- Research article
- Open Access
Tomato SR/CAMTA transcription factors SlSR1 and SlSR3L negatively regulate disease resistance response and SlSR1L positively modulates drought stress tolerance
© Li et al.; licensee BioMed Central Ltd. 2014
- Received: 2 August 2014
- Accepted: 15 October 2014
- Published: 28 October 2014
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.
- Tomato (Solanum lycopersicum)
- Disease resistance response
- Drought stress
Plants are vulnerable to various biotic and abiotic stresses but have evolved to equip with sophisticated signaling networks to precisely regulate defense response to unfavorable stresses. Upon perception of environmental stress, a set of early signaling events including changes in the cytosolic free calcium (Ca2+) concentration signatures (i.e. oscillations varying in cellular location, amplitude, duration or frequency) is often activated and integrated into different signaling pathways, which ultimately initiate transcriptional reprogramming leading to expression of a large set of stress-responsive genes -. Extensive biochemical and genetic studies have demonstrated that cellular Ca2+ as a universal second messenger plays critical roles in regulating defense responses to diverse biotic and abiotic stresses -.
Cellular Ca2+ changes can be sensed and interpreted by calcium-binding proteins (CaBPs) including calmodulin (CaM), calcineurin B-like proteins and calcium-dependent protein kinases -. These CaBPs regulate cellular responses through two distinct pathways upon sensing different biotic and abiotic signals -. Firstly, CaBPs trigger rapid responses by direct binding to cytosolic target proteins and modulating their activity. Alternatively, CaBPs modulate indirect and relatively slow cellular responses by interacting with transcription factors to regulate gene expression. Recently, a class of CaM-binding transcription factors (CAMTA for CaM-binding transcription activator) has been identified in plants -. The CAMTA proteins, also called signal-responsive (SR) proteins, are present in all plant and animal species examined to date and are highly conserved in their protein structures. Typically, the SR/CAMTA proteins contain a CG-1 DNA-binding domain (binding to specific cis-elements in promoter regions of the target genes) at the N-terminus, a TIG domain (an immunoglobulin-like fold involved in nonspecific DNA binding), three ankyrin repeats (implicated in protein-protein interaction) and five putative CaM-binding motifs called as IQ motif -. Biochemical studies with the Arabidopsis AtSR1 and rice OsCBT proteins have identified the primary target of DNA cis-element for SR/CAMTA proteins as CGCG and CGTG motifs in promoter regions of the target genes ,.
The SR/CAMTA proteins represent a small family of transcription activators in plants. For instance, six genes encoding for SR/CAMTA proteins were identified in Arabidopsis . The SR/CAMTA genes were shown to be responsive to multiple abiotic and biotic stresses including cold, wounding, drought and pathogen attack, as well as to stress-related hormones like ethylene, auxin, methyl jasmonate (MeJA) and salicylic acid (SA) ,,-. Recent genetic studies with loss-of-function and gain-of-function mutants have shown that members of the SR/CAMTA family play important roles in plant response to abiotic and biotic stresses. The Arabidopsis AtSR1 knockout mutant showed enhanced disease resistance against multiple pathogens with different infection styles including Pseudomonas syringae pv. tomato (Pst), Botrytis cinerea and Golovinomyces cichoracearum- but decreased resistance against insect herbivores ,. By contrast, overexpression of AtSR1 conferred an increased susceptibility to Pst DC3000, B. cinerea and G. cichoracearum,. Similarly, the rice oscbt mutant exhibited significant resistance to blast fungal pathogen Magnaporthe grisea and leaf blight bacterial pathogen Xanthomonas oryzae pv. oryzae. On the other hand, it was recently shown that the Arabidopsis AtSR1 and AtSR2 also play important roles in regulating tolerance to low temperature , and drought stress . The function of SR/CAMTA proteins in plant biotic and abiotic stress response is achieved mainly through regulating expression of genes whose promoter regions contain the CGCG boxes ,,,.
Seven SlSRs/CAMTAs (hereafter referred to as SlSRs for convenience) genes were identified in tomato and were shown to be developmentally regulated during fruit development and ripening and induced by ethylene ,. Further detailed analysis revealed that the SlSR genes showed differential expression patterns in tomato fruit in response to low temperature, mechanical injury, infection of the necrotrophic fungal pathogen B. cinerea, and treatments with the signaling molecules SA and MeJA . However, direct genetic evidence supporting the involvement of the tomato SlSRs in biotic and abiotic stress responses is still lacking. In the present study, we investigated the possible functions of SlSRs in disease resistance and drought stress tolerance using virus-induced gene silencing (VIGS) approach. Our VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L are negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L is a positive regulator of drought stress response.
Expression patterns of SlSRsin response to pathogen infection
Silencing of SlSR1 and SlSR3L conferred an increased resistance to B. cinerea and Pst DC3000
Silencing of SlSR1 and SlSR3L resulted in constitutive defense response
We next examined whether silencing of SlSR1 or SlSR3L led to constitutive expression of defense genes. The expression levels of five representative defense genes that are regulated through different defense signaling pathways and three marker genes for pathogen-associated molecular patterns-triggered immunity (PTI) were analyzed and compared between the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants and the pTRV2-GUS-infiltrated plants. As shown in Figure 6B, the expression levels of PR1a, PR1b and PR-P2, which are thought to be regulated by the SA-mediated signaling pathway , and of PIN2 and LapA, which are considered to be regulated by the JA/ET signaling pathway , were increased significantly in the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants, leading to 8?~?25 folds of increases for the PR1a, PR1b and PR-P2 genes and 1?~?11 folds of increases for the PIN2 and LapA genes, as compared with those in the pTRV2-GUS-infiltrated plants. Similarly, the expression levels of Pti5, Lrr22 and WRKY28, three PTI marker genes in tomato ,, in the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants were significantly higher than those in the pTRV2-GUS-infiltrated plants, resulting in increases of 0.5?~?3.0 folds in the expression levels (Figure 6C). These data demonstrate that silencing of either SlSR1 or SlSR3L activates constitutively the expression of the defense and PTI genes, leading to constitutive defense response in the SlSR1- and SlSR3L-silenced plants.
