Plant resistance to parasitic plants: molecular approaches to an old foe.

Parasitic weeds pose severe constraint on major agricultural crops. Varying levels of resistance have been identified and exploited in the breeding programmes of several crops. However, the level of protection achieved to date is either incomplete or ephemeral. Resistance is mainly determined by the coexistence of several mechanisms controlled by multigenic and quantitative systems. Efficient control of the parasite requires a better understanding of the interaction and their associated resistance mechanisms at the histological, genetic and molecular levels. Application of postgenomic technologies and the use of model plants should improve the understanding of the plant-parasitic plant interaction and drive not only breeding programmes through either marker-assisted selection (MAS) or transgenesis but also the development of alternative methods to control the parasite. This review presents the current approaches targeting the characterization of resistance mechanisms and explores their potentiality to control parasitic plants.

Systemic acquired resistance in sunflower (Helianthus annuus L.).

Systemic acquired resistance (SAR) to infection by Botrytis cinerea in the leaves of sunflower (Helianthus annuus L.) plants was induced following cotyledon inoculation with B. cinerea or treatment with abiotic inducers. Salicylic acid (SA), benzo-(1,2,3)-thiadiazole-7-carbothioic S-methyl ester (BTH), 2,6-dichloroisonicotinic acid (INA) or EDTA protected sunflower plants against Botrytis infection, that was revealed by a reduction in the number and area of the necrotic lesions in upper leaves after challenge inoculation with the pathogen. SA and BTH were more potent inducers than INA, EDTA or pre-inoculation with the fungus. In addition to resistance to B. cinerea, the upper leaves have also developed resistance to maceration by a mixture of cell wall-degrading enzymes. Calcium nitrate inhibited both the protective effect and the resistance of leaf discs to cell-wall degrading enzymes. All the tested chemicals increased the synthesis and excretion of sunflower phytoalexins--coumarins scopoletin and ayapin and induced the PR-proteins chitinase and 1,3-beta-glucanase, being the inducer effect of each activator correlated with the level of protection against B. cinerea (BTH > SA > INA > EDTA). Thus, SAR induction is mediated by general increase of plant defence responses. This is the first report on SAR in sunflower.

Sunflower (Helianthus annuus L.) response to broomrape (Orobanche cernua Loefl.) parasitism: induced synthesis and excretion of 7-hydroxylated simple coumarins.

The interaction of the parasitic plant Orobanche cernua with resistant and susceptible cultivars of Helianthus annuus L. was investigated. Using different bioassays to evaluate the early stages of the parasite life cycle (germination, attachment, penetration, and establishment), differences were observed between O. cernua-resistant and O. cernua-susceptible sunflower varieties. Germination of O. cernua seeds in the presence of resistant sunflower roots was approximately half that of germination in the presence of susceptible roots, and germinated seeds displayed enhanced browning symptoms. Parasite radicles or host-tissue around the contact point turned brown after O. cernua attachment to sunflower roots, especially in the resistant varieties. These observations suggested the possible accumulation of toxic compounds as a defence strategy in the resistant sunflower varieties. Sunflower 7-hydroxylated simple coumarins may play a defensive role against O. cernua parasitism by preventing successful germination, penetration and/or connection to the host vascular system. This hypothesis is supported by the following data: (i) coumarins inhibited the in vitro germination of O. cernua seeds induced by the strigol analogue GR(24) and caused a browning reaction in germinated seeds and (ii) resistant sunflowers accumulated higher levels of coumarins in roots and excreted greater amounts than susceptible varieties in response to O. cernua infection.

Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination.

Six sunflower sesquiterpene lactone models which share structural features of the lactone rings of strigol and its synthetic analogues, the GR family, with different conformational flexibilities were tested as Orobanche cumana germination stimulants. Among them, parthenolide and 3,5-dihydroxydehydrocostus-lactone significantly increased O. cumana germination, presenting higher activity than GR-24, used as a standard in the germination bioassay. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (O. crenata, O. ramosa and O. aegyptiaca). Data presented are discussed in terms of a structure-activity relationship.

Kinetic properties of phenylalanine ammonia-lyase from sunflower (Helianthus annuus L.) hypocotyls.

Phenylalanine ammonia-lyase (PAL) from sunflower hypocotyls has been partially purified by selective precipitation with ammonium sulfate and molecular gel filtration on Sephacryl S-300. Kinetic assays carried out with this partially purified PAL preparation revealed that the enzyme did not show a homogeneous kinetic behaviour. The observed kinetic pattern and parameters (Km and Vmax) depended on the assay conditions used and the protein concentration added to the assay mixture. PAL displayed Michaelian or negative cooperativity kinetics. Such behaviour can be explained by the existence of an association-dissociation process of PAL-protein subunits. The presence of mono-, tri- and tetrameric forms of PAL has been assessed by molecular gel filtration on Sephacryl S-200, using different elution conditions.

Stress Responses in Alfalfa (Medicago sativa L.): II. Purification, Characterization, and Induction of Phenylalanine Ammonia-Lyase Isoforms from Elicitor-Treated Cell Suspension Cultures.

l-Phenylalanine ammonia-lyase has been purified from elicitor-treated alfalfa (Medicago sativa L.) cell suspension cultures using two protocols based on different sequences of chromatofocusing and hydrophobic interaction chromatography. Three distinct forms of the intact enzyme were separated on the basis of affinity for Octyl-Sepharose, with isoelectric points in the range pH 5.1 to 5.4. The native enzyme was a tetramer of M(r) 311,000; the intact subunit M(r) was about 79,000, although polypeptides of M(r) 71,000, 67,000 and 56,000, probably arising from degradation of the intact subunit, were observed in all preparations. Two-dimensional gel analysis revealed the presence of several subunit isoforms of differing isoelectric points. The purified isoforms of the native enzyme had different K(m) values for l-phenylalanine in the range 40 to 110 micromolar, although mixtures of the forms in crude preparations exhibited apparent negative rate cooperativity. The enzyme activity was induced approximately 16-fold within 6 hours of exposure of alfalfa cells to a fungal elicitor or yeast extract. Analysis by hydrophobic interaction chromatography revealed different proportions of the different active enzyme isoforms, depending upon either time after elicitation or the elicitor used. The elicitor-induced increase in enzyme activity was associated with increased translatable phenylalanine ammonia-lyase mRNA activity in the polysomal fraction.