Temporal and subcellular localization of PR-1 proteins in tomato stem tissues infected by virulent and avirulent isolates of Phytophthora capsici

Summary. Immunoblot analysis and immunogold labeling of PR-1 protein (pathogenesis-related protein 1) in tomato (Lycopersicon esculentum Mill.) were performed to examine the temporal and spatial expression patterns of PR-1 protein induced by Phytophthora capsici infection. Soluble proteins with molecular masses of 10, 17, 25, 27 and 75 kDa were induced and accumulated in P. capsici-infected stem tissues during the compatible and incompatible interactions. Western blot analysis revealed that expression of PR-1 protein (17 kDa), at 12 to 24 h after inoculation, occurred earlier in the incompatible than in the compatible interaction. Immunogold labeling of PR-1 proteins occurred over cell walls and cytoplasm of the host and the oomycete pathogen and at the interface between host and oomycete cell walls at 24 h after inoculation in the compatible interaction. In the incompatible interaction, numerous PR-1 proteins accumulated predominantly over oomycete cell walls and at the interface between host and oomycete cell walls. The quantity of PR-1 proteins deposited in both host and oomycete cells was much less in the compatible than the incompatible interaction. Healthy tomato stem tissue was nearly free of immunogold labeling of PR-1 proteins. Received October 9, 2001 Accepted January 18, 2002     

A <Emphasis Type="Italic">Colletotrichum gloeosporioides</Emphasis>-induced esterase gene of nonclimacteric pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) fruit during ripening plays a role in resistance against fungal infection

Ripe fruits of pepper (Capsicum annuum) are resistant to the anthracnose fungus, Colletotrichum gloeosporioides, whereas unripe-mature fruits are susceptible. A pepper esterase gene (PepEST) that is highly expressed during an incompatible interaction between the ripe fruit of pepper and C. gloeosporioides was previously cloned. Deduced amino acid sequence of PepEST cDNA showed homology to both esterases and lipases, and contained -HGGGF- and -GXSXG- motifs and a catalytic triad. Inhibition of PepEST activity by a specific inhibitor of serine hydrolase demonstrated that a serine residue is critical for the enzyme activity. Expression of PepEST gene was fruit-specific in response to C. gloeosporioides inoculation, and up-regulated by wounding or jasmonic acid treatment during ripening. PepEST mRNA and protein was differentially accumulated in ripe vs. unripe fruit from 24 h after inoculation when C. gloeosporioides isinvading into fruits. Immunochemical examination revealed that PepEST accumulation was localized inepidermal and cortical cell layers in infected ripe fruit, but rarely even in epidermal cells in infected unripe one. Over-expression of PepEST in transgenic Arabidopsis plants caused restriction of Alternaria brassicicola colonization by inhibition of spore production, resulting in enhanced resistance against A.brassicicola. These results suggest that PepEST is involved in the resistance of ripe fruit against C.gloeosporioides infection.^†These authors contributed equally to the work     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Induction of enhanced disease resistance and oxidative stress tolerance by overexpression of pepper basic PR-1 gene in Arabidopsis

The pathogen- and ethylene-inducible pepper-basic pathogenesis-related (PR)-1 gene, CABPR1 , was strongly expressed in pepper leaves by osmotic and oxidative stresses. The pepper CABPR1 was introduced into the Arabidopsis plants under the control of the cauliflower mosaic virus 35S promoter. Polymerase chain reaction-amplification with the Arabidopsis genomic DNA and Northern blot analyses confirmed that the pepper CABPR1 gene was integrated into the Arabidopsis genome, where it was overexpressed in the transgenic Arabidopsis plants under normal growth conditions. The constitutive overexpression of CABPR1 induced the expression of the Arabidopsis PR-genes including PR-4 , PR-5 and PDF1.2 . Enhanced resistance to phytopathogenic bacteria, Pseudomonas syringae pv. tomato DC3000, was also observed in the transgenic Arabidopsis plants. CABPR1 overexpression in the transgenic Arabidopsis caused enhanced seed germination under NaCl (ionic) and mannitol (non-ionic) osmotic stresses. Enhanced tolerances to high salinity and dehydration stresses during seed germination of the transgenic plants were not found at the early seedling stage. The transgenic Arabidopsis plants exhibited a higher tolerance to oxidative stress by methyl viologen at the seed germination, seedling and adult plant stages. These results suggest that the CABPR1 gene may function in the enhanced disease resistance and oxidative stress tolerance of transgenic Arabidopsis plants.     

