Formerly the "Center for Desert Agriculture"

Recent Submissions

  • The Arabidopsis homolog of human G3BP1 is a key regulator of stomatal and apoplastic immunity

    Abulfaraj, Aala A.; Mariappan, Kiruthiga; Bigeard, Jean; Manickam, Prabhu; Blilou, Ikram; Guo, Xiujie; Al-Babili, Salim; Pflieger, Delphine; Hirt, Heribert; Rayapuram, Naganand (Life Science Alliance, LLC, 2018-05-31)
    Mammalian Ras-GTPase–activating protein SH3-domain–binding proteins (G3BPs) are a highly conserved family of RNA-binding proteins that link kinase receptor-mediated signaling to RNA metabolism. Mammalian G3BP1 is a multifunctional protein that functions in viral immunity. Here, we show that the Arabidopsis thaliana homolog of human G3BP1 negatively regulates plant immunity. Arabidopsis g3bp1 mutants showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv. tomato. Pathogen resistance was mediated in Atg3bp1 mutants by altered stomatal and apoplastic immunity. Atg3bp1 mutants restricted pathogen entry into stomates showing insensitivity to bacterial coronatine–mediated stomatal reopening. AtG3BP1 was identified as a negative regulator of defense responses, which correlated with moderate up-regulation of salicylic acid biosynthesis and signaling without growth penalty.
  • Candidate enzymes for saffron crocin biosynthesis are localized in multiple cellular compartments

    Demurtas, Olivia Costantina; Frusciante, Sarah; Ferrante, Paola; Diretto, Gianfranco; Azad, Noraddin Hosseinpour; Pietrella, Marco; Aprea, Giuseppe; Taddei, Anna Rita; Romano, Elena; Mi, Jianing; Al-Babili, Salim; Frigerio, Lorenzo; Giuliano, Giovanni (American Society of Plant Biologists (ASPB), 2018-05-29)
    Saffron is composed of the dried stigmas of Crocus sativus and is the most expensive spice on Earth. Its red color is due to the apocarotenoid glycosides, crocins, which accumulate in the vacuole and reach up to 10% of the stigma dry weight. We have previously characterized the first dedicated enzyme in crocin biosynthesis, CsCCD2, which cleaves zeaxanthin to yield crocetin dialdehyde. In this work, we identified six putative aldehyde dehydrogenase (ALDH) transcripts expressed in saffron stigmas. When expressed in E. coli, only one of corresponding proteins (CsALDH3I1), was able to convert crocetin dialdehyde into the crocin precursor, crocetin. CsALDH3I1 carries a C-terminal hydrophobic domain, similar to that of a Neurospora membrane-associated apocarotenoid dehydrogenase, YLO-1. We also characterized a UDP-glycosyltransferase enzyme, CsUGT74AD1, able to convert crocetin to crocins 1 and 2'. In vitro assays showed high specificity of CsALDH3I1 for crocetin dialdehyde and long chain apocarotenals, and of CsUGT74AD1 for crocetin. Upon extract fractionation, the CsCCD2, CsALDH3I1 and CsUGT74AD1 enzymes partitioned in the insoluble fraction, suggesting that they are associated to membranes or to large insoluble complexes. Immunogold labeling of saffron stigmas and confocal microscopy of fusions to Green Fluorescent Protein expressed in N. benthamiana leaves revealed that CsCCD2 localizes to plastids, CsALDH3I1 to the endoplasmic reticulum (ER) and CsUGT74AD1 to the cytoplasm, in association with cytoskeletal-like structures. Based on our and on literature data, we propose that the ER and cytoplasm function as
  • In silico exploration of Red Sea Bacillus genomes for natural product biosynthetic gene clusters