To determine whether silencing of SlSR1 and SlSR3L activates defense signaling pathways, we further analyzed and compared the expression levels of the key genes encoding important components involved in the SA- and JA/ET-mediated signaling pathways between the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants and the pTRV2-GUS-infiltrated plants. The expression levels of NPR1, EDS1 and TGA1, known to be critical components in the SA-mediated signaling pathway , in the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants were significantly increased, especially for the expression levels of the EDS1 and TGA1 genes, showing 1-fold increase relative to those in the pTRV2-GUS-infiltrated plants (Figure 6C). Similarly, the expression levels of ETR4 and ERF1, known to be associated with ET signaling pathway ,, in the pTRV2-SlSR1- and pTRV2-SlSR3L-infiltrated plants showed 1?~?2-fold increase over those in the pTRV2-GUS-infiltrated plants (Figure 6C). By contrast, the expression level of JAZ1, known to be associated with JA signaling pathway , in the pTRV2-SlSR1- and pTRV-SlSR3L-infiltrated plants was reduced as compared with those in the pTRV2-GUS-infiltrated plants (Figure 6C). These results suggest that silencing of either SlSR1 or SlSR3L can activate both the SA- and ET-mediated signaling pathways but suppress the JA-mediated signaling pathway in the SlSR1- and SlSR3L-silenced plants.
Expression patterns of SlSRsin response to drought stress and ABA
Silencing of SlSR1L reduced drought tolerance in tomato
SlSR1 and SlSR3L are localized in nucleus and have transactivation activity
Previous studies have identified a total of seven SlSRs genes in tomato and found that expression of SlSRs was regulated in tomato fruit by developmental cues and by biotic and abiotic environmental stress signals ,; however, the biological functions of SlSRs in tomato response to biotic and abiotic stress remain unclear. In the present study, data from our VIGS-based functional analyses demonstrate that both SlSR1 and SlSR3L act as negative regulators of defense response against B. cinerea and Pst DC3000 while SlSR1L functions as a positive regulator of drought stress tolerance in tomato. These findings not only demonstrate that members of the small SlSR family play important roles in regulation of defense responses to biotic and abiotic stresses but also extend our understanding on the biological function of SlSRs in regulating stress response.
In plants, members of the SR family have been shown to be induced by infections from different pathogens. For example, the expression of AtSR1 in Arabidopsis was induced by Pst DC3000 and G. cichoracearum. In this study, we observed that the expression of SlSRs could be induced with different patterns by Pst DC3000 and B. cinerea (Figures 1 and 2). In response to infection of B. cinerea, all the SlSRs showed upregulated expression patterns in leaf tissues, especially for SlSR1, SlSR1L, SlSR3L and SlSR4, whose expression levels were significantly upregulated (Figure 1). The B. cinerea-induced expression patterns of SlSRs in leaf tissues are somewhat different from the previous observation that the expressions of SlSR1, SlSR1L, SlSR2L and SlSR3 in wounded tomato fruit were suppressed by B. cinerea, probably due to the tissue-specific expression feature of the SlSRs. In contrast to the B. cinerea-induced expression patterns of SlSRs, expression of SlSR1, SlSR1L, SlSR3 and SlSR3L in leaf tissues was upregulated in response to infection of Pst DC3000 (Figure 2). The Pst DC3000-induced expression of SlSR1 is similar to that of the Arabidopsis AtSR1, closely related to SlSR1, whose expression was induced by Pst DC3000 . However, infection of Pst DC3000 did not affect the expression of SlSR2 and SlSR3 but suppressed the expression of SlSR4 in leaf tissues (Figure 2), which is contrast to the observation that the expression of SlSR4 in fruit tissues was induced by B. cinerea. Therefore, it seems reasonable that the expression of SlSRs is precisely controlled by complex mechanisms in response to infection from different pathogens. On the other hand, the B. cinerea- and Pst DC3000-induced expressions of SlSRs, especially for SlSR1 and SlSR3L, were much evident after 24 hr of inoculation (Figures 1 and 2), which is similar to the observation that the expression of AtSR1 in Arabidopsis leaves was only significantly induced by Pst DC3000 after 24 hr of inoculation . It was also found that the expression of most SlSRs in tomato fruits was markedly induced by exogenous SA and MeJA only after 8 hr of treatment . These observations suggest that most of the SlSRs should belong to late pathogen-responsive genes . The B. cinerea- and Pst DC3000-induced expression of SlSR1 and SlSR3L in leaves (Figures 1 and 2), along with their SA- and MeJA-induced expression patterns in fruits , suggest that SlSR1 and SlSR3L may play important roles in defense response to infection of B. cinerea and Pst DC3000.
In our VIGS-based functional analyses of SlSRs in defense response against different pathogens, we found that silencing of either SlSR1 or SlSR3L resulted in increased disease resistance against B. cinerea and Pst DC3000, as the SlSR1- and SlSR3L-silenced plants exhibited less severity of the diseases and supported less in planta growth of the pathogens than the non-silenced plants (Figures 4 and 5), indicating that loss-of-function of either SlSR1 or SlSR3L confers a broad-spectrum disease resistance against different pathogens with distinct infection styles. SlSR1 is phylogenetically closely related to the Arabidopsis AtSR1 , whose loss-of-function mutant plants showed increased disease resistance against three different pathogens including Pst DC3000, B. cinerea and G. cichoracearum- and gain-of-function mutant plants exhibited compromised systemic acquired resistance and basal immunity ,. Similar results were also observed in the mutant plants of the OsCBT gene, a rice SR family member closely related to AtSR1 and SlSR1, which exhibited significant enhanced resistance to blast fungal pathogen M. grisea and leaf blight bacterial pathogen X. oryzae pv. oryzae. Thus, it is likely that loss-of-function of some of the SR family members can confer broad-spectrum disease resistance in plants. This may have extensive significance that members of the SR family can be used for generating transgenic varieties with broad-spectrum resistance in economically important crops through RNA interference-mediated suppression of expression of target SR genes. Notably, in addition to SR1, the function of other SR family members in disease resistance has not been elucidated so far. In this study, we demonstrated that SlSR3L, like SlSR1 in tomato (Figures 4 and 5) and AtSR1 in Arabidopsis -, acts as a negative regulator of disease resistance against different pathogens in tomato. This finding characterized one member with important function in disease resistance from the relatively small SR family. It will be interesting to examine whether the Arabidopsis homologs of SlSR3L, AtSR3 and AtSR6 , play roles in regulating disease resistance. Although expression of SlSR1L, SlSR2, SlSR2L and SlSR3 was induced by B. cinerea and/or Pst DC3000 (Figures 1 and 2), silencing of each of these SlSR genes had no effect on disease resistance against these two pathogens (Figures 4 and 5). The expression of SlSR4 was induced by B. cinerea but suppressed by Pst DC3000 (Figures 1 and 2); however, silencing of SlSR4 did not lead to any alteration in disease resistance to B. cinerea and Pst DC3000 (Figures 4 and 5). These results indicate that the SlSR genes are highly responsive to pathogen infection but the responsiveness of these SlSR genes may be a side effect caused by pathogen infection but not a true reflection for their function in disease resistance.