Bacillus thuringiensis protein production, signal transduction, and insect control in chemically inducible PR-1a/cry1Ab broccoli plants

In an effort to develop a chemically inducible system for insect management, we studied production of Cry1Ab Bacillus thuringiensis (Bt) protein and control of the diamondback moth (DBM), Plutella xylostella L., in inducer-treated and untreated tissues of a broccoli line transformed with a PR-1a/cry1Ab expression cassette. Spraying leaves of these plants with the inducer acibenzolar-S-methyl (= 1,2,3 benzothiadiazole-7-thiocarboxylic acid-S-methyl-ester) (ASM) triggered expression of the cry1Ab gene and produced a high level of Cry1Ab protein within 2–3 days. Cry1Ab protein persisted in leaves for at least 8 weeks, providing prolonged protection from P. xylostella attack. Signals generated in inducer-treated leaves were transferred to untreated newly emerged leaves or heads, as seen by production of Cry1Ab protein and/or protection from insect damage in these plant parts. Signal transduction proceeded in an attenuated manner up to the sixth newly emerged leaf. No Cry1Ab protein was detectable by ELISA in uninduced young leaves, but small amounts of the protein were present in uninduced leaves older than 3 weeks and caused some insect mortality. Such basal expression of Bt genes without induction may favor the evolution of resistant insect populations and therefore limits the application of the PR-1a/cry1Ab system for insect management. However, the rapid production and steady maintenance of a high level of transgenic protein upon induction, the signal transduction observed, and the fact that the chemical inducer can be used in field conditions make the PR-1a promoter attractive for chemical regulation of other agriculturally or pharmaceutically important genes for which low expression in the absence of induction is not a concern.     

Differential activation of defense-related genes in susceptible and resistant pepper cultivars infected with Phytophthora capsici

This study investigated the expression pattern of genes encoding for a basic PR-1 protein, a basic beta-1,3-glucanase, a peroxidase, and a sesquiterpene cyclase involved in defense responses in three pepper cultivars with different levels of resistance to Phytophthora capsici. All genes were up-regulated in infected stems of the pepper cultivars, with expression being detected 8h post-inoculation. mRNA levels of these genes increased markedly by 24h post-inoculation, and maximal induction levels were observed for the PR-1 and sesquiterpene cyclase genes. PR-1, peroxidase, and sesquiterpene genes were always expressed at higher levels in resistant cultivars than in the susceptible cultivar, although up-regulation was observed in both, suggesting that the differences between these pepper genotypes in susceptibility and resistance are a matter of the timing and magnitude of the defense response.     

Gene expression and insect resistance in transgenic broccoli containing a Bacillus thuringiensis cry1Ab gene with the chemically inducible PR-1a promoter

We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed.     

Early detection of bean infection by Pseudomonas syringae in asymptomatic leaf areas using chlorophyll fluorescence imaging

Chlorophyll fluorescence imaging has been used to analyse the response elicited in Phaseolus vulgaris after inoculation with Pseudomonas syringae pv. phaseolicola 1448A (compatible interaction) and P. syringae pv. tomato DC3000 (incompatible interaction). With the aim of modulating timing of symptom development, different cell densities were used to inoculate bean plants and the population dynamics of both bacterial strains was followed within the leaf tissue. Fluorescence quenching analysis was carried out and images of the different chlorophyll fluorescence parameters were obtained for infected as well as control plants at different timepoints post-infection. Among the different parameters analysed, we observed that non-photochemical quenching maximised the differences between the compatible and the incompatible interaction before the appearance of visual symptom. A decrease in non-photochemical quenching, evident in both infiltrated and non-infiltrated leaf areas, was observed in P. syringae pv. phaseolicola-infected plants as compared with corresponding values from controls and P. syringae pv. tomato-infected plants. No photoinhibitory damage was detected, as the maximum photosystem II quantum yield remained stable during the infection period analysed.     

A cytochrome P450 gene is differentially expressed in compatible and incompatible interactions between pepper (Capsicum annuum) and the anthracnose fungus, Colletotrichum gloeosporioides

The anthracnose fungus, Colletotrichum gloeosporioides, was previously shown to have an incompatible interaction with ripe-red fruit of pepper (Capsicum annuum). However, the fungus had a compatible interaction with unripe-mature-green fruit. Using mRNA differential display, we isolated and characterized a PepCYP gene expressed in the incompatible interaction. The PepCYP gene encodes a protein homologous to cytochrome P450 proteins containing a heme-binding domain. The expression level of PepCYP is higher in the incompatible interaction than in the compatible interaction, and then remains elevated in the incompatible interaction. In the compatible interaction, the expression of PepCYP is transient. The induction of PepCYP gene is up-regulated by wounding or jasmonic acid treatment during ripening. Analysis of PepCYP expression by in situ hybridization shows that the accumulation of PepCYP mRNA is localized in the epidermal cell layers, but not in the cortical cell layers. An examination of transverse sections of the fruits inoculated with the fungus shows that the fungus invades and colonizes the epidermal cell layers of the unripe fruit at 24 and 72 h after inoculation, respectively, but not those of the ripe fruit. These results suggest that the PepCYP gene product plays a role in the defense mechanism when the fungus invades and colonizes the epidermal cells of fruits in the incompatible interaction during the early fungal infection process.     

