Now showing items 1-20 of 105

    • Involvement of Beneficial Microbe-derived Cyclodipeptides (CDPs) in Promoting Plant Tolerance to Abiotic Stresses

      abdulhakim, fatimah (2020-07) [Thesis]
      Advisor: Hirt, Heribert
      Committee members: Merzaban, Jasmeen; Al-Babili, Salim; Saad, Maged M.; Abulfaraj, Alaa
      Cyclodipeptides (CDPs) are the smallest, most stable cyclic peptides that are synthesized as secondary metabolites by bacteria. The aim of this study was to investigate the effect of the Pseudomonas argentinensis (SA190) and four (CDPs), named as cis-cyclo-(Pro-Phe) (Cyclo2), cis-cyclo-(Pro-Leu) (Cyclo3), cis-cyclo-(Pro-Tyr) (Cyclo4) and cis-cyclo-(Pro-Val) (Cyclo5), with three concentrations (1µM, 100nM, and 10nM), on the growth of Arabidopsis thaliana under normal plant growth conditions [1/2MS media], salt conditions [125 mM NaCl] and drought conditions [25% PEG]. Moreover, we determined the most effective CDPs with optimal concentration. It was found that cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 100nM had an effect on the plant growth and can mimic the effect of SA190 under normal [1/2MS media] conditions. Also, cis-cyclo-(Pro-Tyr) (Cyclo4) at a concentration of 1µM can mimic the effect of SA190 under salt conditions [125mM NaCl]. Finally, cis-cyclo-(Pro-Val) (Cyclo5) at a concentration of 1µM can mimic the effect of SA190 under drought conditions [25% PEG].
    • Dissecting the genetic architecture of salt tolerance in the wild tomato Solanum pimpinellifolium

      Morton, Mitchell (2019-10) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Krattinger, Simon G.; McCabe, Matthew; Schafleitner, Roland
      Salt stress severely constrains plant performance and global agricultural productivity. 5% of arable land, 20% of irrigated areas and 98% of water reserves worldwide are saline. Improving the salt tolerance of major crop species could help attenuate yield losses and expand irrigation opportunities and provide in situ relief in areas where poverty, food and water scarcity are prevalent. Increasing the salt tolerance of crops with high commercial and nutritional value, such as tomato (Solanum lycopersicum L.), would provide particularly significant economic and health benefits. However, salt tolerance is a complex trait with a limited genetic repertoire in domesticated crop varieties, including tomato, frustrating attempts to breed and engineer tolerant crop varieties. Here, a genome-wide association study (GWAS) was undertaken, leveraging the rich genetic diversity of the wild, salt tolerant tomato Solanum pimpinellifolium and the latest phenotyping technologies to identify traits that contribute to salt tolerance and the genetic basis for variation in those traits. A panel of 220 S. pimpinellifolium accessions was phenotyped, focusing on image-based high-throughput phenotyping over time in controlled and field conditions in young and mature plants. Results reveal substantial natural variation in salt tolerance over time across many traits. In particular, the use of unmanned aerial vehicle (UAV)-based remote sensing in the field allowed high-resolution RGB, thermal and hyperspectral mapping that offers new insights into plant performance in the field, over time. To empower our GWAS and facilitate the identification of candidate genes, a new S. pimpinellifolium reference genome was generated, 811Mb in size, N50 of ~76kb, containing 25,970 annotated genes. Analysis of this reference genome highlighted potential contributors to salt tolerance, including enrichments in genes with stress response functions and a high copy number of the salt tolerance-associated gene inositol- 3-phosphate synthase (I3PS). A recently completed full genome re-sequencing of the panel, along with a newly available pseudomolecule-level assembly of the S. pimpinellifolium genome with N50 of ~11Mb, will serve to drive a GWAS to identify loci associated with traits that contribute to salt tolerance. Further research including gene validation, breeding, genetic modification and gene editing experiments will drive the development of new salt tolerant tomato cultivars.
    • The role of NAC transcription factors in responses of plants to heat and salt stresses

