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Recent Submissions

  • Does zaxinone counteract strigolactones in shaping rice architecture?

    Wang, Jian You; Braguy, Justine; Al-Babili, Salim (Plant signaling & behavior, 2023-02-28) [Article]
    The cleavage of plant carotenoids leads to apocarotenoids, a group of metabolites including precursors of the hormones strigolactones (SLs) and abscisic acid, regulatory and signaling molecules. Zaxinone is a recently discovered apocarotenoid growth regulator that improves growth and suppress SL biosynthesis in rice (Oryza sativa). To test if zaxinone also counteracts the growth regulatory effects of SLs in rice, we co-supplied zaxinone and the synthetic SL analog rac-GR24 to the rice SL-deficient DWARF17 (d17) mutant. Results showed that co-application of GR24 and zaxinone still rescued d17 phenotype, indicating that zaxinone and GR24 act independently in regulating root and shoot growth and development in rice.
  • Integration of Apocarotenoid Profile and Expression Pattern of Carotenoid Cleavage Dioxygenases during Mycorrhization in Rice

    Votta, Cristina; Wang, Jian You; Cavallini, Nicola; Savorani, Francesco; Liew, Kit Xi; Lanfranco, Luisa; Al-Babili, Salim; Fiorilli, Valentina (Cold Spring Harbor Laboratory, 2023-02-26) [Preprint]
    Carotenoids are susceptible to degrading processes initiated by oxidative cleavage reactions mediated by Carotenoid Cleavage Dioxygenases that break their backbone, leading to products called apocarotenoids. These carotenoid-derived metabolites include the phytohormones abscisic acid and strigolactones, and different signaling molecules and growth regulators, which are utilized by plants to coordinate many aspects of their life. Several apocarotenoids have been recruited for the communication between plants and arbuscular mycorrhizal (AM) fungi and as regulators of the establishment of AM symbiosis. However, our knowledge on their biosynthetic pathways and the regulation of their pattern during AM symbiosis is still limited. In this study, we generated a qualitative and quantitative profile of apocarotenoids in roots and shoots of rice plants exposed to high/low phosphate concentrations, and upon AM symbiosis in a time course experiment covering different stages of growth and AM development. To get deeper insights in the biology of apocarotenoids during this plant-fungal symbiosis, we complemented the metabolic profiles by determining the expression pattern of CCD genes, taking advantage of chemometric tools. This analysis revealed the specific profiles of CCD genes and apocarotenoids across different stages of AM symbiosis and phosphate supply conditions, identifying novel markers at both local and systemic levels.
  • Transcriptome analysis of the phosphate starvation response sheds light on strigolactone biosynthesis in rice

    Haider, Imran; Yunmeng, Zhang; White, Fred; Li, Changsheng; Incitti, Roberto; Alam, Intikhab; Gojobori, Takashi; Ruyter-Spira, Carolien; Al-Babili, Salim; Bouwmeester, Harro J. (The Plant Journal, Wiley, 2023-02-12) [Article]
    Phosphorus (P) is a major element required for plant growth and development. To cope with P shortage, plants activate local and long-distance signaling pathways, such as the increase in the production and exudation of strigolactones (SLs). The role of the latter in mitigating P deficiency is, however, still largely unknown. To shed light on this, we studied the transcriptional response to P starvation and replenishment in wild-type rice and a SL mutant, dwarf10 (d10), and upon exogenous application of the synthetic SL GR24. P starvation resulted in major transcriptional alterations, such as the upregulation of P TRANSPORTER, SYG1/PHO81/XPR1 (SPX) and VACUOLAR PHOSPHATE EFFLUX TRANSPORTER. Gene Ontology (GO) analysis of the genes induced by P starvation showed enrichment in phospholipid catabolic process and phosphatase activity. In d10, P deficiency induced upregulation of genes enriched for sesquiterpenoid production, secondary shoot formation, and metabolic processes, including lactone biosynthesis. Furthermore, several genes induced by GR24 treatment shared the same GO terms with P starvation-induced genes, such as oxidation reduction, heme binding and oxidoreductase activity, hinting at the role that SLs play in the transcriptional reprogramming upon P starvation. Gene co-expression network analysis uncovered a METHYL TRANSFERASE that displayed co-regulation with known rice SL biosynthetic genes. Functional characterization showed that this gene encodes an enzyme catalyzing the conversion of carlactonoic acid to methyl carlactonoate. Our work provides a valuable resource to further studies on the response of crops to P deficiency and reveals a tool for the discovery of new SL biosynthetic genes.
  • The Arabidopsis D27-LIKE1 is a cis/cis/trans-β-carotene Isomerase that Contributes to Strigolactone Biosynthesis and Negatively Impacts Abscisic Acid Level