The observed increased disease resistance against B. cinerea and Pst DC3000 in the SlSR1- and SlSR3L-silenced plants may be attributed to an improved basic immunity resulted from loss-of-function of SlSR1 and SlSR3L. This hypothesis is supported by several lines of evidence obtained from some biochemical and molecular analyses toward the SlSR1- and SlSR3L-silenced plants under pathogen-free conditions. Firstly, the SlSR1- and SlSR3L-silenced plants constitutively accumulated high level of H2O2 in leaves, as revealed by in situ DAB staining (Figure 6A). Similar phenomenon was also observed in the Arabidopsis sr1 mutant plants that accumulated high level of H2O2 in leaves without pathogen infection ,. It was previously found that suppression of SlWfi1 expression resulted in significant decrease of H2O2 accumulation in antisense tomato plants . The expression of SlWfi1 and SlRboh1, encoding plasma membrane-localized NADPH oxidases that are involved in generating ROS , was significantly upregulated in the SlSR1- and SlSR3L-silenced plants (Figure 6B), indicating an accelerated generation of ROS in these plants. By contrast, the ROS scavenging capacity in the SlSR1- and SlSR3L-silenced plants might not be affected significantly as the expression of the SOD gene was not changed (Figure 6B). The upregulated expression of the CAT and APX genes in the SlSR1- and SlSR3L-silenced plants might be a response to the high level of H2O2 accumulated in these plants as H2O2 was found to be capable of mediating the expression of CAT genes under stress conditions ,. An accelerated generation with an unchanged scavenging capacity in the SlSR1- and SlSR3L-silenced plants broke the ROS generating and scavenging balance and favored to accumulate high level of H2O2, which in turn acts as signaling molecules to activate defense response against pathogens. Secondly, the SlSR1- and SlSR3L-silenced plants constitutively expressed defense genes and PTI marker genes. Similar to the observations that the Arabidopsis sr1 and rice oscbt mutant plants constitutively expressed a diverse set of defense genes ,,, constitutive high levels of expression of defense genes including SlPR1a, SlPR1b, SlPR-P2, SlPIN2 and SlLapA, which are regulated by different signaling pathways, were observed in the SlSR1- and SlSR3L-silenced plants (Figure 6B). In addition, the expression of the PTI marker genes including Pti5, Lrr22 and WRKY28, was also significantly upregulated in the SlSR1- and SlSR3L-silenced plants (Figure 6C). These data indicate that both SlSR1 and SlSR3L negatively regulate PTI response. In this regarding, silencing of either SlSR1 or SlSR3L would relieve their suppression on PTI response and thus lead to increased resistance against multiple pathogens including B. cinerea and Pst DC3000 (Figures 4 and 5). This is supported by general knowledge that PTI but not effector-triggered immunity (ETI) plays important roles in regulating immunity against necrotrophic fungal pathogens like B. cinerea while both the PTI and ETI are require for immunity to biotrphic/hemibiotrophic pathogens such as Pst DC3000 . Thirdly, the SlSR1- and SlSR3L-silenced plants constitutively activated the SA- and ET-mediated defense signaling pathways. It was previously found that the Arabidopsis sr1 mutant plants contained high level of SA and had upregulated expression of defense and signaling genes , and that AtSR1 can bind to the CGCG box in the promoters of EDS1, NDR1 and EIN3,, indicating the involvements of AtSR1 in the SA- and ET-mediated signaling pathways. Our qRT-PCR analyses of expression of some defense signaling pathway-associated genes also demonstrated that both SlSR1 and SlSR3L have functions that negatively regulate the SA- and ET-mediated signaling pathways in tomato, as revealed by the upregulated expression of the SlNPR1, SlEDS1, SlTGA1, SlETR4 and SlERF1 genes in the SlSR1- and SlSR3L-silenced plants (Figure 6C). This is partially supported by the facts that the expression of SlSR1 and SlSR3L in tomato fruits was induced rapidly by exogenously applied SA and ET ,. In particular, it was shown that the Arabidopsis AtSR1 could bind to the CGCG box, a characteristic cis-elements for the SR proteins , in the promoter region of EDS1 and suppressed the expression of EDS1, which is critical to biosynthesis of SA. Bioinformatics analysis also identified a CGCG box within 1.3 Kb from the starting codon in the promoter of tomato SlEDS1 gene . It is thus possible that SlSR1 and perhaps SlSR3Ll regulate negatively the expression of SlEDS1 through binding to the CGCG box as the expression level of SlEDS1 was upregulated when either SlSR1 or SlSR3L was silenced (Figure 6C). Furthermore, the expression of SlJAZ1, known to be associated with the JA-mediated signaling pathway , was downregulated in the SlSR1- and SlSR3L-silenced plants (Figure 6C), suggesting a negative impact of SlSR1 and SlSR3L on the JA-mediated signaling pathway. This is in agreement with the observations that the Arabidopsis AtSR1 is a negative regulator for JA biosynthesis and herbivory tolerance ,. Another possibility that the constitutively activated SA-mediated signaling pathway in the SlSR1- and SlSR3L-silenced plants antagonistically suppressed the JA-mediated signaling pathway cannot be ruled out because the antagonistic cross-talk between these two signaling pathways is a common phenomenon occurred in regulating defense response against infection by different pathogens . Taken together, these data suggest that both SlSR1 and SlSR3L negatively regulate basic immunity in tomato through modulating the SA- and ET-mediated signaling pathways.
