Identification of fungal (Magnaporthe grisea) stress-induced genes in wild rice (Oryza minuta)

To identify fungal stress-related genes in wild rice, Oryza minuta, we constructed a subtracted library using suppression subtractive hybridization in combination with mirror orientation selection. DNA chips containing 960 randomly selected cDNA clones were applied by reverse Northern analysis to eliminate false positive clones from the library and to prescreen differentially expressed genes. In total, 377 cDNA clones were selected on the basis of their signal intensities and expression ratios. Sequence analyses of these 377 cDNA fragments revealed that 180 of them (47.7%) represented unique genes. Of these180 cDNAs, 89 clones (49.6%) showed significant homologies to previously known genes, while the remaining 91 did not match any known sequences. The putative functions of the 180 unique ESTs were categorized by aligning them with MIPS data. They were classified into seven different groups using microarray data-derived expression patterns and verified by Northern blotting.Abbreviations ER: Endoplasmic reticulum - EST: Expressed sequence tag - MIPS: Munich Information Center for Protein Sequences - MOS: Mirror orientation selection - NCBI: National Center for Biotechnology Information - omfi: Oryza minuta fungal-stress induced - PCD: Programmed cell death - PDI: Proteins disulfide isomerase - SSH: Suppression subtractive hybridization Communicated by I.S. ChungK.S. Shim and S.K. Cho contributed equally to this work.     

Overexpression of OgPAE1 from wild rice confers fungal resistance against Botrytis cinerea

A full-length cDNA of the OgPAE1 gene encoding the alpha5 subunit of the 20S proteasome was isolated from wild rice (Oryza grandiglumis) treated by wounding or with a fungal elicitor. The deduced amino acid sequence of OgPAE1 comprises 237 amino acids (25.99 kDa), and shows 94.5% homology with Arabidopsis thaliana AtPAE1. Expression of OgPAE1 is regulated by defense-related signaling chemicals such as cantharidin, endothall and jasmonic acid. Overexpression of OgPAE1 in A. thaliana leads to resistance to the fungal pathogen Botrytis cinerea by lowering disease rate and size of necrotic lesions, and by less penetration and colonization of fungal hyphae. The results indicate that the 20S proteasome from wild rice is involved in the B. cinerea defense pathway via an as yet undetermined mechanism.     

Enhanced fungal resistance in Arabidopsis expressing wild rice PR-3 (OgChitIVa) encoding chitinase class IV

Oryza grandiglumis Chitinase IVa (OgChitIVa) cDNA encoding a class IV chitinase was cloned from wild rice (Oryza grandiglumis). OgChitIVa cDNA contains an open reading frame of 867 nucleotides encoding 288 amino acid residues with a predicted molecular weight of 30.4 kDa and isoelectric point of 8.48. Deduced amino acid sequences of OgChitIVa include the signal peptide and chitin-binding domain in the N-terminal domain and conserved catalytic domain. OgChitIVa showed significant similarity at the amino acid level with related monocotyledonous rice and maize chitinase, but low similarity with dicotyledoneous chitinase. Southern blot analysis showed that OgChitIVa genes are present as two copies in the wild rice genome. It was shown that RNA expression of OgChitIVa was induced by defense/stress signaling chemicals, such as jasmonic acid, salicylic acid, and ethephon or cantharidin and endothall or wounding, and yeast extract. It was demonstrated that overexpression of OgChitIVa in Arabidopsis resulted in mild resistance against the fungal pathogen, Botrytis cinerea, by lowering disease rate and necrosis size. RT-PCR analysis showed that PR-1 and PR-2 RNA expression was induced in the transgenic lines. Here, we suggest that a novel OgChitIVa gene may play a role in signal transduction process in defense response against B. cinerea in plants. J.-H. Pak and E.-S. Chung contributed equally to this work.     

Identification and cloning of a submergence-induced gene OsGGT (glycogenin glucosyltransferase) from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) by suppression subtractive hybridization

A submergence-induced gene, OsGGT, was cloned from 7-day submerged rice (Oryza sativa L. plants, FR13A (a submergence-tolerant cultivar, Indica), using suppression subtractive hybridization and both 5- and 3-rapid amplification of cDNA ends (RACE). The full-length OsGGT cDNA contains 1,273 bp with an open reading frame of 1,140 bp (17–1,156) that encodes 379 amino acids. Its deduced amino acid sequence is homologous with glycogenin glucosyltransferase. We found that the OsGGT gene is located in the 17,970–20,077 bp region of genome fragment AAAA01002475.1 of the Indica cultivar and in the 53,293–51,186 bp region of genome fragment AC037426.12 of chromosome 10 of the Japanica cultivar. A time-course study showed that OsGGT-gene expression increased in FR13A during submergence but decreased in IR42 (submergence-intolerant cultivar, Indica). The expression of the OsGGT gene in FR13A was induced by salicylic acid and benzyladenine. The accumulation of OsGGT mRNA in FR13A also increased in response to ethylene, gibberellin, abscisic acid, drought and salt treatment, but methyl jasmonate treatment and cold stress had no effect on expression. These results suggest that the OsGGT gene could be related to submergence stress and associated with a general defensive response to various environmental stresses.The nucleotide sequences of OsGGT cDNA has been submitted to GenBank DDBJ under accession numbers AB164463.     