    Yang, Yu; Abuauf, Haneen W.; Song, Shanshan; Wang, Jian You; Alagoz, Yagiz; Moreno, Juan C.; Mi, Jianing; Ablazov, Abdugaffor; Jamil, Muhammad; Ali, Shawkat; Zheng, Xiongjie; Balakrishna, Aparna; Blilou, Ikram; Al-Babili, Salim (The Plant Journal, Wiley, 2023-01-05) [Article]
    The enzyme DWARF27 (D27) catalyzes the reversible isomerization of all-trans- into 9-cis-β-carotene, initiating strigolactone (SL) biosynthesis. Genomes of higher plants encode two D27-homologs, D27-like1 and -like2, with unknown functions. Here, we investigated the enzymatic activity and biological function of the Arabidopsis D27-like1. In vitro enzymatic assays and expression in Synechocystis sp. PCC6803 revealed a yet not reported 13-cis/15-cis/9-cis- and a 9-cis/all-trans-β-carotene isomerization. Although disruption of AtD27-like1 did not cause SL deficiency phenotypes, overexpression of AtD27-like1 in the d27 mutant restored the more-branching phenotype, indicating a contribution of AtD27-like1 to SL biosynthesis. Accordingly, generated d27 d27like1 double mutants showed more pronounced branching phenotype, compared to d27. The contribution of AtD27-like1 to SL biosynthesis is likely due to its formation of 9-cis-β-carotene that was present at higher levels in AtD27-like1 overexpressing lines. In contrast, AtD27-like1 expression correlated negatively with the content of 9-cis-violaxanthin, a precursor of abscisic acid (ABA), in shoots. Consistently, ABA levels were higher in shoots and also in dry seeds of the d27like1 and d27 d27like1 mutants. Transgenic lines expressing β-glucuronidase (GUS) driven by the AtD27LIKE1 promoter and transcript analysis of hormone-treated Arabidopsis seedlings unraveled that AtD27LIKE1 is expressed in different tissues and regulated ABA and auxin. Taken together, our work revealed a cis/cis-β-carotene isomerase that affects the content of both cis-carotenoid derived plant hormones ABA and SLs.
  • Emerging AI-Powered Technologies for Plant Tissue Imaging and Phenomics

    Lube, Vinicius (2022-12-20) [Dissertation]
    Advisor: Blilou, Ikram
    Committee members: Al-Babili, Salim; Liberale, Carlo; Pieterse, Corné
    Monitoring, tracking, and analyzing the dynamic growth of a living organism is essential to understanding its response to changes in its surrounding environment. Imaging tools to study these dynamics at spatial and temporal scales with optimal resolution rely on high-performance instrumentations. These systems are generally costly, stationary, and not flexible. In addition, performing non-destructive high-throughput phenotyping to extract roots' structural and morphological features remains challenging. We developed the MultipleXLab: a modular, mobile, and cost-effective robotic root imager to tackle these limitations. Among its advantages associated with a large field-of-view, integrated programmable plant-growth lighting, and high magnification with a high resolving power, the system is useful for a wide range of biological applications. We have also created the MultipleXLab Advanced; this configuration turns the system into a mobile environmental chamber by also featuring temperature control and automated irrigation. Another system we developed was the MultipleXLab Advanced Fluorescence to allow fluorescence imaging with a resolution that competes with a fluorescence binocular or even a fluorescence microscope. Furthermore, we have implemented various technologies and techniques to facilitate 3D imaging and quantification, ranging from X-ray micro-Computed Tomography to 3D segmentation of tissues, cells, and cellular compartments within the cell imaged using Confocal Laser Scanning Microscopy. For future research, we have conceptualized an upscaled system named MultipleXLabXL. This larger system will allow tracking, monitoring, and quantifying root growth of a much higher number of seedlings for more extended periods.
  • Are carotenoids the true colors of crop improvement?

