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Item Recent advances in plant gene silencing methods(Springer Nature Publishing AG, 2022) Pandey, Prachi; Mysore, Kirankumar S.; Senthil-Kumar, MuthappaWith the increasing understanding of fundamentals of gene silencing pathways in plants, various tools and techniques for downregulating the expression of a target gene have been developed across multiple plant species. This chapter provides an insight into the molecular mechanisms of gene silencing and highlights the advancements in various gene silencing approaches. The prominent aspects of different gene silencing methods, their advantages and disadvantages have been discussed. A succinct discussion on the newly emerged microRNA-based technologies like microRNA-induced gene silencing (MIGS) and microRNA-mediated virus-induced gene silencing (MIR-VIGS) are also presented. We have also discussed the gene-editing system like CRISPR-Cas. The prominent bottlenecks in gene silencing methods are the off-target effects and lack of universal applicability. However, the tremendous growth in understanding of this field reflects the potentials for improvements in the currently available approaches and the development of new widely applicable methods for easy, fast, and efficient functional characterization of plant genes.Item High-throughput analysis of gene function under multiple abiotic stresses using leaf disks from silenced plants(Springer Nature Publishing AG, 2022) Yamunarani, Ramegowda; Ramegowda, Venkategowda; Senthil-Kumar, Muthappa; Mysore, Kirankumar S.The high throughputness and affordability of “omics” technologies is leading to the identification of a large number of abiotic stress genes, with many of them responsive to multiple stresses. In vivo functional characterization of these genes under multiple stresses is challenging but essential to develop resilient crops for the changing climate. Here we describe a high-throughput Virus-Induced Gene Silencing-based methodology for functional analysis of genes under multiple abiotic stresses using leaf disks. Leaves with maximal silencing, which is localized to only a few leaves and to a short period, can be effectively used for multiple stress imposition and stress affect quantification.Item A novel role of salt and drought induced RING 1 protein in modulating plant defense against hemibiotrophic and necrotrophic pathogens(American Phytopathological Society, 2021) Ramu, Vemanna S.; Oh, Sunhee; Lee, Hee-Kyung; Nandety, Raja Sekhar; Oh, Youngjae; Lee, Seonghee; Nakashima, Jin; Tang, Yuhong; Senthil-Kumar, Muthappa; Mysore, Kirankumar S.Many plant encoded E3 ligases are known to be involved in plant defense. Here we report a novel role of E3 ligase SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1) in plant immunity. Even though SDIR1 is reasonably well-characterized, its role in biotic stress response is not known. The silencing of SDIR1 in Nicotiana benthamiana reduced the multiplication of the virulent bacterial pathogen Pseudomonas syringae pv. tabaci. The Arabidopsis sdir1 mutant is resistant to virulent pathogens, whereas SDIR1 overexpression lines are susceptible to both host and nonhost hemibiotrophic bacterial pathogens. However, sdir1 mutant and SDIR1 overexpression lines showed hypersusceptibility and resistance, respectively, against the necrotrophic pathogen, Erwinia carotovora. The mutant of SDIR1 target protein, SDIR-interacting protein 1 (SDIR1P1), also showed resistance to host and nonhost pathogens. In SDIR1 overexpression plants, transcripts of NAC transcription factors were less accumulated and the levels of JA and abscisic acid (ABA) were increased. In sdir1 mutants, JA signaling genes JAZ7 and JAZ8 were downregulated. These data suggest that SDIR1 is a susceptibility factor, and its activation/overexpression enhances disease caused by P. syringae pv. tomato DC3000 in Arabidopsis. Our results show a novel role of SDIR1 in modulating plant defense gene expression and plant immunity.Item Ribosomal protein QM/RPL10 positively regulates defence and protein translation mechanisms during nonhost disease resistance(John Wiley & Sons, 2020) Ramu, Vemanna S.; Dawane, Akashata; Lee, Seonghee; Oh, Sunhee; Lee, Hee-Kyung; Sun, Liang; Senthil-Kumar, Muthappa; Mysore, Kirankumar S.Ribosomes play an integral part in plant growth, development, and defence responses. We report here the role of ribosomal protein large (RPL) subunit QM/RPL10 in nonhost disease resistance. The RPL10-silenced Nicotiana benthamiana plants showed compromised disease resistance against nonhost pathogen Pseudomonas syringae pv. tomato T1. The RNA-sequencing analysis revealed that many genes involved in defence and protein translation mechanisms were differentially affected due to silencing of NbRPL10. Arabidopsis AtRPL10 RNAi and rpl10 mutant lines showed compromised nonhost disease resistance to P. syringae pv. tomato T1 and P. syringae pv. tabaci. Overexpression of AtRPL10A in Arabidopsis resulted in reduced susceptibility against host pathogen P. syringae pv. tomato DC3000. RPL10 interacts with the RNA recognition motif protein and ribosomal proteins RPL30, RPL23, and RPS30 in the yeast two-hybrid assay. Silencing or mutants of genes encoding these