Patrick Thalouarn

Defense Gene Expression Analysis of Arabidopsis thaliana Parasitized by Orobanche ramosa

ABSTRACT The infection of Arabidopsis thaliana roots with the obligate parasite Orobanche ramosa represents a useful model for a study of the molecular events involved in the host plant response to a parasitic plant attack. To avoid analysis problems due to the subterranean development of O. ramosa, we developed two in vitro co-culture systems: O. ramosa seedlings infesting Arabidopsis plantlet roots and callus tissues. We were then able to investigate the expression patterns of some host plant genes selected among genes known to be involved in metabolic pathways and resistance mechanisms activated during several plant-pathogen interactions including ethylene, isoprenoid, phenylpropanoid, and jasmonate biosynthesis pathways, oxidative stress responses, and pathogenesis-related proteins. Molecular analyses were carried out using polymerase chain reaction amplification methods allowing semiquantitative evaluation of transcript accumulation during early (first hours) and late (15 days) stages of infestation, in whole roots or parts close to the parasite attachment site. In A. thaliana, O. ramosa induced most of the general response signaling pathways in a transient manner even before its attachment to A. thaliana roots. However, no salicylic acid-dependent defense is observed because no activation of systemic acquired resistance markers is detectable, whereas genes, co-regulated by jasmonate and ethylene, do display enhanced expression.

Identification by suppression subtractive hybridization and expression analysis of Arabidopsis thaliana putative defence genes during Orobanche ramosa infection

A suppression subtractive hybridization strategy was used to identify genes that were induced 2 h after infection of Arabidopsis thaliana by broomrape (Orobanche ramosa) seedlings. Among these genes, the expression of twelve was analysed from the first hour to the seventh day of this compatible interaction by cDNA blot analyses. The twelve genes showed a transient expression, which occurred in seven cases as early as the first or second hour of interaction and ended 2 or 3 h later. Most of the proteins encoded by these genes are already known to be involved in different A. thaliana response pathways to pathogen attack. The first two genes to be induced (Rc kinase and ACaM5) encoded a receptor and a calmodulin, and could be involved in signal transduction. The two following genes encoding a sucrose carrier (SUC1) and a protein with a pectin methylesterase inhibitor domain were found to be overexpressed; their roles are consistent with plant defence emergence. The gst1, gst11 and peptide methionine sulfoxide reductase genes, which were turned on early, are known to play a role in the detoxification of reactive oxygen species. The accumulation of mRNAs for lox1, a latex allergen and a myrosinase binding protein could be related to a jasmonate dependent pathway, while genes for a class III peroxidase and a caffeoyl-CoA 3-O-methyltransferase, both likely to be involved in cell wall reinforcement, were also upregulated.

Physiology and histology of resistance to Striga hermonthica in Sorghum bicolor var. Framida

Germination, attachment to host root and growth of Striga hermonthica (Del.) Benth. seeds and seedlings were studied in in vitro co-culture with Sorghum bicolor (L.) Moench and in pot experi- ments. Two varieties, the resistant Framida and the susceptible CK-60B, were used. Histological, mor- phological and physiological studies revealed the key stages of resistance mechanisms involved. Resistance of Framida to Striga does not occur at the germination or the attachment stages, since its roots do not support fewer Striga than does CK-60B. As Framida roots support the lowest number of young Striga stems with scale leaves, its resistance appears to occur during the establishment of a functional haustorium. Metabolite uptake by the haustorium and growth rate of the young parasite were lower on Framida roots than on CK-60B roots, even when similarly developed haustoria were compared. Furthermore, at a later stage of infestation, significant accumulation of a coloured material likely to be rich in phenolic compounds was observed in and around Framida conductive tissues, but not CK-60B tissues. These features indicate the involvement of at least three steps in development of resistance in Framida roots: the first is linked to a partial inhibition of development of the young haustorium; the second could play a role in the physiological events that decrease nutrient translocation towards the haus- toria; and the last seems to be associated with the accumulation of a coloured phenolic-like material.

Divergent evolution of two plastid genes, rbcL and atpB, in a non-photosynthetic parasitic plant

Plastid DNA (ptDNA) regions for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiso) (rbcL) and the β-subunit of ATP synthase (atpB) genes of the holoparasite Lathraea clandestina L. were sequenced. These regions were obtained by cloning either a Bam HI endonuclease generated fragment from the Lathraea ptDNA or polymerase chain reaction (PCR) amplified products. The Lathraea ptDNA contains the entire sequence for the rbcL gene which shares 94.5% homology with the Nicotiana tabacum gene, whereas atpB is maintained as a pseudogene. The intergenic region between divergently transcribed rbcL and atpB genes is shorter (758 bp) in L. clandestina plastid genome in comparison with N. tabacum (823 bp), however they have a noticeable similarity, mainly in the rbcL 5′-upstream region. A low level of the rbcL gene transcription was detected whereas no atpB transcripts were found in Lathraea. The plasmid rbcL gene of the hemiparasite Melampyrum pratense and the autotroph Digitalis purpurea both from the Scrophulariaceae were cloned by PCR amplification and then sequenced. The L. clandestina rbcL gene is highly homologous to the M. pratense and D. purpurea genes. The data indicate that the evolution of the plastid atpB-rbcL region was different in parasites from the Scrophulariaceae and Orobanchaceae families.