    Moreno, Juan C; Al-Babili, Salim (New Phytologist, Wiley, 2022-12-07) [Article]
  • Metabolomics of plant root exudates: From sample preparation to data analysis.

    Salem, Mohamed A; Wang, Jian You; Al-Babili, Salim (Frontiers in plant science, Frontiers Media SA, 2022-12-06) [Article]
    Plants release a set of chemical compounds, called exudates, into the rhizosphere, under normal conditions and in response to environmental stimuli and surrounding soil organisms. Plant root exudates play indispensable roles in inhibiting the growth of harmful microorganisms, while also promoting the growth of beneficial microbes and attracting symbiotic partners. Root exudates contain a complex array of primary and specialized metabolites. Some of these chemicals are only found in certain plant species for shaping the microbial community in the rhizosphere. Comprehensive understanding of plant root exudates has numerous applications from basic sciences to enhancing crop yield, production of stress-tolerant crops, and phytoremediation. This review summarizes the metabolomics workflow for determining the composition of root exudates, from sample preparation to data acquisition and analysis. We also discuss recent advances in the existing analytical methods and future perspectives of metabolite analysis.
  • Perspectives on the metabolism of strigolactone rhizospheric signals

    Wang, Jian You; Braguy, Justine; Chen, Guan Ting Erica; Jamil, Muhammad; Balakrishna, Aparna; Berqdar, Lamis; Al-Babili, Salim (Frontiers in Plant Science, Frontiers Media SA, 2022-11-24) [Article]
    Strigolactones (SLs) are a plant hormone regulating different processes in plant development and adjusting plant’s architecture to nutrition availability. Moreover, SLs are released by plants to communicate with beneficial fungi in the rhizosphere where they are, however, abused as chemical cues inducing seed germination of root parasitic weeds, e.g. Striga spp., and guiding them towards host plants in their vicinity. Based on their structure, SLs are divided into canonical and non-canonical SLs. In this perspective, we describe the metabolism of root-released SLs and SL pattern in rice max1-900 mutants, which are affected in the biosynthesis of canonical SLs, and show the accumulation of two putative non-canonical SLs, CL+30 and CL+14. Using max1-900 and SL-deficient d17 rice mutants, we further investigated the metabolism of non-canonical SLs and their possible biological roles. Our results show that the presence and further metabolism of canonical and non-canonical SLs are particularly important for their role in rhizospheric interactions, such as that with root parasitic plants. Hence, we proposed that the root-released SLs are mainly responsible for rhizospheric communications and have low impact on plant architecture, which makes targeted manipulation of root-released SLs an option for rhizospheric engineering.
  • Canonical strigolactones are not the major determinant of tillering but important rhizospheric signals in rice

