Tomato SlSAP3, a member of the stress-associated protein family, is a positive regulator of immunity against Pseudomonas syringae pv. tomato DC3000.
ABSTRACT: Tomato stress-associated proteins (SAPs) belong to A20/AN1 zinc finger protein family, some of which have been shown to play important roles in plant stress responses. However, little is known about the functions and underlying molecular mechanisms of SAPs in plant immune responses. In the present study, we reported the function of tomato SlSAP3 in immunity to Pseudomonas syringae pv. tomato (Pst) DC3000. Silencing of SlSAP3 attenuated while overexpression of SlSAP3 in transgenic tomato increased immunity to Pst DC3000, accompanied with reduced and increased Pst DC3000-induced expression of SA signalling and defence genes, respectively. Flg22-induced reactive oxygen species (ROS) burst and expression of PAMP-triggered immunity (PTI) marker genes SlPTI5 and SlLRR22 were strengthened in SlSAP3-OE plants but were weakened in SlSAP3-silenced plants. SlSAP3 interacted with two SlBOBs and the A20 domain in SlSAP3 is critical for the SlSAP3-SlBOB1 interaction. Silencing of SlBOB1 and co-silencing of all three SlBOB genes conferred increased resistance to Pst DC3000, accompanied with increased Pst DC3000-induced expression of SA signalling and defence genes. These data demonstrate that SlSAP3 acts as a positive regulator of immunity against Pst DC3000 in tomato through the SA signalling and that SlSAP3 may exert its function in immunity by interacting with other proteins such as SlBOBs, which act as negative regulators of immunity against Pst DC3000 in tomato.
Project description:Plants intensely modulate respiration when pathogens attack, but the function of mitochondrial respiration-related genes in plant-bacteria interaction is largely unclear. Here, the functions of ?-ketoglutarate dehydrogenase (?-kGDH) E2 subunit and alternative oxidase (AOX) were investigated in the interaction between tomato and the virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pst). Pst inoculation suppressed the transcript abundance of ?-kGDH E2, but enhanced AOX expression and salicylic acid (SA) accumulation. Gene silencing and transient overexpression approaches revealed that plant susceptibility to Pst was significantly reduced by silencing ?-kGDH E2 in tomato, but increased by overexpressing ?-kGDH E2 in Nicotiana benthamiana, whereas silencing or overexpressing of AOX1a did not affect plant defense. Moreover, silencing octanoyltransferase (LIP2), engaged in the lipoylation of ?-kGDH E2, significantly reduced disease susceptibility and hydrogen peroxide accumulation. Use of transgenic NahG tomato plants that cannot accumulate SA as well as the exogenous SA application experiment evidenced that ?-kGDH E2 acts downstream of SA defense pathway. These results demonstrate tomato ?-kGDH E2 plays a negative role in plant basal defense against Pst in an AOX-independent pathway but was associated with lipoylation and SA defense pathways. The findings help to elucidate the mechanisms of mitochondria-involved plant basal immunity.
Project description:S-adenosylhomocysteine hydrolase (SAHH), catalyzing the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine, is a key enzyme that maintain the cellular methylation potential in all organisms. We report here the biological functions of tomato SlSAHHs in stress response. The tomato genome contains three SlSAHH genes that encode SlSAHH proteins with high level of sequence identity. qRT-PCR analysis revealed that SlSAHHs responded with distinct expression induction patterns to Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea as well as to defense signaling hormones such as salicylic acid, jasmonic acid and a precursor of ethylene. Virus-induced gene silencing-based knockdown of individual SlSAHH gene did not affect the growth performance and the response to Pst DC3000. However, co-silencing of three SlSAHH genes using a conserved sequence led to significant inhibition of vegetable growth. The SlSAHH-co-silenced plants displayed increased resistance to Pst DC3000 but did not alter the resistance to B. cinerea. Co-silencing of SlSAHHs resulted in constitutively activated defense responses including elevated SA level, upregulated expression of defense-related and PAMP-triggered immunity marker genes and increased callose deposition and H2O2 accumulation. Furthermore, the SlSAHH-co-silenced plants also exhibited enhanced drought stress tolerance although they had relatively small roots. These data demonstrate that, in addition to the functions in growth and development, SAHHs also play important roles in regulating biotic and abiotic stress responses in plants.