The involvement of the SR family members in abiotic stress was recently investigated using Arabidopsis knockout mutant lines ,. Our VIGS-based functional analyses discovered that silencing of SlSR1L resulted in decreased drought stress tolerance (Figures 7E and 8A). SlSR1L is phylogenetically related to the Arabidopsis AtSR2 , which was shown to regulate drought stress responses . Thus, it is likely that SlSR1L plays an important role in regulation of drought stress tolerance in tomato. The expression of SlSR1L was induced significantly by drought stress in detached leaves and in whole plants (Figure 7A and D). The decreased drought tolerance in the SlSR1L-silenced plants might be caused by multiple factors including morphological, physiological and molecular changes, which are affected by loss-of-function of SlSR1L. Like the stunted primary root in the Arabidopsis sr2 mutant plants , the SlSR1L-silenced plants had limited root system and biomass under normal growth condition (Figure 8C and D). Although the exact function of SlSR1L in development of the root system in tomato needs to be further investigated, the limited root system in the SlSR1L-silenced plants may result in reduced capacity of water uptake. Another fact that affected the water status in the SlSR1L-silenced plants was the accelerated rate of water loss, as revealed in the detached leaves (Figure 8B). Furthermore, the expression of SlAREB1, SlDREB, SlSpUSP, SlAREB2 and SGN-213276 was suppressed in the SlSR1L-silenced plants (Figure 8E), indicating that SlSR1L may regulate the expression of a large set of drought stress-responsive genes. Recent microarray-based analyses of gene expression profiling between the Arabidopsis sr1 and wild type plants revealed that hormone-mediated signaling such as ABA-mediated signaling plays important roles in AtSR1-regulated abiotic stress response ,. However, exogenous ABA did not induce the expression of SlSR1L in tomato (Figure 7B) and AtSR2 in Arabidopsis . Thus, it is likely that, as a transcription factor, SlSR1L acts in a yet-unknown signaling pathway, in which some hormones such as ABA are involved, to regulate drought stress response in tomato.
Our data presented in this study clearly demonstrate that SlSR1/SlSR3L and SlSR1L play important roles in biotic and abiotic stress responses, respectively. However, several questions regarding the mechanism of action of SlSR1/SlSR3L and SlSR1L in biotic and abiotic stress response need to be addressed. Further identification of downstream target genes regulated by SlSR1/SlSR3L and SlSR1L will help to elucidate the molecular mechanisms and the signaling pathways involved in the SlSR1/SlSR3L-regulated defense response against pathogens and the SlSR1L-regulated drought stress response. Of particular, the SlSR3L will be a priority for further study because the function of its orthologs in other plants such as the Arabidopsis AtSR3 and AtSR6  has not been defined yet. On the other hand, biochemical studies have shown that the Arabidopsis AtSR2/CAMTA1 , rice OsCBT  and tomato SlSR1 and SlSR3L (Figure 9) are functionally transcriptional activators in yeast. However, the Arabidopsis AtSR1 was shown to bind to the CGCG box in the promoters of the EDS1, NDR1 and EIN3 genes and repress their expression ,. Thus, the biochemical mechanism regarding how the SR proteins as transcriptional activators repress the expression of the target genes after binding to the CGCG box in the promoters of these genes needs to be further investigated.
Tomato genome encodes seven SlSR genes and expression of SlSR1 and SlSR3L was significantly induced by B. cinerea and Pst DC3000. Silencing of either SlSR1 or SlSR3L resulted in enhanced resistance to B. cinerea and Pst DC3000 and led to constitutive accumulation of H2O2, elevated expression of defense genes, PTI marker genes and regulatory genes involved in the SA- and ET-mediated signaling pathways. Meanwhile, expression of SlSR1L was significantly induced by drought stress and silencing of SlSR1L led to decreased drought stress tolerance. These results 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.
Plant growth and treatments
Tomato (Solanum lycopersicum) cv. Suhong 2003 was used for all experiments. Seeds were scarified on moist filter paper in Petri dishes for 2 days and the sprouted seeds were transferred into a mixture of perlite: vermiculite: plant ash (1:6:2). All tomato plants were grown in a growth room at 22–24°C with 60% relative humidity (RH) under a 14 hr light (350 μmol · s-1 · m-2 photons m-2 sec-1)/10 hr dark cycle. Two-week-old plants were used for VIGS assays and four-week-old plants were used for other experiments. For analysis of gene expression in response to pathogen infection, whole plant inoculation assays for B. cinerea and vacuum infiltrated inoculation assays for Pst DC3000 along with corresponding mock-inoculation controls were performed (see below). For analysis of gene expression in drought stress, drought stress was applied to the plants by stopping watering for a period until wilting symptom appeared and normally watered plants were used as controls in the whole plant drought stress assays. Alternatively, fully expanded leaves were detached and subjected to drought stress treatment by placing on lab blench or on water-saturated filter papers in Petri dishes as controls in detached leaf assays. For ABA treatment, a solution of ABA (100 ?M) or same volume of water as a control was sprayed onto leaf surface of the tomato plants. Leaf samples were collected at indicated time points after treatment or inoculation and stored at -80°C until use.
Plant inoculation and disease assays
Pst DC3000 was grown overnight in King’s B (KB) liquid medium containing rifampicin at 50 ?g/ml. The bacteria were collected and resuspended in 10 mM MgCl2 to OD600?=?0.0002 for plant inoculation. Four-week-old plants were vacuum infiltrated with suspension of Pst DC3000 or with MgCl2 solution as a mock inoculation control. The inoculated plants were kept in a sealed container to maintain high humidity (RH?>?90%) and disease progress was observed daily. Leaf samples were collected from at least six Pst DC3000-inoculated or mock-inoculated plants at different time points after inoculation and used for analysis of gene expression and in planta bacterial growth. For measurement of bacterial growth, leaf discs (6 mm in diameter) were surface sterilized in 70% ethanol for 10 s, homogenized in 200 ?l of 10 mM MgCl2, diluted in 10 mM MgCl2, and plated on KB agar plates containing rifampicin at 50 ?g/ml. The plates were incubated at 28°C and the bacterial numbers were counted 3 days after incubation.