    Ito, Shinsaku; Braguy, Justine; Wang, Jian You; Yoda, Akiyoshi; Fiorilli, Valentina; Takahashi, Ikuo; Jamil, Muhammad; Felemban, Abrar; Miyazaki, Sho; Mazzarella, Teresa; Chen, Guan-Ting Erica; Shinozawa, Akihisa; Balakrishna, Aparna; Berqdar, Lamis; Rajan, Chakravarty; Ali, Shawkat; Haider, Imran; Sasaki, Yasuyuki; Yajima, Shunsuke; Akiyama, Kohki; Lanfranco, Luisa; Zurbriggen, Matias D.; Nomura, Takahito; Asami, Tadao; Al-Babili, Salim (Science Advances, American Association for the Advancement of Science (AAAS), 2022-11-02) [Article]
    Strigolactones (SLs) are a plant hormone inhibiting shoot branching/tillering and a rhizospheric, chemical signal that triggers seed germination of the noxious root parasitic plant Striga and mediates symbiosis with beneficial arbuscular mycorrhizal fungi. Identifying specific roles of canonical and noncanonical SLs, the two SL subfamilies, is important for developing Striga-resistant cereals and for engineering plant architecture. Here, we report that rice mutants lacking canonical SLs do not show the shoot phenotypes known for SL-deficient plants, exhibiting only a delay in establishing arbuscular mycorrhizal symbiosis, but release exudates with a significantly decreased Striga seed–germinating activity. Blocking the biosynthesis of canonical SLs by TIS108, a specific enzyme inhibitor, significantly lowered Striga infestation without affecting rice growth. These results indicate that canonical SLs are not the determinant of shoot architecture and pave the way for increasing crop resistance by gene editing or chemical treatment.
  • Effect of heat stress on in vitro pollen germination and pollen tube elongation of Chenopodium quinoa and wild relatives

    Morris, Angel (2022-11) [Thesis]
    Advisor: Tester, Mark A.
    Committee members: Blilou, Ikram; Chodasiewicz, Monika
    Climate change is one of the biggest challenges facing agriculture today. Transient or prolonged heat stress can be detrimental to plant reproductive development. The male gametophyte, pollen, is particularly sensitive to heat stress, resulting in sterile pollen (pre-anthesis) or deformed/stunted pollen tubes (post-anthesis). Quinoa (Chenopodium quinoa Willd.) has recently seen a rise in global interest due to its nutritional qualities, but global expansion of quinoa is partially hindered by its susceptibility to heat. It has been hypothesized that introgression of heat tolerance traits from wild relatives that occupy warmer environments can increase thermotolerance in quinoa. The goal of this research was to investigate the effect of heat stress on mature pollen grains from quinoa and its wild relatives, C. berlandieri and C. hircinum. To answer this question, several experiments were performed: 1. The nuclear number of the pollen of wild relatives was assessed to determine whether mature pollen grains were released at the trinucleate stage. All pollen was found to be trinucleate. 2. Pollen germination medium was optimized for four accessions: C. quinoa (QQ74; PI 614886), C. berlandieri var. zschackei (CB; BYU14118), and C. hircinum (CHA; Hircinum-069 and CHC; BYU17105). Optimal sucrose and PEG concentrations were determined to be: 5% sucrose/20% PEG for QQ74 and CB; 20% sucrose/0% PEG for CHA; and 10% sucrose/20% PEG for CHC. 3. Temperature optima for pollen germination for QQ74 was 32°C-36°C; CB was 30°C-34°C; CHA was 36°C; and CHC was 32°C -34°C. Overall, pollen from wild relatives was not found to be more heat-tolerant than pollen from domesticated quinoa. 4. Pollen tube elongation over time was observed for all four accessions at 34°C and 38°C, with CHA and QQ74 having the lowest decrease in rate at 38°C (35 and 45%, respectively). This study provides a new method for pollen collection for quinoa and its wild relatives, further optimizes the pollen germination media for QQ74, introduces pollen germination media for three wild accessions, and investigates the effect of heat stress on mature pollen grains. These observations can be employed in future studies investigating heat stress response of pollen in quinoa and its wild relatives.
  • A single amino acid change can alter the specificity of the multi-allelic wheat stem rust resistance locus SR9

