Detection of Secreted Effector Proteins from Phelipanche aegyptiaca During Invasion of Melon Roots.

Parasites can interact with their host plants through the induction and delivery of secreted effector proteins that facilitate plant colonization by decomposing plant cell walls and inhibiting plant immune response to weaken the defense ability of the host. Yet effectors mediating parasitic plant-host interactions are poorly understood. Phelipanche aegyptiaca is an obligate root parasite plant causing severe yield and economic losses in agricultural fields worldwide. Host resistance against P. aegyptiaca occurred during the attachment period of parasitism. Comparative transcriptomics was used to assess resistant and susceptible interactions simultaneously between P. aegyptiaca and two contrasting melon cultivars. In total, 2,740 secreted proteins from P. aegyptiaca were identified here. Combined with transcriptome profiling, 209 candidate secreted effector proteins (CSEPs) were predicted, with functional annotations such as cell wall degrading enzymes, protease inhibitors, transferases, kinases, and elicitor proteins. A heterogeneous expression system in Nicotiana benthamiana was used to investigate the functions of 20 putatively effector genes among the CSEPs. Cluster 15140.0 can suppress BAX-triggered programmed cell death in N. benthamiana. These findings showed that the prediction of P. aegyptiaca effector proteins based on transcriptomic analysis and multiple bioinformatics software is effective and more accurate, providing insights into understanding the essential molecular nature of effectors and laying the foundation of revealing the parasite mechanism of P. aegyptiaca, which is helpful in understanding parasite-host plant interaction.

First Report of Phelipanche aegyptiaca on Plectranthus scutellarioides in Xinjiang, China.