B. cinerea was grown on 2× V8 agar (36% V8 juice, 0.2% CaCO3, and 2% agar) at 22°C and spores were collected and resuspended in 1% maltose buffer to 2 × 105 spores/ml for whole plant inoculation and 1 × 105 spores/ml for detached leaf inoculation. Whole plant inoculation and detached leaf inoculation assays were performed according to previously reported procedure ,. In the detached leaf inoculation assays, leaves were detached from at least 10 four-week-old VIGS-infiltrated plants and inoculated by dropping 5 ?l of spore suspension on leaf surface. In the whole plant inoculation assays, four-week-old plants were inoculated by foliar spraying with spore suspension of B. cinerea or with same volume of 1% maltose buffer as a mock-inoculation control and leaf samples were collected at different time points after inoculation for analysis of gene expression and in planta fungal growth. The inoculated leaves and plants were kept at 22°C in sealed containers to retain the moist conditions favorable for disease development. In planta fungal growth was analyzed by the amplification of the transcripts of a B. cinerea Actin gene as a marker , using a pair of primers BcActin-F and BcActin-R (Additional file 2). Relative fungal growth was expressed as folds of the transcript levels of BcActin vs the transcript levels of a tomato Actin gene.
Construction of vectors and VIGS assays
Based on the cDNA sequences of all seven SlSR genes , fragments of 386–462 bp for each SlSR gene (Additional file 3) were amplified with gene-specific primers (Additional file 2) from cDNAs synthesized from total RNA prepared from tomato leaf samples. After cloning and sequencing, these VIGS fragments were cloned into pTRV2 vector , yielding pTRV2-SlSRs. These pTRV2-SlSR constructs were then introduced into Agrobacterium tumefaciens strain GV3101 by electroporation using GENE PULSER II Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA). Agrobacteria carrying pTRV2-GUS (as a negative control), pTRV2-PDS (as a positive control for silencing efficiency examination) or pTRV2-SlSRs were cultivated in YEP medium (10 g/l peptone, 10 g/l yeast extract, 5 g NaCl/l, 50 ?g/ml rifampicin, 50 ?g/ml kanamycin and 25 ?g/ml gentamicin) for 36 hr with continuous shaking in a 28°C incubator. Cells were centrifuged and resuspended in infiltration buffer (10 mM MgCl2, 150 ?M acetosyringone, MES, pH5.7). The agrobacteria carrying pTRV2-GUS, pTRV2-PDS or pTRV2-SlSR were mixed with the agrobacteria carrying pTRV1 in a ratio of 1:1 and maintained at OD600?=?1.5 for 3 hr at room temperature. The mixed agrobacteria suspension was infiltrated into the abaxial surface of the 2-week-old seedlings using 1 mL needleless syringes. Efficiency of the silencing procedure was evaluated based on the appearance of bleaching phenotype in the pTRV2-PDS-infiltrated plants . When more than 90% of the pTRV2-PDS-infiltrated plants showed bleaching phenotype, the pTRV2-GUS- or pTRV2-SlSR-infiltrated plants in an independent experiment with same VIGS procedure were used for further study. Leaf samples were collected at 2 weeks after VIGS infiltration and used for analysis of the silencing efficiency by qRT-PCR.
The coding sequences of SlSR1 and SlSR3L were PCR amplified using pairs of gene-specific primers SlSR1-3F/SlSR1-3R and SlSR3L-3F/SlSR3L-3R, respectively (Additional file 2). After confirmation by sequencing, the coding regions of the SlSR1 and SlSR3L genes were cloned into pFGC-Egfp. The recombinant plasmids pFGC-Egfp-SlSR1, pFGC-Egfp-SlSR3L and pFGC-Egfp were transformed into A. tumefacies strain GV3101 and the transformed agrobacteria were infiltrated individually into leaves of four-week-old N. benthamiana plants using 1-ml needless syringes. These agroinfiltrated plants were allowed to grow in a growth chamber at 25°C for 48 hr, and the GFP fluorescence was examined under a Leica TCS SP5 laser confocal microscope with excitation wavelength of 488 nm.
Transcription activation assay in yeast
The coding sequences of SlSR1 and SlSR3L were amplified using gene-specific primers (Additional file 2) and cloned into pBD-GAL4Cam vector to yield pBD-SlSR1 and pBD-SlSR3L. These two recombinant plasmids and the pBD empty vector (as a negative control) were transformed into yeast strain AH109. The transformed yeasts were confirmed by colony PCR and then cultivated on the SD/–Trp and SD/–Trp/–His medium for 3 days at 28°C, followed by addition of x-?-gal (5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside). Transactivation activity of the fused proteins was evaluated according to the growth situation and production of blue pigments after the addition of x-?-gal of the transformed yeast cells on the SD/–Trp/–His medium.
Detection of H2O2 accumulation
Detection of in situ H2O2 was carried out using DAB staining method as described previously . Leaves were collected from pTRV2-SlSR1- or pTRV2-SlSR3L-infiltrated and pTRV2-GUS-infiltrated plants and dipped into DAB solution (1 mg/ml, pH3.8) and incubated for 8 hr in dark at room temperature. Thereafter, leaves were placed into acetic acid/glycerol/ethanol (1:1:1, vol/vol/vol) and boiled for 5 min in a water bath. After several changes of the solution, then leaves were maintained in 60% glycerol and accumulation of H2O2 in leaves was photographed using a digital camera.
Drought tolerance assays
At least 10 individual pTRV2-SlSRs- or pTRV2-GUS-infiltrated plants were used in each experiment. Drought stress was applied to the tomato plants by stopping watering for a certain period of 10–12 days or until the wilting symptoms were obvious. To assess the ratio of water loss, fully expanded leaves from 6 individual pTRV2-SlSR1L- or pTRV2-GUS-infiltrated plants were detached and placed on the bench top. The weights of the leaves were recorded at different time points after detachment and the average water loss ratio was calculated by comparing with the initial weights. Roots from 6 individual pTRV2-SlSR1L- or pTRV2-GUS-infiltrated plants were cut, cleaned and dried in 70°C oven for 24 hr and the final weight was calculated and compared. Leaf samples were collected from the pTRV2-SlSRs- or pTRV2-GUS-infiltrated plants at 0 hr (as unstressed control) and 7 days after stopping watering and were subjected to analysis of gene expression.
qRT-PCR analysis of gene expression
Total RNA was extracted from frozen leaf samples using TRIzol reagent (Invitrogen, Shanghai, China) and treated with RNase-free DNase (TaKaRa, Dalian, China) to erase any genomic DNA in the RNA samples. First-strand cDNA was synthesized from 0.6 ?g total RNA using AMV reverse transcriptase (TaKaRa, Dalian, China) according to the manufacturer’s recommendations. Each qRT-PCR reaction contained 12.5 ?l SYBR Premix Ex Taq (TaKaRa, Dalian, China), 0.1 ?g cDNA and 7.5 pmol of each gene-specific primer (Additional file 2) in a final volume of 25 ?l, and run on three independent biological replicates. The qRT-PCR was performed in a CFX96 real-time PCR detection system (BioRad, Hercules, CA, USA) and relative expression levels were calculated using the 2-??CT method. The expression level of a tomato actin gene was used as an internal control to normalize the expression data for the target genes. Relative expression levels of the target genes were shown as folds of the expression level of the actin gene or as folds of the expression levels in treated plants/control plants.
