conferred resistance to two R. solani anastomosis groups. Both Arabidopsis and rice plants overexpressing BSR2 had slower growth and produced longer seeds than wild-type control plants. In contrast, BSR2-knockdown rice plants were more susceptible to R. solani and displayed faster growth and shorter seeds in comparison with the control. These results indicate that BSR2 is associated with disease resistance, growth rate and seed size in rice and suggest that its function is evolutionarily conserved in both monocot rice and dicot Arabidopsis.

Potential transgenic routes to increase tree biomass.

Biomass is a prime target for genetic engineering in forestry because increased biomass yield will benefit most downstream applications such as timber, fiber, pulp, paper, and bioenergy production. Transgenesis can increase biomass by improving resource acquisition and product utilization and by enhancing competitive ability for solar energy, water, and mineral nutrients. Transgenes that affect juvenility, winter dormancy, and flowering have been shown to influence biomass as well. Transgenic approaches have increased yield potential by mitigating the adverse effects of prevailing stress factors in the environment. Simultaneous introduction of multiple genes for resistance to various stress factors into trees may help forest trees cope with multiple or changing environments. We propose multi-trait engineering for tree crops, simultaneously deploying multiple independent genes to address a set of genetically uncorrelated traits that are important for crop improvement. This strategy increases the probability of unpredictable (synergistic or detrimental) interactions that may substantially affect the overall phenotype and its long-term performance. The very limited ability to predict the physiological processes that may be impacted by such a strategy requires vigilance and care during implementation. Hence, we recommend close monitoring of the resultant transgenic genotypes in multi-year, multi-location field trials.

Production of indole alkaloids by metabolic engineering of the tryptophan pathway in rice.

Tryptophan decarboxylase (TDC) converts tryptophan (Trp) into tryptamine, consequently increasing the metabolic flow of tryptophan derivatives into the production of secondary metabolites such as indole alkaloids. We inserted an expression cassette containing OsTDC, a putative tryptophan decarboxylase gene from rice, into an expression plasmid vector containing OASA1D, the feedback-resistant anthranilate synthase alpha-subunit mutant (OASA1D). Overexpression of OASA1D has been reported to significantly increase Trp levels in rice. The co-expression of OsTDC and OASA1D in rice calli led to almost complete depletion of the Trp pool and a consequent increase in the tryptamine pool. This indicates that TDC inactivity is a contributory factor for the accumulation of Trp in rice transgenics overexpressing OASA1D. Metabolic profiling of the calli expressing OsTDC and OASA1D revealed the accumulation of serotonin and serotonin-derived indole compounds (potentially pharmacoactive ß-carbolines) that have not been reported from rice. Rice calli overexpressing OASA1D:OASA1D is a novel system for the production of significant amounts of pharmacologically useful indole alkaloids in rice.

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice.

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.

A new allele of flower color gene W1 encoding flavonoid 3'5'-hydroxylase is responsible for light purple flowers in wild soybean Glycine soja.

BACKGROUND: Glycine soja is a wild relative of soybean that has purple flowers. No flower color variant of Glycine soja has been found in the natural habitat. RESULTS: B09121, an accession with light purple flowers, was discovered in southern Japan. Genetic analysis revealed that the gene responsible for the light purple flowers was allelic to the W1 locus encoding flavonoid 3'5'-hydroxylase (F3'5'H). The new allele was designated as w1-lp. The dominance relationship of the locus was W1 >w1-lp >w1. One F2 plant and four F3 plants with purple flowers were generated in the cross between B09121 and a Clark near-isogenic line with w1 allele. Flower petals of B09121 contained lower amounts of four major anthocyanins (malvidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside and delphinidin 3-O-glucoside) common in purple flowers and contained small amounts of the 5'-unsubstituted versions of the above anthocyanins, peonidin 3,5-di-O-glucoside, cyanidin 3,5-di-O-glucoside and cyanidin 3-O-glucoside, suggesting that F3'5'H activity was reduced and flavonoid 3'-hydroxylase activity was increased. F3'5'H cDNAs were cloned from Clark and B09121 by RT-PCR. The cDNA of B09121 had a unique base substitution resulting in the substitution of valine with methionine at amino acid position 210. The base substitution was ascertained by dCAPS analysis. The polymorphism associated with the dCAPS markers co-segregated with flower color in the F2 population. F3 progeny test, and dCAPS and indel analyses suggested that the plants with purple flowers might be due to intragenic recombination and that the 65 bp insertion responsible for gene dysfunction might have been eliminated in such plants. CONCLUSIONS: B09121 may be the first example of a flower color variant found in nature. The light purple flower was controlled by a new allele of the W1 locus encoding F3'5'H. The flower petals contained unique anthocyanins not found in soybean and G. soja. B09121 may be a useful tool for studies of the structural and functional properties of F3'5'H genes as well as investigations on the role of flower color in relation to adaptation of G. soja to natural habitats.