      Alshareef, Nouf Owdah Hameed (2019-08) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Blilou, Ikram; Pain, Arnab; Balazadeh, Salma
      Soil salinity and heat stress are two major abiotic stresses affecting plant growth and yield. Transcription factors (TFs) are key regulators in stress responses. They link stress sensing with many tolerance mechanisms by translating stress signals into changes in gene expression that ultimately contribute to stress tolerance. The NAC (NAM, ATAF and CUC) TF family have been found to be involved in responses to biotic and abiotic stresses. In this PhD project, the role of NAC TFs in response to heat and salt stress was studied in the model system Arabidopsis thaliana (Arabidopsis), and in two agriculturally relevant species, Solanum lycopersicum (tomato) and Chenopodium quinoa (quinoa). Plants have the ability to acquire thermotolerance if they are pre-exposed to a mild, non-lethal high temperature. The maintenance of acquired thermotolerance for several days is known as thermomemory. Here we investigated the role of NAC TFs in thermotolerance. The expression profiles of 104 Arabidopsis NAC TFs were measured and compared between primed and unprimed plants. Some NACs with a distinctive expression pattern in response to thermopriming were selected for further phenotypic analysis. Knock-out (KO) mutants of the ATAF1 gene showed an enhanced thermomemory phenotype compared with wild type plants (WT) and from this work, the functions of the ATAF1 gene were studied further. RNAseq co-expression analyses of ATAF1 overexpressor and ataf1 KO plants found that ANAC055 expression was co-regulated with that of ATAF1. JUBGBRUNNEN1 (JUB1) is another NAC TF involved in responses to heat, drought and salinity. In this study, the role of AtJUB1 overexpression in salinity was investigated in tomato plants. AtJUB1 overexpression resulted in higher proline levels and improved maintenance of water content and biomass in AtJUB1-overexpressing plants grown hydroponically under salinity compared with WT plants. Quinoa has recently gained much attention because of its high nutritional value and high tolerance to several stresses including drought and salinity. NAC TFs are hypothesized to play a major role in quinoa’s tolerance to abiotic stresses. In this study, the NAC TFs family were identified and investigated in the genome of quinoa. 107 NAC TF genes were identified and their transcriptional responses to different stresses including salt, drought and heat were investigated.
    • Genetics of Salinity Tolerance in Rice

      Al Tamimi, Nadia (2019-05) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Al-Babili, Salim; Gojobori, Takashi; McCouch, Susan
      For more than half of the world’s population, rice (Oryza sativa L.), the most saltsensitive cereal, is a dietary staple. Soil salinity is a major constraint to rice production worldwide. Thus, to feed 9 billion people by 2050, we need to increase rice production while facing the challenges of rapid global environmental changes. To meet some of these challenges, there is a vital requirement to significantly increase rice production in salinized land and improve photosynthetic efficiency. Exposure of plants to soil salinity rapidly reduces their growth and transpiration rates (TRs) due to the ‘osmotic component’ of salt stress (sensu Munns and Tester), which is hypothesized to be related to sensing and signaling mechanisms. Over time, toxic concentrations of Na+ and Cl− accumulate in the cells of the shoot, known as the ‘ionic component’ of salt stress, which causes premature leaf senescence. Both osmotic and ionic components of salinity stress are likely to impact yield. Despite significant advances in our understanding of the ionic components of salinity tolerance, little is known about the early responses of plants to salinity stress. In my PhD project, the aim was to analyze naturally occurring variation in salinity tolerance of rice and identify key genes related to higher salinity tolerance using high-throughput phenomics and field trials. I used a forward genetics approach, with two rice diversity panels (indica and aus) and recently published sequencing data (McCouch et al., 2017). Indica and aus were phenotyped under controlled conditions, while the indica diversity panel was also further studied under field conditions for salinity tolerance. I also examined previously unexplored traits associated with salinity tolerance, in particular the effects of salinity on transpiration and transpiration use efficiency. The non-destructive high-throughput experiments conducted under controlled conditions gave insights into the understudied shoot ion-independent component of salinity tolerance. In parallel, the field experiments increased our understanding of the genetic control of further components of salinity tolerance, including the maintenance of yield under saline conditions. Importantly, this project also aimed to improve the current association methods of GWAS by exploring and testing novel Mixed Linear Models. One major benefit of this Ph.D. project was the development of a more holistic approach that recognizes the complexity of the genotype–phenotype interaction. The purpose of my work was to shed more light on the genetic mechanisms of salinity tolerance in rice and discover genes associated with traits contributing to higher photosynthetic activity under both controlled and field conditions. This will ultimately lead to further exploration of the genetic diversity present in the PRAY indica panel, in order to develop higher yielding rice varieties.
    • How does light affect the heat stress response in Arabidopsis?

