Fusarium species cause serious diseases in cereal staple food crops such as wheat and maize. Currently, the mechanisms underlying resistance to Fusarium-caused diseases are still largely unknown. In the present study, we employed a combined proteomic and transcriptomic approach to investigate wheat genes responding to F. graminearum infection that causes Fusarium head blight (FHB). We found a total of 163 genes and 37 proteins that were induced by infection. These genes and proteins were associated with signaling pathways mediated by salicylic acid (SA), jasmonic acid (JA), ethylene (ET), calcium ions, phosphatidic acid (PA), as well as with reactive oxygen species (ROS) production and scavenging, antimicrobial compound synthesis, detoxification, and cell wall fortification. We compared the time-course expression profiles between FHB-resistant Wangshuibai plants and susceptible Meh0106 mutant plants of a selected set of genes that are critical to the plants' resistance and defense reactions. A biphasic phenomenon was observed during the first 24 h after inoculation (hai) in the resistant plants. The SA and Ca2+ signaling pathways were activated within 6 hai followed by the JA mediated defense signaling activated around 12 hai. ET signaling was activated between these two phases. Genes for PA and ROS synthesis were induced during the SA and JA phases, respectively. The delayed activation of the SA defense pathway in the mutant was associated with its susceptibility. After F. graminearum infection, the endogenous contents of SA and JA in Wangshuibai and the mutant changed in a manner similar to the investigated genes corresponding to the individual pathways. A few genes for resistance-related cell modification and phytoalexin production were also identified. This study provided important clues for designing strategies to curb diseases caused by Fusarium.
Citation: Ding L, Xu H, Yi H, Yang L, Kong Z, Zhang L, et al. (2011) Resistance to Hemi-Biotrophic F. graminearum Infection Is Associated with Coordinated and Ordered Expression of Diverse Defense Signaling Pathways. PLoS ONE 6(4): e19008. https://doi.org/10.1371/journal.pone.0019008
Editor: Miguel A. Blazquez, Instituto de Biología Molecular y Celular de Plantas, Spain
Received: November 21, 2010; Accepted: March 16, 2011; Published: April 20, 2011
Copyright: © 2011 Ding et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was partially supported by ‘863’ program (2006AA10A104), ‘973’ program (2004CB117205), NSFC programs (31030054, 30430440, 30025030, 30721140555), and ‘111’ project of MOE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
Competing interests: The authors have declared that no competing interests exist.
Plants have evolved multiple layers of passive and active defense mechanisms to combat microbial pathogen attack in order to maintain their growth or survival. Passive defense takes advantage of preexisting structures  and preformed antimicrobial or toxic secondary metabolites, proteins, or peptides . Active defenses, such as oxidative burst induction , hypersensitive response (HR) , accumulation of toxic compounds , and fortification of cell walls , are triggered rapidly and directly in response to pathogen attack. The timing of defense reaction activation and the strength of the defense response determine the resistance level.
Active plant defense is finely regulated to survive adversity at a minimum expense to growth. This regulation is multifaceted and might vary depending on the plant taxa and the pathogen lifestyle. The innate plant immunity system known as pathogen-associated molecular patterns (PAMP), or PAMP-triggered immunity (PTI) contributes to the first line of active defense through the Ca2+ signaling pathway, MAPK cascade, and transcriptome reprogramming that activate appropriate defense responses –. When PTI becomes ineffective because of pathogen effector generation, the second line of active defense, referred to as effector-triggered immunity (ETI), is required for resistance meditated by the interactions of host resistance (R) genes and pathogen effectors . ETI, currently exclusively found in resistance against biotrophic pathogens that derive nutrients from living host tissues, is generally race-specific and results in HR at the infection site. Concomitant with the accumulation of salicylic acid (SA), systemic acquired resistance (SAR), a kind of broad-spectrum disease resistance that develops throughout the whole plant, is activated . PAMPs may also contribute to SAR initiation in Arabidopsis .
For diseases caused by necrotrophic pathogens that derive nutrients from dead or dying cells and hemi-biotrophic pathogens that obtain nutrients from both living and dead tissues, R gene-mediated HR is only beneficial for their growth, and therefore alternative defense mechanisms exist. Jarosch et al.  reported that barley (Hordeum vulgare L.) plants carrying the mlo mutant gene that confers a durable resistance against powdery mildew are hypersusceptible to the hemi-biotroph rice blast fungus Magnaporthe grisea. A gene conferring resistance to the Puccinia coronata biotroph in oat (Avena sativa) controls susceptibility to the Cochliobolus victoriae necrotroph . It has been speculated that toxins or damage to host cells generated by necrotrophic pathogens may initiate defense responses . A number of studies have demonstrated that jasmonic acid (JA) and ethylene (ET) signaling pathways play important roles in resistance against necrotrophic pathogens and hemi-biotrophic pathogens. Mutation of the JA receptor protein COI1 gene alters resistance to necrotrophic pathogens such as Alternaria brassicicola and Botrytis cinerea . Similarly, Arabidopsis ET insensitive mutants, such as etr1 and ein2-1, are susceptible to necrotrophic pathogens . The ERF1 and ORA59 transcription factors integrate these two pathways and activate expression of defense-related genes such as PDF1.2 , . In addition to the JA/ET pathways, signaling pathways mediated by heterotrimeric G proteins, such as those involving ABA and DELLA proteins, show positive effects to necrotroph resistance –.
The mechanisms underlying resistance to necrotrophic and hemi-biotrophic pathogens are complicated. The SA and JA/ET defense pathways generally interact antagonistically in the resistance response. Many mutants compromised in SA signaling are susceptible to biotrophic pathogens and display an enhanced resistance to some, if not all, necrotrophic pathogens. Mutants compromised in JA/ET signaling usually show the contrary . However, exceptions to these findings have been reported in a number of studies. For example, SA signaling is not required for resistance of Arabidopsis to Verticillium longisporum , but is essential for the resistance to the soil-borne pathogenic Pythium irregulare and Fusarium oxysporum oomycetes , . The latter has a lifestyle and infection strategies similar to V. longisporum. Cell death lesions resulting from reaction oxidative species (ROS) are associated with F. oxysporum infection in the Arabidopsis cpr5/hys1 mutant , but with growth restriction of F. oxysporum f. sp. Asparagi in asparagus . Arabidopsis resistance to A. brassicicola is not affected by disturbance of ET signaling, but requires JA signaling . These results suggest that resistance to non-biotrophic pathogens might be host-pathogen specific.
Most current knowledge regarding plant disease resistance derives from studies of dicotyledonous species (e.g. Arabidopsis thaliana), with little knowledge having been derived from monotyledonous plants such as rice, wheat, maize, and sorghum. This discrepancy not only limits our understanding of plant resistance strategies, but also limits our capacity to curb diseases affecting agricultural productivity. Here, we investigated the molecular response of wheat to F. graminearum attack, with the aim of identifying key resistance-associated genes and the molecular events governing early resistance reactions.
Like F. oxysporum, F. graminearum Schwabe [teleomorph Gibberella zeae (Schweinitz) Petch] is a ubiquitous filamentous fungus. It is the main causal agent of head blight or ‘scab’ in wheat (Triticum), barley (Hordeum), rice (Oryza), and oats (Avena), and Gibberella stalk and ear rot disease in maize (Zea). Fusarium species also cause root rot, seedling blight, and foot rot . Fusarium head blight (FHB), or ear rot, occurs mainly in areas with high humidity during flowering, and has emerged as the most serious wheat and barley disease in China, North America, and Europe. Wheat FHB epidemics often result in 10–15%, and sometimes even >50%, yield loss . Furthermore, the presence of mycotoxins including nivalenol and deoxynivalenol (DON) in the infected grains are detrimental to both humans and livestock . Nganje et al.  estimated that the direct and secondary economic losses due to FHB for all crops in the Northern Great Plains and Central United States amounted to $2.7 billion from 1998 to 2000 alone. Because of the serious threat to food security and safety imposed by scab disease, improving scab resistance of wheat cultivars has become a major focus in wheat genetics and breeding. Although a great deal of effort has been made to genetically improve disease resistance, the progress has been slow and inefficient because of its complex genetic nature.
F. graminearum can infect cereal florets through natural openings and by direction penetration of the epidermal cuticle and cell wall. It produces various hydrolyzing enzymes to facilitate penetration . Cell wall reinforcement, accumulation of plant defense compounds, and a higher transcription level of xylanase inhibitors, chitinase, glucanase, and pathogenesis-related (PR) genes have been associated with the attack response –. Some of the early signaling events associated with infection of dicot plants by necrotrophic pathogens, such as oxidative burst and scavenging, and JA/ET dependent defense signaling were also related to the F. graminearum-wheat interaction –. However, their exact roles and the coordinated regulations in FHB resistance have yet to be well defined. A few studies have shown that a cautious approach must be taken in extrapolating data from dicot plants to cereals. For example, the Arabidopsis PR1 gene is only responsive to SA treatment, while its wheat homolog PR1.1 is only responsive to methyl JA treatment . Although disruption of SA signaling in dicots could enhanced resistance to some necrotrophic pathogens, over-expression of Arabidopsis NPR1 enhanced FHB resistance in wheat, likely together with faster and stronger SAR activation .
Here we focused on the clarification of the early signaling events in wheat responding to F. graminearum by employing an integrated proteomics and transcriptomics approach and expression profile investigation of genes involved in these events in FHB-resistant Wangshuibai and its susceptible mutant. We demonstrated that there is a biphasic signaling event that involving Ca2+, SA, and JA/ET pathways and their coordinated and ordered activation are critical to the occurrence of the resistance. These findings are helpful to shed light on mechanisms underlying resistance to hemi-biotrophic pathogens in monotyledonous plants, and to provide critical clues to curb crop diseases caused by Fusarium species.
Contrasting phenotypes in resistance to F. graminearum infection and spread within spikes
In this study, we employed two lines with contrasting response to F. graminearum infection. As shown in the phenotyping study, fifteen days after spraying inoculation, only small chlorotic/necrotic lesions confined to the infection points of the spikes were noted in Wangshuibai, a germplasm with a high level of FHB resistance. However, the FHB-susceptible Meh0106 mutant displayed severe disease symptoms in the inoculated spikes, with chlorosis and necrosis reaching almost the entire inoculated spikes (Figure 1). The compromised resistance of Meh0106 to infection and the spread of F. graminearum within Meh0106 spikes were validated in repeated trials (Table 1).
Distinct protein profiles after infection with F. graminearum
To investigate the proteome-level responses of scab-resistant Wangshuibai and FHB-susceptible Meh0106 plants to initial infection of F. graminearum, we compared their protein profiles in spikes 12 h after spraying inoculation (hai). Total proteins were subjected to 2-DE analysis with 17-cm gel strips (Figure 2). In three independent experiments, over 600 protein spots were detected reproducibly after silver staining, mostly distributed from 20 to 100 kDa with pI values in the range of 4 to 9. Compared to the mock treatment, there were 77 protein spots showing at least 1.5-fold volume changes in one or both of the genotypes, more than 70% of which exhibited at least 2-fold changes (Figure S1). Although most spots showed quantitative changes, seven of them showed qualitative changes after infection, of which four were only detected in the infected spikes in both lines (spots 12, 17, 36, 40), one was detected in the infected mutant (spot 38), and two were only detected in spikes without inoculation (spots 47, 73). There were 47 spots up-regulated (Figure 3A) and 30 down-regulated by the infection (Figure 3B), of which a total 52 (67%) showed similar variation patterns between Wangshuibai and the mutant (Figures 3A and 3B). It was noticeable that 25 spots showed up- or down-regulation only in one of the two lines, of which 12 (19%) spots showed line-specific volume changes in Wangshuibai, with one down-regulation spot and 13 (20%) spots showed line-specific volume changes in the mutant. These results implied that substantial cellular events in response to F. graminearum infection occur in both Wangshuibai and the mutant.
This is a representative image from three technical and three biological replicates.
