Pyramiding of two rice bacterial blight resistance genes, Xa3 and Xa4, and a closely linked cold-tolerance QTL on chromosome 11.

KEY MESSAGE: We fine mapped the Xa4 locus and developed a pyramided rice line containing Xa3 and Xa4 R - alleles and a cold-tolerance QTL. This line will be valuable in rice breeding. Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is a destructive disease of cultivated rice. Pyramiding BB resistance genes is an essential approach for increasing the resistance level of rice varieties. We selected an advanced backcross recombinant inbred line 132 (ABL132) from the BC3F7 population derived from a cross between cultivars Junam and IR72 by K3a inoculation and constructed the mapping population (BC4F6) to locate the Xa4 locus. The Xa4 locus was found to be delimited within a 60-kb interval between InDel markers InDel1 and InDel2 and tightly linked with the Xa3 gene on chromosome 11. After cold (4 °C) treatment, ABL132 with introgressions of IR72 in chromosome 11 showed lower survival rate, chlorophyll content, and relative water content compared to Junam. Genetic analysis showed that the cold stress-related quantitative trait locus (QTL) qCT11 was located in a 1.3-Mb interval close to the Xa4 locus. One line, ABL132-36, containing the Xa3 resistance allele from Junam, the Xa4 resistance allele from IR72, and the cold-tolerance QTL from Junam (qCT11), was developed from a BC4F6 population of 250 plants. This is the first report on the pyramiding of Xa3 and Xa4 genes with a cold-tolerance QTL. This region could provide a potential tool for improving resistance against BB and low-temperature stress in rice-breeding programs.

Genetic analysis and molecular mapping of low amylose gene du12(t) in rice (Oryza sativa L.).

KEY MESSAGE: We obtained interesting results for genetic analysis and molecular mapping of the du12(t) gene. Control of the amylose content in rice is the major strategy for breeding rice with improved quality. In this study, we conducted genetic analysis and molecular mapping to identify the dull gene in the dull rice, Milyang262. A single recessive gene, tentatively designated as du12(t), was identified as the dull gene that leads to the low amylose character of Milyang262. To investigate the inheritance of du12(t), genetic analysis on an F2 population derived from a cross between the gene carrier, Milyang262, and a moderate amylose content variety, Junam, was conducted. A segregation ratio of 3:1 (χ (2) = 1.71, p = 0.19) was observed, suggesting that du12(t) is a single recessive factor that controls the dull character in Milyang262. Allelism tests confirmed that du12(t) is not allelic to other low amylose controlling genes, wx or du1. Recessive class analysis was performed to localize the du12(t) locus. Mapping of du12(t) was conducted on F2 and F3 populations of Baegokchal/Milyang262 cross. Linkage analysis of 120 F2 plants revealed that RM6926 and RM3509 flank du12(t) at a 2.38-Mb region. To refine the du12(t) locus position, 986 F2 and 289 F3 additional normal plants were screened by the flanking markers. Twenty-six recombinant plants were identified and later genotyped with four additional adjacent markers located between RM6926 and RM3509. Finally, du12(t) was mapped to an 840-kb region on the distal region of the long arm of chromosome 6, delimited by SSR markers RM20662 and RM412, and co-segregated by RM3765 and RM176.

Molecular cloning and characterization of OsUPS, a U-box containing E3 ligase gene that respond to phosphate starvation in rice (Oryza sativa).

The ubiquitin-26S proteasome system is important in the quality control of intracellular proteins. The ubiquitin-26S proteasome system includes the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes. U-box proteins are a derived version of RING-finger domains, which have E3 enzyme activity. Here, we present the isolation of a novel U-box protein, U-box containing E3 ligase induced by phosphate starvation (OsUPS), from rice (Oryza sativa). The cDNA encoding the O. sativa U-box protein (OsUPS) comprises 1338 bp, with an open reading frame of 445 amino acids. The amino acid sequence of OsUPS cDNA shows 41-79% identity with other plant U-box homologous genes. The open reading frame of the OsUPS protein is comprised of notable domains: a single ~70-amino acid domain and a GKL domain that contains conserved glycine, lysine/arginine residues and leucine-rich feature. We found that full-length expression of OsUPS was up-regulated in both rice plants and cell culture in the absence of inorganic phosphate (P(i)). A self-ubiquitination assay indicated that the bacterially expressed OsUPS protein had E3 ligase activity, and subcellular localization results showed that OsUPS was located in the chloroplast. These results support the notion that OsUPS plays an important role in the P(i) signaling pathway through the ubiquitin-26S proteasome system.