      Kim, Eunje (2018-11) [Thesis]
      Advisor: Tester, Mark A.
      Committee members: Hirt, Heribert; Voolstra, Christian R.
      Light and temperature are two of the most important environmental factors regulating plant development. Although heat stress has been well studied, little is known about the interaction between light and temperature. In this study, we performed phenotypic assays comparing seedling responses to heat under light and dark conditions. Seedlings exposed to heat in the dark show lower survival rates than seedlings stressed in the light. To identify transcriptional changes underlying light-dependent heat tolerance, we used RNA-sequencing. The light-dependent heat stress responses involved a plethora of genes which could be potential candidate genes for light-induced heat tolerance, including transcription factors (bHLH) and genes commonly associated with biotic stress. By using the latest high-throughput phenotyping facility, we found that the light-dependent heat tolerance is reflected more on the maintenance of photosynthetic capacity, rather than leaf temperature. These results provide insights into how light increases heat stress tolerance in Arabidopsis seedlings and suggest its underlying mechanisms.
    • The Role of Chromatin Associated Proteins in Plant Innate Immunity and Jasmonic Acid Signaling

      Jarad, Mai (2018-11) [Dissertation]
      Advisor: Hirt, Heribert
      Committee members: Blilou, Ikram; Arold, Stefan T.; Gust, Andrea
      Pathogen-associated molecular pattern (PAMP) recognition occurs by plasma membrane located receptors that induce among other processes nuclear gene expression. The plant FLS2-BAK1 receptor complex binds the bacterial PAMP, flg22 and induces a series of defense responses. The resulting signal transduction events occur through the activation of two MAPK signaling cascades, which trigger a rapid and strong activation of MPK3, MPK4 and MPK6. Cellular responses to pathogens are regulated by the activated MAPKs, which lead to the eventual phosphorylation of cytoplasmic and nuclear substrates. These MAPK substrates in turn respond to phosphorylation by reprogramming the expression of defense genes. A large scale phosphoproteomics screen of nuclear proteins in wild type and mpk mutant plants in response to flg22 revealed several novel putative targets of MAP kinases. This thesis is aimed at identifying the role of two of these chromatin associated proteins in plant immunity and their signaling mechanisms. The chromatin associated proteins we chose to study here are LITTLE NUCLEI/CROWDED NUCLEI (LINC/CRWN), LINC1 and the AT-HOOK MOTIF CONTANING NUCLEAR LOCALIZED 13 (AHL13) proteins. We demonstrate that these two chromatin associated proteins play a positive regulatory role in jasmonic acid signaling and immunity. Knock out mutants for both genes exhibit impairment in early and late innate immune reposes to both PAMP and hemibiotrophic pathogen strains. We also demonstrate that these mutants are compromised in regulating the expression of genes involved in jasmonic acid (JA) signaling and responses and genes involved in the biosynthesis both the indole and aliphatic glucosinolate (GS) pathways. Moreover, Pst DC3000 hrcC triggers JA and JAIle accumulation in these mutants, whereas salicylic acid (SA) levels are unchanged. We were also able to identify and validate two novel MAPK targeted phosphosites in AHL13 that affect the protein stability of AHL13 and we establish its role as a MPK6 substrate that affects jasmonic acid biosynthesis and PTI responses. Together this work identifies two novel signaling components involved in the regulation of jasmonic acid homeostasis and immunity.
    • Identifying the genetic basis of new components of salinity tolerance in barley