BRASSINOSTEROID UPREGULATED1, encoding a helix-loop-helix protein, is a novel gene involved in brassinosteroid signaling and controls bending of the lamina joint in rice.

Brassinosteroids (BRs) are involved in many developmental processes and regulate many subsets of downstream genes throughout the plant kingdom. However, little is known about the BR signal transduction and response network in monocots. To identify novel BR-related genes in rice (Oryza sativa), we monitored the transcriptomic response of the brassinosteroid deficient1 (brd1) mutant, with a defective BR biosynthetic gene, to brassinolide treatment. Here, we describe a novel BR-induced rice gene BRASSINOSTEROID UPREGULATED1 (BU1), encoding a helix-loop-helix protein. Rice plants overexpressing BU1 (BU1:OX) showed enhanced bending of the lamina joint, increased grain size, and resistance to brassinazole, an inhibitor of BR biosynthesis. In contrast to BU1:OX, RNAi plants designed to repress both BU1 and its homologs displayed erect leaves. In addition, compared to the wild type, the induction of BU1 by exogenous brassinolide did not require de novo protein synthesis and it was weaker in a BR receptor mutant OsbriI (Oryza sativa brassinosteroid insensitive1, d61) and a rice G protein alpha subunit (RGA1) mutant d1. These results indicate that BU1 protein is a positive regulator of BR response: it controls bending of the lamina joint in rice and it is a novel primary response gene that participates in two BR signaling pathways through OsBRI1 and RGA1. Furthermore, expression analyses showed that BU1 is expressed in several organs including lamina joint, phloem, and epithelial cells in embryos. These results indicate that BU1 may participate in some other unknown processes modulated by BR in rice.

The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production.

The upregulation of the tryptophan (Trp) pathway in rice leaves infected by Bipolaris oryzae was indicated by: (i) enhanced enzyme activity of anthranilate synthase (AS), which regulates metabolic flux in the Trp pathway; (ii) elevated levels of the AS (OASA2, OASB1, and OASB2) transcripts; and (iii) increases in the contents of anthranilate, indole, and Trp. The measurement of the contents of Trp-derived metabolites by high-performance liquid chromatography coupled with tandem mass spectrometry revealed that serotonin and its hydroxycinnamic acid amides were accumulated in infected leaves. Serotonin accumulation was preceded by a transient increase in the tryptamine content and by marked activation of Trp decarboxylase, indicating that enhanced Trp production is linked to the formation of serotonin from Trp via tryptamine. Feeding of radiolabeled serotonin to inoculated leaves demonstrated that serotonin is incorporated into the cell walls of lesion tissue. The leaves of a propagating-type lesion mimic mutant (sl, Sekiguchi lesion) lacked both serotonin production and deposition of unextractable brown material at the infection sites, and showed increased susceptibility to B. oryzae infection. Treating the mutant with serotonin restored deposition of brown material at the lesion site. In addition, the serotonin treatment suppressed the growth of fungal hyphae in the leaf tissues of the sl mutant. These findings indicated that the activation of the Trp pathway is involved in the establishment of effective physical defenses by producing serotonin in rice leaves.

Integrated metabolomic and transcriptomic analyses of high-tryptophan rice expressing a mutant anthranilate synthase alpha subunit.

