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PubMed2004

Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins.

Partially tree-ripened ripe fruit of peach (Prunus persica L.) were stored for 1-4 weeks at 5 degrees C and then ripened at 20 degrees C for 3 d to induce chilling injury. With increasing cold storage the incidence and severity of mealiness symptoms increased progressively, manifested as reduced quantities of free juice and internal flesh browning. Relative to juicy fruit, tissue of mealy fruit showed altered intercellular adhesion when examined by microscopy and, upon crushing, a higher proportion of cells remained intact and did not release cellular contents. Substantial alterations in the metabolism of cell wall polysaccharides were observed. Chelator-soluble polyuronides from mealy fruit were partially depolymerized during cold storage in a manner dissimilar to that in unripe or ripe juicy fruit, and were not depolymerized further during the ripening period. The solubility of these high molecular weight pectins remained low, and did not show the increase characteristic of juicy fruit. Furthermore, in mealy fruit the dramatic decline in the polymeric Ara content of base-soluble, matrix glycan-enriched fractions occurring during normal ripening was absent, indicating diminished disassembly of an arabinan-rich polysaccharide firmly attached to cellulose. A corresponding rise in the polymeric Ara content of the most soluble pectin fraction was also absent, as was a decline in the Gal content of this extract. The depolymerization of matrix glycans showed only minor differences between juicy and mealy fruit. After cold storage and ripening, the activities of endo-1,4-beta-glucanase (EC 3.2.1.4), endo-1,4-beta-mannanase (EC 3.2.1.78), beta-galactosidase (EC 3.2.1.23), alpha-arabinosidase (EC 3.2.1.55), and particularly endo-polygalacturonase (EC 3.2.1.15) were lower in mealy fruit than in juicy fruit, whereas pectin methylesterase activity (EC 3.1.1.11) was lower in slightly mealy and higher in very mealy fruit. The data suggest that cold storage affects the activities of numerous cell wall-modifying enzymes, with important consequences for pectin metabolism. These changes alter the properties of the primary wall and middle lamella, resulting in tissue breakage along enlarged air spaces, rather than across cells, which reduces the amount and availability of free juice upon tissue fragmentation.

Cell Fractionation↗

PubMed2004

European, Chinese and Japanese pear fruits exhibit differential softening characteristics during ripening.

Softening characteristics were investigated in three types of pear fruit, namely, European pear 'La France', Chinese pear 'Yali', and Japanese pear 'Nijisseiki'. 'La France' fruit softened dramatically and developed a melting texture during ripening, while 'Yali' fruit with and without propylene treatment showed no change in flesh firmness and texture during ripening. Non-treated 'Nijisseiki' did not show a detectable decrease in flesh firmness, whereas continuous propylene treatment caused a gradual decrease in firmness resulting in a mealy texture. In 'La France', the analysis of cell wall polysaccharides revealed distinct solubilization and depolymerization of pectin and hemicellulose during fruit softening. In 'Nijisseiki', propylene treatment led to the solubilization and depolymerization of pectic polysaccharides to a limited extent, but not of hemicellulose. In 'Yali', hemicellulose polysaccharides were depolymerized during ripening, but there was hardly any change in pectic polysaccharides except in the water-soluble fraction. PC-PG1 and PC-PG2, two polygalacturonase (PG) genes, were expressed in 'La France' fruit during ripening, while only PC-PG2 was expressed in 'Nijisseiki' and neither PC-PG1 or PC-PG2 was expressed in 'Yali'. The expression pattern of PC-XET1 was constitutive during ripening in all three pear types. PG activity measured by the reducing sugar assay increased in all three pears during ripening. However, viscometric measurements showed that the levels of endo-PG activity were high in 'La France', low in 'Nijisseiki', and undetectable in 'Yali' fruits. These results suggest that, in pears, cell wall degradation is correlated with a decrease in firmness during ripening and the modification of both pectin and hemicellulose are essential for the development of a melting texture. Furthermore, the data suggest that different softening behaviours during ripening among the three pear fruits may be caused by different endo-PG activity and different expression of PG genes.

China↗

PubMed2006

PpEG4 is a peach endo-beta-1,4-glucanase gene whose expression in climacteric peaches does not follow a climacteric pattern.

