Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots.

AIM: We report the construction of a Virus-Induced Gene Silencing (VIGS) vector and an agroinoculation protocol for gene silencing in cassava (Manihot esculenta Crantz) leaves and roots. The African cassava mosaic virus isolate from Nigeria (ACMV-[NOg]), which was initially cloned in a binary vector for agroinoculation assays, was modified for application as VIGS vector. The functionality of the VIGS vector was validated in Nicotiana benthamiana and subsequently applied in wild-type and transgenic cassava plants expressing the uidA gene under the control of the CaMV 35S promoter in order to facilitate the visualization of gene silencing in root tissues. VIGS vectors were targeted to the Mg2+-chelatase gene in wild type plants and both the coding and promoter sequences of the 35S::uidA transgene in transgenic plants to induce silencing. We established an efficient agro-inoculation method with the hyper-virulent Agrobacterium tumefaciens strain AGL1, which allows high virus infection rates. The method can be used as a low-cost and rapid high-throughput evaluation of gene function in cassava leaves, fibrous roots and storage roots. BACKGROUND: VIGS is a powerful tool to trigger transient sequence-specific gene silencing in planta. Gene silencing in different organs of cassava plants, including leaves, fibrous and storage roots, is useful for the analysis of gene function. RESULTS: We developed an African cassava mosaic virus-based VIGS vector as well as a rapid and efficient agro-inoculation protocol to inoculate cassava plants. The VIGS vector was validated by targeting endogenous genes from Nicotiana benthamiana and cassava as well as the uidA marker gene in transgenic cassava for visualization of gene silencing in cassava leaves and roots. CONCLUSIONS: The African cassava mosaic virus-based VIGS vector allows efficient and cost-effective inoculation of cassava for high-throughput analysis of gene function in cassava leaves and roots.

FLOWERING LOCUS T Triggers Early and Fertile Flowering in Glasshouse Cassava (Manihot esculenta Crantz).

Accelerated breeding of plant species has the potential to help challenge environmental and biochemical cues to support global crop security. We demonstrate the over-expression of ArabidopsisFLOWERING LOCUS T in Agrobacterium-mediated transformed cassava (Manihot esculenta Crantz; cultivar 60444) to trigger early flowering in glasshouse-grown plants. An event seldom seen in a glasshouse environment, precocious flowering and mature inflorescence were obtained within 4-5 months from planting of stem cuttings. Manual pollination using pistillate and staminate flowers from clonal propagants gave rise to viable seeds that germinated into morphologically typical progeny. This strategy comes at a time when accelerated crop breeding is of increasing importance to complement progressive genome editing techniques.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.

Insights into the formation of carlactone from in-depth analysis of the CCD8-catalyzed reactions.

Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis-configured ß-carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used 13 C and 18 O-labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC-MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.

On the substrate- and stereospecificity of the plant carotenoid cleavage dioxygenase 7.

Strigolactones are phytohormones synthesized from carotenoids via a stereospecific pathway involving the carotenoid cleavage dioxygenases 7 (CCD7) and 8. CCD7 cleaves 9-cis-ß-carotene to form a supposedly 9-cis-configured ß-apo-10'-carotenal. CCD8 converts this intermediate through a combination of yet undetermined reactions into the strigolactone-like compound carlactone. Here, we investigated the substrate and stereo-specificity of the Arabidopsis and pea CCD7 and determined the stereo-configuration of the ß-apo-10'-carotenal intermediate by using Nuclear Magnetic Resonance Spectroscopy. Our data unequivocally demonstrate the 9-cis-configuration of the intermediate. Both CCD7s cleave different 9-cis-carotenoids, yielding hydroxylated 9-cis-apo-10'-carotenals that may lead to hydroxylated carlactones, but show highest affinity for 9-cis-ß-carotene.

The path from ß-carotene to carlactone, a strigolactone-like plant hormone.

Strigolactones, phytohormones with diverse signaling activities, have a common structure consisting of two lactones connected by an enol-ether bridge. Strigolactones derive from carotenoids via a pathway involving the carotenoid cleavage dioxygenases 7 and 8 (CCD7 and CCD8) and the iron-binding protein D27. We show that D27 is a ß-carotene isomerase that converts all-trans-ß-carotene into 9-cis-ß-carotene, which is cleaved by CCD7 into a 9-cis-configured aldehyde. CCD8 incorporates three oxygens into 9-cis-ß-apo-10'-carotenal and performs molecular rearrangement, linking carotenoids with strigolactones and producing carlactone, a compound with strigolactone-like biological activities. Knowledge of the structure of carlactone will be crucial for understanding the biology of strigolactones and may have applications in combating parasitic weeds.

In vitro characterization of Synechocystis CYP120A1 revealed the first nonanimal retinoic acid hydroxylase.