    Zhang, Jianping; Nirmala, Jayaveeramuthu; Chen, Shisheng; Jost, Matthias; Steuernagel, Burkhard; Karafiátová, Miroslava; Hewitt, Timothy; Li, Hongna; Edae, Erena; Sharma, Keshav; Hoxha, Sami; Bhatt, Dhara; Antoniou-Kourounioti, Rea; Dodds, Peter; Wulff, Brande B. H.; Doležel, Jaroslav; Ayliffe, Michael; Hiebert, Colin; McIntosh, Robert; Dubcovsky, Jorge; Zhang, Peng; Rouse, Matthew; Lagudah, Evans (Research Square Platform LLC, 2022-10-30) [Preprint]
    Most resistance genes thus far isolated from wheat have a very limited number of functional alleles, with the exception of the powdery mildew PM3 resistance locus. Here we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9, representing the largest multi-allelic rust resistance locus in common wheat. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) at the locus were characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation were used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1, Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing a previously unreported motif at their N termini and an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differed from SR9B by a single amino acid. SR9B and SR9G resistance proteins were also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences.
  • New function of JKD in plant development and defense

    Zhang, Yang (2022-10-19) [Dissertation]
    Advisor: Blilou, Ikram
    Committee members: Hirt, Heribert; Liberale, Carlo; Bendahmane, Abdelhafid
    For optimal growth, plants have evolved strategies to integrate environmental signals to coordinate complex developmental and defensive processes to cope with the changing surroundings. Under challenges, plants prioritize their defense over growth. This trade-off involves complex interactions between multiple hormonal pathways and developmental networks. We discovered that JACKDAW (JKD), the core component of the SHORTROOT (SHR)-SCARECROW (SCR)- JKD plant developmental regulatory network is linking defense responses to the developmental programming. Unlike the well-studied function of JKD in root development, its function in leaves is yet to be understood. We found that JKD is expressed on the abaxial side of the leaf ground tissue. It has conserved functions in promoting SHR nuclear retention and restricting cyclinD6 expression in the leaf. Additionally, JKD has a function in leaf internal architecture establishment, including suppression of the bundle sheath cell division and shaping of the leaf ground tissue. We also found that SHR is a universal asymmetric cell division (ACD) activator, as ectopic SHR expression in the leaf ground tissue promotes stomata development via promoting the ACD to produce more stomata precursor cells. We showed that the knockout mutant of JKD has larger rosettes and better photosynthesis capacity, while the basal defense level and resistance to Botrytis cinerea, a necrotrophic pathogen, are enhanced. Our transcriptome and transcription studies revealed that JKD suppresses the expression of the plant defense hormone Jasmonic acid (JA) response genes and is itself downregulated by JA. This suggests that JKD is involved in the JA signaling, which mediates defense responses for wounding and herbivore attacks. Together, our study indicates that the loss of JKD uncoupled the plant growth-defense trade-off. JKD is a new link between plant development and defense. To verify whether this function of JKD is conserved in crops, JKD orthologues in tomatoes are identified, CRISPR-Cas9 and TILLING mutants are created and analyzed. The results showed that the functions of JKD in root development and resistance to botrytis are conserved. The broad presence of JKD orthologs makes them a great target for molecular breeding to generate crops that do not have to sacrifice their normal growth to defense response.
  • ZAXINONE SYNTHASE 2 regulates growth and arbuscular mycorrhizal symbiosis in rice

    Ablazov, Abdugaffor; Votta, Cristina; Fiorilli, Valentina; Wang, Jian You; Aljedaani, Fatimah R.; Jamil, Muhammad; Balakrishna, Aparna; Balestrini, Raffaella; Liew, Kit Xi; Rajan, Chakravarthy; Berqdar, Lamis; Blilou, Ikram; Lanfranco, Luisa; Al-Babili, Salim (Plant Physiology, Oxford University Press (OUP), 2022-10-12) [Article]
    Carotenoid cleavage, catalyzed by CAROTENOID CLEAVAGE DIOXYGENASEs (CCDs), provides signaling molecules and precursors of plant hormones. Recently, we showed that zaxinone, a apocarotenoid metabolite formed by the CCD ZAXINONE SYNTHASE (ZAS), is a growth regulator required for normal rice (Oryza sativa) growth and development. The rice genome encodes three OsZAS homologs, called here OsZAS1b, OsZAS1c, and OsZAS2, with unknown functions. Here, we investigated the enzymatic activity, expression pattern, and subcellular localization of OsZAS2 and generated and characterized loss-of-function CRISPR/Cas9-Oszas2 mutants. We show that OsZAS2 formed zaxinone in vitro. OsZAS2 was predominantly localized in plastids and mainly expressed under phosphate starvation. Moreover, OsZAS2 expression increased during mycorrhization, specifically in arbuscule-containing cells. Oszas2 mutants contained lower zaxinone content in roots and exhibited reduced root and shoot biomass, fewer tillers, and higher strigolactone (SL) levels. Exogenous zaxinone application repressed SL biosynthesis and partially rescued the growth retardation of the Oszas2 mutant. Consistent with the OsZAS2 expression pattern, Oszas2 mutants displayed a lower frequency of AM colonization. In conclusion, OsZAS2 is a zaxinone-forming enzyme that, similar to previously reported OsZAS, determines rice growth, architecture and SL content and is required for optimal mycorrhization.
  • Molecular perception and metabolic rewiring of the host plant by beneficial microbe Enterobacter sp. SA187