    Othoum, Ghofran K; Bougouffa, Salim; Razali, Rozaimi; Bokhari, Ameerah; Alamoudi, Soha; Antunes, André; Gao, Xin; Hoehndorf, Robert; Arold, Stefan T.; Gojobori, Takashi; Hirt, Heribert; Mijakovic, Ivan; Bajic, Vladimir B.; Lafi, Feras Fawzi; Essack, Magbubah (Springer Nature, 2018-05-22)
    BackgroundThe increasing spectrum of multidrug-resistant bacteria is a major global public health concern, necessitating discovery of novel antimicrobial agents. Here, members of the genus Bacillus are investigated as a potentially attractive source of novel antibiotics due to their broad spectrum of antimicrobial activities. We specifically focus on a computational analysis of the distinctive biosynthetic potential of Bacillus paralicheniformis strains isolated from the Red Sea, an ecosystem exposed to adverse, highly saline and hot conditions.ResultsWe report the complete circular and annotated genomes of two Red Sea strains, B. paralicheniformis Bac48 isolated from mangrove mud and B. paralicheniformis Bac84 isolated from microbial mat collected from Rabigh Harbor Lagoon in Saudi Arabia. Comparing the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 with nine publicly available complete genomes of B. licheniformis and three genomes of B. paralicheniformis, revealed that all of the B. paralicheniformis strains in this study are more enriched in nonribosomal peptides (NRPs). We further report the first computationally identified trans-acyltransferase (trans-AT) nonribosomal peptide synthetase/polyketide synthase (PKS/ NRPS) cluster in strains of this species.ConclusionsB. paralicheniformis species have more genes associated with biosynthesis of antimicrobial bioactive compounds than other previously characterized species of B. licheniformis, which suggests that these species are better potential sources for novel antibiotics. Moreover, the genome of the Red Sea strain B. paralicheniformis Bac48 is more enriched in modular PKS genes compared to B. licheniformis strains and other B. paralicheniformis strains. This may be linked to adaptations that strains surviving in the Red Sea underwent to survive in the relatively hot and saline ecosystems.
  • Optimizing FRET-FLIM Labeling Conditions to Detect Nuclear Protein Interactions at Native Expression Levels in Living Arabidopsis Roots

    Long, Yuchen; Stahl, Yvonne; Weidtkamp-Peters, Stefanie; Smet, Wouter; Du, Yujuan; Gadella, Theodorus W. J.; Goedhart, Joachim; Scheres, Ben; Blilou, Ikram (Frontiers Media SA, 2018-05-15)
    Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study. With an effective optimization strategy, we were able to detect the interaction between the stem cell regulators SHORT-ROOT and SCARECROW at endogenous expression levels in the root pole of living Arabidopsis embryos and developing lateral roots by FRET-FLIM. Using this approach we show that the spatial profile of interaction between two transcription factors can be highly modulated in reoccurring and structurally resembling organs, thus providing new information on the dynamic redistribution of nuclear protein complex configurations in different developmental stages. In principle, our optimization procedure for transcription factor complexes is applicable to any biological system.
  • Boosting Alfalfa (Medicago sativa L.) Production With Rhizobacteria From Various Plants in Saudi Arabia

    Daur, Ihsanullah; Saad, Maged; Eida, Abdul Aziz; Ahmad, Shakeel; Shah, Zahid Hussain; Ihsan, Muhammad Z.; Muhammad, Yasir; Sohrab, Sayed S.; Hirt, Heribert (Frontiers Media SA, 2018-04-04)
    This study focused on rhizobacteria to promote sustainable crop production in arid regions of Saudi Arabia. The study isolated 17 tightly root-adhering rhizobacteria from various plants at Hada Al Sham in Saudi Arabia. All 17 rhizobacterial isolates were confirmed as plant growth promoting rhizobacteria by classical biochemical tests. Using 16S rDNA gene sequence analyses, the strains were identified as Bacillus, Acinetobacter and Enterobacter. Subsequently, the strains were assessed for their ability to improve the physiology, nutrient uptake, growth, and yield of alfalfa plants grown under desert agriculture conditions. The field trials were conducted in a randomized complete block design. Inoculation of alfalfa with any of these 17 strains improved the relative water content; chlorophyll a; chlorophyll b; carotenoid contents; nitrogen (N), phosphorus, and potassium contents; plant height; leaf-to-stem ratio; and fresh and dry weight. Acinetobacter pittii JD-14 was most effective to increase fresh and dry weight of alfalfa by 41 and 34%, respectively, when compared to non-inoculated control plants. Nevertheless, all strains enhanced crop traits when compared to controls plants, indicating that these desert rhizobacterial strains could be used to develop an eco-friendly biofertilizer for alfalfa and possibly other crop plants to enhance sustainable production in arid regions.
  • CRISPR/Cas13 as a Tool for RNA Interference

