CRISPR/Cas9-mediated targeted T-DNA integration in rice

Key message

Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.

Abstract

Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50?80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

     

Variation in stem radial growth of the Australian conifer, Callitris columellaris, across the world’s driest and least fertile vegetated continent

Climate change could alter the biogeography of many tree species. However, there have been few studies of tree growth across climatic gradients at a continental scale. Callitris columellaris is a widespread conifer that spans many climates and landscape positions across Australia. Our aim was to determine how stem radial growth of C. columellaris varies with tree size and with the biogeographic factors of rainfall, temperature, soil fertility and inter-tree competition. We sampled cores from trees at 85 sites in biomes ranging from tropical savanna to arid desert and temperate forest, and measured widths of the 100 outermost growth rings. We analysed ring width in relation to changes in tree age and diameter, and also evaluated the influence of the biogeographic factors on the width of the ten most recently formed rings. The average width of outermost rings varied only slightly with stem diameter, because the decrease in ring width with age and diameter within trees is offset by an increase with diameter among trees. Our analyses thus explain the weak, inconsistent relationships often observed between stem diameter and growth rate amongst trees. The most important biogeographic factors were the climatic ones: across Australia, ring width increased with both mean annual rainfall and mean annual temperature. These relationships were largely driven by continental scale differences between the tropical and the southern (arid plus temperate) sites, while relationships within climate zones were comparatively weak. Ring width decreased with intense inter-tree competition but showed little correlation with available soil nitrogen or phosphorus.     

21/21 translocation: Correlation of banding with meiotic results

Summary Meiotic studies of a boy, both of whose sibs also had Down's syndrome, were suggestive of a 21/21 chromosome rearrangement. Banding of somatic chromosomes from his mother and affected sister validated this interpredation and indicated a 21/21 translocation, not an isochromosome.

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Zusammenfassung Meiose-Untersuchungen an einem Jungen, dessen beide Geschwister ebenfalls das Down-Syndrom hatten, ließen ein 21/21-Chromosomen-Rearrangement vermuten. Die Bandenmuster somatischer Chromosomen seiner Mutter und seiner ebenfalls kranken Schwester sprachen für diese Interpretation und wiesen auf eine 21/21-Translokation, nicht auf ein Isochromosom hin.

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These studies were supported by NIH Grants AM No. 13173 and HD No. 05082 (Dr. Hecht) and RR-62 and No. 5 SO 1 RR054-11 (Dr. Seely).     

Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.     

Environmental fate of the chemical mixtures: Crude oil,JP‐5, mineral spirits,and diesel fuel

Petroleum hydrocarbon mixtures in soils and groundwater present unique challenges in the estimation of potential human exposures and subsequent health risks. A major component of risk assessment affected by mixtures is the evaluation of environmental fate. The fate of petroleum mixtures may be evaluated by using either of three approaches: (1) the evaluation of the fate of indicator chemical(s), (2) the evaluation of the fate of the mixture as a whole with a surrogate, and (3) the evaluation of the fate of the hydrocarbon mixture as a whole. The limiting factor in the selection of an approach is the availability of information on specific chemical constituents in the mixture. The evaluation of environmental fate requires quantitative information regarding the distribution, mobility, and degradation/transformation as represented by various physicochemical properties. In addition to the availability of this information, the selection of the evaluation method should be consistent with the goals of the project, as each approach will produce different results. This presentation discusses the issues related to the identification and implementation of each of the approaches to the evaluation of the environmental fate of four petroleum mixtures (crude oil, JP‐5, mineral spirits, and diesel) for risk assessment purposes.     

Understorey thinning and burning trials are needed in conservation reserves: The case of Tuart (Eucalyptus gomphocephala D.C.)

Summary Management interventions are needed to reverse the decline of Tuart (Eucalyptus gomphocephala) woodland in the Yalgorup area of south‐west Western Australia where the largest intact remaining example of this ecosystem is located. Although the cause of the decline is uncertain and several factors may be involved, management action should not be withheld because the decline process is not fully understood. We contend that the reduction in fire frequency over the last 50 years has led to an increase in understorey density, particularly of Western Australian Peppermint (Agonis flexuosa), resulting in greater competition for resources, which may in turn have increased the susceptibility of healthy woodland to decline. In contrast to Tuart regeneration, which is usually tied to fire, Western Australian Peppermint can establish readily in unburnt woodland. Further, once Western Australian Peppermint seedlings develop to the lignotuberous stage, they can resprout vigorously after fire. Therefore, a combination of fire and the physical removal of understorey in sites where this species has formed extensive thickets is required to: (i) provide an opportunity for regeneration of Tuart in both healthy and declining stands; (ii) improve the chances of sustained recovery of Tuart trees in declining stands; and (iii) ensure heterogeneity in the vegetation at multiple scales, a recognized strategy for conserving biodiversity and increasing ecosystem resilience. We propose that this approach may also be relevant to other tree decline syndromes in southern Australia. However, fostering community support for active intervention using thinning and fire in conservation reserves and staging the operations within an experimental framework will be important for such action to gain both the social and scientific acceptance necessary for it to be applied widely.