A pathogen- and salicylic acid-induced WRKY DNA-binding activity recognizes the elicitor response element of the tobacco class I chitinase gene promoter

Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.     

Isolation and characterization of two pathogen- and salicylic acid-induced genes encoding WRKY DNA-binding proteins from tobacco

A pathogen- and salicylic acid (SA)-induced DNA-binding activity has been recently identified in tobacco that is related to a previously identified class of WRKY DNA-binding proteins. To identify members of the WRKY gene family associated with this DNA-binding activity, we have attempted to isolate those WRKY genes that are induced by pathogen infection. Using a domain-specific differential display procedure, we have isolated two tobacco WRKY genes, tWRKY3 and tWRKY4, that are rapidly induced in resistant tobacco plants after infection by tobacco mosaic virus (TMV). Both tWRK3 and tWRKY4 encode proteins with a single WRKY domain that contain the conserved WRKYGQK sequence. Unlike other isolated WRKY proteins that contain the Cys₂His₂ zinc motif, tWRKY3 and tWRKY4 appear to contain the Cys₂HisCys zinc motif. Nonetheless, both tWRKY3 and tWRKY4 are capable of binding DNA molecules with the W-box (TTGAC) element recognized by other WRKY proteins. Expression of the tWRKY3 and tWRKY4 genes could be rapidly induced not only by TMV infection but also by SA or its biologically active analogues that are capable of inducing pathogenesis-related genes and enhanced resistance. Interestingly, induction of both genes by TMV infection was still observed in resistant tobacco plants expressing the bacterial salicylate hydroxylase gene (nahG), although the levels of induction appeared to be reduced. Identification of pathogen- and SA-induced genes encoding WRKY DNA-binding proteins should facilitate future studies on the regulation and functions of this novel group of DNA-binding proteins.     

An HR-induced tobacco peroxidase gene is responsive to spermine, but not to salicylate, methyl jasmonate, and ethephon

In Tobacco mosaic virus (TMV)-infected tobacco plants carrying the N resistance gene, a hypersensitive reaction or response (HR) occurs to enclose the virus in the infected tissue. Although a contribution of peroxidases to the resistance has been proposed, no evidence has been presented that tobacco peroxidase genes respond to HR. Here, we describe the HR-induced expression of a tobacco peroxidase gene (tpoxC1) whose induction kinetics were slightly different from those of acidic and basic tobacco pathogenesis-related (PR) protein genes. Interestingly, tpoxC1 was insensitive to the inducers of PR genes such as salicylic acid, methyl jasmonate, and ethephon. Spermine activated tpoxC1 gene expression at a low level and both acidic and basic PR gene expression at a considerably higher level. These results indicate that the induced expression of tpoxC1 is regulated differently from that of classical tobacco PR genes in the N gene-mediated self-defense system in tobacco plants.     

Induction of resistance and expression of defense-related genes in tobacco leaves infiltrated with Ralstonia solanacearum

Ralstonia solanacearum 8107 (8107) is non-pathogenic to tobacco and elicits the hypersensitive response (HR). In Nicotiana tabacum cv. Samsun NN leaves infiltrated with 8107, acquired resistance to challenging tobacco mosaic virus (TMV) was induced 2-6 d after 8107-infiltration. hsr203J and hin1 genes were expressed only in the 8107-infiltrated area. On the other hand, the expression of PR-1a and PR-1b genes was not detected in the 8107-infiltrated area, but in areas other than that developing the HR. Expression of these PR-1 genes was regulated simultaneously and the kinetics of the expression was dependent on the distance from the infiltration area. Therefore, diffusible signal(s) might be produced in HR-causing cells and transmitted to peripheral cells resulting in expression of PR genes. In NahG10 tobacco infiltrated with 8107, the HR was induced but resistance to TMV was not. Analysis using NahG10 tobacco also showed that the salicylic acid (SA)-dependent signal regulated the expression of hsr203J and PR-1a, but not that of hin1 and PR-1b. These results suggest that resistance of tobacco to 8107 is SA-independent and involves a quite different mechanism from acquired resistance to TMV induced by 8107-infiltration which is SA-dependent.     

Function of a mitogen-activated protein kinase pathway in N gene-mediated resistance in tobacco

The active defense of plants against pathogens often includes rapid and localized cell death known as hypersensitive response (HR). Protein phosphorylation and dephosphorylation are implicated in this event based on studies using protein kinase and phosphatase inhibitors. Recent transient gain-of-function studies demonstrated that the activation of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco mitogen-activated protein kinases (MAPKs) by their upstream MAPK kinase (MAPKK), NtMEK2 leads to HR-like cell death. Here, we report that the conserved kinase interaction motif (KIM) in MAPKKs is required for NtMEK2 function. Mutation of the conserved basic amino acids in this motif, or the deletion of N-terminal 64 amino acids containing this motif significantly compromised or abolished the ability of NtMEK2DD to activate SIPK/WIPK in vivo. These mutants were also defective in interacting with SIPK and WIPK, suggesting protein-protein interaction is required for the functional integrity of this MAPK cascade. To eliminate Agrobacterium that is known to activate a number of defense responses in transient transformation experiments, we generated permanent transgenic plants. Induction of NtMEK2DD expression by dexamethasone induced HR-like cell death in both T1 and T2 plants. In addition, by using PVX-induced gene silencing, we demonstrated that the suppression of all three known components in the NtMEK2-SIPK/WIPK pathway attenuated N gene-mediated TMV resistance. Together with previous report that SIPK and WIPK are activated by TMV in a gene-for-gene-dependent manner, we conclude that NtMEK2-SIPK/WIPK pathway plays a positive role in N gene-mediated resistance, possibly through regulating HR cell death.     

