Broomrapes ( Phelipanche aegyptiaca and Orobanche spp.) are obligate plant parasites that cause extreme damage to crop plants. The parasite seeds have strict requirements for germination, involving preconditioning and exposure to specific chemicals strigolactones [SLs] exuded by the host roots. SLs are plant hormones derived from plant carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase 8 ( CCD8) . Having no effective means to control parasitic weeds in most crops, and with CRISPR/Cas9 being an effective gene-editing tool, here we demonstrate that CRISPR/Cas9-mediated mutagenesis of the CCD8 gene can be used to develop host resistance to the parasitic weed P . aegyptiaca . Cas9/single guide (sg) RNA constructs were targeted to the second exon of CCD8 in tomato ( Solanum lycopersicum L .) plants. Several CCD8 Cas9 mutated tomato lines with variable insertions or deletions in CCD8 were obtained with no identified off-targets. Genotype analysis of T1 plants showed that the introduced CCD8 mutations are inherited. Compared to control tomato plants, the CCD8 Cas9 mutant had morphological changes that included dwarfing, excessive shoot branching and adventitious root formation. In addition, SL-deficient CCD8 Cas9 mutants showed a significant reduction in parasite infestation compared to non-mutated tomato plants. In the CCD8 Cas9 mutated lines, orobanchol (SL) content was significantly reduced but total carotenoids level and expression of genes related to carotenoid biosynthesis were increased, as compared to control plants. Taking into account, the impact of plant parasitic weeds on agriculture and difficulty to constitute efficient control methods, the current study offers insights into the development of a new, efficient method that could be combined with various collections of resistant tomato rootstocks.
Root parasitic weeds infect numerous economically important crops, affecting total yield quantity and quality. A lack of an efficient control method limits our ability to manage newly developing and more virulent races of root parasitic weeds. To control the parasite induced damage in most host crops, an innovative biotechnological approach is urgently required. Strigolactones (SLs) are plant hormones derived from carotenoids via a pathway involving the Carotenoid Cleavage Dioxygenase (CCD) 7, CCD8 and More Axillary Growth 1 (MAX1) genes. SLs act as branching inhibitory hormones and strictly required for the germination of root parasitic weeds. Here, we demonstrate that CRISPR/Cas9-mediated targted editing of SL biosynthetic gene MAX1, in tomato confers resistance against root parasitic weed Phelipanche aegyptiaca. We designed sgRNA to target the third exon of MAX1 in tomato plants using the CRISPR/Cas9 system. The T0 plants were edited very efficiently at the MAX1 target site without any non-specific off-target effects. Genotype analysis of T1 plants revealed that the introduced mutations were stably passed on to the next generation. Notably, MAX1-Cas9 heterozygous and homozygous T1 plants had similar morphological changes that include excessive growth of axillary bud, reduced plant height and adventitious root formation relative to wild type. Our results demonstrated that, MAX1-Cas9 mutant lines exhibit resistance against root parasitic weed P. aegyptiaca due to reduced SL (orobanchol) level. Moreover, the expression of carotenoid biosynthetic pathway gene PDS1 and total carotenoid level was altered, as compared to wild type plants. Taking into consideration, the impact of root parasitic weeds on the agricultural economy and the obstacle to prevent and eradicate them, the current study provides new aspects into the development of an efficient control method that could be used to avoid germination of root parasitic weeds.
Plant parasitic weed Phelipanche aegyptiaca and Orobanche spp. are obligate plant parasites that cause heavy damage to agricultural crop plants. Germination of the parasite seeds require exposure to specific chemical known as strigolactone [SL].The plant hormone SL is derived from plant carotenoids via cleavage by CCD7 and CCD8 enzymes and exuded by the host roots to the rhizosphere. Here, we provide evidence that CRISPR/Cas9 mediated targeted mutagenesis of two homologues ATP Binding cassette (ABC) protein in tomato (Solyc08g067610 and Solyc08g067620), significantly reduced the germination of the parasitic weed P.aegyptiaca.Constructs harboring specific single guide RNA were prepared and targeted against conserved region in the above tomato genes (Solyc08g067610 and Solyc08g067620). Selected T0-mutated tomato plants showed different type of deletions at both locuses. Furthermore, genotype analysis of T1 plants showed that the introduced mutations stably inherited to next generation with no identified off-targets. Mutated tomato lines, showed more branching, enhanced growth of axillary buds, reduced length of primary stems and significantly reduced development of the parasitic weed P. aegyptiaca as compared to wild type plants. Moreover, in ABC Cas9 mutants the expression level of the genes (CCD8 and MAX1) related to SL biosynthesis were decreased, without alteration in the root extract orobanchol [SL] as compared to control plants.Development of genetic resistance based on genome editing of host key genes are novel approaches for enhancing host-parasite resistance. The current study offers insights into ABC protein homologs and mutagenesis of ABC protein that could be used in the development of efficient method to reduce parasitic weed germination.