      saade, stephanie (2018-10) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Muehlbauer, Gary; Aranda, Manuel; Krattinger, Simon G.
      Barley is a resilient crop that performs better than other cereal plants under abiotic stress conditions, including salinity stress. The understanding of salinity tolerance in crops is a major milestone to increase yield in areas affected by soil salinity. In barley, some components of salinity tolerance have been elucidated, (e.g. HVP10, which is involved in tissue tolerance), yet little research has explored the discovery of other components contributing to salinity tolerance. In this PhD project, a forward genetics approach was used, whereby two barley populations were phenotyped under controlled and field conditions for salinity tolerance. The first population is a diversity panel of two-row European spring barley, and the second population is a nested association mapping barley population with wild donors from the Fertile Crescent. The use of non-destructive high-throughput experiments conducted under controlled conditions provided insight into the understudied shoot ion-independent component of salinity tolerance. In addition, the previously known association HvHKT1;5 was detected under controlled conditions. In parallel, the field experiments increased our understanding of new components of salinity tolerance, such as the maintenance of yield and yield-related traits under saline conditions. This strategy was successful with the identification of a locus on chromosome 2H (140-145 cM), where the allele from one of the wild donors of the nested association mapping population increased yield under saline conditions in the field. When re-evaluating lines homozygous at the 2H locus, ear length, ear number per plant, yield and harvest index were all significantly higher under saline conditions for the lines carrying the wild allele. Furthermore, another interesting locus on chromosome 7H that was responsive to salt treatment and co-localized with HVP1 was identified using the diversity panel population. Loci with known flowering genes were also shown to be involved in salinity tolerance. To conclude, this PhD project shed more light on the genetic mechanisms of salinity tolerance in barley, a knowledge that can benefit breeding programs and can be extended to other crops such as wheat.
    • In-field characterization of salt stress responses of chlorophylls a and b and carotenoid concentrations in leaves of Solanum pimpinellifolium

      Ilies, Dragos-Bogdan (2018-10) [Thesis]
      Advisor: Tester, Mark A.
      Committee members: Al-Babili, Salim; McCabe, Matthew
      Food security is a major concern of the 21st century, given climate change and population growth. In addition, high salt concentrations in soils affect ~20% of irrigated land and cause a substantial reduction in crop yield. Cultivating salt-tolerant crops could enable the use of salt-affected agricultural land, reduce the use of fresh water and alleviate yield losses. Innovative methods need to be developed to study traditional and novel traits that contribute to salinity tolerance and accurately quantify them. These studies would eventually serve for developing new salt tolerant crops, adapted to the harsh arid and semi-arid climate conditions. A study of 200 accessions of the wild tomatoes (Solanum pimpinellifolium) was conducted in field conditions with phenotyping using an unmanned aerial vehicle (UAV)-mounted hyperspectral camera. Six genotypes with different levels of salt tolerance were sampled for leaf pigment analyses, revealing a clear pattern for the high salt tolerant accession M007, where pigment content in the salt-treated plants significantly increased compared to their control counterparts only in harvesting campaigns 3 and 6, each performed two days after the first and second salt stress application events. Moreover, the light harvesting capacity was found to be better maintained under salt stress in the medium (M255) and highly salt tolerant (M007 and M061) accessions. Pigment quantitation data will contribute towards the groundtruthing of hyperspectral imaging for the development of remote sensing-based predictive pigment mapping methods. This work establishes a reliable quantification protocol for correlating pigment content with vegetation indices. Hence, pigment content captured by imaging techniques and validated using biochemical analysis would serve in developing a high-throughput method for pigment quantitation in the field using UAV-based hyperspectral imaging. This would serve as a tool for measuring pigment content in large number of genotypes in the field which would eventually lead to new salt-tolerant genes.
    • Mapping early responses to salt stress in Arabidopsis thaliana

      Awlia, Mariam (2018-09) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Al-Babili, Salim; Arold, Stefan T.; Korte, Arthur
      Salt stress is a global problem that limits agricultural production. The early responses to salinity, independent of toxic shoot-ion accumulation, are still largely unknown. Here, optimised salt treatment and high-throughput phenotyping protocols were developed and used to examine the natural variation in the early responses to salt stress of 191 Arabidopsis thaliana accessions. Common and novel traits of plants grown under salt treatment were captured through time using RGB and chlorophyll fluorescence imaging. Phenotypic data was combined with the Arabidopsis 10M SNP markers for genome-wide association studies to identify genetic components underlying the early responses to salt stress. The most promising candidate loci were selected based on association strength, allele frequency and number of traits associating to the same locus. In silico analysis highlighted interesting allelic variations across the identified loci, and by phenotypically characterising the candidate gene mutants under salt stress, the associations were experimentally validated. This work comprises a detailed study of the natural variation in the early responses to salt stress, which can give insights into the mechanisms contributing to salinity tolerance and provide the fundaments for crop improvements under saline conditions across the globe.
    • Engineering Plant Immunity via CRISPR/Cas13a System

