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  • 2′,3′-cAMP treatment mimics the stress molecular response in Arabidopsis thaliana

    Chodasiewicz, Monika; Kerber, Olga; Gorka, Michal; Moreno, Juan C; Maruri Lopez, Israel; Minen, Romina I; Sampathkumar, Arun; Nelson, Andrew D L; Skirycz, Aleksandra (Plant Physiology, Oxford University Press (OUP), 2022-01-19) [Article]
    The role of the RNA degradation product 2’,3’-cyclic adenosine monophosphate (2’,3’-cAMP) is poorly understood. Recent studies have identified 2’,3’-cAMP in plant material and determined its role in stress signaling. The level of 2’,3’-cAMP increases upon wounding, in the dark, and under heat, and 2’,3’-cAMP binding to an RNA-binding protein, Rbp47b, promotes stress granule (SG) assembly. To gain further mechanistic insights into the function of 2’,3’-cAMP, we used a multi-omics approach by combining transcriptomics, metabolomics, and proteomics to dissect the response of Arabidopsis (Arabidopsis thaliana) to 2’,3’-cAMP treatment. We demonstrated that 2’,3’-cAMP is metabolized into adenosine, suggesting that the well-known cyclic nucleotide–adenosine pathway of human cells might also exist in plants. Transcriptomics analysis revealed only minor overlap between 2’,3’-cAMP- and adenosine-treated plants, suggesting that these molecules act through independent mechanisms. Treatment with 2’,3’-cAMP changed the levels of hundreds of transcripts, proteins, and metabolites, many previously associated with plant stress responses, including protein and RNA degradation products, glucosinolates, chaperones, and SG components. Finally, we demonstrated that 2’,3’-cAMP treatment influences the movement of processing bodies, confirming the role of 2’,3’-cAMP in the formation and motility of membraneless organelles.
  • A manipulation of carotenoid metabolism influence biomass partitioning and fitness in tomato.

    Mi, Jianing; Vallarino, José G; Petřík, Ivan; Novák, Ondřej; Correa, Sandra Marcela; Chodasiewicz, Monika; Havaux, Michel; Rodriguez-Concepcion, Manuel; Al-Babili, Salim; Fernie, Alisdair R; Skirycz, Aleksandra; Moreno, Juan C (Metabolic engineering, Elsevier BV, 2022-01-15) [Article]
    Improving yield, nutritional value and tolerance to abiotic stress are major targets of current breeding and biotechnological approaches that aim at increasing crop production and ensuring food security. Metabolic engineering of carotenoids, the precursor of vitamin-A and plant hormones that regulate plant growth and response to adverse growth conditions, has been mainly focusing on provitamin A biofortification or the production of high-value carotenoids. Here, we show that the introduction of a single gene of the carotenoid biosynthetic pathway in different tomato cultivars induced profound metabolic alterations in carotenoid, apocarotenoid and phytohormones pathways. Alterations in isoprenoid- (abscisic acid, gibberellins, cytokinins) and non-isoprenoid (auxin and jasmonic acid) derived hormones together with enhanced xanthophyll content influenced biomass partitioning and abiotic stress tolerance (high light, salt, and drought), and it caused an up to 77% fruit yield increase and enhanced fruit's provitamin A content. In addition, metabolic and hormonal changes led to accumulation of key primary metabolites (e.g. osmoprotectants and antiaging agents) contributing with enhanced abiotic stress tolerance and fruit shelf life. Our findings pave the way for developing a new generation of crops that combine high productivity and increased nutritional value with the capability to cope with climate change-related environmental challenges.
  • Genome sequences of three Aegilops species of the section Sitopsis reveal phylogenetic relationships and provide resources for wheat improvement