Protein identities of spots showing volume changes responding to F. graminearum infection
Protein identities for 60 of the 77 spots were determined by querying their MALDI-TOF MS data against the Triticeae peptide database (Tables 2 and S1). Five spots (spots 6, 20, 28, 47, and 52) were matched to proteins without any functional information. Nine up-regulated Wangshuibai-specific spots had determined protein identity: four are proteins for the defense signal molecules in ET or JA biosynthesis, four are proteins related to disease defense reactions, and one is related to stress response. Five up-regulated mutant-specific spots had determined protein identity: one is for lysine synthesis that is beneficial for fungal growth, and three are involved in protein degradation. 22 spots showed up-regulation in both Wangshuibai and the mutant, and 18 have been associated with disease resistance or defense (Tables 2 and S1). Among them, spots 12 and 41 are for basal resistance, spots 67 and 71 are related to ET synthesis, spots 1 and 39 are for ROS production, and spots 11, 46, and 48 are for antioxidant defense. These results implied that JA/ET defense signaling and the ROS production and scavenging system are key players in Wangshuibai scab resistance.
Spot 60 was the only down-regulated Wangshuibai-specific spot and was identified as a 2OG-Fe(II) oxygenase family protein. One member protein of the 2OG-Fe(II) oxygenase family in Arabidopsis is required for susceptibility to downy mildew . Of the six down-regulated mutant-specific spots with determined protein identity, four are related to cell morphogenesis or cell wall formation, implying that disturbance of cell wall structure compromised mutant scab resistance. Among the 17 down-regulated spots occurring in both Wangshuibai and the mutant with determined protein identity, five might be related to reduction of oxidative burst. The remaining spots are mostly related to photosynthesis or energy metabolism.
Comparative transcriptome identification of genes differentially expressed in response to F. graminearum infection
To more comprehensively understand the wheat response to F. graminearum infection, we identified up-regulated genes by examining changes at the transcriptome level using an in silico strategy. Up-regulated genes may include genes expressed only after pathogen attack and those expressed in higher abundance after pathogen attack. To identify genes in the former category, we compared 20,878 ESTs from libraries prepared with young wheat spikes (flowering tissues) inoculated with F. graminearum to 1.4 million ESTs from libraries prepared with different tissues of wheat, barley, and other Triticeae species not affected by biotic stress using a cutoff of ≥90% homology in at least 100 bp sequence overlap, or ESTs from other cereal plants not affected by biotic stress deposited in the NCBI EST database using a cutoff of ≥80% homology. After removing poor quality sequences and duplicated copies, we obtained 696 ESTs that were only expressed after the pathogen attack. Using 90% homology as the cutoff, these ESTs were assembled into 533 unigenes including 117 contigs and 416 singletons. Functional annotations were obtained for 113 of them through BLASTx similarity searches.
To identify genes in the second category, all wheat ESTs except those in the first category were grouped using a cutoff of <90% homology. From the 85,051 groups generated by this process, 5,015 that had at least two ESTs from the F. graminearum-induced libraries (ESTs from SSH libraries prepared with F. graminearum-infected tissues were not counted) were subjected to further analysis. In each of these groups, we counted the ESTs from the F. graminearum-induced libraries (excluding the SSH ones) and those from libraries prepared with flower tissues not subjected to Fusarium inoculation at stages starting from meiosis to 24 hai post-pollination. The latter included a total of 25 libraries and 115,795 ESTs. ESTs bearing the same clone name were counted once. By assuming each group represented one unigene, we used the EST frequency of occurrence in the flower tissue libraries without pathogen attack as the estimate of its normal expression rate. The probability of sampling a certain number of ESTs in a specific group from the F. graminearum-induced libraries was then calculated. One hundred and thirty nine unigenes were considered to be up-regulated, since they had twice the respective EST frequency from the induced libraries than the frequency in each of the non-induced libraries and also had <0.001 sampling probability. The homologs of these unigenes in other cereal plants were also expressed less abundantly/frequently in the corresponding non-stressed flowering tissues. Functional annotations were obtained for 106 of them through BLASTx similarity searches.
Of the 219 up-regulated unigenes with annotation, 163 (74%) were functionally associated with defense against pathogen attack (Tables 3 and S2). We then examined the expression profiles of 55 Arabidopsis homologs using the microarray data from treatments of six types of biotic stresses downloaded from Bio-Array Resource (http://www.bar.utoronto.ca/). Most of them, 46/55 (84%), had a higher expression level under at least one kind of biotic stress, and 32/55 (58%) had a higher expression level under at least three kinds of biotic stresses (Figure 4). These results confirmed that the strategy used for identification of genes associated with response to F. graminearum infection was effective.
Of the 163 up-regulated defense related unigenes, 117 were classified into ten functional categories based on their putative functions (Tables 3 and S2). Many of the remaining 46 functionally un-classified unigenes are JA/ET-inducible and thus could be placed into some of these categories as more information becomes available.
Eight of the up-regulated unigenes (IGS001–IGS008) are related to SA defense pathways, of which seven code for PR1, PR2, and PR5, the SAR marker genes in dicot plants. IGS002 codes for a R2R3 Myb-like protein, which is associated with HR and programmed cell death (PCD) regulation in response to pathogen attack through induction of SA biosynthesis . Ten unigenes (IGS009–IGS018) were associated with JA/ET biosynthesis or JA/ET signaling: IGS009 codes for JASMONATE INSENSITIVE 1-like protein and IGS016 codes for AtPFT1-like transcription coactivator, both of which are required for JA-dependent defense signaling , ; IGS017 codes for a ERF1-like protein, where ERF1 is a positive regulator of disease resistance responses in the JA/ET pathways . IGS010 and IGS018, coding for short-chain alcohol dehydrogenase and iron/ascorbate-dependent oxidoreductase, respectively, might be related to JA and ET biosynthesis , . It is noteworthy that there were three unigenes (IGS019–IGS021) related to PA signaling and four (IGS104–IGS107) related to Ca2+ signaling, suggesting their involvement in the response to F. graminearum infection. IGS022 and IGS023 encode BRI1-associated receptor kinase (BAK1)-like proteins that are associated with integration of diverse perception events into downstream PAMP responses leading to systemic immunity . IGS024 encodes a MAP kinase phosphatase (MKP) whose homolog in Arabidopsis is a negative regulator of the MAPK cascade, repressing ICS1 mediated SA biosynthesis through calmodulin (CaM) binding , implying that they were components for fine-tuning of the SA signaling pathway. Thus, these three unigenes are related to crosstalk of signaling pathways. Transcriptome analysis also revealed fifteen disease resistance gene analogs (RGAs) (IGS075–IGS089) and fourteen protein kinases (IGS090–IGS103). Strikingly, most of these unigenes were expressed only after pathogen attack. They could be important for regulation of crosstalk between signaling pathways mediated by phytohormones in response to pathogen infection .
Other up-regulated unigenes include 35 (IGS025–IGS059) that encode proteins involved in antimicrobial compound synthesis or detoxification, 15 (IGS060–IGS074) that encode proteins involved in anti-oxidative stress, 6 (IGS108–IGS113) that encode proteins associated with defense-related post-transcriptional regulation, and 4 (IGS114–IGS117) that encode cell wall modifying enzymes. Interestingly, 16 of the unigenes related to antimicrobial compound synthesis or detoxification, and almost all the unigenes related to defense-related post-transcriptional regulation were found selectively within spikes under attack, suggesting that Fusarium infection directly activated defense-related secondary metabolism and a complicated signaling network regulated at multiple levels.
Defense-related signaling pathways were expressed differently in Wangshuibai and the susceptible mutant
Transcriptomic analysis showed that wheat responded to F. graminearum infection by turning on several defense-related signaling pathways, including those mediated by SA, JA, ET, Ca2+, and PA. Proteomic analysis also showed that the proteins related to JA/ET signaling were expressed differentially between Wangshuibai and susceptible mutant plants. Here, we examined the time-course expression profiles of key genes involved in these pathways through quantitative real-time reverse transcriptase PCR (qRT-PCR) of samples collected from infected flowering tissues of Wangshuibai and the susceptible Meh0106 mutant to verify these findings and to examine the crosstalk between different pathways.
SA signaling pathway.
To see how the SA signaling pathway, which governs local resistance and SAR, reacted to F. graminearum infection, we investigated the expression of phenylalanine ammonia lyase gene (PAL), isochorismate synthase gene (ICS1), and β-(1,3; 1,4)-glucanase-2 (Glu2) within 36 hai. PAL is the key enzyme for SA biosynthesis via the phenylpropanoid pathway . Wildermuth et al.  reported that, in Arabidopsis, most SA is synthesized via the isochrorismate pathway, employing ICS1 as the key enzyme. ICS1 homologs have since been identified in a number of plants including wheat. In the qRT-PCR analysis, we noted that PAL transcription was induced rapidly in Wangshuibai, peaking within 3 hai, while PAL induction in the susceptible mutant was markedly slower and less efficient (Figure 5A). The expression profiles for Glu2, which responds exclusively to SA signaling , were the same as those for PAL (Figure 5B). Meanwhile, ICS1 was down-regulated after F. graminearum infection and did not differ considerably between Wangshuibai and the mutant (Figure 5C). Thus, the F. graminearum infection caused increased PAL, but not ICS1, expression, which could result in SA accumulation via the phenylpropanoid pathway. These results suggest that early activation of SA signaling could be a critical factor in scab resistance and that a delay in activation of this pathway may be associated with susceptibility.
The expression levels were relative to no inoculation (0 h) after normalization of the qRT-PCR outputs with the wheat tubulin gene output. RT-PCR was performed using gene-specific primers (Table S4). The experiment was repeated three times with similar results. Data were presented as average + S.D with n = 3.
Ca2+ signaling pathway.
Calcium-transporting ATPase is a major regulator of intracellular Ca2+ concentrations in support of proper cell signaling. We investigated the expression profiles of the differentially expressed IGS104 that encodes a calcium-transporting ATPase-like protein and the wheat homolog of the rice calcium-transporting ATPase 1. Though the RT-PCR of IGS104 was not successful, the calcium-transporting ATPase 1 homolog showed significant transient increases at 3 hai in both resistant and non-resistant plants (Figure 5D), even though to a much greater degree in the resistant plants. The wheat homolog of maize calmodulin1, a key regulatory protein in Ca2+ signal transduction, exhibited the same expression pattern as the calcium-transporting ATPase 1 homolog (Figure 5E). These results imply that the Ca2+ signaling pathway participated in the early reaction of wheat to F. graminearum infection and is related to basal resistance. Hence, the mutant plant' basal defense may be compromised.
PA signaling pathway.
IGS019 encoding diacylglycerol kinase (DGK) and IGS020 encoding phospholipase D (PLD) were only identified in the F. graminearum-infected flowering tissues. DGK and PLD are key enzymes in different phosphatidic acid (PA) synthesis pathways. PA is a lipid second messenger in plants whose involvement in biotic and abiotic stress responses is gaining attention. DGK was rapidly activated after infection in Wangshuibai and that PLD was activated about 3 hai later and a little more efficiently (Figures 5F and 5G). Their expression patterns were consistent with pathogen-induced biphasic PA accumulation, first via the PLC-DGK pathway then via the PLD pathway . In the mutant, DGK and PLD induction were not efficient (Figures 5F and 5G) and the slight change in their expression levels might suggest a lack of PA accumulation. The DGK expression profile was similar to that of PAL (Figures 5A and 5F), which is not surprising since DGK is inducible by the SA analog benzothiadiazole (BTH) . By SA addition to Arabidopsis cell suspensions, Krinke et al.  demonstrated that PLD activation is also an early component of the SA signaling pathway. Thus, the association of the PA signaling pathway with scab resistance may have resulted from SA signaling, and be involved in PLC/DGK-mediated signaling in oxidative burst and hypersensitive cell death .
JA/ET signaling pathway.
Both transcriptomic and proteomic analyses showed that the JA/ET pathways were actively involved in the response to F. graminearum infection. We found that the gene for 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) (spot 33) enzyme catalyzing the oxidation of ACC to ethylene, and the gene for jasmonate biosynthesis isoenzyme 12-oxophytodienoate reductase 3 (12-OPR3) (spot 58) were both only elevated in Wangshuibai (Table S1). Similarly, the induction was only noted in this resistance material in the time course studies of their expression (Figures 5H and 5I). Maximum 12-OPR3 expression occurred around 12 hai. Compared with 12-OPR3, ACO was activated more rapidly, with the transcripts peaking around 6 hai.