Mechanism of salt tolerance in wild rice (Oryza coarctata Roxb)

Summary Oryza coarctata, a highly salt-resistant wild rice species, is commonly found on the banks of coastal rivers in India. This species can also withstand saline water (20 to 40 dSm⁻¹ E.C) submergence for quite a long period. It was revealed thatO. coarctata has some special unicellular salt hairs (trichomes) on the adaxial surface of the leaves, by which they efficiently maintain a low concentration of toxic salts in the plant tissue. Sodium and chloride were the dominant ions in the excreted material but they also excrete potassium, magnesium and calcium. With the increase in soil salinity sodium, magnesium and chloride excretion increased.O. coarctata maintained the optimum mineral concentration in its tissues. Maximum accumulation of potassium was observed in the leaves. With the increase in salt stress total biomass production and osmotic potential increased over control but there was no change in the moisture percentage of leaves.     

Comparative RFLP mapping of an allotetraploid wild rice species (Oryza latifolia) and cultivated rice (O. sativa)

The purpose of this study was to construct a comparative RFLP map of an allotetraploid wild rice species, Oryza latifolia, and to study the relationship between the CCDD genome of O. latifolia and the AA genome of O. sativa. A set of RFLP markers, which had been previously mapped to the AA genome of cultivated rice, were used to construct the comparative map. Fifty-eight F₂ progeny, which were derived from a single F₁ plant, were used for segregation analysis. The comparative RFLP map contains 149 DNA markers, including 145 genomic DNA markers from cultivated rice, 3 cDNA markers from oat, and one known gene (waxy, from maize). Segregation patterns reflected the allotetraploid ancestry of O. latifolia, and the CC and DD genomes were readily distinguished by most probes tested. There is a high degree of conservation between the CCDD genome of O. latifolia and the AA genome of O. sativa based on our data, but some inversions and translocations were noted.     

Transfer of bacterial blight and blast resistance from the tetraploid wild rice Oryza minuta to cultivated rice,Oryza sativa

Summary Oryza minuta J. S. Presl ex C. B. Presl is a tetraploid wild rice with resistance to several insects and diseases, including blast (caused by Pyricularia grisea) and bacterial blight (caused by Xanthomonas oryzae pv. oryzae). To transfer resistance from the wild species into the genome of cultivated rice (Oryza sativa L.), backcross progeny (BC₁, BC₂, and BC₃) were produced from interspecific hybrids of O. sativa cv IR31917-45-3-2 (2n=24, AA genome) and O. minuta Acc. 101141 (2n=48, BBCC genomes) by backcrossing to the O. sativa parent followed by embryo rescue. The chromosome numbers ranged from 44 to 47 in the BC₁ progeny and from 24 to 37 in the BC₂ progeny. All F₁ hybrids were resistant to both blast and bacterial blight. One BC₁ plant was moderately susceptible to blast while the rest were resistant. Thirteen of the 16 BC₂ progeny tested were resistant to blast; 1 blast-resistant BC₂, plant 75-1, had 24 chromosomes. A 3 resistant: 1 susceptible segregation ratio, consistent with the action of a major, dominant gene, was observed in the BC₂F₂ and BC₂F₃ generations. Five of the BC₁ plants tested were resistant to bacterial blight. Ten of the 21 BC₂ progeny tested were resistant to Philippine races 2, 3, and 6 of the bacterial blight pathogen. One resistant BC₂, plant 78-1, had 24 chromosomes. The segregation of reactions of the BC₂F₂, BC₂F₃, and BC₂F₄ progenies of plant 78-1 suggested that the same or closely linked gene(s) conferred resistance to races 2, 3, 5, and 6 of the bacterial blight pathogen from the Philippines.     

Accessing genes in the tertiary gene pool of rice by direct introduction of total DNA fromZizania palustris (wild rice)

Transfer of useful genes from wild relatives of crop plants has relied upon successful conventional crossing or the availability of the cloned gene. Co-bombardment of rice callus with total genomic DNA from wild rice (Zizania palustris) and a plasmid containing a gene confirming hygromycin resistance allowed recovery under selection of transgenic plants with grain characteristics from wild rice. Amplified Fragment Length Polymorphism (AFLP) analysis suggested that a significant amount of DNA fromZizania was introduced by this procedure. One plant had 16 of a possible 122Zizania specific AFLP markers detected with the primers used. This approach may have potential for introgression of genes from wild relatives in other cases where highly efficient transformation methods are available.     