    Avni, Raz; Lux, Thomas; Minz-Dub, Anna; Millet, Eitan; Sela, Hanan; Distelfeld, Assaf; Deek, Jasline; Yu, Guotai; Steuernagel, Burkhard; Pozniak, Curtis; Ens, Jennifer; Gundlach, Heidrun; Mayer, Klaus F. X.; Himmelbach, Axel; Stein, Nils; Mascher, Martin; Spannagl, Manuel; Wulff, Brande B. H.; Sharon, Amir (The Plant Journal, Wiley, 2022-01-08) [Article]
    Aegilops is a close relative of wheat (Triticum spp.), and Aegilops species in the section Sitopsis represent a rich reservoir of genetic diversity for improvement of wheat. To understand their diversity and advance their utilization, we produced whole-genome assemblies of Ae. longissima and Ae. speltoides. Whole-genome comparative analysis, along with the recently sequenced Ae. sharonensis genome, showed that the Ae. longissima and Ae. sharonensis genomes are highly similar and most closely related to the wheat D subgenome. By contrast, the Ae. speltoides genome is more closely related to the B subgenome. Haplotype block analysis supported the idea that Ae. speltoides is the closest to the wheat B subgenome and highlighted variable and similar genomic regions between the three Aegilops species and wheat. Genome-wide analysis of nucleotide-binding leucine-rich repeat (NLR) genes revealed species-specific and lineage-specific NLR genes and variants, demonstrating the potential of Aegilops genomes for wheat improvement.
  • CS-Cells: A CRISPR-Cas12 DNA Device to Generate Chromosome-Shredded Cells for Efficient and Safe Molecular Biomanufacturing

    Pantoja Angles, Aarón; Ali, Zahir; Mahfouz, Magdy M. (ACS Synthetic Biology, American Chemical Society (ACS), 2022-01-03) [Article]
    Synthetic biology holds great promise for translating ideas into products to address the grand challenges facing humanity. Molecular biomanufacturing is an emerging technology that facilitates the production of key products of value, including therapeutics and select chemical compounds. Current biomanufacturing technologies require improvements to overcome limiting factors, including efficient production, cost, and safe release; therefore, developing optimum chassis for biomolecular manufacturing is of great interest for enabling diverse synthetic biology applications. Here, we harnessed the power of the CRISPR-Cas12 system to design, build, and test a DNA device for genome shredding, which fragments the native genome to enable the conversion of bacterial cells into nonreplicative, biosynthetically active, and programmable molecular biomanufacturing chassis. As a proof of concept, we demonstrated the efficient production of green fluorescent protein and violacein, an antimicrobial and antitumorigenic compound. Our CRISPR-Cas12-based chromosome-shredder DNA device has built-in biocontainment features providing a roadmap for the conversion of any bacterial cell into a chromosome-shredded chassis amenable to high-efficiency molecular biomanufacturing, thereby enabling exciting and diverse biotechnological applications.
  • On the effects of CO2 atmosphere in the pyrolysis of Salicornia bigelovii

    Aljaziri, Jinan; Gautam, Ribhu; Alturkistani, Sultan H.; Fiene, Gabriele; Tester, Mark A.; Sarathy, Mani (Bioresource Technology Reports, Elsevier BV, 2022-01) [Article]
    This study focuses on understanding the effects of a CO$_2$ atmosphere on the pyrolysis of $\textit{Salicornia bigelovii}$ by performing a detailed kinetic analysis and investigating the pyrolysis products. In comparison to N$_2$ pyrolysis, CO$_2$ pyrolysis increased the amounts of acids, phenols, amines/amides and N-aromatics in the bio-oil. Biochar showed a 6.5% increase in carbon and a 5.8% decrease in oxygen due to the presence of CO$_2$ in the pyrolysis atmosphere. CO$_2$ also inhibited the volatilization of certain functional groups, such as phenols, tertiary alcohols and aromatics from the biochar, and the surface area of the biochar was 12 times larger than pyrolysis in N$_2$ atmosphere. Pyrolysis in CO$_2$ led to an increase in the average apparent activation energy from 146.5 kJ mol$^{−1}$ in N$_2$ to 163.4 kJ mol$^{−1}$. The kinetic equation was found to conform to a three dimensional diffusion mechanism. Finally, the pre-exponential factor was determined for each reaction.
  • Exploring the Diversity and Regulation of Apocarotenoid Metabolic Pathways in Plants