Coleus (Plectranthus scutellarioides [L.] R.Br.[syn.: Solenostemon scutellarioides]) is a perennial plant in the Lamiaceae family. It produces variegated leaves of various colors. It is commonly cultivated as an ornamental plant or grown in commercial greenhouses (Garibaldi et al. 2019). Phelipanche aegyptiaca Pers. is a dicotyledonous holoparasitic flowering plant that parasitizes more than 30 food crops (e.g., tomato, sunflower, and chickpea), ornamental crops, and others in different parts of the world, causing heavy economic losses (Nosratti et al. 2020). In 2016 and 2017, broomrape was observed parasitizing coleus in the greenhouse (86° 3' 36" E, 44° 18' 36" N, 500 m elevation) in Shihezi, Xinjiang, China (Supplementary Figure 1A-D). A single coleus plant could be parasitized by average 6-10 broomrape plants, and 20% of coleus plants were infested. The infection was confirmed by verifying the attachment of the broomrape to the coleus root. The inflorescences of the broomrape were normal and healthy and produced germinable seeds (germination rate: 80-90%). The morphological characteristics of the coleus are shown in Supplementary Figures 6 and 7. The main botanical features of the broomrape are as follows: (i) stem 20.65±7.07 cm tall, erect, branched, frail, rather hairy, bulbous at the base with secondary roots; (ii) inflorescence usually many-flowered, lax and cylindrical; (iii) bracts 6.87±0.93 mm long, ovate to lanceolate; (iv) calyx 1.09±0.09 cm long, shortly campanulate; (v) corolla 3.38±0.19 cm long, erect to suberect, white at the base, blue-purple in the upper part, sparsely glandular-villous; (vi) stamens 4, filaments inserted 5-6 mm from the base of the corolla, 1.26±0.11 cm long, anthers with villous; (vii) pistil 2.9±0.15 cm long, ovary glabrous, style with short glandular hairs, stigma bilobed, white (Supplementary Figure 2) (Teimoury et al. 2012; Piwowarczyk et al. 2019). For molecular identification, total genomic DNA was extracted from the flowers of the broomrape (found parasitizing coleus plants), and the ribosomal protein S2 (rps2) and ribosomal DNA internal transcribed spacer (ITS) region were amplified by PCR using the primer pairs rps2F/rps2R, ITS1/ITS4 (Table 1) (Park et al. 2007; Anderson et al. 2004). Two sequences with 580 bp (ITS) and 443 bp (rps2) were obtained (GenBank accession No. MW811482 and MW883573). BLAST analysis showed that the ITS sequence was most similar (identity 100%) to P. aegyptiaca (KC811171) and the rps2 sequence (identity 99%) also matched that of P. aegyptiaca (KC814957). Phylogenetic analysis of the ITS regions and rps2 genes showed that broomrape was fallen into P. aegyptiaca groups (Supplementary Figure 3). Morphological and molecular findings strongly support the conclusion that the broomrape on coleus was P. aegyptiaca. In order to verify that coleus was a host of P. aegyptiaca, coleus seedlings were collected and moved to 1.5-L pots containing a mixture of compost-vermiculite-sand (1:1:1 v:v:v) and seeds of P. aegyptiaca harvested from the host coleus (50 mg of P. aegyptiaca seeds per 1 kg of the substrate). Another three coleus seedlings were transplanted into pots of the same size containing the same mixture as above without P. aegyptiaca seeds. These served as controls. After 90 days of inoculation, the leaves of the infected hosts were lighter in color than those of uninfected hosts (Supplementary Figures 4A, 6). The roots of coleus and P. aegyptiaca were carefully washed with water, and an average of 3-4 emerged broomrape shoots and 50-60 underground attachments were observed on coleus roots (Supplementary Figure 4B). P. aegyptiaca can develop normally in the root of the coleus plant, from germination through attachment to host roots and development of tubercles (Supplementary Figure 5 A-E). Longitudinal and transverse sections of the parasite and host roots at the tubercle stage revealed that the endophytic tissues of P. aegyptiaca had reached and connected to the host vascular bundle (Supplementary Figure 5F-I), confirming the normal biological development and function of P. aegyptiaca haustoria. To the best of our knowledge, this is the first report of P. aegyptiaca parasitizing coleus in Xinjiang, China. Coleus is a very widely cultivated horticultural ornamental plant, and it grows in the same environments favored by P. aegyptiaca; so, the plant can aid the transmission of P. aegyptiaca to previously clear regions. It is necessary to improve the management of coleus in places where P. aegyptiaca is prevalent so as to reduce its spread. References: Garibaldi, A., et al. 2019. Plant Dis. 104:590. https://doi.org/10.1094/PDIS-07-19-1399-PDN Crossref, ISI, Google Scholar Nosratti, I., et al. 2020. Weed Sci. 68:555-564. https://doi.org/10.1017/wsc.2020.61 Crossref, ISI, Google Scholar Teimoury, M., et al. 2012. Plant Dis. 96:1232. https://doi.org/10.1094/PDIS-01-12-0068-PDN Crossref, ISI, Google Scholar Piwowarczyk, R., et al. 2019. Phytotaxa. 386:001-106. https://doi.org/10.11646/phytotaxa.386.1.1 Crossref, ISI, Google Scholar Park, J. M., et al. 2007. Mol. Phylogenet. Evol. 43: 974-985. https://doi.org/10.1016/j.ympev.2006.10.011 Crossref, ISI, Google Scholar Anderson, I. C., et al. 2004. Environ. Microbiol. 6: 769-779. https://doi.org/10.1111/j.1462-2920.2004.00675.x Crossref, ISI, Google Scholar.

Low Infection of Phelipanche aegyptiaca in Micro-Tom Mutants Deficient in CAROTENOIDCLEAVAGE DIOXYGENASE 8.

