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In JoVE (1)
Other Publications (11)
- The Journal of Biological Chemistry
- Current Opinion in Plant Biology
- Genome / National Research Council Canada = Génome / Conseil National De Recherches Canada
- Cell
- Cellular Microbiology
- Genome / National Research Council Canada = Génome / Conseil National De Recherches Canada
- Nature
- Annual Review of Phytopathology
- The Plant Journal : for Cell and Molecular Biology
- Molecular Plant-microbe Interactions : MPMI
- Current Opinion in Biotechnology
Articles by Maeli Melotto in JoVE
JoVE Immunology and Infection
Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging
Biology, University of Texas at Arlington
We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.
Other articles by Maeli Melotto on PubMed
Regulation of Plant Arginase by Wounding, Jasmonate, and the Phytotoxin Coronatine
In mammalian cells, induced expression of arginase in response to wound trauma and pathogen infection plays an important role in regulating the metabolism of L-arginine to either polyamines or nitric oxide (NO). In higher plants, which also utilize arginine for the production of polyamines and NO, the potential role of arginase as a control point for arginine homeostasis has not been investigated. Here, we report the characterization of two genes (LeARG1 and LeARG2) from Lycopersicon esculentum (tomato) that encode arginase. Phylogenic analysis showed that LeARG1 and -2, like all other plant arginases, are more similar to agmatinase than to arginases from vertebrates, fungi, and bacteria. Nevertheless, recombinant LeARG1 and -2 exhibited specificity for L-arginine over agmatine and related guanidino substrates. The plant enzymes, like mammalian arginases, were inhibited (K(i) approximately 14 microM) by the NO precursor N(G)-hydroxy-L-arginine. These results indicate that plant arginases define a distinct group of ureohydrolases that function as authentic L-arginases. LeARG1 and LeARG2 transcripts accumulated to their highest levels in reproductive tissues. In leaves, LeARG2 expression and arginase activity were induced in response to wounding and treatment with jasmonic acid (JA), a potent signal for plant defense responses. Wound- and JA-induced expression of LeARG2 was not observed in the tomato jasmonic acid-insensitive1 mutant, indicating that this response is strictly dependent on an intact JA signal transduction pathway. Infection of wild-type plants with a virulent strain of Pseudomonas syringae pv. tomato also up-regulated LeARG2 expression and arginase activity. This response was mediated by the bacterial phytotoxin coronatine, which exerts its virulence effects by co-opting the host JA signaling pathway. These results highlight striking similarities in the regulation of arginase in plants and animals and suggest that stress-induced arginase may perform similar roles in diverse biological systems.
Suppression of Host Defense in Compatible Plant-Pseudomonas Syringae Interactions
Despite impressive advances in the study of plant resistance to pathogens, little is known about the molecular basis of plant susceptibility to virulent pathogens. Recent progress in susceptible plant-Pseudomonas syringae interactions has provided a glimpse into the battles fought between plants and bacterial pathogens. A key step for pathogenesis appears to be the suppression of host defenses. Suppression of host defenses, including basal defense, gene-for-gene resistance and nonhost resistance, is a key step for pathogenesis. Defense suppression is mediated by bacterial effector proteins, which are secreted through the type III secretion system, and by coronatine, a bacterial toxin that structurally and functionally mimics methyl jasmonate, a plant defense signaling molecule.
Comparative Bioinformatic Analysis of Genes Expressed in Common Bean (Phaseolus Vulgaris L.) Seedlings
To rapidly and cost-effectively generate gene expression data, we developed an annotated unigene database of common bean (Phaseolus vulgaris L.). In this study, 3 cDNA libraries were constructed from the bean breeding line SEL1308, 1 from young leaf and 2 from seedlings inoculated or not inoculated with the fungal pathogen Colletotrichum lindemuthianum (Sacc. & Magnus) Briosi & Cavara, which causes anthracnose in common bean. To this date, 5255 single-pass sequences have been included in the database after selection based on sequence quality. These ESTs were trimmed and clustered using the computer programs Phred and CAP3 to form a unigene collection of 3126 unique sequences. Within clusters, 318 single nucleotide polymorphisms (SNPs) and 68 insertions-deletions (indels) were found, indicating the presence of paralogous gene families in our database. Each unigene sequence was analyzed for possible function using their similarity to known genes represented in the GenBank database and classified into 14 categories. Only 314 unigenes showed significant similarities to Phaseolus genomic sequences and P. vulgaris ESTs, which indicates that 90% (2818 unigenes) of our database represent newly discovered common bean genes. In addition, 12% (387 unigenes) were shown to be specific to common bean. This study represents a first step towards the discovery of novel genes in beans and a valuable source of molecular markers for expressed gene tagging and mapping.