Transgenic rice plants overexpressing a mutant rice gene for anthranilate synthase alpha subunit (OASA1D) accumulate large amounts of free tryptophan (Trp) with few adverse effects on the phenotype, except for poor germination and weak seedling growth. Metabolic profiling of 8-d-old seedlings of Nipponbare and two high-Trp lines, HW1 and HW5, by high performance liquid chromatography-photo diode array (HPLC-PDA) confirmed that, relative to Nipponbare, only the peak attributed to Trp was significantly changed in the profiles of the OASA1D lines. More detailed and targeted analysis using HPLC coupled with tandem mass spectrometry revealed that the OASA1D lines had higher levels of anthranilate, tryptamine, and serotonin than Nipponbare, but these metabolites were at much lower levels than free Trp. The levels of phenylalanine (Phe) and tyrosine (Tyr) were not affected by the overproduction of Trp. Transcriptomic analysis by microarray validated by quantitative Real-Time PCR (qRT-PCR) revealed that at least 12 out of 21 500 genes showed significant differential expression among genotypes. Except for the OASA1D transgene and a putative IAA beta-glucosyltransferase, these were not related to Trp metabolism. Most importantly, the overexpression of the OASA1D and the consequent accumulation of Trp in these lines had little effect on the overall transcriptome, consistent with the minimal effects on growth and the metabolome. Integrated analysis of the metabolome and transcriptome of these OASA1D transgenic lines indicates that the over-accumulation of free Trp may be partly due to the low activity of Trp decarboxylase or other metabolic genes that directly utilize Trp as a substrate.

Isolation and molecular characterization of a Spotted leaf 18 mutant by modified activation-tagging in rice.

A lesion mimic mutant that we designated Spotted leaf 18 (Spl18) was isolated from 13,000 activation-tagging lines of rice produced by our modified activation-tagging vector and further characterized. Spl18 was dominant and its phenotype was linked to the T-DNA insertion. An ORF was located about 500 bp downstream of the inserted T-DNA, and the deduced protein, designated OsAT1, showed sequence similarity to an acyltransferase whose expression is induced by hypersensitive reaction in tobacco. The transcriptional level of OsAT1 was very low in the WT leaf blade but high in Spl18 leaf blade. In wild-type rice, OsAT1 was transcribed mainly in the young panicle, in the panicle just after heading, and in the leaf sheath. In addition, transcription of the genes for PR protein was upregulated in Spl18, accumulation of phytoalexins (both momilactone A and sakuranetin) was increased, and resistance to blast disease was improved. We then combined OsAT1 genomic DNA downstream of the modified 35S promoter and re-transformed it into rice. Lesion mimic and blast resistance phenotypes were detected in the transgenic lines produced, clearly indicating that overexpression of OsAT1 caused the Spl18 phenotypes. In addition, plants overexpressing OsAT1 showed resistance to bacterial blight.

Structure-based in vitro engineering of the anthranilate synthase, a metabolic key enzyme in the plant tryptophan pathway.

Rice (Oryza sativa) anthranilate synthase alpha-subunit, OASA2, was modified by in vitro mutagenesis based on structural information from bacterial homologs. Twenty-four amino acid residues, predicted as putative tryptophan binding sites or their proximal regions in the OASA2 sequence, were selected and 36 mutant OASA2 genes were constructed by PCR-based site-directed mutagenesis. Corresponding mutant proteins were synthesized in a combination of two in vitro systems, transcription with a bacteriophage SP6 RNA polymerase and translation with a wheat-embryo cell-free system. Enzymatic functions of the mutant proteins were simultaneously examined, and we found six mutants with elevated catalytic activity and five mutants with enhanced tolerance to feedback inhibition by tryptophan. Moreover, we observed that some sets of specific combinations of the novel mutations additively conferred both characteristics to the mutant enzymes. The functions of the mutant enzymes were confirmed in vivo. The free tryptophan content of mutant rice calli expressing OASA2 enzyme with a double mutation was 30-fold of that of untransformed calli. Thus, our in vitro approach utilizing structural information of bacterial homologs is a potent technique to generate designer enzymes with predefined functions.

OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression.

The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stress-inducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses.

DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression.

DRE/CRT is a cis-acting element that is involved in gene expression responsive to drought and low-temperature stress in higher plants. DREB1A/CBF3 and DREB2A are transcription factors that specifically bind to DRE/CRT in Arabidopsis. We precisely analyzed the DNA-binding specificity of DREBs. Both DREBs specifically bound to six nucleotides (A/GCCGAC) of DRE. However, these proteins had different binding specificities to the second or third nucleotides of DRE. Gel mobility shift assay using mutant DREB proteins showed that the two amino acids, valine and glutamic acid conserved in the ERF/AP2 domains, especially valine, have important roles in DNA-binding specificity. In the Arabidopsis genome, 145 DREB/ERF-related proteins are encoded. These proteins were classified into five groups-AP-2 subfamily, RAV subfamily, DREB subfamily, ERF subfamily, and others. The DREB subfamily included three novel DREB1A- and six DREB2A-related proteins. We analyzed expression of novel genes for these proteins and discuss their roles in stress-responsive gene expression.