Elongator and its epigenetic role in plant development and responses to abiotic and biotic stresses

Elongator, a six-subunit protein complex, was initially isolated as an interactor of hyperphosphorylated RNA polymerase II in yeast, and was subsequently identified in animals and plants. Elongator has been implicated in multiple cellular activities or biological processes including tRNA modification, histone modification, DNA demethylation or methylation, tubulin acetylation, and exocytosis. Studies in the model plant Arabidopsis thaliana suggest that the structure of Elongator and its functions are highly conserved between plants and yeast. Disruption of the Elongator complex in plants leads to aberrant growth and development, resistance to abiotic stresses, and susceptibility to bacterial pathogens. The morphological and physiological phenotypes of Arabidopsis Elongator mutants are associated with decreased histone acetylation and/or altered DNA methylation. This review summarizes recent findings related to the epigenetic function of Elongator in plant development and responses to abiotic and biotic stresses

A rapid biosensor-based method for quantification of free and glucose-conjugated salicylic acid

<p>Abstract</p> <p>Background</p> <p>Salicylic acid (SA) is an important signalling molecule in plant defenses against biotrophic pathogens. It is also involved in several other processes such as heat production, flowering, and germination. SA exists in the plant as free SA and as an inert glucose conjugate (salicylic acid 2-<it>O</it>-β-D-glucoside or SAG). Recently, Huang <it>et al</it>. developed a bacterial biosensor that responds to free SA but not SAG, designated as <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>. In this paper we describe an improved methodology for <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.</p> <p>Results</p> <p>On the basis of the original biosensor-based method, we optimized extraction and quantification. SAG content was determined by treating crude extracts with β-glucosidase, then measuring the released free SA with the biosensor. β-glucosidase treatment released more SA in acetate buffer extract than in Luria-Bertani (LB) extract, while enzymatic hydrolysis in either solution released more free SA than acid hydrolysis. The biosensor-based method detected higher amounts of SA in pathogen-infected plants than did a GC/MS-based method. SA quantification of control and pathogen-treated wild-type and <it>sid2 </it>(SA induction-deficient) plants demonstrated the efficacy of the method described. Using the methods detailed here, we were able to detect as little as 0.28 μg SA/g FW. Samples typically had a standard deviation of up to 25% of the mean.</p> <p>Conclusion</p> <p>The ability of <it>Acinetobacter </it>sp. ADPWH_<it>lux </it>to detect SA in a complex mixture, combined with the enzymatic hydrolysis of SAG in crude extract, allowed the development of a simple, rapid, and inexpensive method to simultaneously measure free and glucose-conjugated SA. This approach is amenable to a high-throughput format, which would further reduce the cost and time required for biosensor-based SA quantification. Possible applications of this approach include characterization of enzymes involved in SA metabolism, analysis of temporal changes in SA levels, and isolation of mutants with aberrant SA accumulation.</p

A high-throughput method for isolation of salicylic acid metabolic mutants

<p>Abstract</p> <p>Background</p> <p>Salicylic acid (SA) is a key defense signal molecule against biotrophic pathogens in plants. Quantification of SA levels in plants is critical for dissecting the SA-mediated immune response. Although HPLC and GC/MS are routinely used to determine SA concentrations, they are expensive and time-consuming. We recently described a rapid method for a bacterial biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA quantification, which enables high-throughput analysis. In this study we describe an improved method for fast sample preparation, and present a high-throughput strategy for isolation of SA metabolic mutants.</p> <p>Results</p> <p>On the basis of the previously described biosensor-based method, we simplified the tissue collection and the SA extraction procedure. Leaf discs were collected and boiled in Luria-Bertani (LB), and then the released SA was measured with the biosensor. The time-consuming steps of weighing samples, grinding tissues and centrifugation were avoided. The direct boiling protocol detected similar differences in SA levels among pathogen-infected wild-type, <it>npr1 </it>(nonexpressor of pathogenesis-related genes), and <it>sid2 </it>(SA induction-deficient) plants as did the previously described biosensor-based method and an HPLC-based approach, demonstrating the efficacy of the protocol presented here. We adapted this protocol to a high-throughput format and identified six <it>npr1 </it>suppressors that accumulated lower levels of SA than <it>npr1 </it>upon pathogen infection. Two of the suppressors were found to be allelic to the previously identified <it>eds5 </it>mutant. The other four are more susceptible than <it>npr1 </it>to the bacterial pathogen <it>Pseudomonas syringae </it>pv. <it>maculicola </it>ES4326 and their identity merits further investigation.</p> <p>Conclusions</p> <p>The rapid SA extraction method by direct boiling of leaf discs further reduced the cost and time required for the biosensor <it>Acinetobacter </it>sp. ADPWH_<it>lux</it>-based SA estimation, and allowed the screening for <it>npr1 </it>suppressors that accumulated less SA than <it>npr1 </it>after pathogen infection in a high-throughput manner. The highly efficacious SA estimation protocol can be applied in genetic screen for SA metabolic mutants and characterization of enzymes involved in SA metabolism. The mutants isolated in this study may help identify new components in the SA-related signaling pathways.</p

