Characterization of hsr201 and hsr515, two tobacco genes preferentially expressed during the hypersensitive reaction provoked by phytopathogenic bacteria

During an incompatible interaction between tobacco and the bacterial phytopathogen Pseudomonas solanacearum, 2 classes of genes, the so-called hsr (hypersensitivity-related) genes, activated preferentially during the hypersensitive reaction, and the str (sensitivity-related) genes, expressed strongly during compatible and incompatible interactions, have been identified. In this report, two hsr cDNA clones, hsr515 and hsr201, as well as their expression patterns are presented. Hsr515 was found to encode a P450 monooxygenase and is most similar to the ripening-related avocado gene CYP71A1 (40.6% amino acid identity). Hsr201 presents 58.6% amino acid identity with pTom36, a tomato gene expressed during fruit maturation. The putative functions of the hsr gene products appear to be quite diverse and their characteristics of activation were found to be very conserved: accumulation of the corresponding mRNAs primarily in leaf areas in contact with the avirulent P. solanacearum strain or with a Pseudomonas fluorescens strain containing the hrpZ gene encoding a necrotizing polypeptide, harpin and absence of expression during normal plant development. Our results also suggest that, in a tobacco line expressing NahG, a lower level of salicylic acid, a compound associated with systemic acquired resistance, and also possibly involved in the development of necrotic lesions characteristic of the HR, does not affect the hsr gene expression.     

Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response

Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.     

Differential in vitro DNA binding activity to a promoter element of the gn1β-1,3-glucanase gene in hypersensitively reacting tobacco plants

In a hypersensitive reaction to pathogen infection, expression of the β-1,3-glucanase gn1 gene is induced in cells surrounding the necrotic lesions. The 5′-flanking sequence of gn1 was examined to investigate the molecular basis controlling activation of gene expression during this plant defense response. Studies on transgenic tobacco plants containing gn1 promoter deletions fused to the β-glucuronidase reporter gene revealed the presence of negative and positive regulatory sequences mediating both the level and the spatial distribution of gn1 expression. Promoter sequences to ?138 bp were sufficient to confer increased gene expression around the necrotic lesions produced in response to Pseudomonas syringae pv. syringae inoculation. It is demonstrated by electrophoretic mobility shift assays that nuclear proteins in both healthy and hypersensitively reacting tobacco leaves interact with DNA sequences within the regulatory elements identified. Among the binding sequences characterized, the promoter region extending from ?250 to ?217 bp contained the DNA motif -GGCGGC- found to be conserved in most if not all promoters of genes encoding pathogenesis-related basic proteins. The activity bound by this promoter sequence was stronger in hypersensitively responding tissues than in healthy untreated tobacco leaves.     

PopW of Ralstonia solanacearum,a new two‐domain harpin targeting the plant cell wall

Harpins are extracellular glycine‐rich proteins eliciting a hypersensitive response (HR). In this study, we identified a new harpin, PopW, from Ralstonia solanacearum strain ZJ3721. This 380‐amino‐acid protein is acidic, rich in glycine and serine, and lacks cysteine. When infiltrated into the leaves of tobacco (non‐host), PopW induced a rapid tissue collapse via a heat‐stable but protease‐sensitive HR‐eliciting activity. PopW has an N‐terminal harpin domain (residues 1–159) and a C‐terminal pectate lyase (PL) domain (residues 160–366); its HR‐eliciting activity depends on its N‐terminal domain. Analyses of subcellular localization and plasmolysis demonstrated that PopW targeted the onion cell wall. This was further confirmed by its ability to specifically bind to calcium pectate, a major component of the plant cell wall. However, PopW had no detectable PL activity. Western blotting revealed that PopW was secreted by the type III secretion system in an hrpB‐dependent manner. Gene sequencing indicated that popW is conserved among 20 diverse strains of R. solanacearum. A popW‐deficient mutant retained the ability of wild‐type strain ZJ3721 to elicit HR in tobacco and to cause wilt disease in tomato (a host). We conclude that PopW is a new cell wall‐associated, hrpB‐dependent, two‐domain harpin that is conserved across the R. solanacearum species complex.     

A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible

Plants activate disease resistance responses when they recognize pathogen-derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro-organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen-inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1-PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.     