The rbcL gene from the non-photosynthetic parasite Lathraea clandestina is not transcribed by a plastid-encoded RNA polymerase

Abstract In the plastome of the obligate root-parasitic plant, Lathraea clandestina, the rbcL gene has been maintained and is expressed, despite the reduced size and gene content of the plastid genome. Some of the plastid genes involved in translation (e. g. transfer RNAs, ribosomal RNAs and ribosomal proteins) have been sequenced and still appear to code for functional ribosomal components. Indeed, the 16S rRNA and rpl20 genes are expressed whilst other necessary tRNA and ribosomal protein-encoding genes have probably been deleted or truncated. Although obtained by PCR, the four rpo genes for Escherichia coli-like plastid encoded RNA polymerase appear to be pseudogenes. Nevertheless, the rbcL gene, with a “–10, –35” prokaryotic-like promoter, is still transcribed. In contrast to photosynthetic plants, rbcL transcripts in Lathraea are larger in their 5′ region and cover the prokaryotic-like promoter. The transcription initiation site is located near the ATG start codon of the atpB pseudogene. Similarity to non-consensus E. coli-like plastid promoters suggests that rbcL transcription is driven by a nuclear-encoded RNA polymerase.

The obligate root parasite Orobanche cumana exhibits several rbcL sequences.

Orobanche species characterization using plastid sequences as molecular markers revealed that O. cumana contains at least two distinct rbcL sequences: one similar in size to the truncated rbcL pseudogene from O. cernua, a closely related species, and another with a size comparable to that of rbcL plastid genes from autotrophic plants. In this work, the nucleotide sequences of these two copies are reported and analysed. The organization of the O. cumana plastid genome was investigated using a long-distance PCR strategy in order to determine their localization. Because of the non-plastid localization of the rbcL larger copy, Southern blot and PCR chromosome-walking experiments were carried out to better characterize this transferred sequence and to identify its localization. Then the mode of multiple transfer of genetic information from plastid to nucleus and the concomitant plastid sequence disorganization and migration during parasitic plant evolution are discussed.

Ribulose 1,5-biphosphate carboxylase activity in the holoparasite Lathraea clandestina L.

Summary A ribulose 1,5-biphosphate carboxylase (EC 4.1.1.39) activity was demonstrated and determined in the organs of the holoparasitic Scrophulariaceae Lathraea clandestina L.The determinations were performed using a crude extract purified through precipitation with 20 % to 65 % saturated ammonium sulfate. Two determination methods were used: one, spectrophotometric, based on NADH oxidation following the incorporation of CO2, the other, radioisotopic, measuring incorporated radioactivity after contact with NaH14CO3.RuBP carboxylase activity was found to be highest in the scale leaves; yet, it is 10 to 20 times lower than in C3-type leaves like those of tobacco or spinach.

Physiological Changes in a Root Hemiparasitic Angiosperm, Thesium humile (Santalaceae), before and after Attachment to the Host Plant (Triticum vulgare)

Summary Thesium humile , an obligate root hemiparasitic angiosperm, can live in an autotrophic-like way during several weeks before attachment to the host. About 6 weeks after germination, a decreasing growth rate and symptoms of senescence of the seedling are observed. Before attachment, Thesium is able to take up water and inorganic ions from the soil, but its mineral content is characterized by very high Na and low P levels. During the same period, rates of net photosynthesis and dark respiration as well as RuBPc and PEPc activities decrease continuously. The expression of a photosynthetic gene like psbA also decreases and the amount of transcripts of the rbcL gene is very low. After attachment to the host, Thesium recovers a more physiological balance by deriving inorganic ions from the host xylem sap. Increasing growth rate, photosynthetic carbon fixation, carboxylase capacities and expression of a photosynthetic gene like rbcL are observed and become fairly similar to those found in non-parasitic C 3 plants. A strong K accumulation in leaves also occurred and that might be an explanation for the observed anomalous stomatal behaviour. Before attachment, the poorly developed root system of Thesium seems unable to selectively absorb inorganic ions from the soil and to maintain a physiological mineral balance. Consequential to the very high Na and the low P contents in the seedling of the parasite, metabolic perturbations affecting also gene expression might result.

Activity of secreted cell wall-modifying enzymes and expression of peroxidase-encoding gene following germination of Orobanche ramosa

Radicle growth of germinated seed of the root parasite O. ramosa is shown to be rapidly accompanied by secretion of proteins including pectinolytic enzymes, polygalacturonase and rhamnogalacturonase. These secretions peaked between 4 to 8 d after induction of germination and remained constant for some further days in the case of polygalacturonases. After 6 d, germinated seeds secreted proteins which exhibit peroxidase activity. The latter may be correlated with expression of OrPOX1, a putative gene encoding for secreted peroxidase. The involvement of these enzymes in host root attack and haustorium formation by the parasite is discussed.

Carbon Nutrition in a Rafflesiaceae Holoparasite Cytinus hypocistis L. Fixed on, or Experimentally Isolated from the Host Cistus Monspeliensis L.

Abstract The host-parasite pair Cistus Monspeliensis-Cytinus hypocistis is fed, via the aerial organs of the host, with 14 CO 2 . The radioactivity measurements show that the main substances transfered from the host to the parasite are sucrose, glutamic acid, and aspartic acid. The parasite isolated from the host is also shown to be capable of fixing atmospheric 14 C thus fixed represents about 12% of the 14 C received from the host by the non-isolated parasite. The nature of the labelled products found in the detached parasite not only confirms the existence of PEP carboxylase activity, but also suggests the intervention of a RuBP carboxylase.