To ascertain the roles of JA and ET pathways in scab resistance, the PR3 marker gene for JA/ET-mediated defense response and the wheat ERF1 homolog were examined. ERF1 is a key element in the integration of JA and ET signaling . The transcript level of the ERF1 homolog showed a gradual increase after infection in Wangshuibai that became significant at 3 hai (Figure 5J). PR3 was induced slightly in Wangshuibai after F. graminearum infection before 6 hai, and then peaked at 12 hai (Figure 5K). The expression levels of both genes remained unchanged in the mutant within 36 hai (Figures 5J and 5K). These results clearly supported the view that there is an association between the JA/ET pathways and scab resistance.
Interactions between SA and JA/ET pathways.
Since SA defense pathways are generally known to be antagonistic to the JA/ET pathways, to investigate their coordination in responding to F. graminearum infection we checked the temporal transcript changes of a few SA regulatory genes including EDS1, MYC2, BIK1, NPR1, MPK4, and WRKY33. EDS1 is an essential component of R gene mediated disease resistance in Arabidopsis and is required for SA-regulated basal resistance by modulating SA accumulation . MYC2 is a pathogen-inducible gene and negatively regulates the expression of JA/ET pathway-associated defense genes in Arabidopsis by acting upstream of ERF1 . BIK1 negatively regulates SA accumulation and has a positive effect on the expression of the JA-responsive PDF1.2 gene . NPR1 positively regulates SA signaling and induces PR gene expression by nuclear localization after pathogen attack . Cytosolic NPR1 has a negative effect on JA signaling . MPK4 and WRKY33 are components of the MAPK cascade. The former negatively regulates the SA pathway and positively regulates the JA/ET pathway through EDS1/PAD4 . The latter is an in vitro substrate of MPK4 and has a similar regulatory role as MPK4 on the SA and JA/ET defense pathways in response to hemi-biotrophic and necrotrophic pathogens .
As shown in Figure 5L, EDS1 was weakly induced at 3 hai in Wangshuibai. The transcript then declined by 6 hai and reached a non-infection level by 12 hai. The MYC2 homolog had a similar expression pattern (Figure 5M). Interestingly, their declines were coincident with a gradual increase in BIK1 transcript levels that peaked at 12 hai (Figure 5N). The NPR1 expression profile was similar to that of EDS1, except that it had a much more efficient induction at 3 hai (Figure 5O). In the mutant, EDS1, MYC2, and NPR1 had similar expression profiles. They showed slight induction at 3 hai, but the induction continued and peaked at 12 hai. In contrast, although BIK1 had the transcription efficiency similar to that of EDS1 at 3 hai in the mutant, it did not show significant induction at 12 hai relative to 6 hai. These results suggest that appropriate defense reactions to F. graminearum attack at different time frames depend on proper activation of particular pathways. The mutant's susceptibility was caused, at least in part, by failed timely positive regulation of the JA/ET pathway due to reduced function of BIK1 and inappropriate high levels of NPR1 and MYC2. Higher EDS1 and NPR1 activity at 12 hai in the mutant may indicate a higher level of SA signaling that is unfavorable to the needed JA signaling.
In contrast to BIK1, WRKY33 had maximum abundance at 12 hai in both Wangshuibai and the mutant (Figure 5P), although the mutant expression level was lower. Similar expression patterns were also observed for IGS024, which encode MKP1-like protein (Figure 5Q), and MPK4 (data not shown). This finding suggests that though the MAPK cascade may be involved in responses to F. graminearum infection, but is not the factor responsible for the differential resistant responses between Wangshuibai and the mutant.
ROS production and the subsequent anti-oxidative activity differed between Wangshuibai and the susceptible mutant
Transcriptomic and proteomic analyses revealed that genes and proteins involved in ROS production and relieving oxidative stress were induced after infection. It is well known that ROS, including the superoxide radical anion (O2−), hydroperoxyl radical (HO2•), hydroxyl radical (HO•), and hydrogen peroxide (H2O2) accumulate during the earliest events in many plants under pathogen attack. However, the role of ROS is far clearer against biotrophic pathogens than against necrotrophic and hemi-necrotrophic pathogens. Since H2O2 is relatively stable among ROS, we examined the association with F. graminearum infection of the polyamine oxidase (PAO) pathway and the NADPH oxidase pathway that play critical roles in H2O2 production in plants , . In the PAO pathway, S-adenosylmethionine decarboxylase (SAMDC) catalyzes polyamine biosynthesis, which is then converted to H2O2 by PAO. In the NADPH oxidase pathway, NADPH oxidase generates superoxide. Superoxide dismutase (SOD) then dismutates superoxide into oxygen and H2O2. A transient increase in NADPH oxidase transcripts, which peaked at 3 hai, was observed in Wangshuibai (Figure 5R), while transcripts of SAMDC and PAO showed a gradual increase in Wangshuibai before 24 hai (Figures 5S and 5T). We noted negligible change in the expression levels of these genes in the susceptible mutant, suggesting that ROS production is related to resistance to F. graminearum infection.
Consistent with the induced expression of NADPH oxidase gene, in Wangshuibai, the activities of SOD and peroxidase (POD), which break down H2O2, more than doubled in the first 6 hai (Figures 6A and 6B). Relative to those in Wangshuibai, the activity changes in the mutant were slower and less prominent.
Genes for cell wall modification and biosynthesis of antimicrobial compounds were differentially expressed in Wangshuibai and the susceptible mutant
In addition to the defense signaling pathways, components in defense-related metabolic pathways were also identified at the proteome and/or transcriptome level, such as cell wall fortification, antimicrobial compound biosynthesis, and PR gene activation. Expression of caffeoyl-CoA 3-O-methyltransferase (CCOMT) (spot 36), a critical in lignin synthesis and functions in structural barrier defense , was almost unchanged in the mutant, but was induced as early as 3 hai in Wangshuibai, peaking 12 hai. Moreover, CCOMT showed a relatively higher level in Wangshuibai than in the susceptible mutant throughout the infection response process (Figure 5U).
With respect to antimicrobial compound biosynthesis, we investigated the expression profiles of IGS057 encoding chalcone synthase (CHS) and IGS031 encoding cytochrome P450 71D8 (CYP71D8), which catalyze biosynthesis of flavonoid type phytoalexins and camalexin type phytoalexins, respectively , . Induction of IGS057 was similar in Wangshuibai and the mutant (Figure 5V). However, IGS031 was only significantly induced in Wangshuibai, peaking at 12 hai (Figure 5W), indicating that synthesis of camalexin type phytoalexins could be important for FHB resistance. Chitinase 1, associated with the protein represented by spot 09, degrades fungal cell walls . The transcription of Chitinase 1 was up-regulated after F. graminearum infection and no significant difference in expression was observed between Wangshuibai and the susceptible mutant (Figure 5X). Like IGS057, Chitinase 1 may participate in the defense response to FHB, without contributing to resistance.
SA and JA contents changed differentially in Wangshuibai and the susceptible mutant after the infection
Since SA and JA signaling are all involved in reactions to F. graminearum infection, the endogenous contents of SA and JA in tissues used in the expression analysis were investigated. The variation patterns of their concentrations across the time points were remarkably similar to the expression profiles of the investigated genes corresponding to the respective pathways (Figure 7). The SA concentration in Wangshuibai increased 3-fold at 3 hai, but declined rapidly at 6 hai; its rise in the susceptible mutant occurred slower and reached the highest point at 24 hai (Figure 7A). In both Wangshuibai and the mutant, the induced JA accumulation started at 3 hai and reached the peak levels at 12 hai (Figure 7B). Interestingly, the SA content was in a much lower basal level in the mutant than in Wangshuibai, implying that the mutant have SA synthesis deficiency.
In the mutant library created with the FHB-resistant Wangshuibai, we identified the Meh0106 mutant that has completely lost FHB resistance. Substantial alternation by mutation of phenotypes governed by QTLs has been reported in other plants as well. For example, the rice moc1 mutant has only the main culm with no tillers ; moc1 is a recessive mutation of MOC that encodes a putative GRAS family nuclear protein regulating the control of tillering. Given the disruption of multiple defense pathways in Meh0106, it is also likely that the loss of resistance is due to mutation of a regulator critical to FHB resistance in Wangshuibai. Measurement of the SA and JA contents in Wangshuibai and the mutant revealed that the latter has much less basal SA. SA is one of most important regulating molecules participating in disease resistance in plants.
In recent years, a number of genes or proteins have been identified through analyzing the expressed products of genes after F. graminearum infection –, –. Their functional annotations suggested that multiple events occurred in the defense response. Nevertheless, little is known about the molecular events contributing to the contrasting performances of resistant and susceptible genotypes to F. graminearum infection because of different genotypes used, limited resolution of the technologies, small size of the investigated cDNA and protein samples, and particularly the lack of genetically different materials only in the resistance-related genes. The availability of Meh0106 made it feasible to explore the molecular mechanisms governing FHB resistance.
Proteome and transcriptome profiling indicated that multiple defense-related signaling and cellular events contribute to FHB resistance
By comparing the spike proteomes of Wangshuibai and its FHB-susceptible mutant without F. graminearum infection with those at 12 hai of F. graminearum, we identified 77 protein spots showing at least 1.5-fold volume change from over 600 protein spots. Although the number might not be extraordinary, our annotation results are quite informative. Most of the up-regulated proteins, either from the resistant Wangshuibai or from the susceptible mutant or from both, are involved in basal resistance or defense (Tables 2 and S1). These results were not different from other similar studies , . The present study is distinguished from others by the identification of proteins involved in JA and ET synthesis and of proteins related to the phenylpropanoid pathway. The lack of up-regulated PR proteins identified in this study can most likely be attributed to the fact that we used a 12-h time interval between inoculation and sampling, while others used at least a 24-h time interval , . Hence, activation of the JA/ET pathway may be a relatively early defense event and the massive production of PR proteins a relatively late defense event. It is noteworthy that four of the nine up-regulated spots found only in Wangshuibai were proteins associated with either ET or JA biosynthesis and one was associated with defense-related lignification. Meanwhile, the six down-regulated spots found only in the mutant were predominantly proteins related to cell morphogenesis or cell wall formation, implying that JA/ET defense signaling and cell wall modification are important resistance reactions in FHB resistance.
Transcriptome analyses of resistant and susceptible genotypes after F. graminearum infection using suppression subtractive hybridization, microarray, and cDNA-RFLP revealed that SA, JA/ET signaling, antioxidative reactions, and defense-related secondary metabolism were associated with defense reactions , , . However, the identified gene numbers and profiles differ substantially between studies due to differences in the genotypes and technologies used, and thus do not constitute a comprehensive description. Moreover, in Li and Yen , more than half of the 608 genes identified as associated with the defense response could not be functionally annotated. In the present study, we did in silico transcriptome comparison of the F. graminearum infected tissues with the corresponding healthy tissues, regardless of the genotypes of the compared ESTs. This resulted in findings that were consistent with our proteome analysis that enabled identification of extra signaling and cellular events contributing to defense reactions. Although only 219 of the 639 identified pathogen responsive genes could be annotated, three quarters of those identifications were functionally defense-related. Notably, besides the findings in the proteome analysis, multiple components of the SA, PA, and Ca2+ signaling pathways, as well as the MAPK cascade, were up-regulated by pathogen infection. Moreover, a number of genes for RGAs and kinase proteins, post-transcriptional regulation, and secondary metabolism such as antimicrobial compound synthesis and detoxification were only found in spikes under pathogen attack, implying their unique roles in defense against F. graminearum. Through expression profiling, we confirmed the involvement of the identified individual pathways and cellular events in scab resistance (Figure 5). A few other studies have also reported that some genes for cytochrome P450 proteins, UDP-glucosyltransferase, PR proteins, JA/ET biosynthesis, and RGAs are differentially expressed in resistant genotypes , , . RGAs and kinase proteins have been related to crosstalk regulation between signaling pathways mediated by plant hormones in response to pathogen infection. For example, R protein Mi-1 mediated aphid resistance in tomato involves both SA and JA signaling . Differentially expressed NB-ARC domain containing RGAs were also expressed differentially in comparisons between the transcriptomes of a pair of wheat near-isogenic lines carrying either the resistant or susceptible allele at the FHB-resistant QTL Fhb1 after infection .