Plant Stomata Function in Innate Immunity Against Bacterial Invasion
Microbial entry into host tissue is a critical first step in causing infection in animals and plants. In plants, it has been assumed that microscopic surface openings, such as stomata, serve as passive ports of bacterial entry during infection. Surprisingly, we found that stomatal closure is part of a plant innate immune response to restrict bacterial invasion. Stomatal guard cells of Arabidopsis perceive bacterial surface molecules, which requires the FLS2 receptor, production of nitric oxide, and the guard-cell-specific OST1 kinase. To circumvent this innate immune response, plant pathogenic bacteria have evolved specific virulence factors to effectively cause stomatal reopening as an important pathogenesis strategy. We provide evidence that supports a model in which stomata, as part of an integral innate immune system, act as a barrier against bacterial infection.
Role of Plant Stomata in Bacterial Invasion
Stomata are microscopic pores in the epidermis of the aerial parts of terrestrial plants. These pores are essential for photosynthesis, as they allow CO(2) to diffuse into the plant. The size of the stomatal pore changes in response to environmental conditions, such as light intensity, air humidity and CO(2) concentrations, as part of the plant's adaptation to maximize photosynthetic efficiency and, at the same time, to minimize water loss. Historically, stomata have been considered as passive portal of entry for plant pathogenic bacteria. However, recent studies suggest that stomata can play an active role in restricting bacterial invasion as part of the plant innate immune system. Some plant pathogens have evolved specific virulence factors to overcome stomata-based defence. Interestingly, many bacterial disease outbreaks require high humidity, rain, or frost damage, which could promote stomatal opening and/or bypass stomatal defence by creating wounds as alternative entry sites. Further studies on microbial and environmental regulation of stomata-based defence should fill gaps in our understanding of bacterial pathogenesis, disease epidemiology and phyllosphere microbiology.
Development, Characterization, and Comparative Analysis of Polymorphism at Common Bean SSR Loci Isolated from Genic and Genomic Sources
Microsatellites or SSRs (single sequence repeats) have been used to construct and integrate genetic maps in crop species, including Phaseolus vulgaris. In the present study, 3 cDNA libraries generated by the Bean EST project (http://lgm.esalq.usp.br/BEST/), comprising a unigene collection of 3126 sequences and a genomic microsatellite-enriched library, were analyzed for the presence of SSRs. A total of 219 expressed sequence tags (ESTs) were found to carry 240 SSRs (named EST-SSR), whereas 714 genomic sequences contained 471 SSRs (named genomic-SSR). A subset of 80 SSRs, 40 EST-SSRs, and 40 genomic-SSRs were evaluated for molecular polymorphism in 23 genotypes of cultivated beans from the Mesoamerican and Andean genetic pools, including Brazilian cultivars and 2 related species. Of the common bean genotypes, 31 EST-SSR loci were polymorphic, yielding 2-12 alleles as compared with 26 polymorphic genomic-SSRs, accounting for 2-7 alleles. Cluster analysis from data using both genic and genomic-SSR revealed a clear separation between Andean and Mesoamerican beans. The usefulness of these loci for distinguishing bean genotypes and genetic mapping is discussed.
JAZ Repressor Proteins Are Targets of the SCF(COI1) Complex During Jasmonate Signalling
Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCF(COI1) ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl-isoleucine (JA-Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCF(COI1) ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCF(COI1)-JAZ1 protein complex as a site of perception of the plant hormone JA-Ile.
Role of Stomata in Plant Innate Immunity and Foliar Bacterial Diseases
Pathogen entry into host tissue is a critical first step in causing infection. For foliar bacterial plant pathogens, natural surface openings, such as stomata, are important entry sites. Historically, these surface openings have been considered as passive portals of entry for plant pathogenic bacteria. However, recent studies have shown that stomata can play an active role in limiting bacterial invasion as part of the plant innate immune system. As a counter-defense, the plant pathogen Pseudomonas syringae pv. tomato DC3000 uses the virulence factor coronatine to actively open stomata. In nature, many foliar bacterial disease outbreaks require high humidity, rain, or storms, which could favor stomatal opening and/or bypass stomatal defense by creating wounds as alternative entry sites. Further studies on microbial and environmental regulation of stomatal closure and opening could fill gaps in our understanding of bacterial pathogenesis, disease epidemiology, and microbiology of the phyllosphere.