BTH-Induced Accumulation of Extracellular Proteins and Blackspot Disease in Rose

Treatment of rose shoots with 50 µM acibenzolar-S-methyl (BTH) resulted in increased protection against Diplocarpon rosae. This was accompanied by the induction and accumulation of a set of extracellular proteins as shown by SDS-PAGE and 2D-PAGE. Some of these proteins have been identified as PR-1, PR-2, PR-3 and PR-5 proteins by immunoblot analysis probed with tobacco antisera against PR-1c, PR-N, PR-Q and PR-S protein. Most of the extracellular proteins activated by BTH were also induced and found to accumulate in leaves upon infection with Diplocarpon rosae. However, their accumulation was much more pronounced in BTH-pretreated leaves than in water-pretreated leaves upon a challenge inoculation with D. rosae, particularly, the 15 kD PR-1, 36 and 37 kD PR-2 proteins. They may be more important in the expression of disease resistance.     

Enzymes regulating the accumulation of active oxygen species during the hypersensitive reaction of bean to Pseudomonas syringae pv. phaseolicola

Changes in the activities of superoxide dismutase (SOD; EC 1.15.1.1), peroxidase (POD; EC 1.11.1.7) and catalase (CAT; EC 1.11.1.6) which regulate the persistence of active oxygen species (AOS) were examined in leaves of bean (Phaseolus vulgaris L. cv. Tendergreen) undergoing compatible and incompatible interactions to race 6 and race 3 strains, respectively, of the halo-blight bacterium Pseudomonas syringae pv. phaseolicola. Resistance of cv. Tendergreen to race 3 is determined by the R3 gene and was expressed by a hypersensitive reaction (HR) which was associated with a rapid increase in lipid peroxidation between 8 and 12 h after inoculation. Five main isoforms of SOD were resolved by native polyacrylamidegel electrophoresis (PAGE). Major changes were found in the activities of the cytosolic Cu, Zn-SOD3 and Cu, ZnSOD5 isoforms, which increased by 6 h after inoculation with race 3, and the possibly peroxisomal MnSOD2 isoform, which decreased rapidly in tissue undergoing the HR. Three further minor isoforms of SOD showed a strong increase in activity during the HR. A low level of extracellular SOD activity was also resolved; two isoforms, one of which increased dramatically in activity during the HR, were detected within intercellular fluids recovered from inoculation sites. Fewer changes in SOD activities were found during the compatible interaction to race 6, and they did not occur until 16 h after inoculation. In tissue around infiltration sites, no decrease in the activity of Mn-SOD2 was observed but slight increases in some other isoforms were found. Four groups of POD isoforms were detected in both 3,3-diaminobenzidine/H₂O₂-and o-dianisidine/H₂O₂-stained PAGE gels. Significant changes in activity were again associated with development of the HR. In particular, by 2 h after inoculation, increases in POD3a, b and c isoforms were detected within total soluble extracts and also in POD3c within intercellular fluids (no other isoform was found in the apoplasm). By contrast, POD1 and POD2 activities generally declined following inoculation. The principal change in activity in tissues surrounding infiltration sites was an increase in POD3 isoforms following inoculation with race 3. Measurements of total activity showed a decrease in CAT activity as early as 2 h after inoculation, followed by a recovery after 8 h and a further decrease as infiltrated tissue collapsed during the HR. A more-gradual decline in CAT activity was observed at sites undergoing the compatible interaction and also in tissue surrounding inoculation sites. The spatial and temporal changes detected in activities of CAT and isoforms of SOD and POD clearly demonstrate the complexity and potential subtlety of control of the production and persistence of AOS in bean following microbial challenge. The generation of AOS through HR-specific, early increases in extra-cellular POD and SOD isoforms is discussed.This work was supported in part by the scientific Research Foundaation (OTKA F 5082), the foundation for Hungarian science, a british council scolership to A.L.A and the U.K. Agricultural and food Reaserch council.