Strigolactones (SLs), a group of plant hormones, induce germination of root-parasitic plants and inhibit shoot branching in many plants. Shoot branching is an important trait that affects the number and quality of flowers and fruits. Root-parasitic plants, such as Phelipanche spp., infect tomato roots and cause economic damage in Europe and North Africa-hence why resistant tomato cultivars are needed. In this study, we found carotenoid cleavage dioxygenase 8-defective mutants of Micro-Tom tomato (slccd8) by the "targeting induced local lesions in genomes" (TILLING) method. The mutants showed excess branching, which was suppressed by exogenously applied SL. Grafting shoot scions of the slccd8 mutants onto wild-type (WT) rootstocks restored normal branching in the scions. The levels of endogenous orobanchol and solanacol in WT were enough detectable, whereas that in the slccd8 mutants were below the detection limit of quantification analysis. Accordingly, root exudates of the slccd8 mutants hardly stimulated seed germination of root parasitic plants. In addition, SL deficiency did not critically affect the fruit traits of Micro-Tom. Using a rhizotron system, we also found that Phelipanche aegyptiaca infection was lower in the slccd8 mutants than in wild-type Micro-Tom because of the low germination. We propose that the slccd8 mutants might be useful as new tomato lines resistant to P. aegyptiaca.

Global Transcriptomic Analysis Reveals the Mechanism of Phelipanche aegyptiaca Seed Germination.

Phelipanche aegyptiaca is one of the most destructive root parasitic plants of Orobanchaceae. This plant has significant impacts on crop yields worldwide. Conditioned and host root stimulants, in particular, strigolactones, are needed for unique seed germination. However, no extensive study on this phenomenon has been conducted because of insufficient genomic information. Deep RNA sequencing, including de novo assembly and functional annotation was performed on P. aegyptiaca germinating seeds. The assembled transcriptome was used to analyze transcriptional dynamics during seed germination. Key gene categories involved were identified. A total of 274,964 transcripts were determined, and 53,921 unigenes were annotated according to the NR, GO, COG, KOG, and KEGG databases. Overall, 5324 differentially expressed genes among dormant, conditioned, and GR24-treated seeds were identified. GO and KEGG enrichment analyses demonstrated numerous DEGs related to DNA, RNA, and protein repair and biosynthesis, as well as carbohydrate and energy metabolism. Moreover, ABA and ethylene were found to play important roles in this process. GR24 application resulted in dramatic changes in ABA and ethylene-associated genes. Fluridone, a carotenoid biosynthesis inhibitor, alone could induce P. aegyptiaca seed germination. In addition, conditioning was probably not the indispensable stage for P. aegyptiaca, because the transcript level variation of MAX2 and KAI2 genes (relate to strigolactone signaling) was not up-regulated by conditioning treatment.

Tomato Mutants Reveal Root and Shoot Strigolactone Involvement in Branching and Broomrape Resistance.

The phytohormones strigolactones (SLs) control root and shoot branching and are exuded from roots into the rhizosphere to stimulate interaction with mycorrhizal fungi. The exuded SLs serve as signaling molecules for the germination of parasitic plants. The broomrape Phelipanche aegyptiaca is a widespread noxious weed in various crop plants, including tomato (Solanum lycopersicum). We have isolated three mutants that impair SL functioning in the tomato variety M82: SHOOT BRANCHING 1 (sb1) and SHOOT BRANCHING 2 (sb2), which abolish SL biosynthesis, and SHOOT BRANCHING 3 (sb3), which impairs SL perception. The over-branching phenotype of the sb mutants resulted in a severe yield loss. The isogenic property of the mutations in a determinate growth variety enabled the quantitative evaluation of the contribution of SL to yield under field conditions. As expected, the mutants sb1 and sb2 were completely resistant to infection by P. aegyptiaca due to the lack of SL in the roots. In contrast, sb3 was more susceptible to P. aegyptiaca than the wild-type M82. The SL concentration in roots of the sb3 was two-fold higher than in the wild type due to the upregulation of the transcription of SL biosynthesis genes. This phenomenon suggests that the steady-state level of root SLs is regulated by a feedback mechanism that involves the SL signaling pathway. Surprisingly, grafting wild-type varieties on sb1 and sb2 rootstocks eliminated the branching phenotype and yield loss, indicating that SL synthesized in the shoots is sufficient to control shoot branching. Moreover, commercial tomato varieties grafted on sb1 were protected from P. aegyptiaca infection without significant yield loss, offering a practical solution to the broomrape crisis.