      Aljedaani, Fatimah R. (2018-05) [Thesis]
      Advisor: Mahfouz, Magdy M.
      Committee members: Blilou, Ikram; Ghaffour, NorEddine
      Viral diseases constitute a major threat to the agricultural production and food security throughout the world. Plants cope with the invading viruses by triggering immune responses and small RNA interference (RNAi) systems. In prokaryotes, CRISPR/Cas systems function as an adaptive immune system to provide bacteria with resistance against invading phages and conjugative plasmids. Interestingly, CRISPR/Cas9 system was shown to interfere with eukaryotic DNA viruses and confer resistance against plant DNA viruses. The majority of the plant viruses have RNA genomes. The aim of this study is to test the ability of the newly discovered CRISPR/Cas13a immune system, that targets and cleaves single stranded RNA (ssRNA) in prokaryotes, to provide resistance against RNA viruses in plants. Here, I employ the CRISPR/Cas13a system for molecular interference against Turnip Mosaic Virus (TuMV), a plant RNA virus. The results of this study established the CRISPR/Cas13a as a molecular interference machinery against RNA viruses in plants. Specifically, my data show that the CRISPR/Cas13a machinery is able to interfere with and degrade the TuMV (TuMV-GFP) RNA genome. In conclusion, these data indicate that the CRISPR/Cas13 systems can be employed for engineering interference and durable resistance against RNA viruses in diverse plant species.
    • 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 in Plant Science, Frontiers Media SA, 2018-03-19) [Article]
      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.
    • 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. (Zoology, Elsevier BV, 2018-02-15) [Article]
      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 (Bioorganic & Medicinal Chemistry Letters, Elsevier BV, 2018-02-09) [Article]
      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.
    • Chromosome-scale comparative sequence analysis unravels molecular mechanisms of genome evolution between two wheat cultivars

      Thind, Anupriya Kaur; Wicker, Thomas; Mueller, Thomas; Ackermann, Patrick M; Steuernagel, Burkhard; Wulff, Brande B.H; Spannagl, Manuel; Twardziok, Sven O; Felder, Marius; Lux, Thomas; Mayer, Klaus F.X; Keller, Beat; Krattinger, Simon G.; International Wheat Genome Sequencing Consortium (Cold Spring Harbor Laboratory, 2018-02-08) [Preprint]
      Background: Recent improvements in DNA sequencing and genome scaffolding have paved the way to generate high-quality de novo assemblies of pseudomolecules representing complete chromosomes of wheat and its wild relatives. These assemblies form the basis to compare the evolutionary dynamics of wheat genomes on a megabase-scale. Results: Here, we provide a comparative sequence analysis of the 700-megabase chromosome 2D between two bread wheat genotypes, the old landrace Chinese Spring and the elite Swiss spring wheat line CH Campala Lr22a. There was a high degree of sequence conservation between the two chromosomes. Analysis of large structural variations revealed four large insertions/deletions (InDels) of >100 kb. Based on the molecular signatures at the breakpoints, unequal crossing over and double-strand break repair were identified as the evolutionary mechanisms that caused these InDels. Three of the large InDels affected copy number of NLRs, a gene family involved in plant immunity. Analysis of single nucleotide polymorphism (SNP) density revealed three haploblocks of 8 Mb, 9 Mb and 48 Mb with a 35-fold increased SNP density compared to the rest of the chromosome. Conclusions: This comparative analysis of two high-quality chromosome assemblies enabled a comprehensive assessment of large structural variations. The insight obtained from this analysis will form the basis of future wheat pan-genome studies.
    • A computational framework for cortical microtubule dynamics in realistically shaped plant cells

