Browsing by Author "Mysore, Kirankumar S."

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Advances in plant gene silencing methods
(Springer, 2015) Pandey, Prachi; Senthil-Kumar, Muthappa; Mysore, Kirankumar S.
Understanding molecular mechanisms of transcriptional and posttranscriptional gene silencing pathways in plants over the past decades has led to development of tools and methods for silencing a target gene in various plant species. In this review chapter, both the recent understanding of molecular basis of gene silencing pathways and advances in various widely used gene silencing methods are compiled. We also discuss the salient features of the different methods like RNA interference (RNAi) and virus-induced gene silencing (VIGS) and highlight their advantages and disadvantages. Gene silencing technology is constantly progressing as reflected by rapidly emerging new methods. A succinct discussion on the recently developed methods like microRNA-mediated virus-induced gene silencing (MIR-VIGS) and microRNA-induced gene silencing (MIGS) is also provided. One major bottleneck in gene silencing approaches has been the associated off-target silencing. The other hurdle has been the lack of a universal approach that can be applied to all plants. For example, we face hurdles like incompatibility of VIGS vectors with the host and inability to use MIGS for plant species which are not easily transformable. However, the overwhelming research in this direction reflects the scope for overcoming the short comings of gene silencing technology.
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, Muthappa
Drought 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.
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.
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.
Impact of concurrent drought stress and pathogen infection on plants
(Springer, 2015) Pandey, Prachi; Sinha, Ranjita; Mysore, Kirankumar S.; Senthil-Kumar, Muthappa
Concurrent abiotic and biotic stress situations greatly limit the crop productivity. The global climate change is predicted to bring forth the frequent incidences of concurrent stresses, predominantly drought and pathogen infections. Thus, understanding the impact of drought on plant–pathogen interaction is important. In this chapter, we review the recent studies that focus on the effect of concurrent drought and pathogen infection on plants. These studies indicate that concurrent stress conditions lead to the activation of unique combat pathways that are otherwise not elicited under independent stresses. Plant responses, thus, seem to be adaptively tailored for combating the combined stresses. Here, we focus on the impact of drought stress on plant–pathogen relations and highlight the different ways by which plant–pathogen interactions are modulated at physiological and molecular level. Various studies reviewed in this chapter show that the stress combinations should be considered as a “unique stress” and a better understanding of plant responses to these conditions is needed. Therefore, we propose that further efforts should be directed to identify the potential pathways conferring concurrent stress tolerance.
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.
Plant Gene Silencing: Methods and Protocols
(Springer, 2015) Mysore, Kirankumar S.; Senthil-Kumar, Muthappa
Gene silencing is being popularly used as a functional genomics tool to determine plant gene function. In addition, gene silencing is also used as one of the plant genetic engineering strategies to produce better crop varieties. It is anticipated that the use of gene silencing technology in commercial plant varieties will increase in the future. This volume will provide a comprehensive overview of various gene silencing methodologies and its applications. Gene silencing can be either transcriptional gene silencing (TGS) or posttranscriptional gene silencing (PTGS). PTGS is more popularly used for gene function analyses. PTGS is commonly achieved by either RNA interference (RNAi) or virus-induced gene silencing (VIGS). RNAi in plants can be achieved by expressing hairpin RNA (hpRNA) that fold back to create a double-stranded RNA (dsRNA). These hpRNAs are potent inducers of PTGS and give rise to 21–23 nucleotides small interfering RNAs (siRNAs) derived from the dsRNA by RNase III-like enzymes called Dicers. Then the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complex (RISC). The siRNA strands subsequently guide the RISCs to complementary mRNA molecules, where they cleave and destroy the cognate mRNA thus causing PTGS. VIGS involves deliv- ery of a fragment of plant gene (intended to be silenced) into plant cells via a recombinant virus. The plant defense mechanism silences both the targeted endogenous plant gene and the virus through PTGS. The plant RNA will be converted into dsRNA by RNA-dependent RNA polymerase. The dsRNA will then be degraded in a similar mechanism as that of RNAi. In addition to RNAi and VIGS, PTGS can also be induced by direct delivery of dsRNA into plants. In addition to siRNA microRNA (miRNA) can also cause PTGS. miRNA-mediated PTGS is a natural targeted gene silencing phenomenon inherent in plants for gene regula- tion during plant development and stress responses. Some miRNAs trigger the production of secondary siRNAs from their targets. miRNA-induced gene silencing (MIGS) is an emerging field to silence more than one gene that may not have overall sequence similarity. This volume will cover historical overview of gene silencing mechanisms in plants, vec- tors and strategies available for plant gene silencing, practical applications of gene silencing, and bioinformatics tools and other resources for plant gene silencing. In addition to these review articles, this volume will include methodology for VIGS in various different plant species, understanding plant stress responses using VIGS, miRNA identification, DNA interference, host-induced gene silencing, use of artificial miRNAs for gene silencing, high throughput RNAi, and others.
