Kerrie Ramm

The ANTHER INDEHISCENCE1 Gene Encoding a Single MYB Domain Protein Is Involved in Anther Development in Rice

Using a two-element iAc/Ds transposon-tagging system, we identified a rice (Oryza sativa L. cv Nipponbare) recessive mutant, anther indehiscence1 (aid1), showing partial to complete spikelet sterility. Spikelets of the aid1 mutant could be classified into three types based on the viability of pollen grains and the extent of anther dehiscence. Type 1 spikelets (approximately 25%) were sterile due to a failure in accumulation of starch in pollen grains. Type 2 spikelets (approximately 55%) had viable pollen grains, but anthers failed to dehisce and/or synchronize with anthesis due to failure in septum degradation and stomium breakage, resulting in sterility. Type 3 spikelets (approximately 20%) had normal fertility. In addition, aid1 mutant plants had fewer tillers and flowered 10 to 15 d later than the wild type. The Ds insertion responsible for the aid1 mutation was mapped within the coding region of the AID1 gene on chromosome 6, which is predicted to encode a novel protein of 426 amino acids with a single MYB domain. The MYB domain of AID1 is closely related to that of the telomere-binding proteins of human, mouse, and Arabidopsis, and of single MYB domain transcriptional regulators in plants such as PcMYB1 and ZmIBP1. AID1 was expressed in both the leaves and panicles of wild-type plants, but not in mutant plants.

Agrobacterium-mediated transformation of Australian rice cultivars Jarrah and Amaroo using modified promoters and selectable markers

We report the first successfulAgrobacterium-mediated transformation of Australian elite rice cultivars, Jarrah and Amaroo, using binary vectors with our improved promoters and selectable markers. Calli derived from mature embryos were used as target tissues. The binary vectors contained hph(encoding hygromycin resistance) or bar (encoding herbicide resistance) as the selectable marker gene and uidA (gus) or sgfpS65T as the reporter gene driven by different promoters. Use of Agrobacterium strain AGL1 carrying derivatives of an improved binary vector pWBVec8, wherein the CaMV35S driven hph gene is interrupted by the castor bean catalase 1 intron, produced a 4- fold higher number of independent transgenic lines compared to that produced with the use of strain EHA101 car-rying the binary vector pIG121-Hm wherein the CaMV35S driven hph is intronless. The Ubiquitin promoter produced 30-fold higher s-glucuronidase (GUS) activity (derivatives of binary vector pWBVec8) in transgenic plants than the CaMV35S promoter (pIG121-Hm). The two modified SCSV promoters produced GUS activity com-parable to that produced by the Ubiquitin promoter. Progeny analysis (R1) for hygromycin resistance and GUS activ-ity with selected lines showed both Mendelian and non-Mendelian segregation. Lines showing very high levels of GUS activity in T0 showed a reduced level of GUS activity in their T1 progeny, while lines with moderate levels of GUS activity showed increased levels in T1 progeny. Stable heritable green fluorescent protein (GFP) expression was also observed in few transgenic plants produced with the binary vector pTO134 which had the CaMV35S promoter-driven selectable marker gene bar and a modified CaMV35S promoter-driven reporter gene sgfpS65T.

An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice

We evaluated a two-component transposon iAc/Ds system for generating a library of insertional mutants in rice. The constructs used have gene or enhancer trapping properties, plasmid rescue and T-DNA/Ds launching pad reporter facilities. Mutagenic iAc/Ds lines were produced by three methods: crossing iAc and Ds containing lines; co-transformation with iAc and Ds constructs; and super-transformation of iAc transgenic calli with Ds constructs. First and second generation screening populations, derived from crosses (F2 and F3) or double transformation (DtT1 and DtT2), were analysed for stable insertion lines containing Ds transposed to locations unlinked to iAc. The average frequencies of putative stable insertion (PSI) lines in the F2, DtT1, F3 and DtT2 populations were 6.61, 5.58, 11.47 and 7.05% respectively, with large variations in these frequencies in screening populations derived from different mutagenic lines. Further analyses indicated that 41, 33, 65 and 64% of the PSI lines, respectively, have Ds transposed to locations unlinked to the original Ds launching pad. Using the plasmid rescue system, sequences flanking Ds from 137 PSI lines were obtained. Sixty-eight of these lines had unique insertions in genomic regions, of which 18 were known sequences. Because the average frequency of proven stable insertion lines in any of our screening populations has been less than 5%, we suggest that additional features should be incorporated in this two-component iAc/Ds system to increase the screening efficiency, and to make it suitable for large-scale insertional mutagenesis and determination of gene function in rice.

Ppd-1 is a key regulator of inflorescence architecture and paired spikelet development in wheat.

The domestication of cereal crops such as wheat, maize, rice and barley has included the modification of inflorescence architecture to improve grain yield and ease harvesting1. Yield increases have often been achieved through modifying the number and arrangement of spikelets, which are specialized reproductive branches that form part of the inflorescence. Multiple genes that control spikelet development have been identified in maize, rice and barley2–5. However, little is known about the genetic underpinnings of this process in wheat. Here, we describe a modified spikelet arrangement in wheat, termed paired spikelets. Combining comprehensive QTL and mutant analyses, we show that Photoperiod-1 (Ppd-1), a pseudo-response regulator gene that controls photoperiod-dependent floral induction, has a major inhibitory effect on paired spikelet formation by regulating the expression of FLOWERING LOCUS T (FT)6,7. These findings show that modulated expression of the two important flowering genes, Ppd-1 and FT, can be used to form a wheat inflorescence with a more elaborate arrangement and increased number of grain producing spikelets.

Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants

To gain further understanding of the mechanisms involved in Agrobacterium-mediated genetic transformation and T-DNA integration, we analysed 156 T-DNA/rice, 69 T-DNA/T-DNA and 11 T-DNA/vector backbone (VB) junctions, which included 171 left borders (LB) and 134 right borders (RB). Conserved cleavage was observed in 6% of the LB and 43% of the RB. Terminal microhomology (1-10bp) was identified in 58% of T-DNA/rice, 43% of T-DNA/T-DNA and 82% of T-DNA/VB junctions, and this occurred particularly at the LB junctions. Approximately 32% of both T-DNA/rice and T-DNA/T-DNA junctions harboured 1-344bp of filler DNA that was derived mainly from the T-DNA region adjacent to the breakpoint and/or from the rice genome flanking the T-DNA integration site. Structure of the filler DNA was more complicated at the T-DNA/T-DNA junction than at the T-DNA/rice junction, indicating the presence of T-DNA recombination or rearrangement prior to or during T-DNA integration. When two T-DNAs were integrated in the inverted repeat configuration, significant truncation was always observed in one of the two T-DNAs whereas with direct repeat configuration, a large truncation was less frequent. Most integration events analysed in this study could be addressed by previously proposed models; however, the characteristics of the T-DNA repeats and the complicated filler DNA between two T-DNA copies imply that multiple mechanisms are involved in the formation of T-DNA repeats as well as in T-DNA integration in plants.

Rice ragged stunt oryzavirus genome segments S7 and S10 encode non-structural proteins of M r 68 025 (Pns7) and M r 32 364 (Pns10)

SummaryThe nucleotide sequences of genome segments S7 and S10 of a Thai-isolate of rice ragged stunt virus (RRSV) were determined. The 1 938 bp S7 sequence contains a single large open reading frame (ORF) spanning nucleotides 20 to 1 843 that is predicted to encode a protein of Mr 68 025. The 1 162 bp S10 sequence has a major ORF spanning nucleotides 142 to 1 032 that is predicted to encode a protein of Mr 32 364. This S10 ORF is preceded by a small ORF (nt 20–55) which is probably a minicistron. Coupled in vitro transcription-translation from the two major ORFs gave protein products of the expected sizes. However, no protein was visualised from S10 when the small ORF sequence was included. Proteins were expressed in Escherichia coli from the full length ORF of S7 (P7) and from a segment of the S10 ORF (P10) fused to the ORF of glutathione S-transferase (GST). Neither fusion protein was recognised by polyclonal antibodies raised against RRSV particles. Furthermore, polyclonal antibodies raised against GST-P7 fusion protein did not recognise any virion structural polypeptides. These data strongly suggest that the proteins P7 and P10 do not form part of RRSV particle. This is further supported by observed sequence homology (though very weak) of predicted RRSV P7 and P10 with those of rice dwarf virus (RDV) non-structural proteins Pns6 and Pns9, respectively.

The tms2 gene as a negative selection marker in rice

A conditional negative selection marker is essential for high throughput insertional mutagenesis with any two-element transposon tagging system. Thetms2 gene encodes indoleacetic acid hydrolase (IAAH) which converts naphthaleneacetamide (NAM) to the potent auxin naphthaleneacetic acid, a phytotoxic derivative. This gene, under the control of the manopine synthase gene 2 promoter fromAgrobacterium tumefaciens and exogenously applied NAM, have been used effectively as a negative selector inAc/Ds insertional mutagenesis ofArabidopsis thaliana (Sundaresan et al., 1995). In this study we show thattms2 can also be used as a negative selector in rice. T1 transgenic seedlings expressing thistms2 gene under the control of themas2’ promoter showed significant reduction in shoot and root growth in the presence of 5–10 μM NAM under specified growth conditions compared to plants not containing this gene.

TEOSINTE BRANCHED1 Regulates Inflorescence Architecture and Development in Bread Wheat (Triticum aestivum L.)

An ortholog of TEOSINTE BRANCHED1 regulates inflorescence architecture and developmental rate in bread wheat in a dosage-dependent manner. The flowers of major cereals are arranged on reproductive branches known as spikelets, which group together to form an inflorescence. Diversity for inflorescence architecture has been exploited during domestication to increase crop yields, and genetic variation for this trait has potential to further boost grain production. Multiple genes that regulate inflorescence architecture have been identified by studying alleles that modify gene activity or dosage; however, little is known in wheat. Here, we show TEOSINTE BRANCHED1 (TB1) regulates inflorescence architecture in bread wheat (Triticum aestivum) by investigating lines that display a form of inflorescence branching known as “paired spikelets.” We show that TB1 interacts with FLOWERING LOCUS T1 and that increased dosage of TB1 alters inflorescence architecture and growth rate in a process that includes reduced expression of meristem identity genes, with allelic diversity for TB1 found to associate genetically with paired spikelet development in modern cultivars. We propose TB1 coordinates formation of axillary spikelets during the vegetative to floral transition and that alleles known to modify dosage or function of TB1 could help increase wheat yields.