Interspecific hybridization of cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl.

Crosses were made between four varieties (Mahsuri, Setanjung, MR84 and MR103) of Oryza sativa L. (2n=24, AA) and one accession of O. minuta (2n= 8, BBCC). The seed set obtained ranged between 9.5% and 25.1% depending on the rice variety used. By rescuing 14-day-old embryos and culturing them on 25%-strength MS medium we obtained a total of 414 F₁ hybrids. The F₁s were vigorous, tillered profusely, were perennial and male-sterile. The hybrids were triploid (ABC) with 36 chromosomes and showed irregular meiosis. The average frequency and range of chromosome associations at metaphase I or early anaphase I pollen mother cells of F₁ plants were 29.31(16–36) Is +3.32(0–10) IIs+0.016(0–1) IIIs+0.002(0–1) IVs. Upon backcrossing the original triploid hybrids and colchicine-treated hybrids to their respective recurrent parents, and further embryo rescue, 17 backcross-1 (BC₁) plants were obtained. Of all the crosses using MR84, no BC₁ plant was obtained even after pollinating 13 894 spikelets of the triploid hybrid. The BC₁s were similar in appearence to the F₁s and were male-sterile, their chromosome number ranged from 44 to 48. By backcrossing these BC₁s and nurturing them through embryo rescue, we obtained 32 BC₂ plants. Of these, however, only 18 plants grew vigorously. One of these plants has 24 chromosomes and the other 17 have chromosome numbers ranging between 30 and 37. The 24-chromosome plant was morphologically similar to the O. sativa parent and was partially fertile with a pollen and spikelet fertility of 58.8% and 12.5% respectively. All of the F₁ and BC₁ plants were found to be resistant to five Malaysian isolates (XO66, XO99, XO100, XO257 and XO319) of Xanthomonas campestris pv oryzae. Amongst the BC₂s, the reaction varied from resistant to moderately susceptible. The 24-chromosome BC₂ plant was resistant to the four isolates and moderately resistant to isolate XO100 to which the O. sativa parent was susceptible.Part of PhD thesis submitted by first author to Universiti Kebangsaan Malaysia, Bangi     

Cloning of a putative monogalactosyldiacylglycerol synthase gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) plants and its expression in response to submergence and other stresses

Suppression subtractive hybridization was used to construct a subtractive cDNA library from plants of non-submerged and 7-day-submerged rice (Oryza sativa L., FR13A, a submergence-tolerant cultivar). One clone of the subtractive cDNA library, S23, was expressed abundantly during submergence. The full length of S23 was amplified using 5- and 3-rapid amplification of cDNA ends, and found to consist of 1,671 bp with an open reading frame of 1,077 bp (181–1257) encoding 358 amino acids. Its deduced amino acid sequence showed a high homology with monogalactosyldiacylglycerol synthase (UDPgalactose: 1,2-diacylglycerol 3--d-galactosyl transferase; EC 2.4.1.46, MGDG synthase) from Arabidopsis thaliana; therefore, we named the gene OsMGD. Time-course studies showed that the expression of OsMGD in the rice cultivars FR13A and IR42 (submergence-susceptive cultivar) during submergence was gradually increased and that expression in FR13A was higher than in IR42. The expression of OsMGD in FR13A was influenced by benzyladenine and illumination. The accumulation of OsMGD mRNA in both FR13A and IR42 was also increased by ethephon, gibberellin, drought and salt treatment, but cold stress had no effect on the expression of the gene. These results suggest that the expression of OsMGD mRNA requires benzyladenine or illumination, and that the process is also mediated by ethephon and gibberellin. Salt and drought stress have an effect similar to that of submergence. Furthermore, the enhanced expression of OsMGD may relate to photosynthesis, and play an important role during submergence.Abbreviations BA N⁶-Benzyladenine - GA Gibberellin - MGD Monogalactosyldiacylglycerol synthase - RACE Rapid amplification of cDNA ends     

Somatic embryogenesis and plant regeneration from cultured young inflorescences of Oryza sativa L. (rice)

Compact calli initiated from young inflorescences of Oryza sativa L. (rice) on the Linsmaier and Skoog's (LS) medium containing 1 to 2.5mg/l of 2,4-dichlorophen-oxyacetic acid (2,4-D) were used for regeneration studies. After smooth and compact nodules appeared, these calli were transferred to the regeneration medium containing indole-3-acetic acid (IAA) and either kinetin or 6-benzylaminopurine (BAP). Somatic embryos developed in ten days and were examined by histological studies. Some of the embryos showed scutellum-like structures and a coleoptile-coleorhiza bipolar organization. Regenerated plants had the normal chromosome number of 2n=24.