Zhonglin Mou

Salicylic Acid and its Function in Plant Immunity

The small phenolic compound salicylic acid (SA) plays an important regulatory role in multiple physiological processes including plant immune response. Significant progress has been made during the past two decades in understanding the SA-mediated defense signaling network. Characterization of a number of genes functioning in SA biosynthesis, conjugation, accumulation, signaling, and crosstalk with other hormones such as jasmonic acid, ethylene, abscisic acid, auxin, gibberellic acid, cytokinin, brassinosteroid, and peptide hormones has sketched the finely tuned immune response network. Full understanding of the mechanism of plant immunity will need to take advantage of fast developing genomics tools and bioinformatics techniques. However, elucidating genetic components involved in these pathways by conventional genetics, biochemistry, and molecular biology approaches will continue to be a major task of the community. High-throughput method for SA quantification holds the potential for isolating additional mutants related to SA-mediated defense signaling.

The Arabidopsis Mediator Complex Subunit16 Positively Regulates Salicylate-Mediated Systemic Acquired Resistance and Jasmonate/Ethylene-Induced Defense Pathways

This article identifies a role for the Arabidopsis thaliana Mediator complex subunit MED16 in bridging between specific transcription activators and the transcriptional machinery in the activation of salicylic acid–responsive defenses, including defense gene transcription, pathogen resistance, and systemic acquired resistance. MED16 also participates in ethylene- and jasmonic acid–induced defenses. Systemic acquired resistance (SAR) is a long-lasting plant immunity against a broad spectrum of pathogens. Biological induction of SAR requires the signal molecule salicylic acid (SA) and involves profound transcriptional changes that are largely controlled by the transcription coactivator NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1). However, it is unclear how SAR signals are transduced from the NPR1 signaling node to the general transcription machinery. Here, we report that the Arabidopsis thaliana Mediator subunit16 (MED16) is an essential positive regulator of SAR. Mutations in MED16 reduced NPR1 protein levels and completely compromised biological induction of SAR. These mutations also significantly suppressed SA-induced defense responses, altered the transcriptional changes induced by the avirulent bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000/avrRpt2, and rendered plants susceptible to both Pst DC3000/avrRpt2 and Pst DC3000. In addition, mutations in MED16 blocked the induction of several jasmonic acid (JA)/ethylene (ET)–responsive genes and compromised resistance to the necrotrophic fungal pathogens Botrytis cinerea and Alternaria brassicicola. The Mediator complex acts as a bridge between specific transcriptional activators and the RNA polymerase II transcription machinery; therefore, our data suggest that MED16 may be a signaling component in the gap between the NPR1 signaling node and the general transcription machinery and may relay signals from both the SA and the JA/ET pathways.

Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis.

Although it is well known that the pyridine nucleotides NAD and NADP function inside the cell to regulate intracellular signaling processes, recent evidence from animal studies suggests that NAD(P) also functions in the extracellular compartment (ECC). Extracellular NAD(P) [eNAD(P)] can either directly bind to plasma membrane receptors or be metabolized by ecto-enzymes to produce cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, and/or may ADP-ribosylate cell-surface receptors, resulting in activation of transmembrane signaling. In this study, we report that, in plants, exogenous NAD(P) induces the expression of pathogenesis-related (PR) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Chelation of Ca(2+) by EGTA significantly inhibits the induction of PR genes by exogenous NAD(P), suggesting that exogenous NAD(P) may induce PR genes through a pathway that involves Ca(2+) signaling. We show that exogenous application of NAD(P) causes accumulation of the defense signal molecule salicylic acid (SA), and induces both SA/NPR1-dependent and -independent PR gene expression and disease resistance. Furthermore, we demonstrate that NAD(P) leaks into the plant ECC after mechanical wounding and pathogen infection, and that the amount of NAD(P) leaking into the ECC after P. syringae pv. tobacco DC3000/avrRpt2 infection is sufficient for induction of both PR gene expression and disease resistance. We propose that NAD(P) leakage from cells losing membrane integrity upon environmental stress may function as an elicitor to activate plant defense responses. Our data provide evidence that eNAD(P) functions in plant signaling, and illustrate the potential importance of eNAD(P) in plant innate immunity.

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

BackgroundSalicylic 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-O-β-D-glucoside or SAG). Recently, Huang et al. developed a bacterial biosensor that responds to free SA but not SAG, designated as Acinetobacter sp. ADPWH_lux. In this paper we describe an improved methodology for Acinetobacter sp. ADPWH_lux-based free SA quantification, enabling high-throughput analysis, and present an approach for the quantification of SAG from crude plant extracts.ResultsOn 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 sid2 (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.ConclusionThe ability of Acinetobacter sp. ADPWH_lux 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.

Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker

Citrus canker, caused by the bacterial pathogen Xanthomonas citri subsp. citri (Xcc), is a serious leaf and fruit spotting disease affecting many important citrus cultivars including grapefruit and certain sweet oranges. Currently, efficacious and economical disease control measures for highly susceptible citrus cultivars are lacking. Development of commercial cultivars with greater field resistance to citrus canker is the optimum strategy for effective disease management. In this study, we generated transgenic ‘Duncan’ grapefruit (DG) and ‘Hamlin’ sweet orange (Ham) expressing the Arabidopsis NPR1 gene (AtNPR1), which is a key positive regulator of the long-lasting broad-spectrum resistance known as systemic acquired resistance (SAR). Our results indicate that over-expression of AtNPR1 in citrus increases resistance to citrus canker and that the resistance is related with the expression levels of AtNPR1 in the transgenic plants. The line (DG 42-2) with the highest expression level of AtNPR1 was also the most resistant, which developed significant fewer lesions accompanied by a ten-fold reduction in Xcc population. The lesions developed on DG 42-2 were smaller and darker than those on the control and lacked callus formation. These lesion phenotypes resemble those on canker resistant kumquats and canker susceptible citrus trees treated with SAR-inducing compounds. Therefore, over-expression of AtNPR1 in citrus is a promising approach for development of more resistant cultivars to citrus canker.

The Arabidopsis Elongator Complex Subunit2 Epigenetically Regulates Plant Immune Responses

This work identifies a role for the Arabidopsis Elongator complex subunit ELP2 in somatic DNA demethylation/methylation and suggests that ELP2-mediated epigenetic regulation plays a vital function in plant immune responses. The Arabidopsis thaliana Elongator complex subunit2 (ELP2) genetically interacts with NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1), a key transcription coactivator of plant immunity, and regulates the induction kinetics of defense genes. However, the mechanistic relationship between ELP2 and NPR1 and how ELP2 regulates the kinetics of defense gene induction are unclear. Here, we demonstrate that ELP2 is an epigenetic regulator required for pathogen-induced rapid transcriptome reprogramming. We show that ELP2 functions in a transcriptional feed-forward loop regulating both NPR1 and its target genes. An elp2 mutation increases the total methylcytosine number, reduces the average methylation levels of methylcytosines, and alters (increases or decreases) methylation levels of specific methylcytosines. Interestingly, infection of plants with the avirulent bacterial pathogen Pseudomonas syringae pv tomato DC3000/avrRpt2 induces biphasic changes in DNA methylation levels of NPR1 and PHYTOALEXIN DEFICIENT4 (PAD4), which encodes another key regulator of plant immunity. These dynamic changes are blocked by the elp2 mutation, which is correlated with delayed induction of NPR1 and PAD4. The elp2 mutation also reduces basal histone acetylation levels in the coding regions of several defense genes. Together, our data demonstrate a new role for Elongator in somatic DNA demethylation/methylation and suggest a function for Elongator-mediated chromatin regulation in pathogen-induced transcriptome reprogramming.

Elongator subunit 2 is an accelerator of immune responses in Arabidopsis thaliana

Immune responses in eukaryotes involve rapid and profound transcriptional reprogramming. Although mechanisms regulating the amplitude of defense gene expression have been extensively characterized, those controlling the speed of defense gene induction are not well understood. Here, we show that the Arabidopsis Elongator subunit 2 (AtELP2) regulates the kinetics of defense gene induction. AtELP2 is required for rapid defense gene induction and the establishment of full basal and effector-triggered immunity (ETI). Surprisingly, biological or chemical induction of systemic acquired resistance (SAR), a long-lasting plant immunity against a broad spectrum of pathogens, restores pathogen resistance to Atelp2 mutant plants. Simultaneous removal of AtELP2 and NPR1, a transcription coactivator essential for full-scale expression of a subset of defense genes and the establishment of SAR, completely abolishes resistance to two different ETI-inducing pathogens. These results demonstrate that AtELP2 is an accelerator of defense gene induction, which functions largely independently of NPR1 in establishing plant immunity.