Molecular characterization of phenylalanine ammonia lyase gene from Cistanche deserticola.

We cloned the gene, CdPAL1, from Cistanche deserticola callus using RACE PCR with degenerate primers that were designed based on a multiple sequence alignment of known PAL genes from other plant species. The gene shows high homology to other known PAL genes registered in GenBank. The recombinant protein exhibited Michaelis-Menten kinetics with a Km of 0.1013 mM, Vmax of 4.858 µmol min(-1), Kcat of 3.36 S(-1), and Kcat/Km is 33,168 M(-1) S(-1). The enzyme had an optimal pH of 8.5 and an activation energy of 38.92 kJ mol(-1) when L-Phenylalanine was used as a substrate; L-tyrosine cannot be used as substrate for this protein. The optimal temperature was 55°C, and the thermal stability results showed that, after a treatment at 70°C for 20 min, the protein retained 87% activity, while a treatment at 75°C for 20 min resulted in a loss of over 85% of the enzyme activity. Treatment with heavy metal ions (Hg2+, Pb2+, and Zn2+) showed remarkable inhibitory effects. Among the intermediates from the lignin (cinnamyl alcohol, cinnamyl aldehyde, coniferyl aldehyde, coniferyl alcohol), phenylpropanoid (cinnamic acid, coumaric acid, caffeic acid, and chlorogenic acid) and phenylethanoid (tyrosol and salidroside) biosynthetic pathways, only cinnamic acid showed strong inhibitory effects against CdPAL1 activity with a Ki of 8 µM. Competitive inhibitor AIP exhibited potent inhibition with Ki=0.056 µM.

Effects of 2-aminoindan-2-phosphonic acid treatment on the accumulation of salidroside and four phenylethanoid glycosides in suspension cell culture of Cistanche deserticola.

2-Aminoindan-2-phosphonic acid (AIP), a specific competitive phenylalanine ammonia lyase (PAL) inhibitor was applied to a suspension cell culture of Cistanche deserticola. The effects of AIP treatment on cell growth, PAL activity, contents and yields of total phenolic compound, salidroside and four phenylethanoid glycosides (PheGs) are investigated. The results demonstrated that, 0.5 and 2.0 µM AIP treatments had similar effects on the measurements investigated in this study. AIP treatment resulted in significant decreases in PAL activity, total phenolic compounds content, and PheGs content. Linear regression analysis showed that PAL activity had a high correlation coefficient with the total phenolic compound content and the four PheGs contents. Total PAL activity-time area under curve (AUC) had a high correlation coefficient with the total phenolic compound yield and the yields of five tested compounds in untreated cell samples. In AIP-treated cells, total PAL activity-time AUC retained a high correlation with the total phenolic compound yield and the yields of three tested compounds, echinacoside, acteoside, and tubuloside A, but not salidroside and cistanoside A. The difference could be caused by the different biosynthetic origins of each of the tested compounds. These results demonstrate the important role of PAL in the biosynthesis of PheGs in the suspension cell culture of C. deserticola.

Mapping of a Major QTL, qBK1Z, for Bakanae Disease Resistance in Rice.

Bakanae disease is a fungal disease of rice (Oryza sativa L.) caused by the pathogen Gibberella fujikuroi (also known as Fusarium fujikuroi). This study was carried out to identify novel quantitative trait loci (QTLs) from an indica variety Zenith. We performed a QTL mapping using 180 F2:9 recombinant inbred lines (RILs) derived from a cross between the resistant variety, Zenith, and the susceptible variety, Ilpum. A primary QTL study using the genotypes and phenotypes of the RILs indicated that the locus qBK1z conferring bakanae disease resistance from the Zenith was located in a 2.8 Mb region bordered by the two RM (Rice Microsatellite) markers, RM1331 and RM3530 on chromosome 1. The log of odds (LOD) score of qBK1z was 13.43, accounting for 30.9% of the total phenotypic variation. A finer localization of qBK1z was delimited at an approximate 730 kb interval in the physical map between Chr01_1435908 (1.43 Mbp) and RM10116 (2.16 Mbp). Introducing qBK1z or pyramiding with other previously identified QTLs could provide effective genetic control of bakanae disease in rice.

Molecular characterization of OsPAP2: transgenic expression of a purple acid phosphatase up-regulated in phosphate-deprived rice suspension cells.

