Overexpression of CsSAMT in Citrus sinensis Induces Defense Response and Increases Resistance to Xanthomonas citri subsp. citri (original) (raw)
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Scientific Reports
salicylic acid (sA) and its methyl ester, methyl salicylate (MesA) are well known inducers of local and systemic plant defense responses, respectively. MesA is a major mediator of systemic acquired resistance (sAR) and its conversion back into sA is thought to be required for sAR. In many plant species, conversion of MeSA into SA is mediated by MeSA esterases of the SABP2 family. Here we show that the Citrus sinensis SABP2 homologue protein CsMES1 catalyzes the hydrolysis of MeSA into SA. Molecular modeling studies suggest that CsMES1 shares the same structure and SA-binding mode with tobacco SABP2. However, an amino acid polymorphism in the active site of CsMES1-related proteins suggested an important role in enzyme regulation. We present evidence that the side chain of this polymorphic residue directly influences enzyme activity and SA binding affinity in CsMES proteins. We also show that SA and CsMES1 transcripts preferentially accumulate during the incompatible interaction between Xanthomonas aurantifolii pathotype C and sweet orange plants. Moreover, we demonstrate that sA and MesA inhibited citrus canker caused by Xanthomonas citri, whereas an inhibitor of CsMES1 enhanced canker formation, suggesting that CsMES1 and SA play a role in the local defense against citrus canker bacteria. Salicylic acid (SA), also known as 2-hydroxybenzoic acid, is a plant hormone that has long been recognized as a defense signaling molecule involved in both local and systemic acquired resistance (SAR) against microbial pathogens in many plant species 1-4. In response to pathogen infection, SA binds to and activates NPR1, a master regulator of the SA-mediated defense response. NPR1 functions together with the transcription factor TGA to directly promote transcription of an array of defense-related genes 4,5. In several plant species, SA is synthesized from chorismate by two biosynthetic routes, the isochorismate synthase (ICS) and phenylalanine ammonia-lyase (PAL) pathways. Endogenous SA can also undergo a series of chemical modifications including hydroxylation, glycosylation, methylation and amino acid conjugation 3,6-8. Such modifications directly affect the biochemical properties of the SA derivatives, thus altering their mode of action and interaction with protein ligands. For instance, while glycosylation and amino acid conjugation are important for SA inactivation, accumulation and storage, methylation of the SA carbonyl group into a methyl ester (MeSA) greatly enhances the volatility of this molecule, making MeSA an efficient long distance and systemic defense signal 3,9,10. MeSA is synthesized by carbonyl methyltransferases of the SABATH family, including the S-adenosyl-L-methionine (SAM) methyl transferases (SAMT), which uses SAM as a methyl donor 11,12. Although regarded as one of the main mediators of SAR against numerous microbial pathogens, MeSA is thought to be biologically inert and its conversion back into SA appears to be necessary for the activation of plant defenses at distal sites of pathogen attack 2,3,9,13. This is consistent with the role played by the SA-methylesterases that catalyze the hydrolysis of MeSA into SA. These enzymes have been characterized in a few plant species, including tobacco, potato, Arabidopsis and poplar 9,14-17. Brazilian Biosciences national Laboratory (LnBio), Brazilian center for Research in energy and Materials (cnPeM),
Tree physiology, 2017
In order to clarify whether high linalool content in citrus leaves alone induces strong field resistance to citrus canker caused by Xanthomonas citri subsp. citri (Xcc), and to assess whether this trait can be transferred to a citrus type highly sensitive to the bacterium, transgenic 'Hamlin' sweet orange (Citrus sinensis L. Osbeck) plants over-expressing a linalool synthase gene (CuSTS3-1) were generated. Transgenic lines (LIL) with the highest linalool content showed strong resistance to citrus canker when spray inoculated with the bacterium. In LIL plants inoculated by wounding (multiple-needle inoculation), the linalool level was correlated with the repression of the bacterial titer and up-regulation of defense-related genes. The exogenous application of salicylic acid, methyl jasmonate or linalool triggered responses similar to those constitutively induced in LIL plants. The linalool content in Ponkan mandarin leaves was significantly higher than that of leaves from six...
Overexpression of a citrus NDR1 ortholog increases disease resistance in Arabidopsis
Frontiers in plant science, 2013
Emerging devastating diseases, such as Huanglongbing (HLB) and citrus canker, have caused tremendous losses to the citrus industry worldwide. Genetic engineering is a powerful approach that could allow us to increase citrus resistance against these diseases. The key to the success of this approach relies on a thorough understanding of defense mechanisms of citrus. Studies of Arabidopsis and other plants have provided a framework for us to better understand defense mechanisms of citrus. Salicylic acid (SA) is a key signaling molecule involved in basal defense and resistance (R) gene-mediated defense against broad-spectrum pathogens. The Arabidopsis gene NDR1 (NON-RACE-SPECIFIC DISEASE RESISTANCE 1) is a positive regulator of SA accumulation and is specifically required for signaling mediated by a subset of R genes upon recognition of their cognate pathogen effectors. Our bioinformatic analysis identified an ortholog of NDR1 from citrus, CsNDR1. Overexpression of CsNDR1 complemented s...
Over-expression of the Arabidopsis NPR1 gene in citrus increases resistance to citrus canker
European Journal of Plant Pathology, 2010
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.