Elongator subunit 3 positively regulates plant immunity through its histone acetyltransferase and radical S-adenosylmethionine domains

BackgroundPathogen infection triggers a large-scale transcriptional reprogramming in plants, and the speed of this reprogramming affects the outcome of the infection. Our understanding of this process has significantly benefited from mutants that display either delayed or accelerated defense gene induction. In our previous work we demonstrated that the Arabidopsis Elongator complex subunit 2 (AtELP2) plays an important role in both basal immunity and effector-triggered immunity (ETI), and more recently showed that AtELP2 is involved in dynamic changes in histone acetylation and DNA methylation at several defense genes. However, the function of other Elongator subunits in plant immunity has not been characterized.ResultsIn the same genetic screen used to identify Atelp2, we found another Elongator mutant, Atelp3-10, which mimics Atelp2 in that it exhibits a delay in defense gene induction following salicylic acid treatment or pathogen infection. Similarly to AtELP2, AtELP3 is required for basal immunity and ETI, but not for systemic acquired resistance (SAR). Furthermore, we demonstrate that both the histone acetyltransferase and radical S-adenosylmethionine domains of AtELP3 are essential for its function in plant immunity.ConclusionOur results indicate that the entire Elongator complex is involved in basal immunity and ETI, but not in SAR, and support that Elongator may play a role in facilitating the transcriptional induction of defense genes through alterations to their chromatin.

Nuclear localization of NPR1 is required for regulation of salicylate tolerance, isochorismate synthase 1 expression and salicylate accumulation in Arabidopsis

Plant systemic acquired resistance (SAR) is a broad-spectrum immune response in which pathogen infection in local tissue induces resistance in systemic leaves. Activation of SAR requires the signal molecule salicylic acid (SA), which is primarily synthesized from chorismate via isochorismate through the action of isochorismate synthase 1 (ICS1) and a putative isochorismate pyruvate lyase. The Arabidopsis transcription coactivator NPR1 is a key regulator of SAR, which functions at multiple nodes in the SA signaling network. NPR1 not only acts downstream of SA to activate SAR, but also upstream of SA to suppress the expression of ICS1, thus inhibiting SA biosynthesis. NPR1 also positively regulates SA tolerance and plays a role in SA-mediated negative regulation of jasmonic acid (JA) signaling. The NPR1 protein contains a functional bipartite nuclear localization signal (NLS). It has been shown that the NLS and nuclear localization of NPR1 are required for activation of pathogenesis-related gene expression, whereas modulation of the crosstalk between SA- and JA-dependent defense pathways is mediated by cytosolic NPR1. In this study we used two transgenic lines, one expressing a mutated npr1 with a dysfunctional NLS and the other in which NPR1 nuclear localization can be induced by dexamethasone treatment, to test whether nuclear localization is required for other functions of NPR1. We found that prevention of NPR1 nuclear localization renders transgenic seedlings sensitive to the toxicity of high levels of SA and causes over-accumulation of ICS1 transcripts and SA in response to pathogen infection. Induction of NPR1 nuclear localization restores SA tolerance and normal accumulation of ICS1 transcripts and SA. These results indicate that the NLS and nuclear localization of NPR1 are required for regulation of SA tolerance, ICS1 expression and SA accumulation.

Characterization of Arabidopsis 6-Phosphogluconolactonase T-DNA Insertion Mutants Reveals an Essential Role for the Oxidative Section of the Plastidic Pentose Phosphate Pathway in Plant Growth and Development

Arabidopsis PGL1, PGL2, PGL4 and PGL5 are predicted to encode cytosolic isoforms of 6-phosphogluconolactonase (6PGL), whereas PGL3 is predicted to encode a 6PGL that has been shown to localize in both plastids and peroxisomes. Therefore, 6PGL may exist in the cytosol, plastids and peroxisomes. However, the function of 6PGL in these three subcellular locations has not been well defined. Here we show that PGL3 is essential, whereas PGL1, PGL2 and PGL5 are individually dispensable for plant growth and development. Knockdown of PGL3 in the pgl3 mutant leads to a dramatic decrease in plant size, a significant increase in total glucose-6-phosphate dehydrogenase activity and a marked decrease in cellular redox potential. Interestingly, the pgl3 plants exhibit constitutive pathogenesis-related gene expression and enhanced resistance to Pseudomonas syringae pv. maculicola ES4326 and Hyaloperonospora arabidopsidis Noco2. We found that although pgl3 does not spontaneously accumulate elevated levels of free salicylic acid (SA), the constitutive defense responses in pgl3 plants are almost completely suppressed by the npr1 and sid2/eds16/ics1 mutations, suggesting that the pgl3 mutation activates NPR1- and SID2/EDS16/ICS1-dependent defense responses. We demonstrate that plastidic (not peroxisomal) localization and 6PGL activity of the PGL3 protein are essential for complementing all pgl3 phenotypes, indicating that the oxidative section of the plastidic pentose phosphate pathway (PPP) is required for plant normal growth and development. Thus, pgl3 provides a useful tool not only for defining the role of the PPP in different subcellular compartments, but also for dissecting the SA/NPR1-mediated signaling pathway.