    Zheng, Xiongjie; Yang, Yu; Al-Babili, Salim (Frontiers in Plant Science, Frontiers Media SA, 2021-12-10) [Article]
    In plants, carotenoids are subjected to enzyme-catalyzed oxidative cleavage reactions as well as to non-enzymatic degradation processes, which produce various carbonyl products called apocarotenoids. These conversions control carotenoid content in different tissues and give rise to apocarotenoid hormones and signaling molecules, which play important roles in plant growth and development, response to environmental stimuli, and in interactions with surrounding organisms. In addition, carotenoid cleavage gives rise to apocarotenoid pigments and volatiles that contribute to the color and flavor of many flowers and several fruits. Some apocarotenoid pigments, such as crocins and bixin, are widely utilized as colorants and additives in food and cosmetic industry and also have health-promoting properties. Considering the importance of this class of metabolites, investigation of apocarotenoid diversity and regulation has increasingly attracted the attention of plant biologists. Here, we provide an update on the plant apocarotenoid biosynthetic pathway, especially highlighting the diversity of the enzyme carotenoid cleavage dioxygenase 4 (CCD4) from different plant species with respect to substrate specificity and regioselectivity, which contribute to the formation of diverse apocarotenoid volatiles and pigments. In addition, we summarize the regulation of apocarotenoid metabolic pathway at transcriptional, post-translational, and epigenetic levels. Finally, we describe inter- and intraspecies variation in apocarotenoid production observed in many important horticulture crops and depict recent progress in elucidating the genetic basis of the natural variation in the composition and amount of apocarotenoids. We propose that the illustration of biochemical, genetic, and evolutionary background of apocarotenoid diversity would not only accelerate the discovery of unknown biosynthetic and regulatory genes of bioactive apocarotenoids but also enable the identification of genetic variation of causal genes for marker-assisted improvement of aroma and color of fruits and vegetables and CRISPR-based next-generation metabolic engineering of high-value apocarotenoids.
  • Rational design of Striga hermonthica-specific seed germination inhibitors

    Zarban, Randa Alhassan Yahya; Hameed, Umar Farook Shahul; Jamil, Muhammad; Ota, Tsuyoshi; Wang, Jian You; Arold, Stefan T.; Asami, Tadao; Al-Babili, Salim (Plant Physiology, Oxford University Press (OUP), 2021-11-27) [Article]
    The obligate hemiparasitic weed Striga hermonthica grows on cereal roots and presents a severe threat to global food security by causing enormous yield losses, particularly in Sub-Saharan Africa. The rapidly increasing Striga seed bank in infested soils provides a major obstacle in controlling this weed. Striga seeds require host derived strigolactones (SLs) for germination, and corresponding antagonists could be used as germination inhibitors. Recently, we demonstrated that the common detergent Triton X-100 is a specific inhibitor of Striga seed germination by binding non-covalently to its receptor, Striga hermonthica HYPO-SENSITIVE TO LIGHT 7 (ShHTL7), without blocking the rice (Oryza sativa) SL receptor DWARF14 (OsD14). Moreover, triazole ureas, the potent covalently binding antagonists of rice SL perception with much higher activity towards OsD14, showed inhibition of Striga but were less specific. Considering that Triton X-100 is not suitable for field application and by combining structural elements of Triton and triazole urea, we developed two hybrid compounds, KK023-N1 and KK023-N2, as potential Striga-specific germination inhibitors. Both compounds blocked the hydrolysis activity of ShHTL7 but did not affect that of OsD14. Binding of KK023-N1 diminished ShHTL7 interaction with Striga hermonthica MORE AXILLARY BRANCHING 2 (ShMAX2), a major component in SL signal transduction, and increased ShHTL7 thermal specificity. Docking studies indicate that KK023-N1 binding is not covalent but is caused by hydrophobic interactions. Finally, in vitro and greenhouse tests revealed specific inhibition of Striga seed germination, which led to a 38% reduction in Striga infestation in pot experiments. These findings reveal that KK023-N1 is a potential candidate for combating Striga and a promising basis for rational design and development of further Striga-specific herbicides.
  • The barley immune receptor Mla recognizes multiple pathogens and contributes to host range dynamics