Ralstonia solanacearum type III secretion system effector Rip36 induces a hypersensitive response in the nonhost wild eggplant Solanum torvum

Ralstonia solanacearum is a Gram‐negative soil‐borne bacterium that causes bacterial wilt disease in more than 200 plant species, including economically important Solanaceae species. In R. solanacearum, the hypersensitive response and pathogenicity (Hrp) type III secretion system is required for both the ability to induce the hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. Recently, 72 effector genes, called rip (Ralstonia protein injected into plant cells), have been identified in R. solanacearum RS1000. RS1002, a spontaneous nalixidic acid‐resistant derivative of RS1000, induced strong HR in the nonhost wild eggplant Solanum torvum in an Hrp‐dependent manner. An Agrobacterium‐mediated transient expression system revealed that Rip36, a putative Zn‐dependent protease effector of R. solanacearum, induced HR in S. torvum. A mutation in the putative Zn‐binding motif (E149A) completely abolished the ability to induce HR. In agreement with this result, the RS1002‐derived Δrip36 and rip36E149A mutants lost the ability to induce HR in S. torvum. An E149A mutation had no effect on the translocation of Rip36 into plant cells. These results indicate that Rip36 is an avirulent factor that induces HR in S. torvum and that a putative Zn‐dependent protease motif is essential for this activity.     

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpinpss-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS)

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin_pss, an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin_pss-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coliwas able to significantly increase active oxygen species (AOS) generation when mixed with harpin_pss in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin_pss in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflpgene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin_pss-mediated hypersensitive cell death in vivo. In response to harpin_pss, suspension cells derived from Pflptransgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl₃) were used to study the involvement of AOS in harpin_pss-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin_pss inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.     

Developmental and pathogen-induced activation of an msr gene, str246C, from tobacco involves multiple regulatory elements

A family of genes, the so-called msr genes (multiple stimulus response), has recently been identified on the basis of sequence homology in various plant species. Members of this gene family are thought to be regulated by a number of environmental or developmental stimuli, although it is not known whether any one member responds more specifically to one stimulus, or whether each gene member responds to various environmental stimuli. In this report, we address this question by studying the tobacco msr gene str246C. Using transgenic tobacco plants containing 2.1 kb of 5 flanking DNA sequence from the str246C gene fused to the -glucuronidase (GUS) coding region, the complex expression pattern of the str246C promoter has been characterized. Expression of the str246C promoter is strongly and rapidly induced by bacterial, fungal and viral infection and this induction is systemic. Elicitor preparations from phytopathogenic bacteria and fungi activate the str246C promoter to high levels, as do wounding, the application of auxin, auxin and cytokinin, salicylic acid or copper sulfate, indicating the absence of gene specialization within the msr gene family, at least for str246C. In addition, GUS activity was visualized. histochemically in root meristematic tissues of tobacco seedlings and is restricted to roots and sepals of mature plants. Finally, analysis of a series of 5 deletions of the str246C promoter-GUS gene fusion in transgenic tobacco plants confirms the involvement of multiple regulatory elements. A region of 83 by was found to be necessary for induction of promoter activity in response to Pseudomonas solanacearum, while auxin inducibility and root expression are apparently not controlled by this element, since its removal does not abolish either response. An element of the promoter with a negative effect on promoter activation by P. solanacearum was also identified.Joint first authors     

Indole-3-acetic acid reverses the harpin-induced hypersensitive response and alters the expression of hypersensitive-response-related genes in tobacco

Harpin proteins stimulate hypersensitive response (HR) in plants. However, the mechanism by which HR is regulated is not clear. The role of the auxin, indole-3-acetic acid (IAA), in the control of harpin-stimulated HR was investigated. IAA was used to inhibit HR that was stimulated by purified fusion harpin_Xoo protein in tobacco. Semi-quantitative PCR and qRT-PCR were employed to detect the expression of HR related genes. IAA at 100 μM reversed harpin-induced HR which was inhibited by 500 μM 2,3,5-triiodobenzoic acid (TIBA). Semi-quantitative PCR and qRT-PCR showed the combined application of 100 μM IAA and harpin protein from Xanthomonas oryzae enhanced the expression of HR marker gene, hsr203J, but weakened the expression of the disease-defense gene, chia5. TIBA also decreased the expression of hsr203J but increased the expression of chia5. Thus, the auxin can reverse harpin_Xoo-induced HR.