All experiments were repeated independently three times and data were collected from experiments on three biological samples. All data obtained were subjected to statistical analysis according to the Student’s t-test and the probability values of p?<?0.05 were considered as significant difference.
Accession numbers for SlSRs
The tomato SlSR gene sequences used in this study were retrieved from GenBank under the following accession numbers: SlSR1, GU170838; SlSR1L, JN558810; SlSR2, JN566047; SlSR2L, JN566048; SlSR3, JN566049; SlSR3L, JN566051; SlSR4, JN566050. These SlSR gene sequences were deposited by Yang et al. .
Availability of supporting data
XL, LH, YZ, ZO, YH, HZ and DL carried out the experiments. XL and FS designed the experiments. FS and XL wrote the paper. All authors read and approved the final manuscript.
This work was supported by the National High-Tech R & D Program (No. 2012AA101504 and No. 2012AA101505), the National Basic Research Program of China (2009CB119005) and the Research Fund for the Doctoral Program of Higher Education of China (20120101110070).
- Kudla J, Batistic O, Hashimoto K: Calcium signals: the lead currency of plant information processing. Plant Cell. 2010, 22 (3): 541-563. 10.1105/tpc.109.072686.PubMed CentralView ArticlePubMedGoogle Scholar
- McAinsh MR, Pittman JK: Shaping the calcium signature. New Phytol. 2009, 181 (2): 275-294. 10.1111/j.1469-8137.2008.02682.x.View ArticlePubMedGoogle Scholar
- Plieth C: Signal percolation through plants and the shape of the calcium signature. Plant Signal Behav. 2010, 5 (4): 379-385. 10.4161/psb.5.4.10717.PubMed CentralView ArticlePubMedGoogle Scholar
- Ma W, Berkowitz GA: The grateful dead: calcium and cell death in plant innate immunity. Cell Microbiol. 2007, 9 (11): 2571-2585. 10.1111/j.1462-5822.2007.01031.x.View ArticlePubMedGoogle Scholar
- Reddy ASN, Ali GS, Celesnik H, Day IS: Coping with stresses: roles of calcium- and calcium/calmodulin-regulated gene expression. Plant Cell. 2011, 23 (6): 2010-2032. 10.1105/tpc.111.084988.PubMed CentralView ArticlePubMedGoogle Scholar
- Sarwat M, Ahmad P, Nabi G, Hu X: Ca2+ signals: the versatile decoders of environmental cues. Crit Rev Biotechnol. 2013, 33 (1): 97-109. 10.3109/07388551.2012.672398.View ArticlePubMedGoogle Scholar
- Poovaiah BW, Du LQ, Wang HZ, Yang TB: Recent advances in calcium/calmodulin-mediated signaling with an emphasis on plant-microbe interactions. Plant Physiol. 2013, 163 (2): 531-542. 10.1104/pp.113.220780.PubMed CentralView ArticlePubMedGoogle Scholar
- Zielinski RE: Calmodulin and calmodulin-binding proteins in plants. Annu Rev Plant Biol. 1998, 49: 697-725. 10.1146/annurev.arplant.49.1.697.View ArticleGoogle Scholar
- Bouche N, Yellin A, Snedden WA, Fromm H: Plant-specific calmodulin-binding proteins. Annu Rev Plant Biol. 2005, 56: 435-466. 10.1146/annurev.arplant.56.032604.144224.View ArticlePubMedGoogle Scholar
- Luan S, Kudla J, Rodriguez-Concepcion M, Yalovsky S, Gruissem W: Calmodulins and calcineurin Bac like proteins: calcium sensors for specific signal response coupling in plants. Plant Cell. 2002, 14 (Suppl): S389-S400.PubMed CentralPubMedGoogle Scholar
- Bouche N, Scharlat A, Snedden W, Bouchez D, Fromm H: A novel family of calmodulin-binding transcription activators in multicellular organisms. J Biol Chem. 2002, 277 (24): 21851-21861. 10.1074/jbc.M200268200.View ArticlePubMedGoogle Scholar
- Yang TB, Poovaiah BW: A calmodulin-binding/CGCG box DNA-binding protein family involved in multiple signaling pathways in plants. J Biol Chem. 2002, 277 (47): 45049-45058. 10.1074/jbc.M207941200.View ArticlePubMedGoogle Scholar
- Finkler A, Ashery-Padan R, Fromm H: CAMTAs: calmodulin-binding transcription activators from plants to human. FEBS Lett. 2007, 581 (21): 3893-3898. 10.1016/j.febslet.2007.07.051.View ArticlePubMedGoogle Scholar
- Choi MS, Kim MC, Yoo JH, Moon BC, Koo SC, Park BO, Lee JH, Koo YD, Han HJ, Lee SY, Chung WS, Lim CO, Cho MJ: Isolation of a calmodulin-binding transcription factor from rice (Oryza sativa L.). J Biol Chem. 2005, 280 (49): 40820-40831. 10.1074/jbc.M504616200.View ArticlePubMedGoogle Scholar
- Reddy ASN, Reddy VS, Golovkin M: A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif. Biochem Biophys Res Commun. 2000, 279 (3): 762-769. 10.1006/bbrc.2000.4032.View ArticlePubMedGoogle Scholar
- Yang TB, Poovaiah BW: An early ethylene up-regulated gene encoding a calmodulin-binding protein involved in plant senescence and death. J Biol Chem. 2000, 275 (49): 38467-38473. 10.1074/jbc.M003566200.View ArticlePubMedGoogle Scholar
- Koo SC, Choi MS, Chun HJ, Shin DB, Park BS, Kim YH, Park HM, Seo HS, Song JT, Kang KY, Yun DJ, Chung WS, Cho MJ, Kim MC: The calmodulin-binding transcription factor OsCBT suppresses defense responses to pathogens in rice. Mol Cells. 2009, 27 (5): 563-570. 10.1007/s10059-009-0081-4.View ArticlePubMedGoogle Scholar
