CYP707As are effectors of karrikin and strigolactone signalling pathways in Arabidopsis thaliana and parasitic plants.

Karrikins stimulate Arabidopsis thaliana germination, whereas parasitic weeds of the Orobanchaceae family have evolved to respond to host-exuded compounds such as strigolactones, dehydrocostus lactone, and 2-phenylethyl isothiocyanate. In Phelipanche ramosa, strigolactone-induced germination was shown to require one of the CYP707A proteins involved in abscisic acid catabolism. Here, germination and gene expression were analysed to investigate the role of CYP707As in germination of both parasitic plants and Arabidopsis upon perception of germination stimulants, after using pharmacological inhibitors and Arabidopsis mutants disrupting germination signals. CYP707A genes were up-regulated upon treatment with effective germination stimulants in both parasitic plants and Arabidopsis. Obligate parasitic plants exhibited both intensified up-regulation of CYP707A genes and increased sensitivity to the CYP707A inhibitor abscinazole-E2B, whereas Arabidopsis cyp707a mutants still positively responded to germination stimulation. In Arabidopsis, CYP707A regulation required the canonical karrikin signalling pathway KAI2/MAX2/SMAX1 and the transcription factor WRKY33. Finally, CYP707As and WRKY33 also modulated Arabidopsis root architecture in response to the synthetic strigolactone rac-GR24, and wrky33-1 exhibited a shoot hyperbranched phenotype. This study suggests that the lack of host-independent germination in obligate parasites is associated with an exacerbated CYP707A induction and that CYP707As and WRKY33 are new players involved in a variety of strigolactone/karrikin responses.

Quinone oxidoreductase 2 is involved in haustorium development of the parasitic plant Phtheirospermum japonicum.

The family Orobanchaceae includes many parasitic plant species. Parasitic plants invade host vascular tissues and form organs called haustoria, which are used to obtain water and nutrients. Haustorium formation is initiated by host-derived chemicals including quinones and flavonoids. Two types of quinone oxidoreductase (QR) are involved in signal transduction leading to haustorium formation; QR1 mediates single-electron transfers and QR2 mediates 2-electron transfers. In the facultative parasite Triphysaria versicolor, QR1 is involved in haustorium induction signaling, while this role is played by QR2 in the model plant Phtheirospermum japonicum. Our results suggest that there is functional diversification in haustorium signaling molecules among different species of the Orobanchaceae.

Mechanism of glyphosate control of Phelipanche aegyptiaca.

MAIN CONCLUSION: Despite its total reliance on its host plant, the holoparasite Phelipanche aegyptiaca suffers from a deficiency of aromatic amino acids upon exposure to glyphosate. The herbicide glyphosate inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a key enzyme in the biosynthesis of aromatic amino acids. However, the functionality of the EPSPS pathway in the obligate root holoparasite Phelipanche aegyptiaca is not straightforward because of the parasite's total dependence on the host plant. Despite the importance of glyphosate as a means of controlling P. aegyptiaca, the mechanism of action of the herbicide in this parasite is not clearly understood. We characterized glyphosate control of P. aegyptiaca by using a glyphosate-resistant tomato (GRT) genotype as the host plant and evaluating the activity of EPSPS and the levels of free aromatic amino acids in the parasite. The viability of the parasite's tissues deteriorated within the first 40 h after treatment (HAT) with glyphosate. In parallel, shikimate accumulation in the parasite was first detected at 24 HAT and increased over time. However, shikimate levels in the GRT host did not increase, indicating that the host was indeed glyphosate tolerant. Free phenylalanine and tyrosine levels decreased by 48 HAT in the parasite, indicating a deficiency of aromatic amino acids. The use of GRT as the host enabled us to observe, in an in situ experimental system, both endogenous EPSPS inhibition and a deficiency of aromatic amino acids in the parasite. We thus provided evidence for the presence of an active EPSPS and aromatic amino acid biosynthesis pathway in P. aegyptiaca and pinpointed this pathway as the target of glyphosate action in this parasite.

PLANT EVOLUTION. Convergent evolution of strigolactone perception enabled host detection in parasitic plants.