Some of the identified genes did not display differential expression patterns between resistant and susceptible genotypes, such as CHS (IGS057) and chitinase 1 (spot 09) (Figures 5V and 5X), suggesting that they are probably involved in general defense reactions. This phenomenon seems to be common among biotic stress-related genes , . Moreover, even though WRKY33, MPK4, and the MKP1-like gene (IGS024) of the MAPK cascade were induced by F. graminearum infection, they were not associated with the mutant's susceptibility. Overexpressing these genes might still be useful for enhancing resistance .
The early defense reactions to F. graminearum infection had a biphasic strategy
For resistance to biotrophic pathogens, SA is the central player in HR and SAR, while Ca2+, nitric oxide, and PA are also associated with early signaling events , , , . They work together to trigger oxidative burst and activate a specific set of PR genes. For resistance to F. graminearum, an arguably hemi-biotrophic pathogen , , the expression profiles of SAR associated markers, such as PR1, PR2, and PR5, after the pathogen infection indicate that SAR is part of the early defense reaction , , . In wheat, faster activation of the defense response and significant enhancement of FHB resistance occurred when expressing AtNPR1, the key positive regulator of Arabidopsis SAR . Genes involved in the SA pathway, including PAL, EDS1, NPR1, and Glu2, were rapidly induced in Wangshuibai (Figures 5A, 5B, 5L and 5O). Their maximum expression occurred at 3 hai and declined thereafter. The induction in the mutant was much slower and less substantial. The change patterns of SA content after the infection in both lines were consistent with the expression profiles of these genes. Moreover, the mutant has less basal SA. These results suggested the importance of SA signaling pathway in scab resistance and the SA synthesis deficiency in the mutant might be the causal factor of its susceptibility. Recently, Makandar et al.  and Cuzick et al.  reported that SA signaling through NPR1 is important for limiting disease severity caused by F. graminearum and F. culmorum in Arabidopsis. These results all support the role of SA in FHB resistance. However, Pritsch et al.  and Li and Yen  reported conflicting results. They noted that the SAR marker genes were induced by pathogen infection, but showed no difference in expression between the resistant and susceptible genotypes. The requirement for SA signaling is similar to the reaction of Arabidopsis to infection of F. oxysporum , which is consistent with the initial biotrophic process of both pathogens. It is likely that SA signaling is an early general defense response or part of the innate immune reaction to F. graminearum infection regardless of genotype, but is also an important prerequisite for later resistance development. Nevertheless, it should be noted that SA signaling is not required for resistance to all hemi-biotrophic pathogens . Plants can synthesize SA via PAL or ICS1 , . However, only PAL was responsive to F. graminearum infection. This observation is similar to that in Arabidopsis plants, which synthesize SA via the PAL pathway in response to the B. cinerea necrotrophic fungi .
Ca2+ signaling is critical for transcriptional reprogramming in plant innate immunity . The variation in cytosolic Ca2+ concentration upon pathogen infection mediates the signaling process. A few studies have demonstrated that a transient change in Ca2+ permeability of the plasma membrane is a common early event key to plant defense signaling . In the response to F. graminearum infection, the examined calcium-transporting ATPase 1 gene and CaM gene were rapidly but transiently induced, similar to the SA signaling-related genes (Figures 5D and 5E). The transcripts also diminished rapidly after 3 hai, which is consistent with the observation that there is rapid but transient Ca2+ accumulation in the cytoplasm during the initial plant-pathogen interaction stage . The differences between the Wangshuibai and mutant expression profiles are striking. Therefore, we concluded that both SA and Ca2+ signaling are primary concurrent signaling events important for the occurrence of resistance.
It is widely accepted that the JA signaling pathway mediates resistance to necrotrophic and hemi-biotrophic pathogens. The findings of the current study and of Li and Yen  support this notion. The JA content increased at 3 hai and in a much faster rate in the FHB-resistant Wangshuibai than in the mutant (Figure 7B). In accordance with this, all the tested JA signaling-related genes, including 12-OPR3 (spot 58), ERF1, and PR3, were induced strongly in Wangshuibai, but induced only weakly in the mutant (Figures 5I, 5J and 5K), indicating that the JA pathway was suppressed to some extent in the susceptible mutant. This interpretation is consistent with the expression profile of BIK1, which acts as a negative regulator of SA accumulation and a positive regulator of the JA/ET response , and was not turned on in the mutant at 12 hai as in Wangshuibai. The significantly higher level of MYC2 expression in the mutant at 12 hai might also not favor timely activation of the JA defense pathway, as MYC2 plays a negative regulatory role in the JA-mediated defense response against F. oxysporum in Arabidopsis . Unlike the Ca2+ and SA signaling-related genes, maximum induction in Wangshuibai occurred at 12 hai, suggesting that activation of the JA signaling pathway followed activation of the Ca2+ and SA defense pathways.
Interestingly, the expression of DGK (IGS019) and PLD (IGS020) also had a biphasic expression pattern (Figures 5F and 5G), with DGK being induced concurrently with the SA and Ca2+ pathway genes, and PLD induced almost concurrently with the JA pathway genes. Both genes contribute to PA synthesis and are SA-inducible , . PLD is also required for wound-induced JA biosynthesis . These expression profiles suggest that DGK expression may be part of the SA signaling pathway while PLD expression may be associated with pathogen-induced JA biosynthesis, although no evidence is available to support this assertion. There is a biphasic ROS burst in response to avirulent pathogen infection  and PA can promote elicitor-induced biphasic ROS burst in rice suspension cells . Concurrent with the above-mentioned biphasic patterns, the expression of NADPH oxidase gene was rapidly induced, peaking at 3 hai, while the expression of SAMDC and PAO was more slowly induced, peaking at 12 hai (Figures 5R, 5S and 5T). All three genes encode key enzymes for H2O2 production. Lherminier et al.  showed that the first wave of H2O2 production is mediated by NADPH oxidase after challenge of tobacco cells with fungal elicitor cryptogein. The differential gene expressions between Wangshuibai and the mutant suggest that they are all part of an integrated resistance system.
In Wangshuibai, the induced expression of NADPH oxidase was accompanied by rapid elevation of the activities of the enzymes POD and SOD (Figures 6A and 6B), both of which are ROS scavengers. There are also active antioxidative activities after infection, as shown in the omics analyses. We speculate that ROS generation is precisely controlled, which benefits FHB resistance since there is only a transient biotrophic stage in F. graminearum infection  and the subsequent PCD caused by ROS could be favorable for F. graminearum growth. After F. graminearum infection, the plants might use multiple strategies to control PCD. Early induction of calcium-transporting ATPase 1 could help control PCD . NADPH oxidase could also participate in cell death suppression at sites surrounding its activation, thus restricting the spread of PCD . Their low level expression in the mutant could result in uncontrolled PCD, contributing to the susceptibility. Moreover, Kemmerling et al.  reported that BAK1, which is induced after the infection, has a role in controlling infection-induced PCD. The second phase accumulation of ROS generation enzymes concurred with the expression of JA signaling genes. There is evidence that ROS may be required for activating and establishing JA/ET signaling . ROS also takes part in lignin polymerization, which is a resistance-related cell wall modification event when attacked by F. graminearum .
In summary, the sequential induction of endogenous SA and JA accumulations and the altered expression peak time of signaling pathway genes points to adoption of a biphasic strategy in the early wheat defense reactions to F. graminearum infection, with the initiation of Ca2+ and SA signaling preceding JA signaling. This strategy may be a feature of plant resistance to some hemi-biotrophic pathogens. PA and ROS accumulation accompanied these two phases, but probably through different synthetic pathways. We noted significant positive correlations between the expression profiles of PAL, CaM, NADPH oxidase, and DGK in Wangshuibai (Table S3), which imply a close association between the initial events. This relationship was not observed in the mutant. Ca2+ can positively activate PA production, which in turn is implicated in increasing NADPH oxidase activity and ROS production . Thus, Ca2+ signaling could play a positive regulatory role in SA signaling and the initial ROS burst in the first phase of FHB resistance reaction. The ROS burst could result from SA signaling or provide positive feedback to the SA defense pathway , . It is likely that, in the susceptible mutant, mutation of a factor having a similar role as Ca2+ signaling or being part of Ca2+ signaling per se disrupted the initial phase of defense signaling events, resulting in failed activation of ET signaling and the subsequent activation of JA signaling.
The two-phase resistance reactions are well coordinated to ensure the successful occurrence of resistance
We have shown that both SA and JA signaling are associated with FHB resistance. However, these two defense-signaling pathways are known to have an antagonistic relationship. Thus, it would be interesting to examine how they are coordinated in the resistance-related early molecular events. We noted that all expression profiles of the examined genes that positively regulate SA signaling were induced regardless of genotype. However in Wangshuibai, the induction peak was at 3 hai, while in the mutant it was at 12 hai, at the same time as maximum expression of JA signaling-related genes. In addition, the maximum transcript levels were significantly higher in Wangshuibai than in the mutant. Similarly, there was also a common pattern for the expression profiles of the examined genes that positively regulate JA signaling. That is, all were induced in Wangshuibai with their peaks at 12 hai and their induction was attenuated in the mutant. The sequential induction of SA and JA content increase would be an effective coordinative mechanism of the two antagonistic pathways, and the delayed induction of SA accumulation in the mutant disrupts this coordination, which results in susceptibility. Indeed, induction of SA and JA signaling concurrently promoted disease severity caused by F. graminearum in Arabidopsis . We postulated that timely and orderly activation of the SA and JA defense pathways is critical in order for them to coordinately confer resistance. Consistent with this notion, exposure of Arabidopsis to MeJA at the beginning of F. graminearum infection enhances disease severity, while exposure to MeJA at 12 and 24 hai increases resistance . The expression patterns of all crosstalk genes, except those linked to MAPK cascade, and of SA and JA signaling genes provide further evidence supporting this hypothesis. Their maximum expressions occurred at the same time as the expression of genes positively regulated by them, and were almost at background (without infection) levels at the same time as the maximum expression of genes negatively regulated by them (Figures 5L–5Q).
The association of ET synthesis with failed mutant resistance is intriguing. ET signaling has been positively related to FHB resistance in the FHB-resistant Sumai 3 cultivar . We found that the ET biosynthesis gene ACO (spot 33) had maximum expression at the time point between the maximum expression of SA and JA signaling related genes. This intermediate presence could be an important regulatory step in the transition from SA mediated defense to JA mediated defense, since ET signaling can render JA response insensitive to SA antagonism in Arabidopsis . Moreover, the activation of the SA signaling pathway may be a prerequisite for optimal induction of ET synthesis since ozone-induced ET accumulation is compromised in NahG and npr1 plants . ERF1/ORA59 could play a regulatory role in the transition process. The gradual increase in ERF1 expression level in Wangshuibai after infection corresponds to its function (Figure 5J). In susceptible germplasm, this could be a different scenario, since ET signaling could be exploited by F. graminearum and thus increase susceptibility . Different branches of ET signaling might be involved in the resistance/susceptibility reactions, since even though the key ET biosynthesis ACO gene and the downstream ET signaling ERF1 gene showed differential expression between Wangshuibai and the mutant after infection, the expression of EIN2, a key component of the ET pathway, was not altered with infection (data not shown). However, caution should be taken since changes in the ET level could influence EIN2 protein stability in the absence of any effect on EIN2 mRNA levels .
FHB resistance is associated with cell wall reinforcement, antimicrobial compound synthesis, and detoxification
Appropriate activation of early defense signaling events leads to disease resistance, which is implemented by cellular activities such as synthesis of phytoalexins, detoxification enzymes, and cell wall modifications. In the omics analyses, a number of genes related to these activities were induced after F. graminearum infection. Even though the Meh0106 mutant phenotype likely resulted from the mutation of a regulatory gene, it does not present with differential expression of all resistance implementation genes. For example, the expression of CHS (IGS057) (Figure 5V) and CYP99A1 (IGS030) (data not shown) that are associated phytoalexin biosynthesis in plants, and of chitinase 1 (spot 09) (Figure 5X) with the function of degrading fungus cell wall, was induced in both Wangshuibai and the mutant after infection, suggesting that they are involved in basal defense but not resistance. On the other hand, significant induction of CYP71D8 (IGS031) and CCOMT (spot 36) was observed in Wangshuibai but not in the mutant (Figures 5W and 5U), indicating their association with resistance. CYP71D8 contributes to production of the phytoalexin camalexin, which is involved in resistance to necrotrophic pathogens in Arabidopsis . CCOMT contributes to lignin biosynthesis, a phenolic cell wall polymer associated with plant defense against biotic and abiotic stresses and indispensable for plant structure and defense . In an FHB-resistant cultivar, lignin content in the cell wall increased at a higher rate than in susceptible cultivars . UDP-glycosyltransferase that is inducible by F. graminearum infection detoxifies the DON toxin produced by the fungus and may be involved in the Fhb1-associated FHB resistance , .