    Ali, Zahir; Mahas, Ahmed; Mahfouz, Magdy M. (Elsevier BV, 2018-03-28)
    Almost all biological processes involve RNA, making it crucial to develop tools for manipulation of the transcriptome. The bacterial CRISPR/Cas13 system was recently rewired to facilitate RNA manipulation in eukaryotes, including plants. We discuss here the opportunities and limitations of using CRISPR/Cas13 in plants for various types of RNA manipulation.
  • TDZ-Induced Plant Regeneration in Jatropha curcas: A Promising Biofuel Plant

    Kumar, Nitish; Bhatt, Vacha D.; Mastan, Shaik G.; Reddy, Muppala P. (Springer Singapore, 2018-03-23)
    In recent years, Jatropha curcas has pronounced attention due to its capacity of production of biodiesel. Uniform large-scale propagation of J. curcas is one of the significant keys that will eventually decide victory. Direct regeneration is one of the methods which help in the production of uniform and homogenous plant, and TDZ plays an important role in the production of plantlets by direct organogenesis in several number of plant species including J. curcas. Measuring the economical importance of J. curcas and the role of TDZ in shoot regeneration, the present book chapter briefly reviews the impact of TDZ on shoot bud induction from various explants of J. curcas.
  • Ethylene induced plant stress tolerance by Enterobacter sp. SA187 is mediated by 2‐keto‐4‐methylthiobutyric acid production

    Zélicourt, Axel de; Synek, Lukas; Saad, Maged; Alzubaidy, Hanin S.; Jalal, Rewaa Sauod Mohammed; Xie, Yakun; Andres-Barrao, Cristina; Rolli, Eleonora; Guerard, Florence; Mariappan, Kiruthiga; Daur, Ihsanullah; Colcombet, Jean; Benhamed, Moussa; Depaepe, Thomas; Van Der Straeten, Dominique; Hirt, Heribert (Public Library of Science (PLoS), 2018-03-19)
    Several plant species require microbial associations for survival under different biotic and abiotic stresses. In this study, we show that Enterobacter sp. SA187, a desert plant endophytic bacterium, enhances yield of the crop plant alfalfa under field conditions as well as growth of the model plant Arabidopsis thaliana in vitro, revealing a high potential of SA187 as a biological solution for improving crop production. Studying the SA187 interaction with Arabidopsis, we uncovered a number of mechanisms related to the beneficial association of SA187 with plants. SA187 colonizes both the surface and inner tissues of Arabidopsis roots and shoots. SA187 induces salt stress tolerance by production of bacterial 2-keto-4-methylthiobutyric acid (KMBA), known to be converted into ethylene. By transcriptomic, genetic and pharmacological analyses, we show that the ethylene signaling pathway, but not plant ethylene production, is required for KMBA-induced plant salt stress tolerance. These results reveal a novel molecular communication process during the beneficial microbe-induced plant stress tolerance.
  • Modify the Histone to Win the Battle: Chromatin Dynamics in Plant–Pathogen Interactions

    Ramirez Prado, Juan Sebastian; Piquerez, Sophie J. M.; Bendahmane, Abdelhafid; Hirt, Heribert; Raynaud, Cécile; Benhamed, Moussa (Frontiers Media SA, 2018-03-19)
    Relying on an immune system comes with a high energetic cost for plants. Defense responses in these organisms are therefore highly regulated and fine-tuned, permitting them to respond pertinently to the attack of a microbial pathogen. In recent years, the importance of the physical modification of chromatin, a highly organized structure composed of genomic DNA and its interacting proteins, has become evident in the research field of plant-pathogen interactions. Several processes, including DNA methylation, changes in histone density and variants, and various histone modifications, have been described as regulators of various developmental and defense responses. Herein, we review the state of the art in the epigenomic aspects of plant immunity, focusing on chromatin modifications, chromatin modifiers, and their physiological consequences. In addition, we explore the exciting field of understanding how plant pathogens have adapted to manipulate the plant epigenomic regulation in order to weaken their immune system and thrive in their host, as well as how histone modifications in eukaryotic pathogens are involved in the regulation of their virulence.
  • The interaction of strigolactones with abscisic acid during the drought response in rice