- Galon Y, Aloni R, Nachmias D, Snir O, Feldmesser E, Scrase-Field S, Boyce JM, Bouche N, Knight MR, Fromm H: Calmodulin-binding transcription activator 1 mediates auxin signaling and responds to stresses in Arabidopsis. Planta. 2010, 232 (1): 165-178. 10.1007/s00425-010-1153-6.View ArticlePubMedGoogle Scholar
- Galon Y, Nave R, Boyce JM, Nachmias D, Knight MR, Fromm H: Calmodulin-binding transcription activator (CAMTA) 3 mediates biotic defense responses in Arabidopsis. FEBS Lett. 2008, 582 (6): 943-948. 10.1016/j.febslet.2008.02.037.View ArticlePubMedGoogle Scholar
- Du LQ, Ali GS, Simons KA, Hou JG, Yang TB, Reddy ASN, Poovaiah BW: Ca2+/calmodulin regulates salicylic-acid-mediated plant immunity. Nature. 2009, 457 (7233): 1154-1158. 10.1038/nature07612.View ArticlePubMedGoogle Scholar
- Nie HZ, Zhao CZ, Wu GH, Wu YY, Chen YF, Tang DZ: SR1, a calmodulin-binding transcription factor, modulates plant defense and ethylene-induced senescence by directly regulating NDR1 and EIN3. Plant Physiol. 2012, 158 (4): 1847-1859. 10.1104/pp.111.192310.PubMed CentralView ArticlePubMedGoogle Scholar
- Laluk K, Prasad KVSK, Savchenko T, Celesnik H, Dehesh K, Levy M, Mitchell-Olds T, Reddy ASN: The calmodulin-binding transcription factor signal responsive1 is a novel regulator of glucosinolate metabolism and herbivory tolerance in Arabidopsis. Plant Cell Physiol. 2012, 53 (12): 2008-2015. 10.1093/pcp/pcs143.PubMed CentralView ArticlePubMedGoogle Scholar
- Qiu Y, Xi J, Du L, Suttle JC, Poovaiah BW: Coupling calcium/calmodulin-mediated signaling and herbivore-induced plant response through calmodulin-binding transcription factor AtSR1/CAMTA3. Plant Mol Biol. 2012, 79 (1–2): 89-99. 10.1007/s11103-012-9896-z.View ArticlePubMedGoogle Scholar
- Jing B, Xu S, Xu M, Li Y, Li S, Ding J, Zhang Y: Brush and spray: a high throughput systemic acquired resistance assay suitable for large scale genetic screening. Plant Physiol. 2011, 157 (3): 973-980. 10.1104/pp.111.182089.PubMed CentralView ArticlePubMedGoogle Scholar
- Doherty CJ, Buskirk HAV, Myers SJ, Thomashow MF: Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. Plant Cell. 2009, 21 (3): 972-984. 10.1105/tpc.108.063958.PubMed CentralView ArticlePubMedGoogle Scholar
- Kim YS, Park SC, Gilmour SJ, Thomashow MF: Roles of CAMTA transcription factors and salicylic acid in configuring the low-temperature transcriptome and freezing tolerance of Arabidopsis. Plant J. 2013, 75 (3): 364-376. 10.1111/tpj.12205.View ArticlePubMedGoogle Scholar
- Pandey N, Ranjan A, Pant P, Tripathi RK, Ateek F, Pandey HP, Patre UV, Sawant SV: CAMTA1 regulates drought responses in Arabidopsis thaliana. BMC Genomics. 2013, 14: 216-10.1186/1471-2164-14-216.PubMed CentralView ArticlePubMedGoogle Scholar
- Zegzouti H, Jones B, Frasse P, Marty C, Maitre B, Latche AA, Pech JC, Bouzayen M: Ethylene-regulated gene expression in tomato fruit: characterization of novel ethylene-responsive and ripening-related genes isolated by differential display. Plant J. 1999, 18 (6): 589-600. 10.1046/j.1365-313x.1999.00483.x.View ArticlePubMedGoogle Scholar
- Yang TB, Peng H, Whitaker BD, Conway WS: Characterization of a calcium/calmodulin regulated SR/CAMTA gene family during tomato fruit development and ripening. BMC Plant Biol. 2012, 12: 19-10.1186/1471-2229-12-19.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang TB, Peng H, Whitaker BD, Jurick WM: Differential expression of calcium/calmodulin-regulated SlSRs in response to abiotic and biotic stresses in tomato fruit. Physiol Plant. 2013, 148 (3): 445-455. 10.1111/ppl.12027.View ArticlePubMedGoogle Scholar
- Kawazu K, Mochizuki A, Sato Y, Sugeno W, Murata M, Seo S, Mitsuhara I: Different expression profiles of jasmonic acid and salicylic acid inducible genes in the tomato plant against herbivores with various feeding modes. Arthropod Plant Interact. 2012, 6: 221-230. 10.1007/s11829-011-9174-z.View ArticleGoogle Scholar
- Liu Y, Schiff M, Dinesh-Kumar SP: Virus-induced gene silencing in tomato. Plant J. 2002, 31 (6): 777-786. 10.1046/j.1365-313X.2002.01394.x.View ArticlePubMedGoogle Scholar
- Marino D, Dunand C, Puppo A, Pauly N: A burst of plant NADPH oxidases. Trends Plant Sci. 2012, 17 (1): 9-15. 10.1016/j.tplants.2011.10.001.View ArticlePubMedGoogle Scholar
- Kim JG, Li X, Roden JA, Taylor KW, Aakre CD, Su B, Lalonde S, Kirik A, Chen Y, Baranage G, McLane H, Martin GB, Mudgett MB: Xanthomonas T3S effector XopN suppresses PAMP-triggered immunity and interacts with a tomato atypical receptor-like kinase and TFT1. Plant Cell. 2009, 21 (4): 1305-1323. 10.1105/tpc.108.063123.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen HP, Chakravarthy S, Velásquez AC, McLane HL, Zeng L, Nakayashiki H, Park DH, Collmer A, Martin GB: Methods to study PAMP-triggered immunity using tomato and Nicotiana benthamiana. Mol Plant-Microbe Interact. 2010, 23 (8): 991-999. 10.1094/MPMI-23-8-0991.View ArticlePubMedGoogle Scholar