Isolation of Haustorium Protoplasts Optimized by Orthogonal Design for Transient Gene Expression in Phelipanche aegyptiaca.

The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in Phelipanche aegyptiaca. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for P. aegyptiaca. Even the parasitic mechanisms of P. aegyptiaca and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in Phelipanche aegyptiaca haustorium. The optimal protoplast yield and activity were 6.2 × 106 protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 µg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts' isolation and transient transformation system of Phelipanche aegyptiaca haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants.

Phenotypic and histological analyses on the resistance of melon to Phelipanche aegyptiaca.

Melon (Cucumis melo L.) is an economically important crop in Xinjiang, China, but its production is constrained by the parasitic plant Phelipanche aegyptiaca that attaches to the roots of many crops and causes severe stunting and loss of yield. Rhizotron, pot, and field experiments were employed to evaluate the resistance of 27 melon cultivars to P. aegyptiaca. Then, the resistant and susceptible cultivars were inoculated with P. aegyptiaca from six populations to assess their resistance stability and broad spectrum. Further microscopic and histological analyses were used to clarify the resistance phenotypes and histological structure. The results showed that Huangpi 9818 and KR1326 were more resistant to P. aegyptiaca compared to other cultivars in the rhizotron, pot, and field experiments. In addition, compared to the susceptible cultivar K1076, Huangpi 9818 and KR1326 showed broad-spectrum resistance to six P. aegyptiaca populations. These two resistant cultivars had lower P. aegyptiaca biomass and fewer and smaller P. aegyptiaca attachments on their roots compared to susceptible cultivar K1076. KR1326 (resistant) and K1076 (susceptible) were selected to further study resistance phenotypes and mechanisms. Germination-inducing activity of root exudates and microscopic analysis showed that the resistance in KR1326 was not related to low induction of P. aegyptiaca germination. The tubercles of parasite on KR1326 were observed slightly brown at 14 days after inoculation (DAI), the necrosis and arrest of parasite development occurred at 23 DAI. Histological analysis of necrosis tubercles showed that the endophyte of parasite had reached host central cylinder, connected with host xylem, and accumulation of secretions and callose were detected in neighbouring cells. We concluded that KR1326 is an important melon cultivar for P. aegyptiaca resistance that could be used to expand the genetic basis of cultivated muskmelon for resistance to the parasite.

Good News for Cabbageheads: Controlling Phelipanche aegyptiaca Infestation under Hydroponic and Field Conditions.

Phelipanche aegyptiaca (Orobanchaceae) is a parasitic weed that causes severe yield losses in field crops around the world. After establishing vascular connections to the host plant roots, P. aegyptiaca becomes a major sink that draws nutrients, minerals, and water from the host, resulting in extensive crop damage. One of the most effective ways to manage P. aegyptiaca infestations is through the use of herbicides. Our main objective was to optimize the dose and application protocol of herbicides that effectively control P. aegyptiaca but do not damage the cabbage crop. The interactions between the cabbage roots and the parasite were first examined in a hydroponic system to investigate the effect of herbicides on initial parasitism stages, e.g., germination, attachment, and tubercles production. Thereafter, the efficacy of glyphosate and ethametsulfuron-methyl in controlling P. aegyptiaca was examined in five cabbage fields naturally infested with P. aegyptiaca. The herbicides glyphosate and ethametsulfuron-methyl were applied on cabbage foliage and in the soil solution, both before and after the parasite had attached to the host roots. A hormesis effect was observed when glyphosate was applied at a dose of 36 g ae ha-1 in a non-infested P. aegyptiaca field. Three sequential herbicide applications (21, 35, and 49 days after planting) effectively controlled P. aegyptiaca without damaging the cabbages at a dose of 72 g ae ha-1 for glyphosate and at all the examined doses for ethametsulfuron-methyl. Parasite control with ethametsulfuron-methyl was also effective when overhead irrigation was applied after the herbicide application.