      Chakrabortty, Bandan; Blilou, Ikram; Scheres, Ben; Mulder, Bela M. (PLOS Computational Biology, Public Library of Science (PLoS), 2018-02-02) [Article]
      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.
    • 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. (Genome Biology, Springer Nature, 2018-01-04) [Article]
      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.
    • Quantitative phosphoproteomic analysis reveals shared and specific targets of Arabidopsis MPK3, MPK4 and MPK6

      Rayapuram, Naganand; Bigeard, Jean; Alhoraibi, Hanna; Bonhomme, Ludovic; Hesse, Anne-Marie; Vinh, Joelle; Hirt, Heribert; Pflieger, Delphine (Molecular & Cellular Proteomics, American Society for Biochemistry & Molecular Biology (ASBMB), 2017-11-22) [Article]
      In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4 and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. While some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4 and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. As a whole, 152 peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar non-fermenting (SnRK) protein kinases.
    • Genetics of Na+ exclusion and salinity tolerance in Afghani durum wheat landraces

      Shamaya, Nawar Jalal; Shavrukov, Yuri; Langridge, Peter; Roy, Stuart John; Tester, Mark A. (BMC Plant Biology, Springer Nature, 2017-11-21) [Article]
      Selecting for low concentration of Na+ in the shoot provides one approach for tackling salinity stress that adversely affects crop production. Novel alleles for Na+ exclusion can be identified and then introduced into elite crop cultivars. We have identified loci associated with lower Na+ concentration in leaves of durum wheat landraces originating from Afghanistan. Seedlings of two F2 populations derived from crossings between Australian durum wheat (Jandaroi) and two Afghani landraces (AUS-14740 and AUS-14752) were grown hydroponically and evaluated for Na+ and K+ concentration in the third leaf. High heritability was found for both third leaf Na+ concentration and the K+/Na+ ratio in both populations. Further work focussed on line AUS-14740. Bulk segregant analysis using 9 K SNP markers identified two loci significantly associated with third leaf Na+ concentration. Marker regression analysis showed a strong association between all traits studied and a favourable allele originating from AUS-14740 located on the long arm of chromosome 4B. The candidate gene in the relevant region of chromosome 4B is likely to be the high affinity K+ transporter B1 (HKT1;5-B1). A second locus associated with third leaf Na+ concentration was located on chromosome 3BL, with the favourable allele originating from Jandaroi; however, no candidate gene can be identified.
    • Identification of Proteins Involved in Salinity Tolerance in Salicornia bigelovii

      Salazar Moya, Octavio Ruben (2017-11) [Dissertation]
      Advisor: Tester, Mark A.
      Committee members: Ravasi, Timothy; Hirt, Heribert; Martinoia, Enrico
      With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for Salicornia research by providing a genome, transcriptomes, and organellar proteomes, contributing to salinity tolerance research overall. We identified a set of candidate genes for salinity tolerance with the aim of shedding some light on the mechanisms by which this plant thrives in highly saline environments.
    • Pea early-browning virus -mediated genome editing via the CRISPR/Cas9 system in Nicotiana benthamiana and Arabidopsis

      Ali, Zahir; Eid, Ayman; Ali, Shawkat; Mahfouz, Magdy M. (Virus Research, Elsevier BV, 2017-10-17) [Article]
      The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system has enabled efficient genome engineering in diverse plant species. However, delivery of genome engineering reagents, such as the single guide RNA (sgRNA), into plant cells remains challenging. Here, we report the engineering of Tobacco rattle virus (TRV) and Pea early browning virus (PEBV) to deliver one or multiple sgRNAs into Nicotiana benthamiana and Arabidopsis thaliana (Col-0) plants that overexpress a nuclear localization signal containing Cas9. Our data showed that TRV and PEBV can deliver sgRNAs into inoculated and systemic leaves, and this resulted in mutagenesis of the targeted genomic loci. Moreover, in N. benthamiana, PEBV-based sgRNA delivery resulted in more targeted mutations than TRV-based delivery. Our data indicate that TRV and PEBV can facilitate plant genome engineering and can be used to produce targeted mutations for functional analysis and other biotechnological applications across diverse plant species.Key message: Delivery of genome engineering reagents into plant cells is challenging and inefficient and this limit the applications of this technology in many plant species. RNA viruses such as TRV and PEBV provide an efficient tool to systemically deliver sgRNAs for targeted genome modification.