In peach (Prunus persica L. Batsch.) the degradation of the pectic compounds of the cell wall is considered to be the principal component responsible for fruit softening. Many genes encoding enzymes acting on the different polymers of the pectic matrix have been shown to be highly expressed during the late phases of softening, with polygalacturonase being the most important. Nevertheless, it is known that softening starts well before the ethylene climacteric rise which occurs concomitant with the maximal expression of the pectolytic enzymes. The cloning and characterization of PpEG4, an endo-beta-1,4-glucanase (EGase) gene preferentially expressed in preclimacteric fruits, are presented here. PpEG4 belongs to the group of EGases containing, at their carboxy-terminus, a peptide similar to the cellulose binding domain of microbial origin. This EGase is also expressed during abscission of both leaves and fruits. The effect of exogenous ethylene treatments on PpEG4 transcription is null in young fruits and negative in preclimacteric ones, while it is positive in abscission zones. Thus, the expression of PpEG4 seems to be more dependent on the type of separation process rather than being influenced by a direct hormone action. The ability of the PpEG4 regulatory sequences to drive transcription in cells undergoing separation events is also maintained in tomato, where about 3 kb of the gene promoter could drive the expression of gusA in preclimacteric fruits and in the fruit abscission zones.

Amino Acid Sequence↗

PubMed2006

Effect of girdling above the abscission zone of fruit on 'Bartlett' pear ripening on the tree.

Pear fruit usually soften and develop a melting texture when harvested at the mature green stage and ripened. The reason why the fruit does not fully ripen on the tree is unknown. To clarify this, our attention was directed to the continuous supply of assimilates and/or other substances into the fruit via phloem transport. To determine the effect of inhibiting phloem transport on fruit ripening on the tree, a girdling treatment was applied to the branch above the abscission zone of 'Bartlett' pear (Pyrus communis L.). Girdling significantly enhanced the ethylene production of fruit on day 12 compared with control fruit. Fruit softening was also stimulated by girdling. On day 8, flesh firmness was similar in treated fruit on the tree and in fruit off the tree, and was significantly lower than that of untreated fruit on the tree. The patterns of transcript accumulation for the ethylene biosynthetic [1-aminocyclopropane-1-carboxylate (ACC) synthase (PcACS) and ACC oxidase (PcACO)] and polygalacturonase (PcPG1 and PcPG3) genes showed good correspondence with ethylene production and fruit softening, respectively. Thus, fruit ripening on the tree was stimulated via ethylene by girdling on the branch above the abscission zone of fruit to interrupt phloem transport. Assimilates and/or other substances in phloem sap may prevent fruit ripening on the tree.

Agriculture↗

PubMed2000

hosoba toge toge, a syndrome caused by a large chromosomal deletion associated with a T-DNA insertion in Arabidopsis.

We isolated a T-DNA-tagged mutant named hosoba toge toge (hot) in which a pleiotropic phenotype was observed in both the shoot and root throughout the life cycle. The phenotype and allelism indicated that the mutant has a defect in both the FASCIATA1 (FAS1) gene and the FT gene located on the bottom arm of chromosome 1. Analysis of the junctions between the T-DNA ends and the plant genome suggested the presence of a 75.8-kbp deletion at the insertion site. In addition to FAS1 and FT, 13 genes were predicted to exist in the region corresponding to that deleted in hot. They include homologs of genes for type II inositol-1,4,5-triphosphate 5-phosphatase (IP5Pase), the beta-chain of N-acetyl-beta-glucosaminidase (NAGase), NADPH oxidoreductase of the zeta-crystallin family, polygalacturonase, and endo-1,4-beta-glucanase. Although most aspects of the hot phenotype can be explained by loss of FAS1 and FT functions, some novel phenotypic features which may represent aspects of a mutant phenotype due to loss-of-function of other gene(s) were observed. One "wild-type" ecotype and a previously reported T-DNA insertion line, neither of which has any obvious phenotypic abnormality, carry a possible loss-of-function mutation in the zeta-crystallin homolog and in the NAGase beta chain homolog, respectively.

Arabidopsis↗

PubMed2005

The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the phytopathogenic Chromista Pythium ultimum and promotes cucumber protection.