    Alzayed, Waad S. (2022-10) [Thesis]
    Advisor: Hirt, Heribert
    Committee members: Aranda, Manuel; Wulff, Brande B. H.; Sheikh, Arsheed
    Among abiotic stresses, salinity is considered the main limiting stress that negatively affects plant growth and reduces productivity worldwide. To overcome this challenge, a sustainable solution such as plant growth-promoting bacteria (PGPB) can be used to meet the increasing demand for food. The desert microbe Enterobacter sp SA187, an endophytic PGPB, induces salt tolerance in both model plant and crops. The interaction between SA187 and the host plant triggers the sulfur pathway in the bacteria which then provides multiple sulfur-containing compounds to its host plant. However, the molecular sensor of these compounds in the host plant is not known. Here, we show that SA187 activates the plant target of rapamycin (TOR) pathway. The beneficial effect of SA187 was lost in TOR mutants like raptor, and by the application of TOR inhibitor AZD8055. Next, we show that SA187 modulates the one- carbon (1C) metabolism of the host plant consisting of methionine and folate cycles. The beneficial effect of SA187 was compromised by using chemical inhibitors of folate cycle like Methotrexate (MTX) and Sulfadiazine (SDZ). The intermediates of the 1C metabolism like Homocysteine and S-adenosyl methionine (SAM) showed similar beneficial effects as SA187 colonized plants. Finally, we showed that SA187 enhances 1C metabolism activity by increasing methylation index (SAM/SAH ratio) in the plants. Taken together, we could show that host TOR-1C axis is essential for plant salt tolerance by SA187.
  • 9-cis-β-Apo-10ʹ-carotenal is the precursor of strigolactones in planta

    Chen, Guan-Ting Erica; Wang, Jian You; Jamil, Muhammad; Braguy, Justine; Al-Babili, Salim (Planta, Springer Science and Business Media LLC, 2022-09-24) [Article]
    Strigolactones (SLs) are plant hormone that regulates plant architecture and mediates rhizospheric communications. Previous in vitro studies using heterogously produced enzymes unraveled the conversion of all-trans-β-carotene via the intermediate 9-cis-β-apo-10ʹ-carotenal into the SL precursor carlactone. However, a direct evidence for the formation of SLs from 9-cis-β-apo-10ʹ-carotenal is still missing. To provide this evidence, we supplied rice seedlings with 13C-labeled 9-cis-β-apo-10ʹ-carotenal and analyzed their SLs by LC–MS. Our results show that 9-cis-β-apo-10ʹ-carotenal is the SL precursor in planta and reveal, for the first time, the application of labeled long-chain apocarotenoids as a promising approach to investigate apocarotenoid metabolism and the genesis of carotenoid-derived growth regulators and signaling molecules.
  • De novo assembly of the Tamarindus indica genome as part of the Kingdom of Saudi Arabia Native Genome Project

