Kirankumar S. Mysore

Large‐scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Medicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190,000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R(1) lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14,000-16,000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500,000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium.

Abstract We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant calli. The results of transient GUS (β-glucuronidase) assays suggest that some of these mutants are blocked at an early step in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus. Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.

The Phytotoxin Coronatine Contributes to Pathogen Fitness and Is Required for Suppression of Salicylic Acid Accumulation in Tomato Inoculated with Pseudomonas syringae pv. tomato DC3000

The roles of the phytotoxin coronatine (COR) and salicylic acid (SA)-mediated defenses in the interaction of Pseudomonas syringae pv. tomato DC3000 and tomato (Solanum lycopersicum) were investigated. Unlike findings reported for Arabidopsis thaliana, DC3000 mutants impaired for production of COR or one of its components, coronafacic acid (CFA) or coronamic acid (CMA), induced distinctly different disease lesion phenotypes in tomato. Tomato plants inoculated with the CFA- CMA- mutant DB29 showed elevated transcript levels of SlICS, which encodes isochorismate synthase, an enzyme involved in SA biosynthesis in S. lycopersicum. Furthermore, expression of genes encoding SA-mediated defense proteins were elevated in DB29-inoculated plants compared with plants inoculated with DC3000, suggesting that COR suppresses SlICS-mediated SA responses. Sequence analysis of SlICS revealed that it encodes a protein that is 55 and 59.6% identical to the A. thaliana ICS-encoded proteins AtICS1 and AtICS2, respectively. Tomato plants silenced for SlICS were hypersusceptible to DC3000 and accumulated lower levels of SA after infection with DC3000 compared with inoculated wild-type tomato plants. Unlike what has been shown for A. thaliana, the COR- mutant DB29 was impaired for persistence in SlICS-silenced tomato plants; thus, COR has additional roles in virulence that are SA independent and important in the latter stages of disease development. In summary, the infection assays, metabolic profiling, and gene expression results described in this study indicate that the intact COR molecule is required for both suppression of SA-mediated defense responses and full disease symptom development in tomato.

Regulation of primary plant metabolism during plant-pathogen interactions and its contribution to plant defense

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.

New dimensions for VIGS in plant functional genomics

Virus-induced gene silencing (VIGS) is an efficient tool for gene function studies. It has been used to perform both forward and reverse genetics to identify plant genes involved in several plant processes. However, this technology has not yet been used to its full potential. This can be attributed to several of its limitations such as inability to silence genes during seed germination and the non-stable nature of silencing. However, several recent studies have shown that these limitations can now be overcome. In this review, we will discuss the limitations of VIGS and suitable solutions. In addition, we also describe the recent improvements and future prospects of using VIGS in plant biology.

Computational Estimation and Experimental Verification of Off-Target Silencing during Posttranscriptional Gene Silencing in Plants

Successful application of posttranscriptional gene silencing (PTGS) for gene function study in both plants and animals depends on high target specificity and silencing efficiency. By computational analysis with genome and/or transcriptome sequences of 25 plant species, we predicted that about 50% to 70% of gene transcripts in plants have potential off-targets when used for PTGS that could obscure experimental results. We have developed a publicly available Web-based computational tool called siRNA Scan to identify potential off-targets during PTGS. Some of the potential off-targets obtained from this tool were tested by measuring the amount of off-target transcripts using quantitative reverse transcription-PCR. Up to 50% of the predicted off-target genes tested in plants were actually silenced when tested experimentally. Our results suggest that a high risk of off-target gene silencing exists during PTGS in plants. Our siRNA Scan tool is useful to design better constructs for PTGS by minimizing off-target gene silencing in both plants and animals.