Project description:Histone H2B monoubiquitination pathway has been shown to play critical roles in regulating growth/development and stress response in Arabidopsis. In the present study, we explored the involvement of the tomato histone H2B monoubiquitination pathway in defense response against Botrytis cinerea by functional analysis of SlHUB1 and SlHUB2, orthologues of the Arabidopsis AtHUB1/AtHUB2.We used the TRV-based gene silencing system to knockdown the expression levels of SlHUB1 or SlHUB2 in tomato plants and compared the phenotype between the silenced and the control plants after infection with B. cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Biochemical and interaction properties of proteins were examined using in vitro histone monoubiquitination and yeast two-hybrid assays, respectively. The transcript levels of genes were analyzed by quantitative real time PCR (qRT-PCR).The tomato SlHUB1 and SlHUB2 had H2B monoubiquitination E3 ligases activity in vitro and expression of SlHUB1 and SlHUB2 was induced by infection of B. cinerea and Pst DC3000 and by treatment with salicylic acid (SA) and 1-amino cyclopropane-1-carboxylic acid (ACC). Silencing of either SlHUB1 or SlHUB2 in tomato plants showed increased susceptibility to B. cinerea, whereas silencing of SlHUB1 resulted in increased resistance against Pst DC3000. SlMED21, a Mediator complex subunit, interacted with SlHUB1 but silencing of SlMED21 did not affect the disease resistance to B. cinerea and Pst DC3000. The SlHUB1- and SlHUB2-silenced plants had thinner cell wall but increased accumulation of reactive oxygen species (ROS), increased callose deposition and exhibited altered expression of the genes involved in phenylpropanoid pathway and in ROS generation and scavenging system. Expression of genes in the SA-mediated signaling pathway was significantly upregulated, whereas expression of genes in the jasmonic acid (JA)/ethylene (ET)-mediated signaling pathway were markedly decreased in SlHUB1- and SlHUB2-silenced plants after infection of B. cinerea.VIGS-based functional analyses demonstrate that both SlHUB1 and SlHUB2 contribute to resistance against B. cinerea most likely through modulating the balance between the SA- and JA/ET-mediated signaling pathways.
Project description:Trehalose and its metabolism have been demonstrated to play important roles in control of plant growth, development, and stress responses. However, direct genetic evidence supporting the functions of trehalose and its metabolism in defense response against pathogens is lacking. In the present study, genome-wide characterization of putative trehalose-related genes identified 11 SlTPSs for trehalose-6-phosphate synthase, 8 SlTPPs for trehalose-6-phosphate phosphatase and one SlTRE1 for trehalase in tomato genome. Nine SlTPSs, 4 SlTPPs, and SlTRE1 were selected for functional analyses to explore their involvement in tomato disease resistance. Some selected SlTPSs, SlTPPs, and SlTRE1 responded with distinct expression induction patterns to Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000 as well as to defense signaling hormones (e.g., salicylic acid, jasmonic acid, and a precursor of ethylene). Virus-induced gene silencing-mediated silencing of SlTPS3, SlTPS4, or SlTPS7 led to deregulation of ROS accumulation and attenuated the expression of defense-related genes upon pathogen infection and thus deteriorated the resistance against B. cinerea or Pst DC3000. By contrast, silencing of SlTPS5 or SlTPP2 led to an increased expression of the defense-related genes upon pathogen infection and conferred an increased resistance against Pst DC3000. Silencing of SlTPS3, SlTPS4, SlTPS5, SlTPS7, or SlTPP2 affected trehalose level in tomato plants with or without infection of B. cinerea or Pst DC3000. These results demonstrate that SlTPS3, SlTPS4, SlTPS5, SlTPS7, and SlTPP2 play roles in resistance against B. cinerea and Pst DC3000, implying the importance of trehalose and tis metabolism in regulation of defense response against pathogens in tomato.