Obligate parasitic plants in the Orobanchaceae germinate after sensing plant hormones, strigolactones, exuded from host roots. In Arabidopsis thaliana, the α/ß-hydrolase D14 acts as a strigolactone receptor that controls shoot branching, whereas its ancestral paralog, KAI2, mediates karrikin-specific germination responses. We observed that KAI2, but not D14, is present at higher copy numbers in parasitic species than in nonparasitic relatives. KAI2 paralogs in parasites are distributed into three phylogenetic clades. The fastest-evolving clade, KAI2d, contains the majority of KAI2 paralogs. Homology models predict that the ligand-binding pockets of KAI2d resemble D14. KAI2d transgenes confer strigolactone-specific germination responses to Arabidopsis thaliana. Thus, the KAI2 paralogs D14 and KAI2d underwent convergent evolution of strigolactone recognition, respectively enabling developmental responses to strigolactones in angiosperms and host detection in parasites.

Trans-specific gene silencing of acetyl-CoA carboxylase in a root-parasitic plant.

Parasitic species of the family Orobanchaceae are devastating agricultural pests in many parts of the world. The control of weedy Orobanchaceae spp. is challenging, particularly due to the highly coordinated life cycles of the parasite and host plants. Although host genetic resistance often provides the foundation of plant pathogen management, few genes that confer resistance to root parasites have been identified and incorporated into crop species. Members of the family Orobanchaceae acquire water, nutrients, macromolecules, and oligonucleotides from host plants through haustoria that connect parasite and host plant roots. We are evaluating a resistance strategy based on using interfering RNA (RNAi) that is made in the host but inhibitory in the parasite as a parasite-derived oligonucleotide toxin. Sequences from the cytosolic acetyl-CoA carboxylase (ACCase) gene from Triphysaria versicolor were cloned in hairpin conformation and introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation. Transgenic roots were recovered for four of five ACCase constructions and infected with T. versicolor against parasitic weeds. In all cases, Triphysaria root viability was reduced up to 80% when parasitizing a host root bearing the hairpin ACCase. Triphysaria root growth was recovered by exogenous application of malonate. Reverse-transcriptase polymerase chain reaction (RT-PCR) showed that ACCase transcript levels were dramatically decreased in Triphysaria spp. parasitizing transgenic Medicago roots. Northern blot analysis identified a 21-nucleotide, ACCase-specific RNA in transgenic M. truncatula and in T. versicolor attached to them. One hairpin ACCase construction was lethal to Medicago spp. unless grown in media supplemented with malonate. Quantitative RT-PCR showed that the Medicago ACCase was inhibited by the Triphysaria ACCase RNAi. This work shows that ACCase is an effective target for inactivation in parasitic plants by trans-specific gene silencing.

Molecular convergence of the parasitic plant species Cuscuta reflexa and Phelipanche aegyptiaca.

The parasitic plant species Cuscuta reflexa and Phelipanche aegyptiaca have independently developed parasitism, the former parasitizing on shoots and the latter attaching to roots. Regardless of these differences, the two species use similar organs, termed haustoria, to attach to the host plant. In this study, we show that this morphological similarity can be extended to the molecular level. An attAGP-promoter from Solanum lycopersicum, which is activated by Cuscuta infections, was also induced after infection by P. aegyptiaca. Furthermore, we show by validation of transcriptome sequencing data that the Phelipanche orthologue of a haustorium-specific Cuscuta gene, which codes for a cysteine proteinase, was activated in the early stages of Phelipanche invasion. Inhibition of the Phelipanche cysteine proteinase was achieved by 35S- or attAGP-promoter-driven expression of its intrinsic inhibitory polypeptide. A reduction in P. aegyptiaca infection rates during experiments in flower pots and in an in vitro polybag system in comparison to controls was recorded.

Role of the sucrose synthase encoding PrSus1 gene in the development of the parasitic plant Phelipanche ramosa L. (Pomel).