The Effect of 10 Crop Plants That Served as Hosts on the Primary Metabolic Profile of the Parasitic Plant Phelipanche aegyptiaca.

Phelipanche aegyptiaca Pers. is a holoparasitic plant that parasitizes various types of host plants. Its penetration into host roots causes a massive reduction in the yield of many crop plants worldwide. The nature of the compounds taken by the parasite from its host is still under debate in the scientific literature. To gain more knowledge about the effect of the hosts on the parasite's primary metabolic profile, GC-MS analyses were conducted on the parasites that developed on 10 hosts from four plant families. There are three hosts from each family: Brassicaceae, Apiaceae and Solanaceae and one host from Fabaceae. The results showed significant differences in the metabolic profiles of P. aegyptiaca collected from the different hosts, indicating that the parasites rely strongly on the host's metabolites. Generally, we found that the parasites that developed on Brassicaceae and Fabaceae accumulated more amino acids than those developed on Apiaceae and Solanaceae that accumulated more sugars and organic acids. The contents of amino acids correlated positively with the total soluble proteins. However, the aromatic amino acid, tyrosine, correlated negatively with the accumulation of the total phenolic compounds. This study contributes to our knowledge of the metabolic relationship between host and parasite.

Simulating sulfosulfuron fate in soil under different weather scenarios to support weed management decisions.

BACKGROUND: Residual herbicides are an important component in many weed control strategies. Their herbicidal activity depends on their fate in soil, with respect to the required concentration for weed control in space and time. In this study, the effect of weather conditions on sulfosulfuron fate in soil, following pre-planting incorporation, and the predicted control efficacy of Egyptian broomrape in tomato, were analyzed for two sites using simulations in Hydrus-1D modeling software. Simulated concentration was compared to measured data from field experiments. RESULTS: Model evaluation against measured data from two fields, with weakly alkaline clay soils, showed high correlations between simulated and measured sulfosulfuron concentrations (r = 0.98 and 0.89). The ratio of measured to simulated concentration was relatively low (1.03) at the top 10-cm layer, in which the mean measured concentration was high (29.6 ng g-1 ). This ratio was higher (12.5) at the 30-60 cm depth, in which the mean measured concentration was lower (0.3 ng g-1 ). Simulations of sulfosulfuron fate in each site, using weather data from the years 2009 to 2019, revealed substantial variations in transport patterns. Thirty days after treatment, 16 out of the 22 years simulated for the two sites (11 at each site) resulted in concentrations lower than the critical value for Egyptian broomrape control throughout the soil profile. The data indicates that variation in sulfosulfuron fate is mainly due to differences in the cumulative precipitation. According to simulation results, cumulative precipitation above 20 or 10 mm during the first 10 or 20 days after treatment, respectively, is expected to reduce the efficiency of broomrape control. CONCLUSION: Considering weather effects when planning herbicide application could optimize herbicide use efficiency. A decision-support tool is presented, whose factors are the time gap and precipitation amount between sulfosulfuron application and tomato planting.

Exploration of resistance to Phelipanche aegyptiaca in tomato.

BACKGROUND: Cultivated tomatoes are highly susceptible to the destructive parasite Phelipanche aegyptiaca. Wild relatives show the potential resistance for genetic improvement. However, their genetic and molecular mechanisms are still unknown. RESULTS: Among 50 wild tomato accessions were evaluated for resistance to P. aegyptiaca, most of the wild relatives exhibited varying degrees of resistance compared to the cultivars. Solanum pennellii LA0716 performed the most promising and solid resistance with very low infection by the broomrape. The resistance involved in LA0716 was further confirmed by cytological analysis, and explored by employing a permanent introgression line (IL) population. Thirteen putative quantitative trait loci (QTLs) conferring the different resistance traits were identified. They are located on chromosomes 1, 2, 3, 4, 6, 8 and 9. The most attractive QTLs are positioned in IL6-2 and overlap with IL6-3. Specially, IL6-2 showed the highest and most consistent resistance for multiple traits and explained the major phenotypic variation of LA0716. Analysis of candidate genes involved in these regions showed that Beta (Solyc06g074240) and P450 (Solyc06g073570, Solyc06g074180 and Solyc06g074420) genes are substantially related to the strigolactone (SL) pathway. Transcript analysis further demonstrated that both Solyc06g073570 and Solyc06g074180 might play an important role in the reduction of P. aegyptiaca infection. CONCLUSION: Germplasms resistant to P. aegyptiaca were found in wild tomato species. QTLs conferring P. aegyptiaca tolerance in LA0716 were identified. IL6-2 is identified as a prospective line possessing the major QTLs. The candidate genes would provide the availability to assist the introgression of the resistance in future breeding programmes. © 2020 Society of Chemical Industry.