2008
Understanding the mechanisms of plant–pathogen interaction is believed to be key to resolve the existing crop disease crisis. Molecular advances have facilitated the discovery and study of genes associated with natural defense pathways in a number of model systems. In our laboratories, citrus homologues of vital defense genes have been identified using comparative analysis and their expression has been characterized. In addition, differential gene expression during infection with citrus canker has been examined. Both approaches have facilitated the study of defense responses in citrus. The better understanding of these natural defense pathways has allowed plant-derived genes to be used to induce disease resistance. These recent discoveries as well as strategies for their practical application in citrus breeding are discussed in this review.
2020
Plant immunity against pathogens and pests is comprised of complex mechanisms orchestrated by signaling pathways regulated by plant hormones [Salicylic acid (SA) and Jasmonic acid (JA)]. Investigations of plant immune response to phytopathogens and phloem-feeders have revealed that SA plays a critical role in reprogramming of the activity and/or localization of transcriptional regulators via post-translational modifications. We explored the contributing effects of herbivory by a phytopathogen vector [Asian citrus psyllid, Diaphorina citri] and pathogen [Candidatus Liberibacter asiaticus (CLas)] infection on response of sweet orange [Citrus sinensis (L.) Osbeck] using manipulative treatments designed to mimic the types of infestations/infections that citrus growers experience when cultivating citrus in the face of Huanglongbing (HLB) disease. A one-time (7 d) inoculation access period with CLas-infected vectors caused SA-associated upregulation of PR-1, stimulating defense response a...
Functional characterization of the citrus canker susceptibility gene CsLOB1
Molecular plant pathology, 2018
Xanthomonas citri ssp. citri (Xcc) is an important plant-pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator-like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306Δp...
Plant Molecular Biology Reporter, 2010
Genetic transformation with genes that code for antimicrobial peptides has been an important strategy used to control bacterial diseases in fruit crops, including apples, pears, and citrus. Asian citrus canker (ACC) caused by Xanthomonas citri subsp. citri Schaad et al. (Xcc) is a very destructive disease, which affects the citrus industry in most citrus-producing areas of the world. Here, we report the production of genetically transformed Natal, Pera, and Valencia sweet orange cultivars (Citrus sinensis L. Osbeck) with the insect-derived attacin A (attA) gene and the evaluation of the transgenic plants for resistance to Xcc. Agrobacterium tumefaciens Smith and Towns-mediated genetic transformation experiments involving these cultivars led to the regeneration of 23 different lines. Genetically transformed plants were identified by polymerase chain reaction, and transgene integration was confirmed by Southern blot analyses. Transcription of attA gene was detected by Northern blot analysis in all plants, except for one Natal sweet orange transformation event. Transgenic lines were multiplied by grafting onto Rangpur lime rootstock plants (Citrus limonia Osbeck) and sprayinoculated with an Xcc suspension (10 6 cfu mL −1 ). Experiments were repeated three times in a completely randomized design with seven to ten replicates. Disease severity was determined in all transgenic lines and in the control (nontransgenic) plants 30 days after inoculation. Four transgenic lines of Valencia sweet orange showed a significant reduction in disease severity caused by Xcc. These reductions ranged from 58.3% to 77.8%, corresponding to only 0.16-0.30% of leaf diseased area as opposed to 0.72% on control plants. One transgenic line of Natal sweet orange was significantly more resistant to Xcc, with a reduction of 45.2% comparing to the control plants, with only 0.14% of leaf diseased area. Genetically transformed Pera sweet orange plants expressing attA gene did not show a significant enhanced resistance to Xcc, probably due to its genetic background, which is naturally more resistant to this pathogen. The potential effect of attacin A antimicrobial peptide to control ACC may be related to the genetic background of each sweet orange cultivar regarding their natural resistance to the pathogen.
Molecular tools have facilitated the discovery and study of genes associated with natural defense pathways in a number of model systems. In our laboratories, citrus homologues of key genes have been identified (for instance, NPR1 and PR1) using comparative analysis and their expression characterized. In addition, differential gene expression during infection with citrus canker has been examined. Both approaches have facilitated the study of defense responses in citrus. The improved understanding of these natural defense pathways in model species has allowed plant-derived genes to be used to induce disease resistance. These recent discoveries as well as strategies for their practical application in citrus breeding are discussed in this review.
Over-expression of the citrus gene CtNH1 confers resistance to bacterial canker disease
Physiological and Molecular Plant Pathology, 2013
Citrus canker is a devastating disease, caused by Xanthomonas axonopodis pv. citri (Xac). It is well established that the NPR1 gene plays a pivotal role in systemic acquired resistance (SAR) in Arabidopsis. In this study, we report the isolation and characterization of an NPR1 homolog from citrus, namely Citrus NPR1 homolog 1 (CtNH1). Sequence alignment and phylogenetic analysis indicate that CtNH1 is closelyrelated to the Arabidopsis NPR1 gene and its orthologs from rice, grapevine, and cacao. When overexpressed in citrus, CtNH1 confers resistance to Xac and leads to constitutive expression of the pathogenesis-related (PR) gene chitinase 1 (Chi1), suggesting that CtNH1 is orthologous to NPR1.