A putative regulatory network for FHB resistance
We have demonstrated that resistance to hemi-biotrophic F. graminearum infection is associated with coordinated and ordered expression of diverse defense signaling pathways and altered secondary metabolism. Based on the content change patterns of defense signaling molecules as well as the sequential events and differential expression profiles of the involved genes in Wangshuibai and the susceptible mutant after F. graminearum infection, we propose a model to illustrate the early cellular events leading to FHB resistance (Figure 8). There were two major phases of resistance reactions during the first 24 hai. The first phase occurred within 6 hai, probably corresponding to a transient biotrophic stage. In this stage, changes or activation of Ca2+ fluxes, Ca2+ signaling, SA signaling, PA signaling, and ROS production and scavenging were the major cellular activities. Since they are all related to HR and PCD and in order to constrain cell death that favors F. graminearum growth, multiple mechanisms controlling PCD were also activated during this stage. The second phase occurred after 6 hai and before 24 hai, probably corresponding to the start of the necrotrophic stage. In this stage, the JA/ET signaling pathway and ROS production via PAO were activated, which lead to a series of resistance reactions. ET signaling activation occurred between the two major phases, facilitating the transition from SA to JA defense signaling, since they are usually antagonistic. The activating order of these signaling events could be critical in forming resistance. Crosstalk genes, such as EDS1, NPR1, BIK1, and ERF1, were actively involved in the regulation processes. The resistance related activities such as PR protein production, cell wall enforcement, and antimicrobial compound synthesis and detoxification could commence soon after infection and peak 6 to 12 hai. Both SA and JA/ET defense signaling pathways can activate these related genes , . We have identified a few genes for resistance-related cell modification and phytoalexin production that could be exploited as a worthy strategy for improving resistance against F. graminearum infection.
PIs, protease inhibitors; XET, xyloglucan endotransglycosylase; Xls, xylanase inhibitors. See the text for other abbreviations. The peripheral solid lines indicate cell wall, the peripheral dotted lines indicate plasma membrane; the solid arrows represent direct interactions, and the dotted arrows represent indirect interactions. Perpendicular line on arrows indicates negative relationship.
Materials and Methods
Plant materials and spike inoculation
Wangshuibai is a common indigenous wheat germplasm of Jiangsu, China. ‘Meh0106’ is a homozygous FHB-susceptible mutant obtained from M2 progenies of Wangshuibai with dry seeds treated with 0.35% (w/v) EMS. They are similar in phenotype except for their resistance level to F. graminearum.
To evaluate resistance to pathogen penetration, spikes at anthesis were inoculated by spraying, and then water was sprayed to maintain moisture. Percentage of diseased spikelets (PDS) and percentage of infected spikes (PIS) were investigated 15 d after the inoculation . To evaluate resistance to pathogen spreading, spikes at anthesis were inoculated through point inoculation, and the number of diseased spikes (NDS) and the length of diseased rachides (LDR) were investigated 15 d post-inoculation in 2006 and 2007 . The inocula were composed of four local virulent strains of F. graminearum (F4, F15, F17, and F34) at a concentration of about 1000 conidiospores per 25 µl. Inoculated spike tissues were harvested at 3, 6, 12, 24, and 36 hai. The control was prepared by mimicking the inoculation with pure water. Tissues for proteomic analysis were prepared in a similar way.
Proteins were extracted from young spikes of Wangshuibai and Meh0106 according to Damerval et al. . Concentration was determined using Bradford's method with bovine serum albumin (BSA) as the standard .
2-D electrophoresis and gel staining
One-hundred-and-twenty-microgram aliquots of protein samples were included in 350 µL rehydration buffer (7 M urea, 2 M thiourea, 32.5 mM CHAPS, 0.5% (v/v) carrier ampholytes pH 3 to 10, 65 mM dithiothreitol and 0.002% (w/v) bromophenol blue). IPG strips (17 cm, pH 3 to 10, nonlinear; Bio-Rad, Hercules, California, USA) were passively rehydrated with rehydration buffer containing the protein sample for 13 h in the Protean system (Bio-Rad). IEF was performed at 250 V for 1 h, 500 V for 1 h, 10 000 V for 2 h, 4000 V for 2 h, all in a linear gradient, and then maintained at 8000 V until at least a total of 56 000 Vh was reached. After IEF, the strips were equilibrated in equilibration buffer I (6 M urea, 69 mM SDS, 0.05 M Tris–HCl, pH 8.8, 20% (v/v) glycerol, 65 mM dithiothreitol) at ambient temperature for 15 min, and then in equilibration buffer II (6 M urea, 69 mM SDS, 0.05 M Tris–HCl, pH 8.8, 20% (v/v) glycerol, 135 mM iodoacetamide) for another 15 min. After the equilibration, the strips were positioned on top of the 2nd-dimension gel and sealed with 1% (w/v) agarose for SDS-PAGE. The 10% SDS-polyacrylamide gels were run in the Ettan DALTtwelve vertical electrophoresis system (Amersham Biosciences, Freiburg, Germany) at 40 mA for 30 min followed by 60 mA for 4.5 h. Silver staining was carried out according to Shevchenko et al. . This experiment was performed with three biological replicates each with three technical triplicates.
The stained gels were scanned with Gel Doc 3000 (Bio-Rad) and the images were analyzed using PDQuest software (Bio-Rad). After alignment, automatic spot matching was performed. The matched spots were then examined manually and falsely matched or unmatched spots were corrected. The quantity values of the spots were exported after detection, standardization, and background elimination. Spots with a quality value over 50 and showing 1.5-fold change in abundance between treatments were selected for further analysis.
De-staining, reduction and iodoacetamide treatments, and trypsin (Promega, Madison, WI, USA) digestion of the selected spots were carried out as described in Shevchenko et al. . Peptides were then extracted from gel pieces once by 5% (w/v) TFA and twice by 2.5% (w/v) TFA in 50% (v/v) acetone (each for 1 h) at 37°C. The resulting peptides were lyophilized completely in a vacuum centrifuge and dissolved in 2 µL 0.5% (w/v) TFA.
MS analysis and protein identification
The peptide solution was mixed with the supernatant of 60% ACN (Fisher Scientific, Springfield, NJ, USA) saturated with a-cyano-4-hydroxycinnamic acid (Sigma, St. Louis, MO, USA), and then air dried on the flat surface of a sample plate. MS analysis was carried out with the MALDI-TOF mass spectrometer Reflex III (Bruker-Daltonics, Billerica, MA) in the positive ion reflector mode and analyzed by the peaklist-generating software FlexControl™ 2.2. Calibration was carried out using autolytic tryptic peptides. The mass spectra data were collected from mono isotopic peaks falling in the m/z range of 750 to 4000 Da with S/N ratio over 10. Outputs resulting from autolysis of trypsin and from commonly occurring keratin contamination were excluded from the subsequent data query.
Using a customized searching tool based on eMowse from EMBOSS (http://emboss.sourceforge.net/), peptide mass fingerprint (PMF) data were matched to wheat polypeptide database constructed from 1,046,811 wheat EST sequences using cTrans , using the setting of monoisotopic molecular weight, missed cleavage = 1, mass tolerance = 50 ppm, fixed modification = carbamidomethyl (Cys) and variable modification = oxidation (Met). ESTs corresponding to the peptide fragments were used to search ESTs with at least 40 base overlap and 94% sequence homology in Triticeae EST database to construct cDNA contigs. Final sequence coverage was calculated by comparing the m/z values obtained by MALDI-TOF MS with the peptide fingerprints obtained by in silico trypsin digestion of the conceptual proteins translated from the contigs using Masspeptide program (http://www.expasy.org). The criteria for a successful identity determination were: at least 3 matched peptides and 20% sequence coverage; isoelectric point differing by at most 2; and molecular weight differing by at most 20% compared with the experimental estimate.
In silico transcriptomic analysis
Extraction of EST sequences from the GenBank database (ftp://ftp.ncbi.nih.gov/genbank/) and vector or adaptor contamination removal were carried out with software cTrans. Fungal DNA contamination was removed by querying the DNA databases of fungi Fusarium graminearum, Fusarium oxysporum, Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Stagonospora nodorum, Ustilago maydis, Puccinia graminis, and Pyrenophora tritici repentis (http://www.broad.mit.edu/annotation/fgi/). All sequence comparisons were carried out with the BLAST tools provided by National Center for Biotechnology Information (NCBI) . CAP3 was used for sequence assembly . The probability to obtain a certain number of ESTs for a gene under the induced condition in the given library size was estimated using the equation , where N = total number of ESTs, n = number of ESTs corresponding to the target gene, f = expression frequency of the target gene under the normal condition. The identified genes were annotated based on Gene Ontology (GO) and information identified in literatures, and classified according to their biological functions.
Microarray data analysis
Homologs of the wheat genes in Arabidopsis were identified through BLASTxing against the Arabidopsis protein database (http://www.arabidopsis.org/) without low complexity filtering, using the threshold of E<1E-4 and >70% similarity. Microarray data produced in six kinds of biotic stresses (http://www.bar.utoronto.ca/) including infections of Botrytis cinerea, Pseudomonas syringae, Phytophtora infestans, and Erysiphe orontii, and treatments of bacterial- and oomycete-derived elicitors, were employed to explore their association with biotic stresses. A homolog was considered to be up-regulated if its peak expression level under a specific biotic stress was at least 1.5-fold higher than the control. Here the expression level was an average of repeated microarray experiments at a specific time point.
RNA was extracted using the Trizol reagent (Invitrogen, USA) following the manufacturer's protocol and quantified with a spectrometer (Ultrospec2100 pro, Amersham Pharmacia, England). First-strand cDNA was synthesized with 3.0 µg total RNA in a 20 µl reaction volume using M-MLV reverse transcriptase (Promega, USA) according to manufacturer's instruction. Two µl of 10× diluted cDNA were used as a template for qRT-PCR analysis. PCR reaction was performed in a total volume of 20 µl, containing 2.0 µl cDNA, 250 M of each primer, and 10 µl of 2 x iQ SYBR Green Supermix (Bio-Rad) on an iCycler iQ fluorescence real time PCR (Bio-Rad). The Q-PCR setting was 1 min at 95°C, followed by 40 cycles of 94°C×15 s, 58°C×25 s and 72°C×30 s. Primers (Table S4) were designed according to the wheat cDNA contigs coding for the corresponding proteins. The relative expression level was normalized with the expression data of wheat tubulin gene and estimated using the 2−ΔΔCT method of Livak and Schmittgen . The PCR was repeated thrice. There were two biological replicates for PCRs of NPR1, EDS1, BIK1, PR3, and MYC2.
SOD and POD assay
Total SOD activity, expressed in units per mg of protein, was assayed according to the method of Beyer and Fridovich . One unit of SOD activity was defined as the sample volume causing 50% inhibition of the absorbance increase, measured at the 560 nm wavelength. POD activity was determined using the guaiacol oxidation method of Chance and Maehly . A unit of peroxidase activity was defined as nmol H2O2 decomposed per minute per mg of protein.
Measurement of SA and JA contents
The tissues used in the measurements were the same as those in expression analysis. Extraction and quantification of SA and JA was performed according to Li et al. . Briefly, 200 mg of tissues was extracted with 0.5 mL of 1-propanol/H2O/ concentrated HCl (2∶1∶0.002, v/v/v). After centrifugation, the supernatants were loaded on C18 solid-phase extraction cartridges (CNWBOND HC-C18, 500 mg, 3 mL). The elutes were used for HPLC-ESI-MS/MS separation in a HPLC (Agilent 1200, Agilent Technologies, CA) and then quantitation in a hybrid triple quadrupole/linear ion trap mass spectrometer (ABI 4000 Q-Trap, Applied Biosystems, CA) using the multiple reaction monitoring (MRM) and information dependent acquisition (IDA) mode. The standard curves for SA and JA quantification were generated using a series of SA and JA (Sigma) dilutions. These experiments were all performed with two biological replicates and each sample was measured three times.