A phosphate starvation-induced, purple, acid phosphatase cDNA was cloned from rice, Oryza sativa. The cDNA encoding the phosphatase (OsPAP2) has 1,893 bp with an open reading frame of 630 amino acid residues. The deduced amino acid sequence of OsPAP2 shows identities of 60-63% with other plant purple acid phosphatases and appears to have five conserved motifs containing the residues involved in metal binding. OsPAP2 expression is up-regulated in the rice plant and in cell cultures in the absence of phosphate (P( i )). The induced expression of OsPAP2 is a specific response to P( i ) starvation, and is not affected by the deprivation of other nutrients. OsPAP2 expression was responsive to the level of P( i )-supply, and transcripts of OsPAP2 were abundant in P( i )-deprived roots. The OsPAP2 cDNA was expressed as a 69 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsPAP2 gene was introduced into Arabidopsis via an Agrobacterium-mediated transformation. Functional expression of the OsPAP2 gene in the transgenic Arabidopsis line was confirmed by northern and western blot analyses, as well as by phosphatase activity assays. These results suggest that the OsPAP2 gene can be used to develop new transgenic dicotyledonous plants that are able to adapt to P( i )-deficient conditions.

Spider Silk Fibroin Protein Heterologously Produced in Rice Seeds Reduce Diabetes and Hypercholesterolemia in Mice.

Silk fibroin proteins are biomaterials with diverse applications. These spider and silkworm proteins have specific biological effects when consumed by mammals; in addition to reducing blood pressure and blood glucose and cholesterol levels, they have anti-human immunodeficiency virus activity. In the present study, rice (Oryza sativa) was engineered to produce the C-terminus of the major ampullate spidroin protein from the spider Araneus ventricosus under the control of a Prolamin promoter. Homozygous transgenic rice lines were identified, and the therapeutic effect of this spider silk fibroin protein on the lipid and glucose metabolism was analyzed in a mouse model. Feeding fat-fed mice, the transgenic rice seeds for four weeks reduced serum concentrations of triglycerides, total cholesterol, low-density lipoprotein cholesterol, glutamic oxaloacetic transaminase, and glutamic pyruvic transaminase, and lowered blood glucose levels. This is the first study to investigate the effects of consumption of rice seeds heterologously expressing spider silk fibroin protein in a mammalian model. Our findings suggest that functional foods containing spider silk fibroin protein might be useful as potential pharmaceutical materials for preventing and treating diabetes, hyperlipidemia, and hypercholesterolemia.

Fine mapping of qBK1, a major QTL for bakanae disease resistance in rice.

BACKGROUND: Bakanae disease is an important fungal disease caused by Gibberella fujikuroi. Incidence of rice bakanae disease creates serious problems in the foremost rice growing countries, and no rice variety has been found to be completely resistant to this disease. However, breeding rice varieties resistant to bakanae disease may be a cost-saving solution preferable to the application of fungicides. In this study, we aimed to determine the exact position and the candidate gene for qBK1, a major resistant quantitative trait locus (QTLs) for bakanae disease. RESULTS: The genotypes/phenotypes of recombinants selected from backcrossed recombinant inbred lines of two rice varieties, Shingwang (resistant) and Ilpum (susceptible), indicated that the locus qBK1, conferring resistance to bakanae disease in Shingwang, was delimited to a 35-kb interval delimited by InDel 18 (23.637 Mbp) and InDel 19-14 (23.672 Mbp). Sequence analysis of this 35-kb region revealed four candidate genes, LOC_Os01g41770, LOC_Os01g41780, LOC_Os01g41790, and LOC_Os01g41800. There were many non-synonymous SNPs in LOC_Os01g41770 and the transcript of LOC_Os01g41790 was early terminated in Shingwang, whereas there were no differences in both LOC_Os01g41780 and LOC_Os01g41800 sequences between Ilpum and Shingwang. Expression profiling of the four candidate genes showed the up-regulation of LOC_Os01g41770, LOC_Os01g41780, and LOC_Os01g41790 in Ilpum and of LOC_Os01g41800 in Shingwang after inoculation of G. fujikuroi. CONCLUSION: Utilization of marker-assisted selection (MAS) with a precise molecular marker on qBK1 could provide an effective tool for breeding rice varieties resistant to bakanae disease. To our knowledge, this is the first report on fine mapping and candidate gene approaches for identifying the gene for qBK1.

Molecular mapping of qBK1 WD , a major QTL for bakanae disease resistance in rice.