    Navarrete Rodriguez, Maria Eugenia (2022-08-10) [Thesis]
    Advisor: Wing, Rod Anthony
    Committee members: Blilou, Ikram; Merzaban, Jasmeen
    The Kingdom of Saudi Arabia Native Genome project aims to generate genomic resources for all the plants, animals, and associated microbiome species in the Kingdom. Tamarindus indica was pointed out by the MEWA as an endangered native species in the KSA and forms part of the first 15 plant species to be studied in the NGP. A voucher tree was identified in the Rijal Almaa region, from which leaf samples were collected. HMW DNA was extracted from this tissue and sequenced using CCS with the Pac-Bio Sequel II platform. The raw data obtained from the sequencing was assembled using HIFIASM, contaminant contigs were removed, and the 15 largest contigs were selected as the primary T. indica assembly. The genome sequence of Sindora glabra was used as reference guide for primary scaffolding, and T. indica optical maps were used for super-scaffolding. Secondary scaffolding utilized Hi-C data to produce a chromosome level assembly of the T. indica genome. Transposable element analysis and a preliminary annotation were performed on the final assembly. This project represents the first step in studying T. indica for the NGP. The final assembly can be used as a foundation for more genetic studies on this species, as a possible reference for other legume species from the Detarioideae family, and for Neo-domestication and reforestation. The pipeline developed for this project can also be used as a template for sequencing and assembling the remaining species in the NGP.
  • Precise genomic deletions and insertions via paired prime editing for crop bioengineering

    Moreno-Ramírez, Jose Luis (2022-08) [Thesis]
    Advisor: Mahfouz, Magdy M.
    Committee members: Blilou, Ikram; Lauersen, Kyle J.
    CRISPR/Cas has been developed for targeted mutagenesis in diverse species, including plants. However, precise genome editing via homology-directed repair (HDR) is inefficient in plants, limiting our ability to make large deletions or insertions in the plant genomes. Prime editing increases the control over the desired editing and allows the precise introduction of all types of mutations, including insertion, deletions, and all possible base conversions, albeit at low efficiencies. Here, we designed a dual prime editing system to generate large deletions and precise insertions of sequences by repairing template complementarity. We coupled dual pegRNA with Cas9 nickase (nCas9) to generate deletions and insertions. In another modality, we used dual pegRNA with wild-type Cas9 to generate double-stranded breaks to improve the editing at the targeted sites. We tested dual pegRNAs to delete the last exon in OsCCD7, delete the microRNA targeted sequence in OsIPA, and insert the T7 promoter in the 3'UTR of OsALS. Our results showed a high frequency of targeted insertion of the T7 promoter sequence in the 3'UTR of OsALS with wtCas9 and nCas9. Sanger sequencing analysis showed partial deletions at the targeted locus. Further improvements in the designs of pegRNAs will increase the precise genome insertions and deletions in plants.
  • Identification of novel, bacteria assisted, iron acquisition strategies in Arabidopsis thaliana

    García Ramírez, Gabriel Xicoténcatl (2022-07) [Thesis]
    Advisor: Hirt, Heribert
    Committee members: Wulff, Brande B. H.; Rosado, Alexandre S.
    Iron is abundant in most agricultural systems, however it is also one of the three most limiting nutrients for crop growth. This can be attributed to iron’s low solubility in aerobic and alkaline conditions, rendering it non-bioavailable for plant uptake. With around 1/3 of the world arable land presenting conditions that limit iron solubility, deciphering the plant machinery behind iron uptake and identifying microbial benefits to iron deficiency are of major interest. In this work, 33 endophytic bacterial isolates previously isolated from three regions in Jordan were tested for iron stress alleviation of Arabidopsis plants with the goal of identifying novel interaction mechanisms. On media with a low concentration of bioavailable iron, 11 isolates were found to be beneficial, while 15 isolates behaved in a pathogenic manner, reducing plant growth in both control as well as limited iron conditions. Beneficial isolates were then tested in plant assays with non-bioavailable iron, we concluded that our strains are also beneficial in these conditions. To further characterize the interaction between the beneficial strains and Arabidopsis plants, gene expression was assessed for plants colonized with select strains. The highest expression of iron-deficiency response genes was at day 6. The increase in expression was also met with an increase in colonization of plants at day 6. Mutant studies revealed that the beneficial effect by the bacterial isolates is dependent on the coumarin pathway, with mutants in FERULOYL-COA 6-HYDROXYLASE 1, f6’h1, showing a drastic decrease in fresh weight compared to wild type counterparts. We also discovered the phytohormone abscisic acid as an important contributor to iron stress alleviation by the beneficial isolates. Colonization assays as well as additional mutant studies will be necessary to further assess the effect of f6’h1 mutants on plant-microbe interaction as well as ABA’s role in plants under iron deficient conditions.
  • Multiple Alr genes exhibit allorecognition-associated variation in the colonial cnidarian Hydractinia