A Critical Role of Two Positively Charged Amino Acids in the Jas Motif of Arabidopsis JAZ Proteins in Mediating Coronatine- and Jasmonoyl Isoleucine-dependent Interactions with the COI1 F-box Protein
SUMMARY: Coronatine is an important virulence factor produced by several pathovars of the bacterial pathogen Pseudomonas syringae. The structure of coronatine is similar to that of a class of plant hormones called jasmonates (JAs). An important step in JA signaling is the SCF(COI1) E3 ubiquitin ligase-dependent degradation of JAZ repressor proteins. We have recently shown that jasmonoyl isoleucine (JA-Ile) promotes physical interaction between Arabidopsis JAZ1 and COI1 (the F-box component of SCF(COI1)) proteins, and that the JA-Ile-dependent COI1-JAZ1 interaction could be reconstituted in yeast cells (i.e. in the absence of other plant proteins). Here we show that coronatine, but not its two biosynthetic precursors, also promotes interaction between Arabidopsis COI1 and multiple JAZ proteins. The C-terminal Jas motif, but not the N-terminal (NT) domain or central ZIM domain of JAZ proteins, is critical for JA-Ile/coronatine-dependent interaction with COI1. Two positively charged amino acid residues in the Jas domain were identified as essential for coronatine-dependent COI1-JAZ interactions. Mutations of these two residues did not affect the ability of JAZ1 and JAZ9 to interact with the transcription factor AtMYC2. Importantly, transgenic Arabidopsis plants expressing JAZ1 carrying these two mutations exhibited JA-insensitive phenotypes, including male sterility and enhanced resistance to P. syringae infection. These results not only suggest that coronatine and JA-Ile target the physical interaction between COI1 and the Jas domain of JAZ repressors, but also illustrate the critical role of positively charged amino acids in the Jas domain in mediating the JA-Ile/coronatine-dependent JAZ interaction with COI1.
Functional Analysis of the N Terminus of the Erwinia Amylovora Secreted Effector DspA/E Reveals Features Required for Secretion, Translocation, and Binding to the Chaperone DspB/F
DspA/E is a type III secreted effector protein required for pathogenicity in the apple and pear pathogen Erwinia amylovora, and DspB/F is a small chaperone protein involved in DspA/E secretion. While the secretion and translocation signals of many type III secretion effector proteins in human enteric pathogens have been characterized extensively, relatively little is known about the translocation requirements of many effectors in plant pathogens, including large DspE-like proteins. In this study, we report a functional analysis of the N terminus of DspE. The minimal requirements for secretion, translocation, and chaperone binding were characterized. Translocation assays using an adenylate cyclase (CyaA) reporter indicated that the first 51 amino acids of DspE were sufficient for translocation and that 150 amino acids were required for optimal translocation levels. The minimal translocation signal corresponded with the requirements for secretion into culture media. Mutations of conserved regions in amino acids 2 through 10 and 31 through 40 were found to influence translocation levels of an N-terminal DspE-CyaA fusion. Yeast two-hybrid and in-vitro pull-down assays revealed a chaperone-binding site within amino acids 51 through 100 of DspE and binding to DspF in this region was disrupted by specific mutations. However, neither disruption of the chaperone-binding domain nor deletion of the dspF gene had a significant impact on translocation levels of N-terminal DspE-CyaA fusions. Our results indicate that the minimal translocation signal of DspE is not coincident with the signal for DspF binding and that translocation of the N terminus of DspE is not dependent on the N-terminal DspF-binding domain.
Plant Stomata: a Checkpoint of Host Immunity and Pathogen Virulence
Stomata are microscopic pores formed by pairs of guard cells in the epidermis of terrestrial plants; they are essential for gas exchange with the environment and controlling water loss. Accordingly, plants regulate stomatal aperture in response to environmental conditions, such as relative humidity, CO(2) concentration, and light intensity. Stomatal openings are also a major route of pathogen entry into the plant and plants have evolved mechanisms to regulate stomatal aperture as an immune response against bacterial invasion. In this review, we highlight studies that begin to elucidate signaling events involved in bacterium-triggered stomatal closure and discuss how pathogens may have exploited environmental conditions or, in some cases, have evolved virulence factors to actively counter stomatal closure to facilitate invasion.