Plant ribosomal proteins, RPL12 and RPL19, play a role in nonhost disease resistance against bacterial pathogens
(Frontiers Media S.A., 2015) Nagaraj, Satish; Senthil-Kumar, Muthappa; Ramu, Vemanna S.; Wang, Keri; Mysore, Kirankumar S.
Characterizing the molecular mechanism involved in nonhost disease resistance is important to understand the adaptations of plant-pathogen interactions. In this study, virus-induced gene silencing (VIGS)-based forward genetics screen was utilized to identify genes involved in nonhost resistance in Nicotiana benthamiana. Genes encoding ribosomal proteins, RPL12 and RPL19, were identified in the screening. These genes when silenced in N. benthamiana caused a delay in nonhost bacteria induced hypersensitive response (HR) with concurrent increase in nonhost bacterial multiplication. Arabidopsis mutants of AtRPL12 and AtRPL19 also compromised nonhost resistance. The studies on NbRPL12 and NbRPL19 double silenced plants suggested that both RPL12 and RPL19 act in the same pathway to confer nonhost resistance. Our work suggests a role for RPL12 and RPL19 in nonhost disease resistance in N. benthamiana and Arabidopsis. In addition, we show that these genes also play a minor role in basal resistance against virulent pathogens.
Recent advances in plant gene silencing methods
(Springer Nature Publishing AG, 2022) Pandey, Prachi; Mysore, Kirankumar S.; Senthil-Kumar, Muthappa
With 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.
Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense
(Frontiers Media S.A., 2014) Rojas, Clemencia M.; Senthil-Kumar, Muthappa; Tzin, Vered; Mysore, Kirankumar S.
Plants are constantly exposed to microorganisms in the environment and, as a result, have evolved intricate mechanisms to recognize and defend themselves against potential pathogens. One of these responses is the downregulation of photosynthesis and other processes associated with primary metabolism that are essential for plant growth. It has been suggested that the energy saved by downregulation of primary metabolism is diverted and used for defense responses. However, several studies have shown that upregulation of primary metabolism also occurs during plant-pathogen interactions. We propose that upregulation of primary metabolism modulates signal transduction cascades that lead to plant defense responses. In support of this thought, we here compile evidence from the literature to show that upon exposure to pathogens or elicitors, plants induce several genes associated with primary metabolic pathways, such as those involved in the synthesis or degradation of carbohydrates, amino acids and lipids. In addition, genetic studies have confirmed the involvement of these metabolic pathways in plant defense responses. This review provides a new perspective highlighting the relevance of primary metabolism in regulating plant defense against pathogens with the hope to stimulate further research in this area.
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.
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, Muthappa
Pyrroline-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.
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.
Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana
(Nature Publishing Group, 2014) Senthil-Kumar, Muthappa; Mysore, Kirankumar S.
Tobacco rattle virus (TRV)-based virus-induced gene silencing (VIGS) is widely used in various plant species to downregulate the expression of a target plant gene. TRV is a bipartite, positive-strand RNA virus with the TRV1 and TRV2 genomes. To induce post-transcriptional gene silencing (PTGS), the TRV2 genome is genetically modified to carry a fragment of the target gene and delivered into the plant (along with the TRV1 genome) by agroinoculation. TRV1- and TRV2-carrying Agrobacterium strains are then co-inoculated into 3-week-old plant leaves by one of three methods: a needleless syringe, the agrodrench method or by pricking with a toothpick. Target gene silencing occurs in the newly developed noninoculated leaves within 2–3 weeks of TRV inoculation. The TRV-VIGS protocol described here takes only 4 weeks to implement, and it is faster and easier to perform than other gene silencing techniques that are currently available. Although we use Nicotiana benthamiana as an example, the protocol is adaptable to other plant species.
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.
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.
Virus-induced gene silencing is a versatile tool for unraveling the functional relevance of multiple abiotic-stress-responsive genes in crop plants
(Frontiers Media S.A., 2014) Ramegowda, Venkategowda; Mysore, Kirankumar S.; Senthil-Kumar, Muthappa
Virus-induced gene silencing (VIGS) is an effective tool for gene function analysis in plants. Over the last decade, VIGS has been successfully used as both a forward and reverse genetics technique for gene function analysis in various model plants, as well as crop plants. With the increased identification of differentially expressed genes under various abiotic stresses through high-throughput transcript profiling, the application of VIGS is expected to be important in the future for functional characterization of a large number of genes. In the recent past, VIGS was proven to be an elegant tool for functional characterization of genes associated with abiotic stress responses. In this review, we provide an overview of how VIGS is used in different crop species to characterize genes associated with drought-, salt-, oxidative- and nutrient-deficiency-stresses. We describe the examples from studies where abiotic stress related genes are characterized using VIGS. In addition, we describe the major advantages of VIGS over other currently available functional genomics tools. We also summarize the recent improvements, limitations and future prospects of using VIGS as a tool for studying plant responses to abiotic stresses.