Biology subjects

Nassar, J. A.

Publications and source records attributed to Nassar, J. A..

3 recordsLinked to original sources

bioRxiv2019

Mutagenesis of two homologs ATP Binding cassette protein in tomato by CRISPR/Cas9 provide resistance against the plant parasite Phelipanche aegyptiaca

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 ABCCas9 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.

plant biology↗

bioRxiv2019

Development of specific molecular markers to distinguish and quantify broomrape species in a soil sample from infected field

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 m) 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.

molecular biology↗

bioRxiv2018

Use of a visible reporter marker- myb-related gene in crop plants to minimize herbicide usage against weeds

Weeds, a main threat to agricultural productivity worldwide, are mostly controlled by herbicides. To minimize herbicide usage by targeting it to weedy areas, we developed a new image-based methodology for robust weed detection that relies on manipulating the crop plants leaf hue, without affecting crop fitness. We generated transgenic tobacco (Nicotiana tabacum Xanthi) lines overexpressing anthocyanin pigment as a traceable marker that differentiates transgenes from the surrounding weeds at an early stage. Transformation with the anthocyanin VlmybA1-2 gene produced purple-colored leaves. Subsequent gene silencing with vector pTRV2:VlmybA1-2 significantly reduced anthocyanin pigments in tobacco leaves 40 days after agroinfiltration, with a concomitant reduction in VlmybA1-2 transcript levels. Purple hue faded gradually, and there were no fitness costs in terms of plant height or leaf number in the silenced vs. non-silenced tobacco transgenes. These results could lead to a new sustainable weed-control method that will alleviate weed-related ecological, agricultural and economic issues.

plant biology↗