    Bettgenhaeuser, Jan; Hernández-Pinzón, Inmaculada; Dawson, Andrew M.; Gardiner, Matthew; Green, Phon; Taylor, Jodie; Smoker, Matthew; Ferguson, John N.; Emmrich, Peter; Hubbard, Amelia; Bayles, Rosemary; Waugh, Robbie; Steffenson, Brian J.; Wulff, Brande B. H.; Dreiseitl, Antonín; Ward, Eric R.; Moscou, Matthew J. (Nature Communications, Springer Science and Business Media LLC, 2021-11-25) [Article]
    Crop losses caused by plant pathogens are a primary threat to stable food production. Stripe rust (Puccinia striiformis) is a fungal pathogen of cereal crops that causes significant, persistent yield loss. Stripe rust exhibits host species specificity, with lineages that have adapted to infect wheat and barley. While wheat stripe rust and barley stripe rust are commonly restricted to their corresponding hosts, the genes underlying this host specificity remain unknown. Here, we show that three resistance genes, Rps6, Rps7, and Rps8, contribute to immunity in barley to wheat stripe rust. Rps7 cosegregates with barley powdery mildew resistance at the Mla locus. Using transgenic complementation of different Mla alleles, we confirm allele-specific recognition of wheat stripe rust by Mla. Our results show that major resistance genes contribute to the host species specificity of wheat stripe rust on barley and that a shared genetic architecture underlies resistance to the adapted pathogen barley powdery mildew and non-adapted pathogen wheat stripe rust.
  • High-efficiency retron-mediated single-stranded DNA production in plants

    Jiang, Wenjun; Rao, Gundra Sivakrishna; Aman, Rashid; Butt, Haroon; Kamel, Radwa; Sedeek, Khalid Elwy Mohamed; Mahfouz, Magdy M. (Submitted to Biotechnology Journal, Authorea, Inc., 2021-11-17) [Preprint]
    Background: Retrons are a class of retroelements that produce multicopy single-stranded DNA (msDNA) and participate in anti-phage defenses in bacteria. Retrons have been harnessed for the over-production of single-stranded DNA (ssDNA), genome engineering, and directed evolution in bacteria, yeast, and mammalian cells. However, no studies have shown retron-mediated ssDNA production in plants, which could unlock potential applications in plant biotechnology. For example, ssDNA can be used as a template for homology-directed repair (HDR) in several organisms. However, current gene editing technologies rely on the physical delivery of synthetic ssDNA, which limits their applications. Main methods and major results: Here, we demonstrated retron-mediated over-production of ssDNA in Nicotiana benthamiana. Additionally, we tested different retron architectures for improved ssDNA production and identified a new retron architecture that resulted in greater ssDNA abundance. Furthermore, co-expression of the gene encoding the ssDNA-protecting protein VirE2 from Agrobacterium tumefaciens with the retron systems resulted in a 10.7-fold increase in ssDNA production in vivo. We also demonstrated CRISPR-retron-coupled ssDNA over-production and targeted HDR in N. benthamiana. Conclusion: We present an efficient approach for in vivo ssDNA production in plants, which can be harnessed for biotechnological applications.
  • In vivo identification of putative CPK5 substrates in Arabidopsis thaliana