Multiple immunity-related genes control susceptibility of Arabidopsis thaliana to the parasitic weed Phelipanche aegyptiaca.

Parasitic weeds represent a major threat to agricultural production across the world. Little is known about which host genetic pathways determine compatibility for any host-parasitic plant interaction. We developed a quantitative assay to characterize the growth of the parasitic weed Phelipanche aegyptiaca on 46 mutant lines of the host plant Arabidopsis thaliana to identify host genes that are essential for susceptibility to the parasite. A. thaliana host plants with mutations in genes involved in jasmonic acid biosynthesis/signaling or the negative regulation of plant immunity were less susceptible to P. aegyptiaca parasitization. In contrast, A. thaliana plants with a mutant allele of the putative immunity hub gene Pfd6 were more susceptible to parasitization. Additionally, quantitative PCR revealed that P. aegyptiaca parasitization leads to transcriptional reprograming of several hormone signaling pathways. While most tested A. thaliana lines were fully susceptible to P. aegyptiaca parasitization, this work revealed several host genes essential for full susceptibility or resistance to parasitism. Altering these pathways may be a viable approach for limiting host plant susceptibility to parasitism.

Enhanced Production of Aromatic Amino Acids in Tobacco Plants Leads to Increased Phenylpropanoid Metabolites and Tolerance to Stresses.

Aromatic amino acids (AAAs) synthesized in plants via the shikimate pathway can serve as precursors for a wide range of secondary metabolites that are important for plant defense. The goals of the current study were to test the effect of increased AAAs on primary and secondary metabolic profiles and to reveal whether these plants are more tolerant to abiotic stresses (oxidative, drought and salt) and to Phelipanche egyptiaca (Egyptian broomrape), an obligate parasitic plant. To this end, tobacco (Nicotiana tabacum) plants were transformed with a bacterial gene (AroG) encode to feedback-insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway. Two sets of transgenic plants were obtained: the first had low expression of the AroG protein, a normal phenotype and minor metabolic changes; the second had high accumulation of the AroG protein with normal, or deleterious morphological changes having a dramatic shift in plant metabolism. Metabolic profiling analysis revealed that the leaves of the transgenic plants had increased levels of phenylalanine (up to 43-fold), tyrosine (up to 24-fold) and tryptophan (up to 10-fold) compared to control plants having an empty vector (EV) and wild type (WT) plants. The significant increase in phenylalanine was accompanied by higher levels of metabolites that belong to the phenylpropanoid pathway. AroG plants showed improved tolerance to salt stress but not to oxidative or drought stress. The most significant improved tolerance was to P. aegyptiaca. Unlike WT/EV plants that were heavily infected by the parasite, the transgenic AroG plants strongly inhibited P. aegyptiaca development, and only a few stems of the parasite appeared above the soil. This delayed development of P. aegyptiaca could be the result of higher accumulation of several phenylpropanoids in the transgenic AroG plants and in P. aegyptiaca, that apparently affected its growth. These findings indicate that high levels of AAAs and their related metabolites have the potential of controlling the development of parasitic plants.

Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolor.

Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds.