    Haider, Imran; Andreo-Jimenez, Beatriz; Bruno, Mark; Bimbo, Andrea; Floková, Kristýna; Abuauf, Haneen Waleed Hamza; Otang Ntui, Valentine; Guo, Xiujie; Charnikhova, Tatsiana; Al-Babili, Salim; Bouwmeester, Harro J; Ruyter-Spira, Carolien (Oxford University Press (OUP), 2018-03-09)
    Both strigolactones (SLs) and abscisic acid (ABA) biosynthetically originate from carotenoids. Considering their common origin, the interaction of these two hormones at the biosynthetic and/or regulatory level may be anticipated. Here, we show in rice that drought simultaneously induces SL production in the root, and ABA production and the expression of SL biosynthetic genes in the shoot. Under control conditions, the ABA concentration was higher in shoots of the SL biosynthetic rice mutants dwarf10 (d10) and d17 than in wild-type plants, while a similar trend was observed for SL-perception mutant d3. These differences were enhanced under drought. However, drought did not result in an increase in leaf ABA content in rice mutant line d27, carrying a mutation in the gene encoding the first committed enzyme in SL biosynthesis, to the same extent as in the other SL mutants and the wild-type. Accordingly, d10, d17 and d3 lines were more drought tolerant than wild-type plants, whereas d27 displayed decreased tolerance. Finally, over-expression of OsD27 in rice resulted in increased levels of ABA when compared with wild-type plants. We conclude that the SL and ABA pathways are connected with each other through D27, which is playing a crucial role in determining ABA and SL content in rice.
  • Thermopriming Triggers Splicing Memory in Arabidopsis

    Ling, Yu; Serano, Natalia Lorena Gorron; Gao, Ge; Atia, Mohamed; Mokhtar, Morad; Woo, Yong H; Bazin, Jeremie; Veluchamy, Alaguraj; Benhamed, Moussa; Crespi, Martin; Gehring, Christoph A; Reddy, A S N; Mahfouz, Magdy M. (Oxford University Press (OUP), 2018-02-20)
    Abiotic and biotic stresses limit crop productivity. Exposure to a non-lethal stress, referred to as priming, can allow plants to survive subsequent and otherwise lethal conditions; the priming effect persists even after a prolonged stress-free period. However, the molecular mechanisms underlying priming are not fully understood. Here, we investigated the molecular basis of heat shock memory and the role of priming in Arabidopsisthaliana. Comprehensive analysis of transcriptome-wide changes in gene expression and alternative splicing in primed and non-primed plants revealed that alternative splicing functions as a novel component of heat shock memory. We show that priming of plants with a non-lethal heat stress results in de-repression of splicing after a second exposure to heat stress. By contrast, non-primed plants showed significant repression of splicing. These observations link ‘splicing memory’ to the ability of plants to survive subsequent and otherwise lethal heat stress. This newly discovered priming-induced splicing memory may represent a general feature of heat stress responses in plants and other organisms as many of the key components of heat shock responses are conserved among eukaryotes. Furthermore, this finding could facilitate the development of novel approaches to improve plant survival under extreme heat stress.
  • Metaorganisms in extreme environments: do microbes play a role in organismal adaptation?

    Bang, Corinna; Dagan, Tal; Deines, Peter; Dubilier, Nicole; Duschl, Wolfgang J.; Fraune, Sebastian; Hentschel, Ute; Hirt, Heribert; Hülter, Nils; Lachnit, Tim; Picazo, Devani; Pita, Lucia; Pogoreutz, Claudia; Radecker, Nils; Saad, Maged; Schmitz, Ruth A.; Schulenburg, Hinrich; Voolstra, Christian R.; Weiland-Bräuer, Nancy; Ziegler, Maren; Bosch, Thomas C.G. (Elsevier BV, 2018-02-15)
    From protists to humans, all animals and plants are inhabited by microbial organisms. There is an increasing appreciation that these resident microbes influence the fitness of their plant and animal hosts, ultimately forming a metaorganism consisting of a uni- or multicellular host and a community of associated microorganisms. Research on host–microbe interactions has become an emerging cross-disciplinary field. In both vertebrates and invertebrates a complex microbiome confers immunological, metabolic and behavioural benefits; conversely, its disturbance can contribute to the development of disease states. However, the molecular and cellular mechanisms controlling the interactions within a metaorganism are poorly understood and many key interactions between the associated organisms remain unknown. In this perspective article, we outline some of the issues in interspecies interactions and in particular address the question of how metaorganisms react and adapt to inputs from extreme environments such as deserts, the intertidal zone, oligothrophic seas, and hydrothermal vents.
  • Synthetic Strigolactone Analogues Reveal Anti-Cancer Activities on Hepatocellular Carcinoma Cells