Project description:Trichoderma species are well known biocontrol agents that are able to induce responses in the host plants against an array of abiotic and biotic stresses. Here, we investigate, when applied to tomato seeds, the potential of Trichoderma strains belonging to three different species, T. parareesei T6, T. asperellum T25, and T. harzianum T34, to control the fully pathogenic strain Pseudomonas syringae pv. tomato (Pst) DC3000, able to produce the coronatine (COR) toxin, and the COR-deficient strain Pst DC3118 in tomato plants, and the molecular mechanisms by which the plant can modulate its systemic defense. Four-week old tomato plants, seed-inoculated, or not, with a Trichoderma strain, were infected, or not, with a Pst strain, and the changes in the expression of nine marker genes representative of salicylic acid (SA) (ICS1 and PAL5) and jasmonic acid (JA) (TomLoxC) biosynthesis, SA- (PR1b1), JA- (PINII and MYC2) and JA/Ethylene (ET)-dependent (ERF-A2) defense pathways, as well as the abscisic acid (ABA)-responsive gene AREB2 and the respiratory burst oxidase gene LERBOH1, were analyzed at 72 hours post-inoculation (hpi) with the bacteria. The significant increase obtained for bacterial population sizes in the leaves, disease index, and the upregulation of tomato genes related to SA, JA, ET and ABA in plants inoculated with Pst DC3000 compared with those obtained with Pst DC3118, confirmed the COR role as a virulence factor, and showed that both Pst and COR synergistically activate the JA- and SA-signaling defense responses, at least at 72 hpi. The three Trichoderma strains tested reduced the DC3118 levels to different extents and were able to control disease symptoms at the same rate. However, a minor protection (9.4%) against DC3000 was only achieved with T. asperellum T25. The gene deregulation detected in Trichoderma-treated plus Pst-inoculated tomato plants illustrates the complex system of a phytohormone-mediated signaling network that is affected by the pathogen and Trichoderma applications but also by their interaction. The expression changes for all nine genes analyzed, excepting LERBOH1, as well as the bacterial populations in the leaves were significantly affected by the interaction. Our results show that Trichoderma spp. are not adequate to control the disease caused by fully pathogenic Pst strains in tomato plants.
Project description:Plants attenuate their responses to a variety of bacterial and fungal pathogens, leading to higher incidences of pathogen infection at night. However, little is known about the molecular mechanism responsible for the light-induced defence response; transcriptome data would likely facilitate the elucidation of this mechanism.In this study, we observed diurnal changes in tomato resistance to Pseudomonas syringae pv. tomato DC3000 (Pto DC3000), with the greatest susceptibility before midnight. Nightly light treatment, particularly red light treatment, significantly enhanced the resistance; this effect was correlated with increased salicylic acid (SA) accumulation and defence-related gene transcription. RNA-seq analysis revealed that red light induced a set of circadian rhythm-related genes involved in the phytochrome and SA-regulated resistance response. The biosynthesis and signalling pathways of multiple plant hormones (auxin, SA, jasmonate, and ethylene) were co-ordinately regulated following Pto DC3000 infection and red light, and the SA pathway was most significantly affected by red light and Pto DC3000 infection. This result indicates that SA-mediated signalling pathways are involved in red light-induced resistance to pathogens. Importantly, silencing of nonexpressor of pathogensis-related genes 1 (NPR1) partially compromised red light-induced resistance against Pto DC3000. Furthermore, sets of genes involved in redox homeostasis (respiratory burst oxidase homologue, RBOH; glutathione S-transferases, GSTs; glycosyltransferase, GTs), calcium (calmodulin, CAM; calmodulin-binding protein, CBP), and defence (polyphenol oxidase, PPO; nudix hydrolase1, NUDX1) as well as transcription factors (WRKY18, WRKY53, WRKY60, WRKY70) and cellulose synthase were differentially induced at the transcriptional level by red light in response to pathogen challenge.Taken together, our results suggest that there is a diurnal change in susceptibility to Pto DC3000 with greatest susceptibility in the evening. The red light induced-resistance to Pto DC3000 at night is associated with enhancement of the SA pathway, cellulose synthase, and reduced redox homeostasis.
Project description:The SR/CAMTA proteins represent a small family of transcription activators that play important roles in plant responses to biotic and abiotic stresses. Seven SlSR/CAMTA genes were identified in tomato as tomato counterparts of SR/CAMTA; however, the involvement of SlSRs/CAMTAs in biotic and abiotic stress responses is not clear. In this study, we performed functional analysis of the SlSR/CAMTA family for their possible functions in defense response against pathogens and tolerance to drought stress.Expression of SlSRs was induced with distinct patterns by Botrytis cinerea and Pseudomonas syringae pv. tomato (Pst) DC3000. Virus-induced gene silencing (VIGS)-based knockdown of either SlSR1 or SlSR3L in tomato resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, marker genes for pathogen-associated molecular pattern-triggered immunity, and regulatory genes involved in the salicylic acid- and ethylene-mediated signaling pathways. Furthermore, the expression of SlSR1L and SlSR2L in detached leaves and whole plants was significantly induced by drought stress. Silencing of SlSR1L led to decreased drought stress tolerance, accelerated water loss in leaves, reduced root biomass and attenuated expression of drought stress responsive genes in tomato. The SlSR1 and SlSR3L proteins were localized in the nucleus of plant cells when transiently expressed in Nicotiana benthamiana and had transcriptional activation activity in yeast.VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L in the tomato SlSR/CAMTA family are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress tolerance in tomato.