Retinoids are C(20) apocarotenoids that have various important functions in metazoans. In addition, several findings suggest their occurrence in eubacteria, including cyanobacteria. It has been shown that the Synechocystis cytochrome P450 enzyme CYP120A1 is a retinoic acid-binding polypeptide. In this work, we determined the reaction catalyzed by CYP120A1 and investigated its substrate specificity in vitro. CYP120A1-containing microsomes generated in yeast converted all-trans-retinoic acid into a compound exhibiting higher polarity in HPLC analysis. Liquid chromatography-MS analysis suggested the introduction of a single hydroxyl group, and NMR analysis of the purified product revealed C16 or C17 as the reaction site. Incubations with cis-retinoic acids, retinal, 3(R)-OH-retinal, retinol, beta-apo-13-carotenone (C(18)) and beta-apo-14'-carotenal (C(22)) resulted in the formation of the corresponding hydroxyl derivatives, as suggested by HPLC and liquid chromatography-MS analyses. Comparisons of the relative product amounts revealed the highest conversion rate for all-trans-retinoic acid, followed by beta-apo-13-carotenone (C(18)). As shown by real-time RT-PCR, CYP120A1 is expressed under normal growth conditions and is slightly induced by high-intensity light. Our work provides the first enzymatic study of a cyanobacterial cytochrome P450, showing it to be the first nonanimal retinoic acid-metabolizing enzyme characterized so far. Moreover, the CYP120A1-catalyzed reaction represents a novel modification of retinoids.

Ustilago maydis accumulates beta-carotene at levels determined by a retinal-forming carotenoid oxygenase.

The basidiomycete Ustilago maydis, the causative agent of corn smut disease, has emerged as a model organism for dimorphism and fungal phytopathogenicity. In this work, we line out the key conserved enzymes for beta-carotene biosynthesis encoded by the U. maydis genome and show that this biotrophic fungus accumulates beta-carotene. The amount of this pigment depended on culture pH and aeration but was not affected by light and was not increased by oxidative stress. Moreover, we identified the U. maydis gene, cco1, encoding a putative beta-carotene cleavage oxygenase. Heterologous overexpression and in vitro analyses of purified enzyme demonstrated that Cco1 catalyzes the symmetrical cleavage of beta-carotene to yield two molecules of retinal. Analyses of beta-carotene and retinal contents in U. maydiscco1 deletion and over-expression strains confirmed the enzymatic function of Cco1, and revealed that Cco1 determines the beta-carotene content. Our data indicate that carotenoid biosynthesis in U. maydis is carried out to provide retinal rather than to deliver protective pigments. The U. maydis genome also encodes three potential opsins, a family of photoactive proteins that use retinal as chromophore. Two opsin genes showed different light-regulated expression patterns, suggesting specialized roles in photobiology, while no mRNA was detected for the third opsin gene in the same experiments. However, deletion of the cco1 gene, which should abolish function of all the retinal-dependent opsins, did not affect growth, morphology or pathogenicity, suggesting that retinal and opsin proteins play no relevant role in U. maydis under the tested conditions.

Dose-dependent RNAi-mediated geminivirus resistance in the tropical root crop cassava.

Cassava mosaic disease is a major constraint for cassava production in Africa, resulting in significant economic losses. We have engineered transgenic cassava with resistance to African cassava mosaic virus (ACMV), by expressing ACMV AC1-homologous hairpin double-strand RNAs. Transgenic cassava lines with high levels of AC1-homologous small RNAs have ACMV immunity with increasing viral load and different inoculation methods. We report a correlation between the expression of the AC1-homologous small RNAs and the ACMV resistance of the transgenic cassava lines. Characterization of the small RNAs revealed that only some of the hairpin-derived small RNAs fall into currently known small interfering RNA classes in plants. The method is scalable to stacking by targeting multiple virus isolates with additional hairpins.

Carotenoid oxygenases involved in plant branching catalyse a highly specific conserved apocarotenoid cleavage reaction.

Recent studies with the high-tillering mutants in rice (Oryza sativa), the max (more axillary growth) mutants in Arabidopsis thaliana and the rms (ramosus) mutants in pea (Pisum sativum) have indicated the presence of a novel plant hormone that inhibits branching in an auxin-dependent manner. The synthesis of this inhibitor is initiated by the two CCDs [carotenoid-cleaving (di)oxygenases] OsCCD7/OsCCD8b, MAX3/MAX4 and RMS5/RMS1 in rice, Arabidopsis and pea respectively. MAX3 and MAX4 are thought to catalyse the successive cleavage of a carotenoid substrate yielding an apocarotenoid that, possibly after further modification, inhibits the outgrowth of axillary buds. To elucidate the substrate specificity of OsCCD8b, MAX4 and RMS1, we investigated their activities in vitro using naturally accumulated carotenoids and synthetic apocarotenoid substrates, and in vivo using carotenoid-accumulating Escherichia coli strains. The results obtained suggest that these enzymes are highly specific, converting the C27 compounds beta-apo-10'-carotenal and its alcohol into beta-apo-13-carotenone in vitro. Our data suggest that the second cleavage step in the biosynthesis of the plant branching inhibitor is conserved in monocotyledonous and dicotyledonous species.