RPL10- interacting proteins in N. benthamiana or Arabidopsis, respectively, also showed compromised disease resistance to nonhost pathogens. These results suggest that QM/ RPL10 positively regulates the defence and translation-associated genes during nonhost pathogen infection.Item Two chloroplast-localized proteins: AtNHR2A and AtNHR2B, contribute to callose deposition during nonhost disease resistance in Arabidopsis(American Phytopathological Society, 2018) Singh, Raksha; Lee, Seonghee; Ortega, Laura; Ramu, Vemanna S.; Senthil-Kumar, Muthappa; Blancaflor, Elison B.; Rojas, Clemencia M.; Mysore, Kirankumar S.Plants are naturally resistant to most pathogens through a broad and durable defense response called nonhost disease resistance. Nonhost disease resistance is a complex process that includes preformed physical and chemical barriers and induced responses. In spite of its importance, many components of nonhost disease resistance remain to be identified and characterized. Using virus-induced gene silencing in Nicotiana benthamiana, we discovered a novel gene that we named NbNHR2 (N. benthamiana nonhost resistance 2). NbNHR2-silenced plants were susceptible to the non-adapted pathogen Pseudomonas syringae pv. tomato T1 that does not cause disease in wild-type or non-silenced N. benthamiana plants. We found two orthologous genes in Arabidopsis thaliana: AtNHR2A and AtNHR2B. Similar to the results obtained in N. benthamiana, Atnhr2a and Atnhr2b mutants were susceptible to the non-adapted bacterial pathogen of A. thaliana, P. syringae pv. tabaci. We further found that these mutants were also defective in callose deposition. AtNHR2A and AtNHR2B fluorescent protein fusions transiently expressed in N. benthamiana localized predominantly to chloroplasts and a few unidentified dynamic puncta. RFP-AtNHR2A and AtNHR2B-GFP displayed overlapping signals in chloroplasts indicating that the two proteins could interact; a notion supported by co-immunoprecipitation studies. We propose that AtNHR2A and AtNHR2B are new components of a chloroplast-signaling pathway that activates callose deposition to the cell wall in response to bacterial pathogens.Item Virus-induced gene silencing database for phenomics and functional genomics in Nicotiana benthamiana(American Society of Plant Biologists, John Wiley & Sons, 2018) Senthil-Kumar, Muthappa; Wang, Mingyi; Chang, Junil; Ramegowda, Venkategowda; Pozo, Olga del; Liu, Yule; Doraiswamy, Vanthana; Lee, Hee-Kyung; Ryu, Choong-Min; Wang, Keri; Xu, Ping; Eck, Joyce Van; Chakravarthy, Suma; Dinesh-Kumar, Savithramma P.; Martin, Gregory B.; Mysore, Kirankumar S.Virus-induced gene silencing (VIGS) is an important forward and reverse genetics method for the study of gene function in many plant species, especially Nicotiana benthamiana. However, despite the widespread use of VIGS, a searchable database compiling the phenotypes observed with this method is lacking. Such a database would allow researchers to know the phenotype associated with the silencing of a large number of individual genes without experimentation. We have developed a VIGS phenomics and functional genomics database (VPGD) that has DNA sequence information derived from over 4,000 N. benthamiana VIGS clones along with the associated silencing phenotype for approximately 1,300 genes. The VPGD has a built-in BLAST search feature that provides silencing phenotype information of specific genes. In addition, a keyword-based search function could be used to find a specific phenotype of interest with the corresponding gene, including its Gene Ontology descriptions. Query gene sequences from other plant species that have not been used for VIGS can also be searched for their homologs and silencing phenotype in N. benthamiana. VPGD is useful for identifying gene function not only in N. benthamiana but also in related Solanaceae plants such as tomato and potato. The database is accessible at http://vigs.noble.org.Item General Control Non-repressible-4 (GCN4) degrades 14-3-3 and the RIN4 complex to regulate stomatal aperture with implications on nonhost disease resistance and drought tolerance(American Society of Plant Biologists, 2017) Kaundal, Amita; Ramu, Vemanna S; Oh, Sunhee; Lee, Seonghee; Pant, Bikram; Lee, Hee-Kyung; Rojas, Clemencia M.; Senthil-Kumar, Muthappa; Mysore, Kirankumar S.Plants have complex and adaptive innate immune responses against pathogen infections. Stomata are key entry points for many plant pathogens. Both pathogens and plants regulate stomatal aperture for pathogen entry and defense, respectively. Not all plant proteins involved in stomatal aperture regulation have been identified. Here we report general control non-repressible-4 (GCN4), an AAA+-ATPase family protein, as one of the key proteins regulating stomatal aperture during biotic and abiotic stress. Silencing of GCN4 in Nicotiana benthamiana and Arabidopsis compromises host and nonhost disease resistance due to open stomata during pathogen infection. AtGCN4 overexpression plants have reduced H+-ATPase activity, less responsive stomata to coronatine or fusicoccin, have reduced pathogen entry, and confers drought tolerance. This study also demonstrates that AtGCN4 interacts with RIN4 and 14-3-3 proteins and suggest that GCN4 may degrade