The Influence of the Plant Growth Regulator Maleic Hydrazide on Egyptian Broomrape Early Developmental Stages and Its Control Efficacy in Tomato under Greenhouse and Field Conditions.

Broomrapes (Phelipanche spp. and Orobanche spp.) are holoparasitic plants that cause tremendous losses of agricultural crops worldwide. Broomrape control is extremely difficult and only amino acid biosynthesis-inhibiting herbicides present an acceptable control level. It is expected that broomrape resistance to these herbicides is not long in coming. Our objective was to develop a broomrape control system in tomato (Solanum lycopersicum L.) based on the plant growth regulator maleic hydrazide (MH). Petri-dish and polyethylene-bag system experiments revealed that MH has a slight inhibitory effect on Phelipanche aegyptiaca seed germination but is a potent inhibitor of the first stages of parasitism, namely attachment and the tubercle stage. MH phytotoxicity toward tomato and its P. aegyptiaca-control efficacy were tested in greenhouse experiments. MH was applied at 25, 50, 75, 150, 300, and 600 g a.i. ha-1 to tomato foliage grown in P. aegyptiaca-infested soil at 200 growing degree days (GDD) and again at 400 GDD. The treatments had no influence on tomato foliage or root dry weight. The total number of P. aegyptiaca attachments counted on the roots of the treated plants was significantly lower at 75 g a.i. ha-1 and also at higher MH rates. Phelipanche aegyptiaca biomass was close to zero at rates of 150, 300, and 600 g a.i. ha-1 MH. Field experiments were conducted to optimize the rate, timing and number of MH applications. Two application sequences gave superior results, both with five split applications applied at 100, 200, 400, 700, and 1000 GDD: (a) constant rate of 400 g a.i. ha-1; (b) first two applications at 270 g a.i. ha-1 and the next three applications at 540 g a.i. ha-1. Based on the results of this study, MH was registered for use in Israel in 2013 with the specified protocol and today, it is widely used by most Israeli tomato growers.

Phloem-Conducting Cells in Haustoria of the Root-Parasitic Plant Phelipanche aegyptiaca Retain Nuclei and Are Not Mature Sieve Elements.

Phelipanche aegyptiaca parasitizes a wide range of plants, including important crops, and causes serious damage to their production. P. aegyptiaca develops a specialized intrusive organ called a haustorium that establishes connections to the host's xylem and phloem. In parallel with the development of xylem vessels, the differentiation of phloem-conducting cells has been demonstrated by the translocation of symplasmic tracers from the host to the parasite. However, it is unclear yet whether haustorial phloem-conducting cells are sieve elements. In this study, we identified phloem-conducting cells in haustoria by the host-to-parasite translocation of green fluorescent protein (GFP) from AtSUC2pro::GFP tomato sieve tubes. Haustorial GFP-conducting cells contained nuclei but not callose-rich sieve plates, indicating that phloem-conducting cells in haustoria differ from conventional sieve elements. To ascertain why the nuclei were not degenerated, expression of the P. aegyptiaca homologs NAC-domain containing transcription factor (NAC45), NAC45/86-dependent exonuclease-domain protein 1 (NEN1), and NEN4 was examined. However, these genes were more highly expressed in the haustorium than in tubercle protrusion, implying that nuclear degradation in haustoria may not be exclusively controlled by the NAC45/86-NEN regulatory pathway. Our results also suggest that the formation of plasmodesmata with large size exclusion limits is independent of nuclear degradation and callose deposition.

Metabolic Investigation of Phelipanche aegyptiaca Reveals Significant Changes during Developmental Stages and in Its Different Organs.