Histograms showing the volume changes of 77 differentially displayed 2-DE spots. Data show a representative experiment from three independent experiments with similar results and three replicates each. The error bars indicate SD of three replicates. Y-axis: relative abundance of the protein; X-axis: spot number. a: Wangshuibai 12 hai with H2O; b: Wangshuibai 12 hai with F. graminearum; c: Meh0106 12 hai with H2O; d: Meh0106 12 hai with F. graminearum.
Protein identities and their functional annotations for the differentially expressed protein spots.
Differentially expressed genes after F. graminearum infection identified by in silico Northerns and their functional annotations.
Expression correlations of the genes examined in Wangshuibai and its susceptible mutant Meh0106 involved in the early signaling events after F. graminearum infection.
We would like to thank Dr. Qifu Li and Dr. Songlin Shi of Academy for Life Science of Xiamen University for MS analysis. We are also grateful to Lili Bian for her technical assistance with transcript profiling experiments. We thank Dr. Hong Chen and Jinhua Peng of Oil Crop Research Institute of Chinese Academy of Agricultural Sciences for SA and JA quantitation.
Conceived and designed the experiments: LD HX ZM. Performed the experiments: LD HX HY LY. Analyzed the data: LD HX ZM. Contributed reagents/materials/analysis tools: HX ZK LZ SX HJ. Wrote the paper: LD HX ZM.
- 1. Martin JT (1964) Role of cuticle in the defense against plant disease. Ann Rev Phytopathol 2: 81100.JT Martin1964Role of cuticle in the defense against plant disease.Ann Rev Phytopathol281100
- 2. Levin DA (1976) The chemical defenses of plants to pathogens and herbivores. Annu Rev Ecol Syst 7: 121–159.DA Levin1976The chemical defenses of plants to pathogens and herbivores.Annu Rev Ecol Syst7121159
- 3. Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48: 251–275.C. LambRA Dixon1997The oxidative burst in plant disease resistance.Annu Rev Plant Physiol Plant Mol Biol48251275
- 4. Watanabe N, Lam E (2006) Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death. Plant J 45: 884–894.N. WatanabeE. Lam2006Arabidopsis Bax inhibitor-1 functions as an attenuator of biotic and abiotic types of cell death.Plant J45884894
- 5. Thomma BP, Nelissen I, Eggermont K, Broekaert WF (1999) Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola. Plant J 19: 163–171.BP ThommaI. NelissenK. EggermontWF Broekaert1999Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola.Plant J19163171
- 6. Bhuiyan NH, Selvaraj G, Wei Y, King J (2009) Role of lignification in plant defense. Plant Signal Behav 4: 158–159.NH BhuiyanG. SelvarajY. WeiJ. King2009Role of lignification in plant defense.Plant Signal Behav4158159
- 7. Boller T, He SY (2009) Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324: 742–744.T. BollerSY He2009Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens.Science324742744
- 8. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, et al. (2002) MAP kinase signaling cascade in Arabidopsis innate immunity. Nature 415: 977–983.T. AsaiG. TenaJ. PlotnikovaMR WillmannWL Chiu2002MAP kinase signaling cascade in Arabidopsis innate immunity.Nature415977983
- 9. Boudsocq M, Willmann MR, McCormack M, Lee H, Shan L, et al. (2010) Differential innate immune signaling via Ca (2+) sensor protein kinases. Nature 464: 418–422.M. BoudsocqMR WillmannM. McCormackH. LeeL. Shan2010Differential innate immune signaling via Ca (2+) sensor protein kinases.Nature464418422
- 10. Nicaise V, Roux M, Zipfel C (2009) Recent advances in PAMP-triggered immunity against bacteria: pattern recognition receptors watch over and raise the alarm. Plant Physiol 150: 1638–1647.V. NicaiseM. RouxC. Zipfel2009Recent advances in PAMP-triggered immunity against bacteria: pattern recognition receptors watch over and raise the alarm.Plant Physiol15016381647
- 11. Durrant WE, Dong X (2004) Systemic acquired resistance. Annu Rev Phytopathol 42: 185–209.WE DurrantX. Dong2004Systemic acquired resistance.Annu Rev Phytopathol42185209
- 12. Mishina TE, Zeier J (2007) Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis. Plant J 50: 500–513.TE MishinaJ. Zeier2007Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis.Plant J50500513
- 13. Jarosch B, Jansen M, Schaffrath U (2003) Acquired resistance functions in mlo barley, which is hypersusceptible to Magnaporthe grisea. Mol Plant Microbe Interact 16: 107–114.B. JaroschM. JansenU. Schaffrath2003Acquired resistance functions in mlo barley, which is hypersusceptible to Magnaporthe grisea.Mol Plant Microbe Interact16107114
- 14. Wolpert TJ, Navarre DA, Moore DL, Macko V (1994) Identification of the 100-kD victorin binding protein from oats. Plant Cell 6: 1145–1155.TJ WolpertDA NavarreDL MooreV. Macko1994Identification of the 100-kD victorin binding protein from oats.Plant Cell611451155
- 15. Pieterse CM, Leon-Reyes A, Van der Ent S, Van Wees SC (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5: 308–316.CM PieterseA. Leon-ReyesS. Van der EntSC Van Wees2009Networking by small-molecule hormones in plant immunity.Nat Chem Biol5308316
- 16. Thomma BP, Eggermont K, Penninckx IA, Mauch-Mani B, Vogelsang R, et al. (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci U S A 95: 15107–15111.BP ThommaK. EggermontIA PenninckxB. Mauch-ManiR. Vogelsang1998Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens.Proc Natl Acad Sci U S A951510715111
- 17. Geraats BP, Bakker PA, van Loon LC (2002) Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana. Mol Plant Microbe Interact 15: 1078–1085.BP GeraatsPA BakkerLC van Loon2002Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana.Mol Plant Microbe Interact1510781085
- 18. Lorenzo O, Piqueras R, Sánchez-Serrano JJ, Solano R (2003) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165–178.O. LorenzoR. PiquerasJJ Sánchez-SerranoR. Solano2003ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense.Plant Cell15165178
- 19. Pré M, Atallah M, Champion A, De Vos M, Pieterse CM, et al. (2008) The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense. Plant Physiol 147: 1347–1357.M. PréM. AtallahA. ChampionM. De VosCM Pieterse2008The AP2/ERF domain transcription factor ORA59 integrates jasmonic acid and ethylene signals in plant defense.Plant Physiol14713471357
- 20. Adie BA, Pérez-Pérez J, Pérez-Pérez MM, Godoy M, Sánchez-Serrano JJ, et al. (2007) ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis. Plant Cell 19: 1665–1681.BA AdieJ. Pérez-PérezMM Pérez-PérezM. GodoyJJ Sánchez-Serrano2007ABA is an essential signal for plant resistance to pathogens affecting JA biosynthesis and the activation of defenses in Arabidopsis.Plant Cell1916651681
- 21. Trusov Y, Sewelam N, Rookes JE, Kunkel M, Nowak E, et al. (2009) Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling. Plant J 58: 69–81.Y. TrusovN. SewelamJE RookesM. KunkelE. Nowak2009Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.Plant J586981
- 22. Navarro L, Bari R, Achard P, Lisón P, Nemri A, et al. (2008) DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling. Curr Biol 18: 650–655.L. NavarroR. BariP. AchardP. LisónA. Nemri2008DELLAs control plant immune responses by modulating the balance of jasmonic acid and salicylic acid signaling.Curr Biol18650655
- 23. Spoel SH, Johnson JS, Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proc Natl Acad Sci U S A 104: 18842–18847.SH SpoelJS JohnsonX. Dong2007Regulation of tradeoffs between plant defenses against pathogens with different lifestyles.Proc Natl Acad Sci U S A1041884218847
- 24. Johansson A, Staal J, Dixelius C (2006) Early responses in the Arabidopsis-Verticillium longisporum pathosystem are dependent on NDR1, JA- and ET-associated signals via cytosolic NPR1 and RFO1. Mol Plant Microbe Interact 19: 958–969.A. JohanssonJ. StaalC. Dixelius2006Early responses in the Arabidopsis-Verticillium longisporum pathosystem are dependent on NDR1, JA- and ET-associated signals via cytosolic NPR1 and RFO1.Mol Plant Microbe Interact19958969
- 25. Berrocal-Lobo M, Molina A (2004) Ethylene response factor 1 mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum. Mol Plant Microbe Interact 17: 763–770.M. Berrocal-LoboA. Molina2004Ethylene response factor 1 mediates Arabidopsis resistance to the soilborne fungus Fusarium oxysporum.Mol Plant Microbe Interact17763770
- 26. Schenk PM, Kazan K, Rusu AG, Manners JM, Maclean DJ (2005) The SEN1 gene of Arabidopsis is regulated by signals that link plant defense responses and senescence. Plant Physiol Biochem 43: 997–1005.PM SchenkK. KazanAG RusuJM MannersDJ Maclean2005The SEN1 gene of Arabidopsis is regulated by signals that link plant defense responses and senescence.Plant Physiol Biochem439971005
- 27. He C, Hsiang T, Wolyn DJ (2001) Activation of defense responses to Fusarium infection in Asparagus densiflorus. Eur J Plant Pathol 107: 473–483.C. HeT. HsiangDJ Wolyn2001Activation of defense responses to Fusarium infection in Asparagus densiflorus.Eur J Plant Pathol107473483
- 28. van Wees SC, Chang HS, Zhu T, Glazebrook J (2003) Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling. Plant Physiol 132: 606–617.SC van WeesHS ChangT. ZhuJ. Glazebrook2003Characterization of the early response of Arabidopsis to Alternaria brassicicola infection using expression profiling.Plant Physiol132606617
- 29. Walter S, Nicholson P, Doohan FM (2010) Action and reaction of host and pathogen during Fusarium head blight disease. New Phytol 185: 54–66.S. WalterP. NicholsonFM Doohan2010Action and reaction of host and pathogen during Fusarium head blight disease.New Phytol1855466
- 30. Shi JR, Wang YZ (1999) The occurrence and control of wheat scab and sharp-eye spot in Jiangsu in 1998. Plant Protection 2: 17–19.JR ShiYZ Wang1999The occurrence and control of wheat scab and sharp-eye spot in Jiangsu in 1998.Plant Protection21719
- 31. Fung F, Clark RF (2004) Health effects of mycotoxins: a toxicological overview. J Toxicol Clin Toxicol 42: 217–234.F. FungRF Clark2004Health effects of mycotoxins: a toxicological overview.J Toxicol Clin Toxicol42217234
- 32. Nganje WE, Bangsund DA, Leistritz FL, Wilson WW, Tiapo NM (2002) Estimating the economic impact of a crop disease: The case of Fusarium Head Blight in U.S. Wheat and Barley. Proceeding of the 2002 National Fusarium Head Blight Forum 275–281.WE NganjeDA BangsundFL LeistritzWW WilsonNM Tiapo2002Estimating the economic impact of a crop disease: The case of Fusarium Head Blight in U.S. Wheat and Barley.Proceeding of the 2002 National Fusarium Head Blight Forum275281
- 33. Li WL, Faris JD, Muthukrishnan S, Liu DJ, Chen PD, et al. (2001) Isolation and characterization of novel cDNA clones of acidic chitinases and β-1, 3-glucanases from wheat spikes infected by Fusarium graminearum. Theor Appl Genet 102: 353–362.WL LiJD FarisS. MuthukrishnanDJ LiuPD Chen2001Isolation and characterization of novel cDNA clones of acidic chitinases and β-1, 3-glucanases from wheat spikes infected by Fusarium graminearum.Theor Appl Genet102353362
- 34. Kang Z, Buchenauer H (2003) Immunocytochemical localization of cell wall-bound thionins and hydroxyproline-rich glycoproteins in Fusarium culmorum-infected wheat spikes. J Phytopathology 151: 120–129.Z. KangH. Buchenauer2003Immunocytochemical localization of cell wall-bound thionins and hydroxyproline-rich glycoproteins in Fusarium culmorum-infected wheat spikes.J Phytopathology151120129
- 35. Golkari S, Gilbert J, Prashar S, Procunier JD (2007) Microarray analysis of Fusarium graminearum-induced wheat genes: identification of organ-specific and differentially expressed genes. Plant Biotechnol J 5: 38–49.S. GolkariJ. GilbertS. PrasharJD Procunier2007Microarray analysis of Fusarium graminearum-induced wheat genes: identification of organ-specific and differentially expressed genes.Plant Biotechnol J53849