Broomrapes (Orobanche and Phelipanche) are obligate holoparasites that cause heavy damage to numerous crops, reducing the yield and its quality. The parasite develops in the soil and exerts the greatest damage prior to its emergence; therefore the majority of field loss may occur before diagnosis of infection. Because of the parasite tiny seed size (200 to 300 μm) and dormancy for several decades in the field, it is very difficult to diagnose the parasite by conventional methods. Therefore, to restrict the parasite seeds spread and contamination to other commercial fields, development of DNA-based molecular markers to identify and quantify broomrape species in a soil sample is much needed. In this study, we developed a specific molecular marker (RbcL-M) based on rbcL (large subunit of the ribulose-bisphosphate carboxylase) gene from Orobanche crenata to differentiate between Orobanche crenata and Orobanche cumana. Likewise, a specific marker (ITS100) based upon unique sequences in the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA of Phelipanche aegyptiaca to quantify three species of the parasite (P. aegyptiaca, O. crenata and O. cumana) in a soil sample was developed. Genomic DNA was extracted from soil samples artificially infested with broomrape seeds or tissue of P. aegyptiaca, O. cumana and O. crenata and subjected to PCR analysis.RbcL-M marker successfully amplified a PCR product (1300bp) when O. crenata seeds or tissues (collected from several locations in Israel) were added to the soil samples. The same marker amplified a PCR product (1000bp) when O. cumana seeds or tissues were added to the soil samples. RbcL-M marker did not amplify soil samples with seeds or tissues of P. aegyptiaca or any soil-borne DNA.Furthermore, using ITS-100 marker and Real-Time PCR analysis, allowed quantitative diagnostic of the parasite in a soil sample from infected sunflower field. As expected the universal internal control primer (UCP-555) amplified a PCR product (555bp) when genomic DNA extracted from soil samples with or without broomrape tissues. The development of an efficient, simple and robust molecular marker to detect and distinguish between broomrape species, has a significant insights on assessment the level of infestation and planning eradication program to the parasite in a field crop.
carotenogenesis has been intensively studied in carrot roots, and transcriptional regulation is thought to be the major factor in carotenoid accumulation in these organs. However, little is known about the transcriptional regulation of carotenoid biosynthetic genes concerning carotenoid accumulation during infestation by the obligate parasite Phelipanche aegyptiaca. HpLc analysis revealed a decrease in carotenoid levels of the different carrot cultivars when parasitized by P. aegyptiaca. Besides, we isolated and analyzed P. aegyptiaca tubercles parasitizing the various carrot root cultivars and show that they accumulate different carotenoids compared to those in non-infested carrot roots. expression analysis of PHYTOENE SYNTHASE (PSY1) and CAROTENOID ISOMERASE (CRTISO) as well as the strigolactone apocarotenoid biosynthetic genes DWARF27 (D27), CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7) and CCD8 revealed that their transcript levels showed significant variation in P. aegyptiaca infested carrot roots. After parasite infestation, the expression of these genes was strongly reduced, as were the carotenoid levels and this was more pronounced in the uncommon non-orange varieties. We also analyzed the parasite genes encoding D27, CCD7 and CCD8 and show that they are expressed in tubercles. this raises important questions of whether the parasite produces its carotenoids and apocarotenoids including strigolactones and whether the latter might have a role in tubercle development. The parasitic weeds of the genera Orobanche, Phelipanche, and Striga (Orobanchaceae) are the most important agricultural weeds in many crops, particularly in carrot, tomato, sunflower, tobacco, and faba bean, causing significant crop losses in many parts of the world 1. These obligate root parasites are completely devoid of chlorophyll and consequently dependent on their host for supply of resources, including water, nutrients, proteins, and oligonucleotides 2-5. The parasite development is divided into pre-parasitic and parasitic stages. The pre-parasitic stage starts with seed pre-conditioning followed by germination. The parasite seed germination is induced by molecules secreted into the rhizosphere by the roots of host plants called germination stimulant 6. Germination stimulants for root parasitic plants have been isolated from host plant root exudates, and the majority of these natural compounds are carotenoid-derived strigolactones 7,8. The parasitic phase begins with the penetration of the parasite haustorium connecting to the host vascular tissues. The haustoria are responsible for host attachment, penetration and resource acquisition 9. The parasite first develops a tubercle, which gives rise to a flowering spike that emerges from the soil 9-12. The flower produces thousands of extremely small seeds, which can survive more than 15 years in a crop field until favorable environmental conditions for germination are obtained 4,9-12 .
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.