    Yip Delormel, Tiffany; Avila-Ospina, Liliana; Davanture, Marlène; Zivy, Michel; Lang, Julien; Valentin, Nicolas; Rayapuram, Naganand; Hirt, Heribert; Colcombet, Jean; Boudsocq, Marie (Plant Science, Elsevier BV, 2021-11-17) [Article]
    Calcium signaling mediates most developmental processes and stress responses in plants. Among plant calcium sensors, the calcium-dependent protein kinases display a unique structure harboring both calcium sensing and kinase responding activities. AtCPK5 is an essential member of this family in Arabidopsis that regulates immunity and abiotic stress tolerance. To understand the underlying molecular mechanisms, we implemented a biochemical approach to identify in vivo substrates of AtCPK5. We generated transgenic lines expressing a constitutively active form of AtCPK5 under the control of a dexamethasone-inducible promoter. Lines expressing a kinase-dead version were used as a negative control. By comparing the phosphoproteome of the kinase-active and kinase-dead lines upon dexamethasone treatment, we identified 5 phosphopeptides whose abundance increased specifically in the kinase-active lines. Importantly, we showed that all 5 proteins were phosphorylated in vitro by AtCPK5 in a calcium-dependent manner, suggesting that they are direct targets of AtCPK5. We also detected several interaction patterns between the kinase and the candidates in the cytosol, membranes or nucleus, consistent with the ubiquitous localization of AtCPK5. Finally, we further validated the two phosphosites S245 and S280 targeted by AtCPK5 in the E3 ubiquitin ligase ATL31. Altogether, those results open new perspectives to decipher AtCPK5 biological functions.
  • Coordinated bacterial and plant sulfur metabolism in Enterobacter sp. SA187–induced plant salt stress tolerance

    Andres-Barrao, Cristina; Alzubaidy, Hanin S.; Jalal, Rewaa S.; Mariappan, Kiruthiga; Zélicourt, Axel de; Bokhari, Ameerah; Artyukh, Olga; Alwutayd, Khairiah Mubarak Saleem; Rawat, Anamika; Shekhawat, Kirti; Almeida-Trapp, Marília; Saad, Maged; Hirt, Heribert (Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, 2021-11-12) [Article]
    Enterobacter sp. SA187 is a root endophytic bacterium that maintains growth and yield of plants under abiotic stress conditions. In this work, we compared the metabolic wirings of Arabidopsis and SA187 in the free-living and endophytic interaction states. The interaction of SA187 with Arabidopsis induced massive changes in bacterial gene expression for chemotaxis, flagellar biosynthesis, quorum sensing, and biofilm formation. Besides modification of the bacterial carbon and energy metabolism, various nutrient and metabolite transporters and the entire sulfur pathway were up-regulated. Under salt stress, Arabidopsis resembled plants under sulfate starvation but not when colonized by SA187, which reprogramed the sulfur regulon of Arabidopsis. In accordance, salt hypersensitivity of multiple Arabidopsis sulfur metabolism mutants was partially or completely rescued by SA187 as much as by the addition of sulfate, L-cysteine, or L-methionine. Many components of the sulfur metabolism that are localized in the chloroplast were partially rescued by SA187. Finally, salt-induced accumulation of reactive oxygen species as well as the hypersensitivity of LSU mutants were suppressed by SA187. LSUs encode a central regulator linking sulfur metabolism to chloroplast superoxide dismutase activity. The coordinated regulation of the sulfur metabolic pathways in both the beneficial microorganism and the host plant is required for salt stress tolerance in Arabidopsis and might be a common mechanism utilized by different beneficial microbes to mitigate the harmful effects of different abiotic stresses on plants.
  • Ultra-high performance liquid chromatography-mass spectrometry analysis of plant apocarotenoids

    Mi, Jianing; Moreno, Juan C.; Alagoz, Yagiz; Liew, Kit Xi; Balakrishna, Aparna; Zheng, Xiongjie; Al-Babili, Salim (Academic Press Inc., 2021-11-04) [Book Chapter]
    Apocarotenoids (APOs) are a class of carotenoid oxidation products with high structural and functional diversity. Apart from serving as precursors of phytohormones, fungal pheromones and vitamin A, several APOs act as signaling molecules involved in stress response and growth as regulators in plants. To comprehensively profile plant APOs, we established an improved ultra-high performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometer (UHPLC-Q-Orbitrap MS) analytical platform. The improved APO analytical platform consists of an optimized sequential APO sample preparation and multiple UHPLC-MS detection methods and was successfully used to identify and quantify multiple subclasses of APOs from tomato fruits. By integrating ultrasound-assisted extraction, solid phase extraction, and chemical derivatization, the improved sequential APOs sample preparation facilitates the simultaneous preparation of different subclasses of APOs from plant materials. In addition, multiple UHPLC-MS detection methods enables high throughput analysis of APOs. Application of this analytical strategy can make important contributions to understanding the function of these compounds and significantly facilitate the elucidation of plant APO metabolism.
  • Population genomic analysis of Aegilops tauschii identifies targets for bread wheat improvement