- 36. Pritsch C, Muehlbauer GJ, Bushnell WR, Somers DA, Vance CP (2000) Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum. Mol Plant Microbe Interact 13: 159–169.C. PritschGJ MuehlbauerWR BushnellDA SomersCP Vance2000Fungal development and induction of defense response genes during early infection of wheat spikes by Fusarium graminearum.Mol Plant Microbe Interact13159169
- 37. Li G, Yen Y (2008) Jasmonate and ethylene signaling pathway may mediate Fusarium Head Blight resistance in wheat. Crop Sci 48: 1888–1896.G. LiY. Yen2008Jasmonate and ethylene signaling pathway may mediate Fusarium Head Blight resistance in wheat.Crop Sci4818881896
- 38. Geddes J, Eudes F, Laroche A, Selinger LB (2008) Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare. Proteomics 8: 545–554.J. GeddesF. EudesA. LarocheLB Selinger2008Differential expression of proteins in response to the interaction between the pathogen Fusarium graminearum and its host, Hordeum vulgare.Proteomics8545554
- 39. Jia H, Cho S, Muehlbauer GJ (2009) Transcriptome analysis of a wheat near-isogenic line pair carrying Fusarium head blight-resistant and -susceptible alleles. Mol Plant Microbe Interact 22: 1366–1378.H. JiaS. ChoGJ Muehlbauer2009Transcriptome analysis of a wheat near-isogenic line pair carrying Fusarium head blight-resistant and -susceptible alleles.Mol Plant Microbe Interact2213661378
- 40. Lu ZX, Gaudet D, Puchalski B, Despins T, Frick M, et al. (2006) Inducers of resistance reduce common bunt infection in wheat seedlings while differentially regulating defense-gene expression. Physiol Mol Plant Pathol 67: 138–148.ZX LuD. GaudetB. PuchalskiT. DespinsM. Frick2006Inducers of resistance reduce common bunt infection in wheat seedlings while differentially regulating defense-gene expression.Physiol Mol Plant Pathol67138148
- 41. Makandar R, Essig JS, Schapaugh MA, Trick HN, Shah J (2006) Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1. Mol Plant Microbe Interact 19: 123–129.R. MakandarJS EssigMA SchapaughHN TrickJ. Shah2006Genetically engineered resistance to Fusarium head blight in wheat by expression of Arabidopsis NPR1.Mol Plant Microbe Interact19123129
- 42. van Damme M, Huibers RP, Elberse J, Van den Ackerveken G (2008) Arabidopsis DMR6 encodes a putative 2OG-Fe(II) oxygenase that is defense-associated but required for susceptibility to downy mildew. Plant J 54: 785–793.M. van DammeRP HuibersJ. ElberseG. Van den Ackerveken2008Arabidopsis DMR6 encodes a putative 2OG-Fe(II) oxygenase that is defense-associated but required for susceptibility to downy mildew.Plant J54785793
- 43. Vailleau F, Daniel X, Tronchet M, Montillet JL, Triantaphylidès C, et al. (2002) A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Proc Natl Acad Sci U S A 99: 10179–10184.F. VailleauX. DanielM. TronchetJL MontilletC. Triantaphylidès2002A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack.Proc Natl Acad Sci U S A991017910184
- 44. Boter M, Ruíz-Rivero O, Abdeen A, Prat S (2004) Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis. Genes Dev 18: 1577–1591.M. BoterO. Ruíz-RiveroA. AbdeenS. Prat2004Conserved MYC transcription factors play a key role in jasmonate signaling both in tomato and Arabidopsis.Genes Dev1815771591
- 45. Kidd BN, Edgar CI, Kumar KK, Aitken EA, Schenk PM, et al. (2009) The mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis. Plant Cell 21: 2237–2252.BN KiddCI EdgarKK KumarEA AitkenPM Schenk2009The mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis.Plant Cell2122372252
- 46. Bogacki P, Oldach KH, Williams KJ (2008) Expression profiling and mapping of defense response genes associated with the barley-Pyrenophora teres incompatible interaction. Mol Plant Pathol 9: 645–660.P. BogackiKH OldachKJ Williams2008Expression profiling and mapping of defense response genes associated with the barley-Pyrenophora teres incompatible interaction.Mol Plant Pathol9645660
- 47. Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nürnberger T, et al. (2007) A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defense. Nature 448: 497–500.D. ChinchillaC. ZipfelS. RobatzekB. KemmerlingT. Nürnberger2007A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defense.Nature448497500
- 48. Bartels S, Anderson JC, González Besteiro MA, Carreri A, Hirt H, et al. (2009) MAP kinase phosphatase1 and protein tyrosine phosphatase1 are repressors of salicylic acid synthesis and SNC1-mediated responses in Arabidopsis. Plant Cell 21: 2884–2897.S. BartelsJC AndersonMA González BesteiroA. CarreriH. Hirt2009MAP kinase phosphatase1 and protein tyrosine phosphatase1 are repressors of salicylic acid synthesis and SNC1-mediated responses in Arabidopsis.Plant Cell2128842897
- 49. Martinez de Ilarduya O, Xie Q, Kaloshian I (2003) Aphid-induced defense responses in Mi-1-mediated compatible and incompatible tomato interactions. Mol Plant Microbe Interact 16: 699–708.O. Martinez de IlarduyaQ. XieI. Kaloshian2003Aphid-induced defense responses in Mi-1-mediated compatible and incompatible tomato interactions.Mol Plant Microbe Interact16699708
- 50. Lee HI, León J, Raskin I (1995) Biosynthesis and metabolism of salicylic acid. Proc Natl Acad Sci U S A 92: 4076–4079.HI LeeJ. LeónI. Raskin1995Biosynthesis and metabolism of salicylic acid.Proc Natl Acad Sci U S A9240764079
- 51. Wildermuth MC, Dewdney J, Wu G, Ausubel FM (2001) Isochorismate synthase is required to synthesize salicylic acid for plant defense. Nature 414: 562–565.MC WildermuthJ. DewdneyG. WuFM Ausubel2001Isochorismate synthase is required to synthesize salicylic acid for plant defense.Nature414562565
- 52. Yamaguchi T, Minami E, Ueki J, Shibuya N (2005) Elicitor-induced activation of phospholipases plays an important role for the induction of defense responses in suspension-cultured rice cells. Plant Cell Physiol 46: 579–587.T. YamaguchiE. MinamiJ. UekiN. Shibuya2005Elicitor-induced activation of phospholipases plays an important role for the induction of defense responses in suspension-cultured rice cells.Plant Cell Physiol46579587
- 53. Chen J, Zhang W, Song F, Zheng Z (2007) Phospholipase C/diacylglycerol kinase-mediated signaling is required for benzothiadiazole-induced oxidative burst and hypersensitive cell death in rice suspension-cultured cells. Protoplasma 230: 13–21.J. ChenW. ZhangF. SongZ. Zheng2007Phospholipase C/diacylglycerol kinase-mediated signaling is required for benzothiadiazole-induced oxidative burst and hypersensitive cell death in rice suspension-cultured cells.Protoplasma2301321
- 54. Krinke O, Flemr M, Vergnolle C, Collin S, Renou JP, et al. (2009) Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis cell suspensions. Plant Physiol 150: 424–436.O. KrinkeM. FlemrC. VergnolleS. CollinJP Renou2009Phospholipase D activation is an early component of the salicylic acid signaling pathway in Arabidopsis cell suspensions.Plant Physiol150424436
- 55. Falk A, Feys BJ, Frost LN, Jones JD, Daniels MJ, et al. (1999) EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc Natl Acad Sci U S A 96: 3292–3297.A. FalkBJ FeysLN FrostJD JonesMJ Daniels1999EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases.Proc Natl Acad Sci U S A9632923297
- 56. Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, et al. (2006) The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens. Plant Cell 18: 257–273.P. VeroneseH. NakagamiB. BluhmS. AbuqamarX. Chen2006The membrane-anchored BOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistance to necrotrophic and biotrophic pathogens.Plant Cell18257273
- 57. Kinkema M, Fan W, Dong X (2000) Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 12: 2339–2350.M. KinkemaW. FanX. Dong2000Nuclear localization of NPR1 is required for activation of PR gene expression.Plant Cell1223392350
- 58. Spoel SH, Koornneef A, Claessens SM, Korzelius JP, Van Pelt JA, et al. (2003) NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol. Plant Cell 15: 760–770.SH SpoelA. KoornneefSM ClaessensJP KorzeliusJA Van Pelt2003NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol.Plant Cell15760770
- 59. Brodersen P, Petersen M, Bjørn Nielsen H, Zhu S, Newman MA, et al. (2006) Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4. Plant J 47: 532–546.P. BrodersenM. PetersenH. Bjørn NielsenS. ZhuMA Newman2006Arabidopsis MAP kinase 4 regulates salicylic acid- and jasmonic acid/ethylene-dependent responses via EDS1 and PAD4.Plant J47532546
- 60. Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48: 592–605.Z. ZhengSA QamarZ. ChenT. Mengiste2006Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens.Plant J48592605
- 61. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, et al. (1998) A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs. Plant Cell 10: 255–266.T. KellerHG DamudeD. WernerP. DoernerRA Dixon1998A plant homolog of the neutrophil NADPH oxidase gp91phox subunit gene encodes a plasma membrane protein with Ca2+ binding motifs.Plant Cell10255266
- 62. Yoda H, Hiroi Y, Sano H (2006) Polyamine oxidase is one of the key elements for oxidative burst to induce programmed cell death in tobacco cultured cells. Plant Physiol 142: 193–206.H. YodaY. HiroiH. Sano2006Polyamine oxidase is one of the key elements for oxidative burst to induce programmed cell death in tobacco cultured cells.Plant Physiol142193206
- 63. Salzman RA, Brady JA, Finlayson SA, Buchanan CD, Summer EJ, et al. (2005) Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiol 138: 352–368.RA SalzmanJA BradySA FinlaysonCD BuchananEJ Summer2005Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid, and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses.Plant Physiol138352368
- 64. Zhou N, Tootle TL, Glazebrook J (1999) Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase. Plant Cell 11: 2419–2428.N. ZhouTL TootleJ. Glazebrook1999Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase.Plant Cell1124192428
- 65. Abeles FB, Bosshart RP, Forrence LE, Habig WH (1971) Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiol 47: 129–134.FB AbelesRP BosshartLE ForrenceWH Habig1971Preparation and purification of glucanase and chitinase from bean leaves.Plant Physiol47129134
- 66. Li X, Qian Q, Fu Z, Wang Y, Xiong G, et al. (2003) Control of tillering in rice. Nature 422: 618–621.X. LiQ. QianZ. FuY. WangG. Xiong2003Control of tillering in rice.Nature422618621
- 67. Kruger WM, Pritsch C, Chao S, Muehlbauer GJ (2002) Functional and comparative bioinformatic analysis of expressed genes from wheat spikes infected with Fusarium graminearum. Mol Plant Microbe Interact 15: 445–455.WM KrugerC. PritschS. ChaoGJ Muehlbauer2002Functional and comparative bioinformatic analysis of expressed genes from wheat spikes infected with Fusarium graminearum.Mol Plant Microbe Interact15445455
- 68. Kong L, Ohm HW, Anderson JM (2007) Expression analysis of wheat defense-related genes in wheat in response to infection by Fusarium graminearum. Genome 50: 1038–1048.L. KongHW OhmJM Anderson2007Expression analysis of wheat defense-related genes in wheat in response to infection by Fusarium graminearum.Genome5010381048
- 69. Zhou W, Kolb FL, Riechers DE (2005) Identification of proteins induced or upregulated by Fusarium head blight infection in the spikes of hexaploid wheat (Triticum aestivum). Genome 48: 770–780.W. ZhouFL KolbDE Riechers2005Identification of proteins induced or upregulated by Fusarium head blight infection in the spikes of hexaploid wheat (Triticum aestivum).Genome48770780