RIN4 and 14-3-3 proteins via a proteasome mediated pathway to reduce the activity of plasma membrane H+-ATPase complex thus reducing proton pump activity to close stomata.Item The small GTPase, nucleolar GTP-binding protein 1 (NOG1), has a novel role in plant innate immunity(Nature Publishing Group, 2017) Lee, Seonghee; Senthil-Kumar, Muthappa; Kang, Miyoung; Rojas, Clemencia M.; Tang, Yuhong; Oh, Sunhee; Choudhury, Swarup Roy; Lee, Hee-Kyung; Ishiga, Yasuhiro; Allen, Randy D.; Pandey, Sona; Mysore, Kirankumar S.Plant defense responses at stomata and apoplast are the most important early events during plant-bacteria interactions. The key components for the signaling of stomatal defense and nonhost resistance have not been fully characterized. Here we report the newly identified small GTPase, Nucleolar GTP-binding protein 1 (NOG1), functions for plant immunity against bacterial pathogens. Virus-induced gene silencing of NOG1 compromised nonhost resistance in N. benthamiana and tomato. Comparative genomic analysis showed that two NOG1 copies are present in all known plant species: NOG1-1 and NOG1-2. Gene downregulation and overexpression studies of NOG1-1 and NOG1-2 in Arabidopsis revealed the novel function of these genes in nonhost resistance and stomatal defense against bacterial pathogens, respectively. Specially, NOG1-2 regulates guard cell signaling in response to biotic and abiotic stimuli through jasmonic acid (JA)- and abscisic acid (ABA)-mediated pathways. The results here provide valuable information on the new functional role of small GTPase, NOG1, in guard cell signaling and early plant defense in response to bacterial pathogens.Item GBF3 transcription factor imparts drought tolerance in Arabidopsis thaliana(Nature Publishing Group, 2017) Ramegowda, Venkategowda; Gill, Upinder Singh; Sivalingam, Palaiyur Nanjappan; Gupta, Aarti; Gupta, Chirag; Govind, Geetha; Nataraja, Karaba N.; Pereira, Andy; Udayakumar, Makarla; Mysore, Kirankumar S.; Senthil-Kumar, MuthappaDrought transcriptome analysis of finger millet (Eleusine coracana) by cDNA subtraction identified drought responsive genes that have a potential role in drought tolerance. Through virus-induced gene silencing (VIGS) in a related crop species, maize (Zea mays), several genes, including a G-BOX BINDING FACTOR 3 (GBF3) were identified as candidate drought stress response genes and the role of GBF3 in drought tolerance was studied in Arabidopsis thaliana. Overexpression of both EcGBF3 and AtGBF3 in A. thaliana resulted in improved tolerance to osmotic stress, salinity and drought stress in addition to conferring insensitivity to ABA. Conversely, loss of function of this gene increased the sensitivity of A. thaliana plants to drought stress. EcGBF3 transgenic A. thaliana results also suggest that drought tolerance of sensitive plants can be improved by transferring genes from far related crops like finger millet. Our results demonstrate the role of GBF3 in imparting drought tolerance in A. thaliana and indicate the conserved role of this gene in drought and other abiotic stress tolerance in several plant species.Item Role of proline and pyrroline-5-carboxylate metabolism in plant defense against invading pathogens(Frontiers Media S.A., 2015) Qamar, Aarzoo; Mysore, Kirankumar S.; Senthil-Kumar, MuthappaPyrroline-5-carboxylate (P5C) is an intermediate product of both proline biosynthesis and catabolism. Recent evidences indicate that proline-P5C metabolism is tightly regulated in plants, especially during pathogen infection and abiotic stress. However, role of P5C and its metabolism in plants has not yet been fully understood. Studies indicate that P5C synthesized in mitochondria has a role in both resistance (R)-gene-mediated and non-host resistance against invading pathogens. Proline dehydrogenase and delta-ornithine amino transferase-encoding genes, both involved in P5C synthesis in mitochondria are implicated in defense response of Nicotiana benthamiana and Arabidopsis thaliana against bacterial pathogens. Such defense response is proposed to involve salicylic acid-dependent pathway, reactive oxygen species (ROS) and hypersensitive response (HR)-associated cell death. Recently HR, a form of programmed cell death (PCD), has been proposed to be induced by changes in mitochondrial P5C synthesis or the increase in P5C levels per se in plants inoculated with either a host pathogen carrying suitable avirulent (Avr) gene or a non-host pathogen. Consistently, A. thaliana mutant plants deficient in P5C catabolism showed HR like cell death when grown in external P5C or proline supplemented medium. Similarly, yeast and plant cells under oxidative stress were shown to increase ROS production and PCD due to increase in P5C levels. Similar mechanism has also been reported as one of the triggers for apoptosis in mammalian cells. This review critically analyzes results from various studies and enumerates the pathways for regulation of P5C levels in the plant cell, especially in mitochondria, during pathogen infection. Further, mechanisms regulating P5C- mediated defense responses, namely HR are outlined. This review also provides new insights into the differential role of proline-P5C metabolism in plants exposed to pathogen infection.