    Gaurav, Kumar; Arora, Sanu; Silva, Paula; Sánchez-Martín, Javier; Horsnell, Richard; Gao, Liangliang; Brar, Gurcharn S.; Widrig, Victoria; John Raupp, W.; Singh, Narinder; Wu, Shuangye; Kale, Sandip M.; Chinoy, Catherine; Nicholson, Paul; Quiroz-Chávez, Jesús; Simmonds, James; Hayta, Sadiye; Smedley, Mark A.; Harwood, Wendy; Pearce, Suzannah; Gilbert, David; Kangara, Ngonidzashe; Gardener, Catherine; Forner-Martínez, Macarena; Liu, Jiaqian; Yu, Guotai; Boden, Scott A.; Pascucci, Attilio; Ghosh, Sreya; Hafeez, Amber; O’Hara, Tom; Waites, Joshua; Cheema, Jitender; Steuernagel, Burkhard; Patpour, Mehran; Justesen, Annemarie Fejer; Liu, Shuyu; Rudd, Jackie C.; Avni, Raz; Sharon, Amir; Steiner, Barbara; Kirana, Rizky Pasthika; Buerstmayr, Hermann; Mehrabi, Ali A.; Nasyrova, Firuza Y.; Chayut, Noam; Matny, Oadi; Steffenson, Brian J.; Sandhu, Nitika; Chhuneja, Parveen; Lagudah, Evans; Elkot, Ahmed F.; Tyrrell, Simon; Bian, Xingdong; Davey, Robert P.; Simonsen, Martin; Schauser, Leif; Tiwari, Vijay K.; Randy Kutcher, H.; Hucl, Pierre; Li, Aili; Liu, Deng-Cai; Mao, Long; Xu, Steven; Brown-Guedira, Gina; Faris, Justin; Dvorak, Jan; Luo, Ming-Cheng; Krasileva, Ksenia; Lux, Thomas; Artmeier, Susanne; Mayer, Klaus F. X.; Uauy, Cristobal; Mascher, Martin; Bentley, Alison R.; Keller, Beat; Poland, Jesse; Wulff, Brande B. H. (Nature Biotechnology, Springer Science and Business Media LLC, 2021-11-01) [Article]
    $\textit{Aegilops tauschii}$, the diploid wild progenitor of the D subgenome of bread wheat, is a reservoir of genetic diversity for improving bread wheat performance and environmental resilience. Here we sequenced 242 $\textit{Ae. tauschii}$ accessions and compared them to the wheat D subgenome to characterize genomic diversity. We found that a rare lineage of $\textit{Ae. tauschii}$ geographically restricted to present-day Georgia contributed to the wheat D subgenome in the independent hybridizations that gave rise to modern bread wheat. Through $\textit{k}$-mer-based association mapping, we identified discrete genomic regions with candidate genes for disease and pest resistance and demonstrated their functional transfer into wheat by transgenesis and wide crossing, including the generation of a library of hexaploids incorporating diverse $\textit{Ae. tauschii}$ genomes. Exploiting the genomic diversity of the $\textit{Ae. tauschii}$ ancestral diploid genome permits rapid trait discovery and functional genetic validation in a hexaploid background amenable to breeding.
  • Multi-omics approaches explain the growth-promoting effect of the apocarotenoid growth regulator zaxinone in rice