The Arabidopsis Elongator Subunit ELP3 and ELP4 Confer Resistance to Bacterial Speck in Tomato

Although production of tomato (Solanum lycopersicum) is threatened by a number of major diseases worldwide, it has been difficult to identify effective and durable management measures against these diseases. In this study, we attempted to improve tomato disease resistance by transgenic overexpression of genes encoding the Arabidopsis thaliana Elongator (AtELP) complex subunits AtELP3 and AtELP4. We show that overexpression of AtELP3 and AtELP4 significantly enhanced resistance to tomato bacterial speck caused by the Pseudomonas syringae pv. tomato strain J4 (Pst J4) without clear detrimental effects on plant growth and development. Interestingly, the transgenic plants exhibited resistance to Pst J4 only when inoculated through foliar sprays but not through infiltration into the leaf apoplast. Although this result suggested possible involvement of stomatal immunity, we found that Pst J4 inoculation did not induce stomatal closure and there were no differences in stomatal apertures and conductance between the transgenic and control plants. Further RNA sequencing and real-time quantitative PCR analyses revealed a group of defense-related genes to be induced to higher levels after infection in the AtELP4 transgenic tomato plants than in the control, suggesting that the enhanced disease resistance of the transgenic plants may be attributed to elevated induction of defense responses. Additionally, we show that the tomato genome contains single-copy genes encoding all six Elongator subunits (SlELPs), which share high identities with the AtELP proteins, and that SlELP3 and SlELP4 complemented the Arabidopsis Atelp3 and Atelp4 mutants, respectively, indicating that the function of tomato Elongator is probably conserved. Taken together, our results not only shed new light on the tomato Elongator complex, but also revealed potential candidate genes for engineering disease resistance in tomato

Exogenous Nicotinamide Adenine Dinucleotide Induces Resistance to Citrus Canker in Citrus

Nicotinamide adenine dinucleotide (NAD) is a universal electron carrier that participates in important intracellular metabolic reactions and signaling events. Interestingly, emerging evidence in animals indicates that cellular NAD can be actively or passively released into the extracellular space, where it is processed or perceived by ectoenzymes or cell-surface receptors. We have recently shown in Arabidopsis thaliana that exogenous NAD induces defense responses, that pathogen infection leads to release of NAD into the extracellular space at concentrations sufficient for defense activation, and that depletion of extracellular NAD (eNAD) by transgenic expression of the human NAD-hydrolyzing ectoenzyme CD38 inhibits plant immunity. We therefore hypothesize that, during plant–microbe interactions, NAD is released from dead or dying cells into the extracellular space where it interacts with adjacent naïve cells’ surface receptors, which in turn activate downstream immune signaling. However, it is currently unknown whether eNAD signaling is unique to Arabidopsis or the Brassicaceae family. In this study, we treated citrus plants with exogenous NAD+ and tested NAD+-induced transcriptional changes and disease resistance. Our results show that NAD+ induces profound transcriptome changes and strong resistance to citrus canker, a serious citrus disease caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc). Furthermore, NAD+-induced resistance persists in new flushes emerging after removal of the tissues previously treated with NAD+. Finally, NAD+ treatment primes citrus tissues, resulting in a faster and stronger induction of multiple salicylic acid pathway genes upon subsequent Xcc infection. Taken together, these results indicate that exogenous NAD+ is able to induce immune responses in citrus and suggest that eNAD may also be an elicitor in this woody plant species