    Hasan, Mohammed Nihal; Choudhry, Hani; Razvi, Syed Shoeb; Moselhy, Said Salama; Kumosani, Taha Abduallah; Zamzami, Mazin A.; Omran, Ziad; Halwani, Majed A.; Al-Babili, Salim; Abualnaja, Khalid Omer; Al-Malki, Abdulrahman Labeed; Alhosin, Mahmoud; Asami, Tadao (Elsevier BV, 2018-02-09)
    Hepatocellular carcinoma (HCC) remains one of the leading causes of death worldwide. The complex etiology is attributed to many factors like heredity, cirrhosis, hepatitis infections or the dysregulation of the different molecular pathways. Nevertheless, the current treatment regimens have either severe side effects or tumors gradually acquire resistance upon prolonged use. Thus, developing a new selective treatment for HCC is the need of the hour. Many anticancer agents derived from plants have been evaluated for their cytotoxicity towards many human cancer cell lines. Strigolactones (SLs)-a newly discovered class of phytohormones, play a crucial role in the development of plant-root and shoot. Recently, many synthetic analogues of SLs have demonstrated pro-apoptotic effects on different cancer cell lines like prostate, breast, colon and lung. In this study, we tested synthetic SLs analogues on HCC cell line-HepG2 and evaluated their capability to induce cell proliferation inhibition and apoptosis. Primary WST-1 assays, followed by annexin-V/7AAD staining, demonstrated the anti-proliferative effects. The SLs analogues TIT3 and TIT7 were found to significantly reduce HepG2 cell viability in a dose- and time-dependent manner and induce apoptosis. Interestingly, though TIT3 and TIT7 strongly affected cancer cell proliferation, both compounds showed moderate anti-proliferative effect on normal cells. Further, migration of cancer cells was suppressed upon treatment with TIT3 and TIT7 in a wound healing assay. In summary, these findings suggest that two SLs analogues TIT3 and TIT7 exert selective inhibitory effects on cancer cells most likely through targeting microtubules. SLs analogues could be used in future as potential anti-cancer candidates in chemotherapy.
  • A computational framework for cortical microtubule dynamics in realistically shaped plant cells

    Chakrabortty, Bandan; Blilou, Ikram; Scheres, Ben; Mulder, Bela M. (Public Library of Science (PLoS), 2018-02-02)
    Plant morphogenesis is strongly dependent on the directional growth and the subsequent oriented division of individual cells. It has been shown that the plant cortical microtubule array plays a key role in controlling both these processes. This ordered structure emerges as the collective result of stochastic interactions between large numbers of dynamic microtubules. To elucidate this complex self-organization process a number of analytical and computational approaches to study the dynamics of cortical microtubules have been proposed. To date, however, these models have been restricted to two dimensional planes or geometrically simple surfaces in three dimensions, which strongly limits their applicability as plant cells display a wide variety of shapes. This limitation is even more acute, as both local as well as global geometrical features of cells are expected to influence the overall organization of the array. Here we describe a framework for efficiently simulating microtubule dynamics on triangulated approximations of arbitrary three dimensional surfaces. This allows the study of microtubule array organization on realistic cell surfaces obtained by segmentation of microscopic images. We validate the framework against expected or known results for the spherical and cubical geometry. We then use it to systematically study the individual contributions of global geometry, cell-edge induced catastrophes and cell-face induced stability to array organization in a cuboidal geometry. Finally, we apply our framework to analyze the highly non-trivial geometry of leaf pavement cells of Arabidopsis thaliana, Nicotiana benthamiana and Hedera helix. We show that our simulations can predict multiple features of the microtubule array structure in these cells, revealing, among others, strong constraints on the orientation of division planes.
  • PCNA Structure and Interactions with Partner Proteins

    Oke, Muse; Zaher, Manal S.; Hamdan, Samir (Springer New York, 2018-01-29)
    Proliferating cell nuclear antigen (PCNA) consists of three identical monomers that topologically encircle double-stranded DNA. PCNA stimulates the processivity of DNA polymerase δ and, to a less extent, the intrinsically highly processive DNA polymerase ε. It also functions as a platform that recruits and coordinates the activities of a large number of DNA processing proteins. Emerging structural and biochemical studies suggest that the nature of PCNA-partner proteins interactions is complex. A hydrophobic groove at the front side of PCNA serves as a primary docking site for the consensus PIP box motifs present in many PCNA-binding partners. Sequences that immediately flank the PIP box motif or regions that are distant from it could also interact with the hydrophobic groove and other regions of PCNA. Posttranslational modifications on the backside of PCNA could add another dimension to its interaction with partner proteins. An encounter of PCNA with different DNA structures might also be involved in coordinating its interactions. Finally, the ability of PCNA to bind up to three proteins while topologically linked to DNA suggests that it would be a versatile toolbox in many different DNA processing reactions.
  • PCNA Loading by RFC, Mechanism of

