Abstract: A library of 29,482 T-DNA enhancer trap lines has been generated in rice cv. Nipponbare. The regions flanking the T-DNA left border from the first 12,707 primary transformants were systematically isolated by adapter anchor PCR and sequenced. A survey of the 7480 genomic sequences larger than 30 bp (average length 250 bp), representing 56.4% of the total readable sequences and matching the rice bacterial artificial chromosome/phage artificial chromosome (BAC/PAC) sequences assembled in pseudomolecules allowed the assigning of 6645 (88.8%) T-DNA insertion sites to at least one position in the rice genome of cv. Nipponbare. T-DNA insertions appear to be rather randomly distributed over the 12 rice chromosomes, with a slightly higher insertion frequency in chromosomes 1, 2, 3 and 6. The distribution of 723 independent T-DNA insertions along the chromosome 1 pseudomolecule did not differ significantly from that of the predicted coding sequences in exhibiting a lower insertion density around the centromere region and a higher density in the subtelomeric regions where the gene density is higher. Further establishment of density graphs of T-DNA inserts along the recently released 12 rice pseudomolecules confirmed this non-uniform chromosome distribution. T-DNA appeared less prone to hot spots and cold spots of integration when compared with those revealed by a concurrent assignment of the Tos17 retrotransposon flanking sequences deposited in the National Center for Biotechnology Information (NCBI). T-DNA inserts rarely integrated into repetitive sequences. Based on the predicted gene annotation of chromosome 1, preferential insertion within the first 250 bp from the putative ATG start codon has been observed. Using 4 kb of sequences surrounding the insertion points, 62% of the sequences showed significant similarity to gene encoding known proteins (E-value < 1.00 e(-05)). To illustrate the in silico reverse genetic approach, identification of 83 T-DNA insertions within genes coding for transcription factors (TF) is presented. Based both on the estimated number of members of several large TF gene families (e.g. Myb, WRKY, HD-ZIP, Zinc-finger) and on the frequency of insertions in chromosome 1 predicted genes, we could extrapolate that 7-10% of the rice gene complement is already tagged by T-DNA insertion in the 6116 independent transformant population. This large resource is of high significance while assisting studies unravelling gene function in rice and cereals, notably through in silico reverse genetics.
Abstract: Transposition of the maize En/Spm system in rice was investigated using a two-component construct consisting of an immobilised transposase source driven by the CaMV 35S-promoter, and a modified I/dSpm transposon. Mobilization of I/dSpm in somatic sectors was demonstrated by sequencing of excision products and isolation of flanking genomic sequences in T0 and T1 progeny plants. Since the transposition efficiency appeared to be considerably lower than that observed in maize or in other heterologous systems like Arabidopsis, we examined En/Spm transcription and splicing in the transgenic rice plants. Northern analysis revealed the presence of transcripts encoding the active TnpA and TnpD transposases, with the latter predominating; this is the reverse of what is seen in maize and Arabidopsis. RT-PCR analysis confirmed the occurrence of correct splicing and the formation of the two other alternatively spliced transcripts (TnpB and TnpC), as previously described for maize. Two alternative splice donor sites at the end of exon 1 were identified in maize at positions 578 and 704. We observe that rice is similar to maize in that TnpA is preferentially spliced at position 578. We also show that in Arabidopsis splicing occurs preferentially at position 704, as in other dicots like tobacco. These observations indicate differences in the splicing of transcripts of the maize En/Spm element between dicot and monocot hosts. Nevertheless, the ratio in which the transcripts for the active transposases are produced seems to determine the efficiency of transposition, irrespective of the host considered. A limiting amount of TnpA might therefore be responsible for the lower transposition activity of En/Spm in rice. Alternatively, reduced mobility of the modified I/dSpm element used may have resulted from the absence of critical sequences necessary for transposition. The influence of endogenous, autonomous, En/Spm -related elements present in the rice genome on the transposition behaviour of the exogenous maize element is also considered.
Abstract: Rice progenies used for the construction of genetic maps permit exhaustive identification and characterization of resistance genes present in their parental cultivars. We inoculated a rice progeny derived from the cross IR64 x Azucena with different Magnaporthe grisea isolates that showed differential responses on the parental cultivars. By QTL mapping, nine unlinked loci conferring resistance to each isolate were identified and named Pi-24( t) to Pi-32( t). They could correspond to nine specific resistance genes. Five of these resistance loci (RLs) were mapped at chromosomal locations where no resistance gene was previously reported, defining new resistance genes. Using degenerate primers of the NBS (nucleotide binding site) motif found in many resistance genes, two resistance gene analogues (RGAs) IR86 and IR14 were identified and mapped closely to two blast RLs (resistance identified in this study, i.e. Pi-29(t) and Pi-30(t) respectively). These two RLs may correspond to the Pi-11 and Pi-a blast resistance genes previously identified. Moreover, the ir86 and ir14 genes have been identified "in silico" on the indica rice cultivar 93-11, recently sequenced by Chinese researchers. Both genes encodes NBS-LRR-like proteins that are characteristics of plant-disease resistance genes.