BACKGROUND: Bakanae or foot rot disease is a prominent disease of rice caused by Gibberella fujikuroi. This disease may infect rice plants from the pre-emergence stage to the mature stage. In recent years, raising rice seedlings in seed boxes for mechanical transplanting has increased the incidence of many seedling diseases; only a few rice varieties have been reported to exhibit resistance to bakanae disease. In this study, we attempted to identify quantitative trait loci (QTLs) conferring bakanae disease resistance from the highly resistant japonica variety Wonseadaesoo. RESULTS: A primary QTL study using the genotypes/phenotypes of the recombinant inbred lines (RILs) indicated that the locus qBK1 WD conferring resistance to bakanae disease from Wonseadaesoo was located in a 1.59 Mb interval delimited on the physical map between chr01_13542347 (13.54 Mb) and chr01_15132528 (15.13 Mb). The log of odds (LOD) score of qBK1 WD was 8.29, accounting for 20.2% of the total phenotypic variation. We further identified a gene pyramiding effect of two QTLs, qBK WD and previously developed qBK1. The mean proportion of healthy plant for 31 F4 RILs that had no resistance genes was 35.3%, which was similar to that of the susceptible check variety Ilpum. The proportion of healthy plants for the lines with only qBK WD or qBK1 was 66.1% and 55.5%, respectively, which was significantly higher than that of the lines without resistance genes and that of Ilpum. The mean proportion of the healthy plant for 15 F4 RILs harboring both qBK WD and qBK1 was 80.2%, which was significantly higher than that of the lines with only qBK WD or qBK1. CONCLUSION: Introducing qBK WD or pyramiding the QTLs qBK WD and qBK1 could provide effective tools for breeding rice with bakanae disease resistance. To our knowledge, this is the first report on a gene pyramiding effect that provides higher resistance against bakanae disease.

Drought-tolerant QTL qVDT11 leads to stable tiller formation under drought stress conditions in rice.

Drought is an important limiting factor for rice production, but the genetic mechanisms of drought tolerance is poorly understood. Here, we screened 218 rice varieties to identify 32 drought-tolerant varieties. The variety Samgang exhibited strong drought tolerance and stable yield in rain-fed conditions and was selected for further study. To identify QTLs for drought tolerance, we examined visual drought tolerance (VDT) and relative water content (RWC) phenotypes in a doubled haploid (DH) population of 101 individuals derived from a cross between Samgang and Nagdong (a drought-sensitive variety). Three QTLs from Samgang were identified for VDT and explained 41.8% of the phenotypic variance. In particular, qVDT11 contributed 20.3% of the phenotypic variance for RWC. To determine QTL effects on drought tolerance in rain-fed paddy conditions, seven DH lines were selected according to the number of QTLs they contained. Of the drought-tolerance-associated QTLs, qVDT2 and qVDT6 did not affect tiller formation, but qVDT11 increased tiller number. Tiller formation was most stable when qVDT2 and qVDT11 were combined. DH lines with both of these drought-tolerance-associated QTLs exhibited the most stable tiller formation. Together, these results suggest that qVDT11 is important for drought tolerance and stable tiller formation in rain-fed paddy fields.

A phosphate starvation-induced acid phosphatase from Oryza sativa: phosphate regulation and transgenic expression.

A phosphate starvation-induced acid phosphatase cDNA was cloned from the rice, Oryza sativa. The cDNA encoding O. sativa acid phosphatase (OsACP1) has 1100 bp with an open reading frame of 274 amino acid residues. The deduced amino acid sequence of OsACP1 cDNA showed 53% identity to tomato acid phosphatase and 46-50% identity to several other plant phosphatases. OsACP1 expression was up-regulated in the rice plant and in cell culture in the absence of phosphate (Pi). The induced expression of OsACP1 was a specific response to Pi starvation, and was not affected by the deprivation of other nutrients. OsACP1 expression was responsive to the level of Pi supply, with transcripts of OsACP1 being abundant in Pi-deprived root. The OsACP1 cDNA was expressed as a 30 kDa polypeptide in baculovirus-infected insect Sf9 cells. In addition, the OsACP1 gene was introduced into Arabidopsis via Agrobacterium-mediated transformation. Functional expression of the OsACP1 gene in the transgenic Arabidopsis lines was confirmed by Northern blot and Western blot analyses, as well as phosphatase activity assays. These results suggest that the OsACP1 gene can be used to develop new transgenic dicotyledonous plants able to adapt to Pi-deficient conditions.