Project description:The Somatic Embryogenesis Receptor Kinase 3 (SERK3)/Brassinosteroid (BR) Insensitive 1-Associated Kinase 1 (BAK1) is required for pattern-triggered immunity (PTI) in Arabidopsis thaliana and Nicotiana benthamiana. Tomato (Solanum lycopersicum) has three SlSERK members. Two of them exhibit particularly high levels of sequence similarity to AtSERK3 and, therefore, were named SlSERK3A and SlSERK3B. To characterize a role for SlSERK3A and SlSERK3B in defense, we suppressed each gene individually or co-silenced both using virus-induced gene silencing (VIGS) in the tomato cv. Moneymaker. Co-silencing SlSERK3A and SlSERK3B resulted in spontaneous necrotic lesions and reduced sensitivity to exogenous BR treatment. Silencing either SlSERK3A or SlSERK3B resulted in enhanced susceptibility to root knot-nematode and to non-pathogenic Pseudomonas syringae pv. tomato (Pst) DC3000 hrcC indicating that both SlSERK3s are positive regulators of defense. Interestingly, silencing SlSERK3B, but not SlSERK3A, resulted in enhanced susceptibility to the pathogenic strain Pst DC3000 indicating distinct roles for these two SlSERK3 paralogs. SlSERK3A and SlSERK3B are active kinases, localized to the plasma membrane, and interact in vivo with the Flagellin Sensing 2 receptor in a flg22-dependent manner. Complementation of the Atserk3/bak1-4 mutant with either SlSERK3A or SlSERK3B partially rescued the mutant phenotype. Thus, SlSERK3A and SlSERK3B are likely to constitute tomato orthologs of BAK1.
Project description:Melatonin is an important secondary messenger in plant innate immunity against the bacterial pathogen Pseudomonas syringe pv. tomato (Pst) DC3000 in the salicylic acid (SA)- and nitric oxide (NO)-dependent pathway. However, the metabolic homeostasis in melatonin-mediated innate immunity is unknown. In this study, comparative metabolomic analysis found that the endogenous levels of both soluble sugars (fructose, glucose, melibose, sucrose, maltose, galatose, tagatofuranose and turanose) and glycerol were commonly increased after both melatonin treatment and Pst DC3000 infection in Arabidopsis. Further studies showed that exogenous pre-treatment with fructose, glucose, sucrose, or glycerol increased innate immunity against Pst DC3000 infection in wild type (Col-0) Arabidopsis plants, but largely alleviated their effects on the innate immunity in SA-deficient NahG plants and NO-deficient mutants. This indicated that SA and NO are also essential for sugars and glycerol-mediated disease resistance. Moreover, exogenous fructose, glucose, sucrose and glycerol pre-treatments remarkably increased endogenous NO level, but had no significant effect on the endogenous melatonin level. Taken together, this study highlights the involvement of sugars and glycerol in melatonin-mediated innate immunity against bacterial pathogen in SA and NO-dependent pathway in Arabidopsis.
Project description:Phosphoinositide 3-kinase (PI3K) plays a vital role in plant response to abiotic stress. However, the role of PI3K in plant immunity is largely unknown. This study showed that PI3K enhanced Arabidopsis resistance to Pseudomonas syringae pv tomato DC3000 (Pst DC3000) and Pst DC3000 (avrRpt2). Overexpression of AtVPS34 promoted stomatal closure while PI3K inhibitors blocked that after spray inoculation. Additionally, gene expression of AtVPS34 was increased upon infection by Pst DC3000 (avrRpt2), and SA upregulated AtVPS34 gene expression in this process. Furthermore, overexpression of AtVPS34 enhanced PR gene expression after syringe infiltration with Pst DC3000 (avrRpt2), while PI3K inhibitors inhibited that. The production of hydrogen peroxide and the expression of gene encoding antioxidant enzyme were both enhanced in AtVPS34 overexpressing lines after spray inoculation or syringe infiltration with Pst DC3000 (avrRpt2). Collectively, these results unraveled a novel and broad role of PI3K in plant immunity which promoted stomatal closure and PR gene expression possibly via regulating ROS production.