Novel Plastid-Nuclear Genome Combinations Enhance Resistance to Citrus Canker in Cybrid Grapefruit

Host disease resistance is the most desirable strategy for control of citrus canker, a disease caused by a gram-negative bacterium Xanthomonas citri subsp. citri. However, no resistant commercial citrus cultivar has been identified. Cybridization, a somatic hybridization approach that combines the organelle and nuclear genomes from different species, was used to create cybrids between citrus canker resistant ‘Meiwa’ kumquat (Fortunella crassifolia Swingle snym. Citrus japonica Thunb.) and susceptible grapefruit (Citrus paradisi Macfad) cultivars. From these fusions, cybrids with grapefruit nucleus, kumquat mitochondria and kumquat chloroplasts and cybrids with grapefruit nucleus, kumquat mitochondria and grapefruit chloroplasts were generated. These cybrids showed a range of citrus canker response, but all cybrids with kumquat chloroplasts had a significantly lower number of lesions and lower Xanthomonas citri subsp. citri populations than the grapefruit controls. Cybrids with grapefruit chloroplasts had a significantly higher number of lesions than those with kumquat chloroplasts. To understand the role of chloroplasts in the cybrid disease defense, quantitative PCR was performed on both cybrid types and their parents to examine changes in gene expression during Xanthomonas citri subsp. citri infection. The results revealed chloroplast influences on nuclear gene expression, since isonuclear cybrids and ‘Marsh’ grapefruit had different gene expression profiles. In addition, only genotypes with kumquat chloroplasts showed an early up-regulation of reactive oxygen species genes upon Xanthomonas citri subsp. citri infection. These cybrids have the potential to enhance citrus canker resistance in commercial grapefruit orchards. They also serve as models for understanding the contribution of chloroplasts to plant disease response and raise the question of whether other alien chloroplast genotypes would condition similar results

Non-Host Defense Response in a Novel Arabidopsis- Xanthomonas citri subsp. citri Pathosystem

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gen

<i>Xcc</i> induces SA production in Arabidopsis.

<p>(<b>A</b>) Free SA levels. (<b>B</b>) Total SA (SA+SAG) levels. Leaves of wild-type Col-0 plants were inoculated with <i>Xcc</i> (OD₆₀₀ = 0.02) or treated with 10 mM MgCl₂. The inoculated leaves were collected at different time points (0, 4, 8, 16, 24, 48, 72, and 96 hpi) for SA measurement by HPLC. Data represent the mean of four independent samples with standard deviation. The experiment was repeated twice with similar results.</p

<i>Xcc</i> does not grow in Arabidopsis.

<p>(<b>A</b>) Growth of syringe-infiltrated <i>Xcc</i> in Col-0. (<b>B</b>) Growth of syringe-infiltrated <i>Xcc</i> in L<i>er</i>. (<b>C</b>) Growth of syringe-infiltrated <i>Xcc</i> in Ws. (<b>D</b>) Growth of syringe-infiltrated <i>Xcc</i> in RLD. (<b>E</b>) Growth of dip-inoculated <i>Xcc</i> in Col-0 and L<i>er</i>. (<b>F</b>) Growth of spray-inoculated <i>Xcc</i> in Col-0 and L<i>er</i>. Four-week-old plants were inoculated with <i>Xcc</i>. Bacterial suspensions with an OD₆₀₀ of 0.002 and 0.02 were used for syringe infiltration and dip/spray inoculation, respectively. The <i>in planta</i> bacterial titers were determined on day 0, 3, 6, 9, 12, and 15 post-inoculation for syringe infiltration, and on day 3, 6, and 9 post-inoculation for dip and spray inoculation (cfu, colony-forming units). Data represent the mean of eight independent samples with standard deviation. The experiment was repeated twice with similar results.</p