    Rodriguez-Valbuena, Henry; Gonzalez-Muñoz, Andrea; Cadavid, Luis F (Immunogenetics, Springer Science and Business Media LLC, 2022-06-27) [Article]
    The genetics of allorecognition has been studied extensively in inbred lines of Hydractinia symbiolongicarpus, in which genetic control is attributed mainly to the highly polymorphic loci allorecognition 1 (Alr1) and allorecognition 2 (Alr2), located within the Allorecognition Complex (ARC). While allelic variation at Alr1 and Alr2 can predict the phenotypes in inbred lines, these two loci do not entirely predict the allorecognition phenotypes in wild-type colonies and their progeny, suggesting the presence of additional uncharacterized genes that are involved in the regulation of allorecognition in this species. Comparative genomics analyses were used to identify coding sequence differences from assembled chromosomal intervals of the ARC and from genomic scaffold sequences between two incompatible H. symbiolongicarpus siblings from a backcross population. New immunoglobulin superfamily (Igsf) genes are reported for the ARC, where five of these genes are closely related to the Alr1 and Alr2 genes, suggesting the presence of multiple Alr-like genes within this complex. Complementary DNA sequence evidence revealed that the allelic polymorphism of eight Igsf genes is associated with allorecognition phenotypes in a backcross population of H. symbiolongicarpus, yet that association was not found between parental colonies and their offspring. Alternative splicing was found as a mechanism that contributes to the variability of these genes by changing putative activating receptors to inhibitory receptors or generating secreted isoforms of allorecognition proteins. Our findings demonstrate that allorecognition in H. symbiolongicarpus is a multigenic phenomenon controlled by genetic variation in at least eight genes in the ARC complex.
  • Evaluation of the Biostimulant Activity of Zaxinone Mimics (MiZax) in Crop Plants

    Wang, Jian You; Jamil, Muhammad; Hossain, Md. Golap; Chen, Guan-Ting Erica; Berqdar, Lamis; Ota, Tsuyoshi; Blilou, Ikram; Asami, Tadao; Al-Solimani, Samir Jamil; Mousa, Magdi Ali Ahmed; Al-Babili, Salim (Frontiers in Plant Science, Frontiers Media SA, 2022-06-16) [Article]
    Global food security is a critical concern that needs practical solutions to feed the expanding human population. A promising approach is the employment of biostimulants to increase crop production. Biostimulants include compounds that boost plant growth. Recently, mimics of zaxinone (MiZax) were shown to have a promising growth-promoting effect in rice (Oryza sativa). In this study, we investigated the effect of MiZax on the growth and yield of three dicot horticultural plants, namely, tomato (Solanum lycopersicum), capsicum (Capsicum annuum), and squash (Cucurbita pepo) in different growth environments, as well as on the growth and development of the monocot date palm (Phoenix dactylifera), an important crop in the Middle East. The application of MiZax significantly enhanced plant height, flower, and branch numbers, fruit size, and total fruit yield in independent field trials from 2020 to 2021. Importantly, the amount of applied MiZax was far less than that used with the commercial compound humic acid, a widely used biostimulant in horticulture. Our results indicate that MiZax have significant application potential to improve the performance and productivity of horticultural crops.

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