- 70. Bernardo A, Bai G, Guo P, Xiao K, Guenzi AC, et al. (2007) Fusarium graminearum-induced changes in gene expression between Fusarium head blight-resistant and susceptible wheat cultivars. Funct Integr Genomics 7: 69–77.A. BernardoG. BaiP. GuoK. XiaoAC Guenzi2007Fusarium graminearum-induced changes in gene expression between Fusarium head blight-resistant and susceptible wheat cultivars.Funct Integr Genomics76977
- 71. Steiner B, Kurz H, Lemmens M, Buerstmayr H (2009) Differential gene expression of related wheat lines with contrasting levels of head blight resistance after Fusarium graminearum inoculation. Theor Appl Genet 118: 753–764.B. SteinerH. KurzM. LemmensH. Buerstmayr2009Differential gene expression of related wheat lines with contrasting levels of head blight resistance after Fusarium graminearum inoculation.Theor Appl Genet118753764
- 72. Anand A, Zhou T, Trick HN, Gill BS, Bockus WW, et al. (2003) Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum. J Exp Bot 54: 1101–1111.A. AnandT. ZhouHN TrickBS GillWW Bockus2003Greenhouse and field testing of transgenic wheat plants stably expressing genes for thaumatin-like protein, chitinase and glucanase against Fusarium graminearum.J Exp Bot5411011111
- 73. de Jong CF, Laxalt AM, Bargmann BO, de Wit PJ, Joosten MH, et al. (2004) Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction. Plant J 39: 1–12.CF de JongAM LaxaltBO BargmannPJ de WitMH Joosten2004Phosphatidic acid accumulation is an early response in the Cf-4/Avr4 interaction.Plant J39112
- 74. Laxalt AM, Raho N, Have AT, Lamattina L (2007) Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells. J Biol Chem 282: 21160–21168.AM LaxaltN. RahoAT HaveL. Lamattina2007Nitric oxide is critical for inducing phosphatidic acid accumulation in xylanase-elicited tomato cells.J Biol Chem2822116021168
- 75. Makandar R, Nalam V, Chaturvedi R, Jeannotte R, Sparks AA, et al. (2010) Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum. Mol Plant Microbe Interact 23: 861–870.R. MakandarV. NalamR. ChaturvediR. JeannotteAA Sparks2010Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum.Mol Plant Microbe Interact23861870
- 76. Cuzick A, Lee S, Gezan S, Hammond-Kosack KE (2008) NPR1 and EDS11 contribute to host resistance against Fusarium culmorum in Arabidopsis buds and flowers. Mol Plant Pathol 9: 697–704.A. CuzickS. LeeS. GezanKE Hammond-Kosack2008NPR1 and EDS11 contribute to host resistance against Fusarium culmorum in Arabidopsis buds and flowers.Mol Plant Pathol9697704
- 77. Ferrari S, Plotnikova JM, De Lorenzo G, Ausubel FM (2003) Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4. Plant J 35: 193–205.S. FerrariJM PlotnikovaG. De LorenzoFM Ausubel2003Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4.Plant J35193205
- 78. Lecourieux D, Ranjeva R, Pugin A (2006) Calcium in plant defense-signaling pathways. New Phytol 171: 249–269.D. LecourieuxR. RanjevaA. Pugin2006Calcium in plant defense-signaling pathways.New Phytol171249269
- 79. Wang C, Zien CA, Afitlhile M, Welti R, Hildebrand DF, et al. (2000) Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in Arabidopsis. Plant Cell 12: 2237–2246.C. WangCA ZienM. AfitlhileR. WeltiDF Hildebrand2000Involvement of phospholipase D in wound-induced accumulation of jasmonic acid in Arabidopsis.Plant Cell1222372246
- 80. Lherminier J, Elmayan T, Fromentin J, Elaraqui KT, Vesa S, et al. (2009) NADPH oxidase-mediated reactive oxygen species production: subcellular localization and reassessment of its role in plant defense. Mol Plant Microbe Interact 7: 868–881.J. LherminierT. ElmayanJ. FromentinKT ElaraquiS. Vesa2009NADPH oxidase-mediated reactive oxygen species production: subcellular localization and reassessment of its role in plant defense.Mol Plant Microbe Interact7868881
- 81. Goswami RS, Kistler HC (2004) Heading for disaster: Fusarium graminearum on cereal crops. Mol Plant Pathol 5: 515–525.RS GoswamiHC Kistler2004Heading for disaster: Fusarium graminearum on cereal crops.Mol Plant Pathol5515525
- 82. Zuppini A, Navazio L, Mariani P (2004) Endoplasmic reticulum stress-induced programmed cell death in soybean cells. J Cell Sci 117: 2591–2598.A. ZuppiniL. NavazioP. Mariani2004Endoplasmic reticulum stress-induced programmed cell death in soybean cells.J Cell Sci11725912598
- 83. Torres MA, Jones JD, Dangl JL (2005) Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana. Nat Genet 37: 1130–1134.MA TorresJD JonesJL Dangl2005Pathogen-induced, NADPH oxidase-derived reactive oxygen intermediates suppress spread of cell death in Arabidopsis thaliana.Nat Genet3711301134
- 84. Kemmerling B, Schwedt A, Rodriguez P, Mazzotta S, Frank M, et al. (2007) The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control. Curr Biol 17: 1116–1122.B. KemmerlingA. SchwedtP. RodriguezS. MazzottaM. Frank2007The BRI1-associated kinase 1, BAK1, has a brassinolide-independent role in plant cell-death control.Curr Biol1711161122
- 85. Penninckx IA, Thomma BP, Buchala A, Métraux JP, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10: 2103–2113.IA PenninckxBP ThommaA. BuchalaJP MétrauxWF Broekaert1998Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis.Plant Cell1021032113
- 86. Olson PD, Varner JE (1993) Hydrogen peroxide and lignification. Plant Cell 4: 887–892.PD OlsonJE Varner1993Hydrogen peroxide and lignification.Plant Cell4887892
- 87. Yu ZL, Zhang JG, Wang XC, Chen J (2008) Excessive copper induces the production of reactive oxygen species, which is mediated by phospholipase D, nicotinamide adenine dinucleotide phosphate oxidase and antioxidant systems. J Integr Plant Biol 50: 157–167.ZL YuJG ZhangXC WangJ. Chen2008Excessive copper induces the production of reactive oxygen species, which is mediated by phospholipase D, nicotinamide adenine dinucleotide phosphate oxidase and antioxidant systems.J Integr Plant Biol50157167
- 88. Leon J, Lawton MA, Raskin I (1995) Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol 108: 1673–1678.J. LeonMA LawtonI. Raskin1995Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco.Plant Physiol10816731678
- 89. Chen Z, Silva H, Klessig DF (1993) Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262: 1883–1886.Z. ChenH. SilvaDF Klessig1993Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid.Science26218831886
- 90. Leon-Reyes A, Du Y, Koornneef A, Proietti S, Körbes AP, et al. (2010) Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic Acid. Mol Plant Microbes Interact 23: 187–197.A. Leon-ReyesY. DuA. KoornneefS. ProiettiAP Körbes2010Ethylene signaling renders the jasmonate response of Arabidopsis insensitive to future suppression by salicylic Acid.Mol Plant Microbes Interact23187197
- 91. Wang KL, Li H, Ecker JR (2002) Ethylene biosynthesis and signaling networks. Plant Cell 14: S131–S151.KL WangH. LiJR Ecker2002Ethylene biosynthesis and signaling networks.Plant Cell14S131S151
- 92. Chen X, Steed A, Travella S, Keller B, Nicholson P (2009) Fusarium graminearum exploits ethylene signaling to colonize dicotyledonous and monocotyledonous plants. New Phytol 182: 975–983.X. ChenA. SteedS. TravellaB. KellerP. Nicholson2009Fusarium graminearum exploits ethylene signaling to colonize dicotyledonous and monocotyledonous plants.New Phytol182975983
- 93. Qiao H, Chang KN, Yazaki J, Ecker JR (2009) Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis. Genes Dev 23: 512–521.H. QiaoKN ChangJ. YazakiJR Ecker2009Interplay between ethylene, ETP1/ETP2 F-box proteins, and degradation of EIN2 triggers ethylene responses in Arabidopsis.Genes Dev23512521
- 94. Kang , Z , Buchenauer H (2000c) Ultrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum. Physiol Mol Plant Pathol 57: 255–268.KangZH. Buchenauer2000cUltrastructural and immunocytochemical investigation of pathogen development and host responses in resistant and susceptible wheat spikes infected by Fusarium culmorum.Physiol Mol Plant Pathol57255268
- 95. Lemmens M, Scholz U, Berthiller F, Dall'Asta C, Koutnik A, et al. (2005) The ability to detoxify the mycotoxin deoxynivalenol co-localizes with a major QTL for Fusarium head blight resistance in wheat. Mol Plant Microbes Interact 18: 1318–1324.M. LemmensU. ScholzF. BerthillerC. Dall'AstaA. Koutnik2005The ability to detoxify the mycotoxin deoxynivalenol co-localizes with a major QTL for Fusarium head blight resistance in wheat.Mol Plant Microbes Interact1813181324
- 96. Poppenberger B, Berthiller F, Lucyshyn D, Sieberer T, Schuhmacher R, et al. (2003) Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP- glucosyltransferase from Arabidopsis thaliana. J Biol Chem 278: 47905–47914.B. PoppenbergerF. BerthillerD. LucyshynT. SiebererR. Schuhmacher2003Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP- glucosyltransferase from Arabidopsis thaliana.J Biol Chem2784790547914
- 97. Lin F, Xue SL, Zhang ZZ, Zhang CQ, Kong ZX, et al. (2006) Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 x Wangshuibai population. II: type I resistance. Theor Appl Genet 112: 528–535.F. LinSL XueZZ ZhangCQ ZhangZX Kong2006Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 x Wangshuibai population. II: type I resistance.Theor Appl Genet112528535
- 98. Lin F, Kong ZX, Zhu HL, Xue SL, Wu JZ, et al. (2004) Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 x Wangshuibai population. I. Type II resistance. Theor Appl Genet 109: 1504–1511.F. LinZX KongHL ZhuSL XueJZ Wu2004Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 x Wangshuibai population. I. Type II resistance.Theor Appl Genet10915041511
- 99. Damerval C, Devienne D, Zivy M, Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedlings proteins. Electrophoresis 7: 52–54.C. DamervalD. DevienneM. ZivyH. Thiellement1986Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedlings proteins.Electrophoresis75254
- 100. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.MM Bradford1976A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem72248254
- 101. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68: 850–858.A. ShevchenkoM. WilmO. VormM. Mann1996Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal Chem68850858
- 102. Xu H, Yang L, Xu P, Tao Y, Ma Z (2007) cTrans: generating polypeptide databases from cDNA sequences. Proteomics 7: 177–179.H. XuL. YangP. XuY. TaoZ. Ma2007cTrans: generating polypeptide databases from cDNA sequences.Proteomics7177179
- 103. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.SF AltschulTL MaddenAA SchäfferJ. ZhangZ. Zhang1997Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.Nucleic Acids Res2533893402
- 104. Huang X, Madan A (1999) CAP3: A DNA sequence assembly program. Genome Res 9: 868–877.X. HuangA. Madan1999CAP3: A DNA sequence assembly program.Genome Res9868877
- 105. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25: 402–408.KJ LivakTD Schmittgen2001Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method.Methods25402408
- 106. Beyer WF Jr, Fridovich I (1987) Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Anal Biochem 161: 559–566.WF Beyer JrI. Fridovich1987Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions.Anal Biochem161559566
- 107. Chance B, Maehly AC (1955) Assay of catalases and peroxidases. Methods Enzymol 2: 764–775.B. ChanceAC Maehly1955Assay of catalases and peroxidases.Methods Enzymol2764775
- 108. Li YH, Wei F, Dong XY, Peng JH, Chen H (2011) Simultaneous analysis of multiple endogenous plant hormones in leaf tissue of oilseed rape by solid-phase extraction coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry. Phytochem Anal, in press. YH LiF. WeiXY DongJH PengH. Chen2011Simultaneous analysis of multiple endogenous plant hormones in leaf tissue of oilseed rape by solid-phase extraction coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.Phytochem Anal, in press