Abstract: A collection of transposon Ac/ Ds enhancer trap lines is being developed in rice that will contribute to the development of a rice mutation machine for the functional analysis of rice genes. Molecular analyses revealed high transpositional activity in early generations, with 62% of the T0 primary transformants and more than 90% of their T1 progeny lines showing ongoing active transposition. About 10% of the lines displayed amplification of the Ds copy number. However, inactivation of Ds seemed to occur in about 70% of the T2 families and in the T3 generation. Southern blot analyses revealed a high frequency of germinal insertions inherited in the T1 progeny plants, and transmitted preferentially over the many other somatic inserts to later generations. The sequencing of Ds flanking sites in subsets of T1 plants indicated the independence of insertions in different T1 families originating from the same T0 line. Almost 80% of the insertion sites isolated showing homology to the sequenced genome, resided in genes or within a range at which neighbouring genes could be revealed by enhancer trapping. A strategy involving the propagation of a large number of T0 and T1 independent lines is being pursued to ensure the recovery of a maximum number of independent insertions in later generations. The inactive T2 and T3 lines produced will then provide a collection of stable insertions to be used in reverse genetics experiments. The preferential insertion of Ds in gene-rich regions and the use of lines containing multiple Ds transposons will enable the production of a large population of inserts in a smaller number of plants. Additional features provided by the presence of lox sites for site-specific recombination, or the use of different transposase sources and selectable markers, are discussed.
Abstract: We investigated the potential of an improved Agrobacterium tumefaciens-mediated transformation procedure of japonica rice ( Oryza sativa L.) for generating large numbers of T-DNA plants that are required for functional analysis of this model genome. Using a T-DNA construct bearing the hygromycin resistance ( hpt), green fluorescent protein ( gfp) and beta-glucuronidase ( gusA) genes, each individually driven by a CaMV 35S promoter, we established a highly efficient seed-embryo callus transformation procedure that results both in a high frequency (75-95%) of co-cultured calli yielding resistant cell lines and the generation of multiple (10 to more than 20) resistant cell lines per co-cultured callus. Efficiencies ranged from four to ten independent transformants per co-cultivated callus in various japonica cultivars. We further analysed the T-DNA integration patterns within a population of more than 200 transgenic plants. In the three cultivars studied, 30-40% of the T(0) plants were found to have integrated a single T-DNA copy. Analyses of segregation for hygromycin resistance in T(1) progenies showed that 30-50% of the lines harbouring multiple T-DNA insertions exhibited hpt gene silencing, whereas only 10% of lines harbouring a single T-DNA insertion was prone to silencing. Most of the lines silenced for hpt also exhibited apparent silencing of the gus and gfp genes borne by the T-DNA. The genomic regions flanking the left border of T-DNA insertion points were recovered in 477 plants and sequenced. Adapter-ligation Polymerase chain reaction analysis proved to be an efficient and reliable method to identify these sequences. By homology search, 77 T-DNA insertion sites were localized on BAC/PAC rice Nipponbare sequences. The influence of the organization of T-DNA integration on subsequent identification of T-DNA insertion sites and gene expression detection systems is discussed.
Abstract: Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1-2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene-rich regions (collectively called the 'gene space') separated by vast gene-empty, repeated sequences. In contrast, in Arabidopsis thaliana, genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T-DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T-DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene-rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.
Abstract: We constructed an alfalfa cDNA library from mRNA extracted from leaves after infection with Pseudomonas syringae (incompatible interaction). Screening with oligodeoxyribonucleotides designed from regions conserved in all known peroxidases allowed the isolation of four cDNAs (Msprx1A, 1B, 1C and 2). Sequence analysis revealed the presence of open reading frames of 351, 355, 358 and 323 amino acids, respectively, with the characteristic consensus sequences of plant peroxidases. Sequence comparison showed that the Msprx2 product is significantly different from the others and, particularly, lacks a C-terminal propeptide which might be required for vacuolar targeting.
Abstract: Infiltration of Medicago sativa leaves with a suspension of Pseudomonas syringae pv. pisi elicits the accumulation of several mRNA classes. A clone, designated as MsPR10-1, encoding a polypeptide exhibiting strong similarity to the class 10 PR protein was isolated and characterized from a cDNA library prepared from leaf mRNA. The corresponding gene was shown to be developmentally regulated: Except in roots, its expression was not detectable in other analyzed organs of healthy plants (hypocotyls, cotyledons, stems, leaves, and flower buds). MsPR10-1 transcript accumulation was especially high in leaf blades during an incompatible interaction: It was already detectable 3 h after infection, reached its maximum level 24 h postinfection, and remained at a high level over a period of at least 72 h. In addition, the expression of this gene was induced by salicylic acid treatment of the leaves. Southern hybridizations showed that this gene belongs to a multigene family. Using a 5' extension technique for cDNA, we demonstrated that during the incompatible interaction with P. syringae pv. pisi several genes or allelic variants of this class were expressed. Measurements of transcript accumulation in both the infiltrated and noninfiltrated zones by Northern and in situ hybridization allowed to demonstrate the "systemic" expression pattern of the MsPR10-1. In situ hybridizations indicated that MsPR10-1 was expressed in the vascular bundles adjacent to and distant from the infection site.
Abstract: Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.