    Wang, Jian You; Alseekh, Saleh; Xiao, Tingting; Ablazov, Abdugaffor; de Souza, Leonardo Perez; Fiorilli, Valentina; Anggarani, Marita; Lin, Pei-Yu; Votta, Cristina; Novero, Mara; Jamil, Muhammad; Lanfranco, Luisa; Hsing, Yue-Ie C.; Blilou, Ikram; Fernie, Alisdair R.; Al-Babili, Salim (Communications Biology, Springer Science and Business Media LLC, 2021-10-25) [Article]
    AbstractThe apocarotenoid zaxinone promotes growth and suppresses strigolactone biosynthesis in rice. To shed light on the mechanisms underlying its growth-promoting effect, we employed a combined omics approach integrating transcriptomics and metabolomics analysis of rice seedlings treated with zaxinone, and determined the resulting changes at the cellular and hormonal levels. Metabolites as well as transcripts analysis demonstrate that zaxinone application increased sugar content and triggered glycolysis, the tricarboxylic acid cycle and other sugar-related metabolic processes in rice roots. In addition, zaxinone treatment led to an increased root starch content and induced glycosylation of cytokinins. The transcriptomic, metabolic and hormonal changes were accompanied by striking alterations of roots at cellular level, which showed an increase in apex length, diameter, and the number of cells and cortex cell layers. Remarkably, zaxinone did not affect the metabolism of roots in a strigolactone deficient mutant, suggesting an essential role of strigolactone in the zaxinone growth-promoting activity. Taken together, our results unravel zaxinone as a global regulator of the transcriptome and metabolome, as well as of hormonal and cellular composition of rice roots. Moreover, they suggest that zaxinone promotes rice growth most likely by increasing sugar uptake and metabolism, and reinforce the potential of this compound in increasing rice performance.
  • Development, validation, and application of an HPLC-MS/MS method for quantification of oxidized fatty acids in plants

    Almeida-Trapp, Marília; Donizetti de Souza, Gezimar; Shekhawat, Kirti; Sheikh, Arsheed Hussain; Mithöfer, Axel; Hirt, Heribert; Rodrigues-Filho, Edson (Journal of Chromatography B, Elsevier BV, 2021-10-22) [Article]
    Oxylipins constitute a huge class of compounds produced by oxidation of long-chain unsaturated fatty acids either chemically (by radicals such as reactive oxygen species, ROS) or enzymatically (by lipoxygenases, LOX; cyclooxygenases, COX; or cytochrome P450 pathways). This process generates fatty acids peroxides, which can then be further modified in a broad range to epoxy, hydroxy, keto, ether fatty acids, and also hydrolyzed to generate small aldehydes and alcohols. In general, oxylipins are present in almost all living organisms and have a wide range of signaling, metabolic, physiological, and ecological roles depending on the particular organism and on their structure. In plants, oxylipins have been extensively studied over the past 35 years. However, these studies have focused mainly on the jasmonates and so-called green leaves volatiles. The function of early LOX products (like keto and hydroxy fatty acids) is yet not well understood in plants, where they are mainly analyzed by indirect methods or by GC–MS what requires a laborious sample preparation. Here, we developed and validated a straightforward, precise, accurate, and sensitive method for quantifying oxylipins in plant tissues using HPLC-MS/MS, with a one-step extraction procedure using low amount of plant tissues. We successfully applied this method to quantify the oxylipins in different plant species and Arabidopsis thaliana plants treated with various biotic and abiotic stress conditions.
  • Cell-to-Cell Communication During Plant-Pathogen Interaction

    Tabassum, Naheed; Blilou, Ikram (Molecular Plant-Microbe Interactions®, Scientific Societies, 2021-10-19) [Article]
    Recognition of pathogen activates cellular signaling such as ROS, MAPK, Ca2+ signaling which ultimately fine-tunes the cell to cell communication. These further coordinates with the hormone signaling to execute the defense response at local and systemic level. Interestingly, phytopathogens have also evolved to manipulate the cellular and hormonal signaling and/or exploit hosts cell to cell connection in multiple ways at multiple levels. Overall, the triumph over the pathogen depends on prime decisions and actions-how the plant maintain, regulate and eventually break the intercellular communication through apoplastic and symplastic routes. Here, we review how intercellular communication in plants is mediated, manipulated and maneuvered during plant-pathogen interaction. Key words: Cell to cell communication, plant defense, plasmodesmata, phytohormones
  • The long road to engineering durable disease resistance in wheat