Phelipanche ramosa L. (Pomel) is a major root-parasitic weed attacking many important crops. Success in controlling this parasite is rare and a better understanding of its unique biology is needed to develop new specific control strategies. In the present study, quantitative polymerase chain reaction experiments showed that sucrose synthase encoding PrSus1 transcripts accumulate at their highest level once the parasite is connected to the host (tomato) vascular system, mainly in the parasite tubercles, which bear numerous adventitious roots. In situ hybridization experiments revealed strong PrSus1 expression in both shoot and root apices, especially in shoot apical meristems and in the vascular tissues of scale leaves and stems, and in the apical meristems and developing xylem in roots. In addition, immunolocalization experiments showed that a sucrose synthase protein co-localized with cell-wall thickening in xylem elements. These findings highlight the role of PrSus1 in the utilization of host-derived sucrose in meristematic areas and in cellulose biosynthesis in differentiating vascular elements. We also demonstrate that PrSus1 is downregulated in response to 2,3,5-triiodobenzoic acid-induced inhibition of polar auxin transport in the host stem, suggesting that PrSus1 activity in xylem maturation is controlled by host-derived auxin.

Invertases involved in the development of the parasitic plant Phelipanche ramosa: characterization of the dominant soluble acid isoform, PrSAI1.

Phelipanche ramosa L. parasitizes major crops, acting as a competitive sink for host photoassimilates, especially sucrose. An understanding of the mechanisms of sucrose utilization in parasites is an important step in the development of new control methods. Therefore, in this study, we characterized the invertase gene family in P. ramosa and analysed its involvement in plant development. Invertase-encoded cDNAs were isolated using degenerate primers corresponding to highly conserved regions of invertases. In addition to enzyme assays, gene expression was analysed using real-time quantitative reverse transcriptase-polymerase chain reaction during overall plant development. The dominant isoform was purified and sequenced using electrospray ionization-liquid chromatography-tandem mass spectrometry (ESI-LC-MS/MS). Five invertase-encoded cDNAs were thus characterized, including PrSai1 which encodes a soluble acid invertase (SAI). Of the five invertases, PrSai1 transcripts and SAI activity were dominant in growing organs. The most active invertase corresponded to the PrSai1 gene product. The purified PrSAI1 displayed low pI and optimal pH values, specificity for ß-fructofuranosides and inhibition by metallic ions and competitive inhibition by fructose. PrSAI1 is a typical vacuolar SAI that is actively involved in growth following both germination and attachment to host roots. In addition, germinated seeds displayed enhanced cell wall invertase activity (PrCWI) in comparison with preconditioned seeds, suggesting the contribution of this activity in the sink strength of infected roots during the subsequent step of root penetration. Our results show that PrSAI1 and, possibly, PrCWI constitute good targets for the development of new transgenic resistance in host plants using proteinaceous inhibitors or silencing strategies.

A single-electron reducing quinone oxidoreductase is necessary to induce haustorium development in the root parasitic plant Triphysaria.

Parasitic plants in the Orobanchaceae develop haustoria in response to contact with host roots or chemical haustoria-inducing factors. Experiments in this manuscript test the hypothesis that quinolic-inducing factors activate haustorium development via a signal mechanism initiated by redox cycling between quinone and hydroquinone states. Two cDNAs were previously isolated from roots of the parasitic plant Triphysaria versicolor that encode distinct quinone oxidoreductases. QR1 encodes a single-electron reducing NADPH quinone oxidoreductase similar to zeta-crystallin. The QR2 enzyme catalyzes two electron reductions typical of xenobiotic detoxification. QR1 and QR2 transcripts are upregulated in a primary response to chemical-inducing factors, but only QR1 was upregulated in response to host roots. RNA interference technology was used to reduce QR1 and QR2 transcripts in Triphysaria roots that were evaluated for their ability to form haustoria. There was a significant decrease in haustorium development in roots silenced for QR1 but not in roots silenced for QR2. The infrequent QR1 transgenic roots that did develop haustoria had levels of QR1 similar to those of nontransgenic roots. These experiments implicate QR1 as one of the earliest genes on the haustorium signal transduction pathway, encoding a quinone oxidoreductase necessary for the redox bioactivation of haustorial inducing factors.