- Ekengren SK, Liu Y, Schiff M, Dinesh-Kumar SP, Martin GB: Two MAPK cascades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato. Plant J. 2003, 36 (6): 905-917. 10.1046/j.1365-313X.2003.01944.x.View ArticlePubMedGoogle Scholar
- Kamiyoshihara Y, Tieman DM, Huber DJ, Klee HJ: Ligand-induced alterations in the phosphorylation state of ethylene receptors in tomato fruit. Plant Physiol. 2012, 160 (1): 488-497. 10.1104/pp.112.202820.PubMed CentralView ArticlePubMedGoogle Scholar
- Di Matteo A, Ruggieri V, Sacco A, Rigana MM, Carriero F, Bolger A, Fernie AR, Frusciante L, Barone A: Identification of candidate genes for phenolics accumulation in tomato fruit. Plant Sci. 2013, 205–206: 87-96. 10.1016/j.plantsci.2013.02.001.View ArticlePubMedGoogle Scholar
- Pauwels L, Goossens A: The JAZ proteins: a crucial interface in the jasmonate signaling cascade. Plant Cell. 2011, 23 (9): 3089-3100. 10.1105/tpc.111.089300.PubMed CentralView ArticlePubMedGoogle Scholar
- Gong PJ, Zhang J, Li H, Yang C, Zhang C, Zhang X, Khurram Z, Zhang Y, Wang T, Fei Z, Ye Z: Transcriptional profiles of drought-responsive genes in modulating transcription signal transduction, and biochemical pathways in tomato. J Exp Bot. 2010, 61 (13): 3563-3575. 10.1093/jxb/erq167.PubMed CentralView ArticlePubMedGoogle Scholar
- Wasilewska A, Vlad F, Sirichandra C, Redko Y, Jammes F, Valon C, Freid it Frey N, Leung J: An update on abscisic acid signaling in plants and more….. Mol Plant. 2008, 1 (2): 198-217. 10.1093/mp/ssm022.View ArticlePubMedGoogle Scholar
- Orellana S, Yanez M, Espinoza A, Verdugo I, Gonzalez E, RuizLara S, Casaretto JA: The transcription factor SlAREB1 confers drought, salt stress tolerance and regulates biotic and abiotic stress-related genes in tomato. Plant Cell Environ. 2010, 33 (12): 2191-2208. 10.1111/j.1365-3040.2010.02220.x.View ArticlePubMedGoogle Scholar
- Li J, Sima W, Ouyang B, Wang TT, Ziaf K, Luo ZD, Liu LF, Li HX, Chen ML, Huang YQ, Feng YQ, Hao YH, Ye ZB: Tomato SlDREB gene restricts leaf expansion and internode elongation by downregulating key genes for gibberellin biosynthesis. J Exp Bot. 2012, 63 (18): 6407-6420. 10.1093/jxb/ers295.PubMed CentralView ArticlePubMedGoogle Scholar
- Loukehaich R, Wang T, Ouyang B, Ziaf K, Li H, Zhang J, Lu Y, Ye Z: SpUSP, an annexin-interacting universal stress protein, enhances drought tolerance in tomato. J Exp Bot. 2012, 63 (15): 5593-5606. 10.1093/jxb/ers220.PubMed CentralView ArticlePubMedGoogle Scholar
- Guo Y, Huang C, Xie Y, Song F, Zhou X: A tomato glutaredoxin gene SlGRX1 regulates plant responses to oxidative, drought and salt stresses. Planta. 2010, 232 (6): 1499-1509. 10.1007/s00425-010-1271-1.View ArticlePubMedGoogle Scholar
- Sagi M, Davydov O, Orazova S, Yesbergenova Z, Ophir R, Stratmann JW, Fluhr R: Plant respiratory burst oxidase homologs impinge on wound responsiveness and development in Lycopersicon esculentum. Plant Cell. 2004, 16 (3): 616-628. 10.1105/tpc.019398.PubMed CentralView ArticlePubMedGoogle Scholar
- Guan LM, Scandalios JG: Hydrogen peroxide-mediated catalase gene expression in response to wounding. Free Radical Bio Med. 2000, 28 (8): 1182-1190. 10.1016/S0891-5849(00)00212-4.View ArticleGoogle Scholar
- Guan LM, Zhao J, Scandalios JG: Cis-elements and trans-factors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H2O2 is the likely intermediary signaling molecule for the response. Plant J. 2000, 22 (2): 87-95. 10.1046/j.1365-313x.2000.00723.x.View ArticlePubMedGoogle Scholar
- Mengiste T: Plant immunity to necrotrophs. Annu Rev Phytopathol. 2012, 50: 267-294. 10.1146/annurev-phyto-081211-172955.View ArticlePubMedGoogle Scholar
- Jones JD, Dangl JL: The plant immune system. Nature. 2006, 444 (7117): 323-329. 10.1038/nature05286.View ArticlePubMedGoogle Scholar
- Glazebrook J: Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol. 2005, 43: 205-227. 10.1146/annurev.phyto.43.040204.135923.View ArticlePubMedGoogle Scholar
- AbuQamar S, Chai MF, Luo H, Song F, Mengiste T: Tomato protein kinase 1b mediates signaling of plant responses to necrotrophic fungi and insect herbivory. Plant Cell. 2008, 20 (7): 1964-1983. 10.1105/tpc.108.059477.PubMed CentralView ArticlePubMedGoogle Scholar
- Li X, Zhang Y, Huang L, Ouyang Z, Hong Y, Zhang H, Li D, Song F: Tomato SlMKK2 and SlMKK4 contribute to disease resistance against Botrytis cinerea. BMC Plant Biol. 2014, 14: 166-10.1186/1471-2229-14-166.PubMed CentralView ArticlePubMedGoogle Scholar
- Benito EP, ten Have A, van’t Klooster JW, van Kan JAL: Fungal and plant gene expression during synchronized infection of tomato leaves by Botrytis cinerea. Eur J Plant Pathol. 1998, 104 (2): 207-220. 10.1023/A:1008698116106.View ArticleGoogle Scholar
- Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB: Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J. 1997, 11 (6): 1187-1194. 10.1046/j.1365-313X.1997.11061187.x.View ArticleGoogle Scholar
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