    Wulff, Brande B. H.; Krattinger, Simon G. (Current Opinion in Biotechnology, Elsevier BV, 2021-09-24) [Article]
    A rich past of generating and configuring genetic structures in wheat (Triticum aestivum) combined with advances in DNA sequencing, bioinformatics and genome engineering has transformed the field of wheat functional genomics. Cloning a gene from the large and complex wheat genome is no longer unattainable; in the past 5 years alone, the molecular identity of 33 wheat disease resistance genes has been elucidated. The next 15 years will see the cloning of most of the 460 known wheat resistance genes and their corresponding effectors. Coupled with mechanistic insights into how resistance genes, effectors and pathogenicity targets interact and are affected by different genetic backgrounds, this will drive systems biology and synthetic engineering studies towards the alluring goal of generating durable disease resistance in wheat.
  • A thermophilic Cas13 enzyme for sensitive and specific one-pot detection of SARS-CoV-2

    Mahas, Ahmed; Marsic, Tin; Masson, Mauricio Lopez Portillo; Wang, Qiaochu; Aman, Rashid; Zheng, Cheng; Ali, Zahir; Ghanem, Bernard; Mahfouz, Magdy M. (Research Square Platform LLC, 2021-09-22) [Preprint]
    Robust, sensitive, and specific diagnostic platforms for early pathogen detection are essential for the identification of infected patients and management of current and future pandemics. CRISPR-Cas systems have been repurposed for SARS-CoV-2 detection in two-pot assays. Two-pot assays require extra steps and are prone to cross-contamination; however, the temperature range of current Cas enzymes limits the development of one-pot assays Here, we characterized TccCas13a, a thermophilic Cas13a enzyme with cis and trans activities from 37–70°C, and HheCas13a, which had a limited range and lower activity. We harnessed TccCas13a in a one-pot SARS-CoV-2 assay with two layers of amplification and TccCas13a-mediated collateral degradation of a single-stranded RNA reporter. This assay showed 95% sensitivity and 100% specificity compared with RT-qPCR on clinical samples. We also developed a mobile phone application to facilitate data reading, collection, and sharing. Our OPTIMA-dx detection module exhibits key features for point-of-care SARS-CoV-2 screening and pathogen detection in general.
  • Strategies for engineering improved nitrogen use efficiency in crop plants via redistribution and recycling of organic nitrogen

    Melino, Vanessa J.; Tester, Mark A.; Okamoto, Mamoru (Current Opinion in Biotechnology, Elsevier BV, 2021-09-22) [Article]
    Global use of nitrogen (N) fertilizers has increased sevenfold from 1960 to 1995 but much of the N applied is lost to the environment. Modifying the temporal and spatial distribution of organic N within the plant can lead to improved grain yield and/or grain protein content for the same or reduced N fertilizer inputs. Biotechnological approaches to modify whole plant distribution of amino acids and ureides has proven successful in several crop species. Manipulating selective autophagy pathways in crops has also improved N remobilization efficiency to sink tissues whilst the contribution of ribophagy, RNA and purine catabolism to N recycling in crops is still too early to foretell. Improved recycling and remobilization of N must exploit N-stress responsive transcriptional regulators, N-sensing or phloem-localized promotors and genetic variation for N-responsive traits.
  • A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics

    Mahas, Ahmed; Wang, Qiaochu; Marsic, Tin; Mahfouz, Magdy M. (ACS Synthetic Biology, American Chemical Society (ACS), 2021-09-21) [Article]
    Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes' RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential <i>in vivo</i> applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.

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