Identification of Arabidopsis rat mutants

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various “reverse genetic” approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

Glycolate Oxidase Modulates Reactive Oxygen Species–Mediated Signal Transduction during Nonhost Resistance in Nicotiana benthamiana and Arabidopsis

This article identifies the peroxisomal enzyme glycolate oxidase (GOX) as an essential element of nonhost resistance and provides evidence that apart from NADPH oxidase–generated H2O2, GOX is an alternate source for H2O2 production and plays a crucial role during both gene-for-gene and nonhost resistance by regulating genes from different defense signal transduction cascades. In contrast to gene-for-gene disease resistance, nonhost resistance governs defense responses to a broad range of potential pathogen species. To identify specific genes involved in the signal transduction cascade associated with nonhost disease resistance, we used a virus-induced gene-silencing screen in Nicotiana benthamiana, and identified the peroxisomal enzyme glycolate oxidase (GOX) as an essential component of nonhost resistance. GOX-silenced N. benthamiana and Arabidopsis thaliana GOX T-DNA insertion mutants are compromised for nonhost resistance. Moreover, Arabidopsis gox mutants have lower H2O2 accumulation, reduced callose deposition, and reduced electrolyte leakage upon inoculation with hypersensitive response–causing nonhost pathogens. Arabidopsis gox mutants were not affected in NADPH oxidase activity, and silencing of a gene encoding NADPH oxidase (Respiratory burst oxidase homolog) in the gox mutants did not further increase susceptibility to nonhost pathogens, suggesting that GOX functions independently from NADPH oxidase. In the two gox mutants examined (haox2 and gox3), the expression of several defense-related genes upon nonhost pathogen inoculation was decreased compared with wild-type plants. Here we show that GOX is an alternative source for the production of H2O2 during both gene-for-gene and nonhost resistance responses.

A GRAS-type transcription factor with a specific function in mycorrhizal signaling

Legumes establish mutualistic associations with mycorrhizal fungi and with nitrogen-fixing rhizobial bacteria. These interactions occur following plant recognition of Nod factor from rhizobial bacteria and Myc factor from mycorrhizal fungi. A common symbiosis signaling pathway is involved in the recognition of both Nod factor and Myc factor and is required for the establishment of these two symbioses. The outcomes of these associations differ, and therefore, despite the commonality in signaling, there must be mechanisms that allow specificity. In Nod factor signaling, a complex of GRAS-domain transcription factors controls gene expression downstream of the symbiosis signaling pathway. Here, we show that a GRAS-domain transcription factor, RAM1, functions in mycorrhizal-specific signaling. Plants mutated in RAM1 are unable to be colonized by mycorrhizal fungi, with a defect in hyphopodia formation on the surface of the root. RAM1 is specifically required for Myc factor signaling and appears to have no role in Nod factor signaling. RAM1 regulates the expression of RAM2, a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. We conclude that mycorrhizal signaling downstream of the symbiosis-signaling pathway has parallels with nodulation-specific signaling and functions to promote mycorrhizal colonization by regulating cutin biosynthesis.

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

Transcriptome profiling of M. truncatula root hairs during the initial stages of rhizobial infection helps to interpret two decades of research on Medicago and provides a foundation for future studies on host-symbiont interactions in the rhizosphere. Nitrogen-fixing rhizobia colonize legume roots via plant-made intracellular infection threads. Genetics has identified some genes involved but has not provided sufficient detail to understand requirements for infection thread development. Therefore, we transcriptionally profiled Medicago truncatula root hairs prior to and during the initial stages of infection. This revealed changes in the responses to plant hormones, most notably auxin, strigolactone, gibberellic acid, and brassinosteroids. Several auxin responsive genes, including the ortholog of Arabidopsis thaliana Auxin Response Factor 16, were induced at infection sites and in nodule primordia, and mutation of ARF16a reduced rhizobial infection. Associated with the induction of auxin signaling genes, there was increased expression of cell cycle genes including an A-type cyclin and a subunit of the anaphase promoting complex. There was also induction of several chalcone O-methyltransferases involved in the synthesis of an inducer of Sinorhizobium meliloti nod genes, as well as a gene associated with Nod factor degradation, suggesting both positive and negative feedback loops that control Nod factor levels during rhizobial infection. We conclude that the onset of infection is associated with reactivation of the cell cycle as well as increased expression of genes required for hormone and flavonoid biosynthesis and that the regulation of auxin signaling is necessary for initiation of rhizobial infection threads.