Phelipanche aegyptiaca Pers. is a root holoparasitic plant considered to be among the most destructive agricultural weeds worldwide. In order to gain more knowledge about the metabolic profile of the parasite during its developmental stages, we carried out primary metabolic and lipid profiling using GC-MS analysis. In addition, the levels of amino acids that incorporate into proteins, total protein in the albumin fraction, nitrogen, reduced sugars, and phenols were determined. For the assays, the whole plants from the four developmental stages-tubercle, pre-emergent shoot, post-emergent shoot, and mature flowering plants-were taken. Thirty-five metabolites out of 66 differed significantly between the various developmental stages. The results have shown that the first three developmental stages were distinguished in their profiles, but the latter two did not differ from the mature stage. Yet, 46% of the metabolites detected did not change significantly during the developmental stages. This is unlike other studies of non-parasitic plants showing that their metabolic levels tend to alter significantly during development. This implies that the parasite can control the levels of these metabolites. We further studied the metabolic nature of five organs (adventitious roots, lower and upper shoot, floral buds, and flowers) in mature plants. Similar to non-parasitic plants, the parasite exhibited significant differences between the vegetative and reproductive organs. Compared to other organs, floral buds had higher levels of free amino acids and total nitrogen, whereas flowers accumulated higher levels of simple sugars such as sucrose, and the putative precursors for nectar synthesis, color, and volatiles. This suggests that the reproductive organs have the ability to accumulate metabolites that are required for the production of seeds and as a source of energy for the reproductive processes. The data contribute to our knowledge about the metabolic behavior of parasites that rely on their host for its basic nutrients.

Primary metabolic profiling of Egyptian broomrape (Phelipanche aegyptiaca) compared to its host tomato roots.

Broomrape (Phelipanche aegyptiaca) is a root holoparasitic plant considered among the most destructive agricultural weeds worldwide. In order to acquire more knowledge about the metabolism of broomrape and its interaction with its tomato host, we performed primary metabolic profiling using GCMS analysis for the early developmental stage of the parasite and of infected and non-infected roots. The analysis revealed that out of 59 metabolites detected, the levels of 37 significantly increased in the parasite while the levels of 10 significantly decreased compared to the infected roots. In addition, the analysis showed that the levels of total protein in the albumin fraction, reducing sugars (representing starch) and total phenols increased by 9.8-, 4.6- and 3.3-fold, respectively, in the parasite compared to the roots. These changes suggest that P. aegyptiaca has its own metabolism that differs significantly in its regulation from those found in their host. In addition, the results have shown that the levels of most of the metabolites in the infected roots were similar to levels detected in the non-infected roots, except for seven metabolites whose levels increased in the infected versus the non-infected roots. This suggests that the parasite did not significantly affect the host primary metabolic pathways.

Cucumber Mosaic Virus as a carotenoid inhibitor reducing Phelipanche aegyptiaca infection in tobacco plants.

Cucumber Mosaic Virus (CMV) is a highly infectious cucumovirus, which infects more than 800 plant species and causes major diseases in greenhouse and field crops worldwide. Parasitic weeds such as Phelipanche aegyptiaca are a major constraint to the production of many crops in the world and the parasite's lifestyle makes control extremely difficult. The parasite seeds can germinate after conditioning and perceiving strigolactones secreted by the host roots. Strigolactones are rhizosphere signaling molecules in plants that are biosynthesized through carotenoid cleavage. In the present study we investigated the possibility of reducing ß-carotene and then strigolactone production in the host roots by blocking carotenoid biosynthesis using CMV-infected tobacco. It was found that CMV downregulated the enzyme phytoene desaturase(PDS) and reduced significantly both carotenoid production and Phelipanche infection in tobacco host roots infected with both CMV and P. aegyptiaca. Based on our results (decrease of ß-carotene and repression of PDS transcripts in tobacco roots), we hypothesized that the reduction of Phelipanche tubercles and shoots occurred due to an effect of CMV on secondary metabolite stimulators such as strigolacetones. Our study indicated that mass production of the host roots was not affected by CMV; however, most inflorescences of Phelipanche grown on CMV-infected tobacco developed abnormally (deformed shoots and short nodes). Carotenoid biosynthesis inhibitors such as CMV can be used to reduce the production of strigolactones, which will lead to decreased Phelipanche attachment. Interestingly, attenuated CMV strains may provide a safe means for enhancing crop resistance against parasitic weeds in a future plan.