    Oke, Muse; Zaher, Manal S.; Hamdan, Samir (Springer New York, 2018-01-29)
    Replicative polymerases achieve highly processive DNA synthesis by binding to a clamp-like processivity factor that is topologically linked to DNA. The eukaryotic processivity clamp, proliferating cell nuclear antigen (PCNA), exists mostly as a closed ring in solution. Replication factor C (RFC), a five-subunit ATP-dependent protein complex, mediates PCNA opening in solution (assembly stage) and closing onto the primer-template (disassembly stage). In the assembly stage, RFC binding to ATP causes conformational changes that trigger RFC to form a complex with PCNA. PCNA is then cracked open at one subunit interface, and both RFC and PCNA adopt an extended spiral structure with a chamber that selects for a primer-template DNA structure. Binding of RFC/PCNA to DNA triggers the disassembly stage by stimulating ATP hydrolysis. Subsequent conformational changes in RFC and PCNA lead to the closing of PCNA onto the primer-template and the dissociation of RFC.
  • Engineering Plant Architecture via CRISPR/Cas9-mediated Alteration of Strigolactone Biosynthesis

    Butt, Haroon; Jamil, Muhammad; Wang, Jian You; Al-Babili, Salim; Mahfouz, Magdy M. (Cold Spring Harbor Laboratory, 2018-01-28)
    Precision plant genome engineering holds much promise for targeted improvement of crop traits via unprecedented single-base level control over the genetic material. Strigolactones (SLs) are a key determinant of plant architecture, known for their role in inhibiting shoot branching (tillering). Here, we used CRISPR/Cas9 in rice (Oryza sativa) for targeted disruption of CAROTENOID CLEAVAGE DIOXYGENASE 7 (CCD7), which controls a key step in SL biosynthesis. The ccd7 mutants exhibited a striking increase in tillering, combined with a dwarf phenotype, which could be rescued by application of the synthetic SL analog GR24. Striga germination assays and liquid chromatography mass spectrometry analysis showed that root exudates of ccd7 mutants were also SL deficient. Taken together, our results show the power of CRISPR/Cas9 for targeted engineering of plant architecture and for elucidating the molecular underpinnings of architecture-related traits.
  • Engineering Plant Immunity by CRISPR/Cas13a

    Aljedaani, Fatimah R.; Mahfouz, Magdy M. (2018-01-24)
  • RNA virus interference via CRISPR/Cas13a system in plants

    Aman, Rashid; Ali, Zahir; Butt, Haroon; Mahas, Ahmed; Aljedaani, Fatimah R.; Khan, Muhammad Zuhaib; Ding, Shouwei; Mahfouz, Magdy M. (Springer Nature, 2018-01-04)
    CRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.CRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.Our data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.
  • Engineering resistance against Tomato yellow leaf curl virus via the CRISPR/Cas9 system in tomato

    Mahfouz, Magdy M.; Tashkandi, Manal; Ali, Zahir; Aljedaani, Fatimah R.; Shami, Ashwag (Cold Spring Harbor Laboratory, 2017-12-22)
    CRISPR/Cas systems confer molecular immunity against phages and conjugative plasmids in prokaryotes. Recently, CRISPR/Cas9 systems have been used to confer interference against eukaryotic viruses. Here, we engineered Nicotiana benthamiana and tomato (Solanum lycopersicum) plants with the CRISPR/Cas9 system to confer immunity against the Tomato yellow leaf curl virus (TYLCV). Targeting the TYLCV genome with Cas9-single guide RNA at the sequences encoding the coat protein (CP) or replicase (Rep) resulted in efficient virus interference, as evidenced by low accumulation of the TYLCV DNA genome in the transgenic plants. The CRISPR/Cas9-based immunity remained active across multiple generations in the N. benthamiana and tomato plants. Together, our results confirmed the efficiency of the CRISPR/Cas9 system for stable engineering of TYLCV resistance in N. benthamiana and tomato, and opens the possibilities of engineering virus resistance against single and multiple infectious viruses in other crops.

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