A virus-inducible tobacco gene encoding a glycine-rich protein shares putative regulatory elements with the ribulose bisphosphate carboxylase small subunit gene

cDNA to an mRNA that is strongly induced in Samsun NN tobacco after tobacco mosaic virus (TMV) infection or salicylic acid treatment was used to probe a genomic blot and to screen a genomic library. The mRNA corresponds to a family of approximately eight genes, four of which were cloned. The sequence of the genes and flanking DNA in two clones was determined. One gene was found to contain an intron of 555 bp; S1-nuclease mapping studies indicated that this gene is expressed. The other gene is interrupted by an intron of 1,954 bp and is probably not expressed after TMV infection. The genes encode a protein of 109 amino acids with a putative N-terminal signal peptide of 26 amino acids. The protein contains a high proportion of glycine (25%) and charged amino acids (29%), suggesting that it may be a cell wall component. A comparison of the upstream sequences of the genes encoding the glycine-rich protein and the pathogenesis-related protein 1a showed only limited homology, although both genes are TMV- and salicylic acid-inducible. However, the upstream sequence of the glycine-rich protein gene contains a 64-bp inverted repeat that occurs in a similar position in the tobacco ribulose bisphosphate carboxylase small subunit gene.     

PeaT1-induced systemic acquired resistance in tobacco follows salicylic acid-dependent pathway

Systemic acquired resistance (SAR) is an inducible defense mechanism which plays a central role in protecting plants from pathogen attack. A new elicitor, PeaT1 from Alternaria tenuissima, was expressed in Escherichia coil and characterized with systemic acquired resistance to tobacco mosaic virus (TMV). PeaT1-treated plants exhibited enhanced systemic resistance with a significant reduction in number and size of TMV lesions on wild tobacco leaves as compared with control. The quantitative analysis of TMV CP gene expression with real-time quantitative PCR showed there was reduction in TMV virus concentration after PeaT1 treatment. Similarly, peroxidase (POD) activity and lignin increased significantly after PeaT1 treatment. The real-time quantitative PCR revealed that PeaT1 also induced the systemic accumulation of pathogenesis-related gene, PR-1a and PR-1b which are the markers of systemic acquired resistance (SAR), NPR1 gene for salicylic acid (SA) signal transduction pathway and PAL gene for SA synthesis. The accumulation of SA and the failure in development of similar level of resistance as in wild type tobacco plants in PeaT1 treated nahG transgenic tobacco plants indicated that PeaT1-induced resistance depended on SA accumulation. The present work suggested that the molecular mechanism of PeaT1 inducing disease resistance in tobacco was likely through the systemic acquired resistance pathway mediated by salicylic acid and the NPR1 gene.     

The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato

Virus-induced gene silencing identified the Avr9/Cf-9 RAPIDLY ELICITED gene ACRE189 as essential for the Cf-9- and Cf-4-mediated hypersensitive response (HR) in Nicotiana benthamiana. We report a role for ACRE189 in disease resistance in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). ACRE189 (herein renamed Avr9/Cf-9-INDUCED F-BOX1 [ACIF1]) encodes an F-box protein with a Leu-rich-repeat domain. ACIF1 is widely conserved and is closely related to F-box proteins regulating plant hormone signaling. Silencing of tobacco ACIF1 suppressed the HR triggered by various elicitors (Avr9, Avr4, AvrPto, Inf1, and the P50 helicase of Tobacco mosaic virus [TMV]). ACIF1 is recruited to SCF complexes (a class of ubiquitin E3 ligases), and the expression of ACIF1 F-box mutants in tobacco compromises the HR similarly to ACIF1 silencing. ACIF1 affects N gene-mediated responses to TMV infection, including lesion formation and salicylic acid accumulation. Loss of ACIF1 function also reduced confluent cell death induced by Pseudomonas syringae pv tabaci. ACIF1 silencing in Cf9 tomato attenuated the Cf-9-dependent HR but not Cf-9 resistance to Cladosporium fulvum. Resistance conferred by the Cf-9 homolog Cf-9B, however, was compromised in ACIF1-silenced tomato. Analysis of public expression profiling data suggests that Arabidopsis thaliana homologs of ACIF1 (VFBs) regulate defense responses via methyl jasmonate- and abscisic acid-responsive genes. Together, these findings support a role of ACIF1/VFBs in plant defense responses.     

Involvement of the small GTPase Rac in the defense responses of tobacco to pathogens

During the hypersensitive response (HR), plants accumulate reactive oxygen species (ROS) that are likely generated at least in part by an NADPH oxidase similar to that found in mammalian neutrophils. An essential regulator of mammalian NADPH oxidase is the small GTP-binding protein Rac. To investigate whether Rac also regulates the pathogen-induced oxidative burst in plants, a dominant negative form of the rice OsRac1 gene was overexpressed in tobacco carrying the N resistance gene. Following infection with Tobacco mosaic virus (TMV), DN-OsRacl plants developed smaller lesions than wild-type plants, accumulated lower levels of lipid peroxidation products, and failed to activate expression of antioxidant genes. These results, combined with the demonstration that superoxide and hydrogen peroxide levels were reduced in DN-OsRacl tobacco developing a synchronous HR triggered by transient expression of the TMV p50 helicase domain or the Pto and AvrPto proteins, suggest that ROS production is impaired. The dominant negative effect of DN-OsRacl could be rescued by transiently overexpressing the wild-type OsRac1 protein. TMV-induced salicylic acid accumulation also was compromised in DN-OsRacl tobacco. Interestingly, while systemic acquired resistance to TMV was not impaired, nonhost resistance to Pseudomonas syringae pv. maculicola ES4326 was suppressed. Thus, the effect DN-OsRac1 expression exerts on the resistance signaling pathway appears to vary depending on the identity of the inoculated pathogen.