The type III secretion system (TTSS) is used by Proteobacteria for pathogenic or symbiotic interaction with plant and animal hosts. Recently, TTSS genes thought to originate from the phytopathogen Pseudomonas syringae were evidenced in Pseudomonas fluorescens KD, which protects cucumber from the oomycete Pythium ultimum (kingdom Chromista/Stramenopila). However, it is not known whether the TTSS contributes to plant protection by the bacterium and, if so, whether it targets the plant or the phytopathogen. Inactivation of TTSS gene hrcV following the insertion of an omega cassette strongly reduced the biocontrol activity of the pseudomonad against P. ultimum on cucumber when compared with the wild type, but had no effect on its root-colonization ability. Analysis of a plasmid-based transcriptional hrpJ'-inaZ reporter fusion revealed that expression in strain KD of the operon containing hrcV was strongly stimulated in vitro and in situ by the oomycete and not by the plant. In vitro, both strain KD and its hrcV mutant reduced the activity level of the pectinase polygalacturonase (a key pathogenicity factor) from P. ultimum, but the reduction was much stronger with the wild type. Together, these results show that the target range of bacterial TTSS is not restricted to plants and animals but also can include members of Chromista/Stramenopila, and suggest that virulence genes acquired horizontally from phytopathogenic bacteria were functionally recycled in biocontrol saprophytic Pseudomonas spp., resulting in enhanced plant protection by the latter.

Artificial Gene Fusion↗

PubMedDate not supplied

Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp. carotovora.

Erwinia carotovora subsp. carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease. In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H. Murata, J. L. McEvoy, A. Chatterjee, A. Collmer, and A. K. Chatterjee, Mol. Plant-Microbe Interact, 4:239-246, 1991). The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis. aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52. The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA. An aepA-lacZ operon fusion was constitutively expressed in E. coli (DH5 alpha) but inducible by pectate and celery extract in E. c. subsp. carotovora (AC5006). These findings suggest that aepA expression may be negatively regulated in E. c. subsp. carotovora. By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription. The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production. Whereas homologs of Ecc71 aepA occur in E. c. subsp. carotovora and E. c. subsp. atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E. c. subsp. carotovora than in E. c. subsp. atroseptica.

Amino Acid Sequence↗

PubMed1996

The RsmA- mutants of Erwinia carotovora subsp. carotovora strain Ecc71 overexpress hrpNEcc and elicit a hypersensitive reaction-like response in tobacco leaves.

Erwinia carotovora subsp. carotovora wild-type strain Ecc71 does not elicit the hypersensitive reaction (HR) in tobacco leaves. By mini-Tn5-Km and chemical mutagenesis we have isolated RsmA- mutants of Ecc71 that produce high basal levels of pectate lyases, polygalacturonase, cellulase, and protease; they also are hypervirulent. The RsmA- mutants, but not their parent strains, elicit an HR-like response in tobacco leaves. This reaction is characterized by the rapid appearance of water soaking followed by tissue collapse and necrosis. The affected areas remain limited to the region infiltrated with bacterial cells, and the symptoms closely resemble a typical HR, e.g., the reactions caused by Pseudomonas syringae pv. pisi. Moreover, low concentrations of cells of the mini-Tn5-Km insertion RsmA- mutant, AC5070, infiltrated into tobacco leaf tissue prevent elicitation of the rapid necrosis by AC5070 or by P. syringae pv. pisi. Elicitation of the HR-like response by the mutants is not affected by the deficiency of N-(3-oxohexanoyl)-L-homoserine lactone, the cell density (quorum) sensing signal. Cloning and sequence analysis have disclosed that E. carotovora subsp. carotovora strain Ecc71 possesses a homolog of E. chrysanthemi hrpN known to encode an elicitor of the HR; the corresponding Ecc71 gene is designated hrpNEcc. Northern (RNA) blot data show that the level of hrpNEcc mRNA is considerably higher in the RsmA- mutants than in the RsmA+ strains. Moreover, a low copy plasmid carrying the rsmA+ allele severely reduces the level of the hrpNEcc transcripts in the RsmA- mutants. These constructs, like the RsmA+ E. carotovora subsp. carotovora strains, do not elicit the HR-like response. These data taken along with the effects of rsmA on exoenzyme production and pathogenicity (A. Chatterjee et al., 1995, Appl. Environ. Microbiol. 61:1959-1967) demonstrate that this global regulator gene plays a critical role in plant interaction of E. carotovora subsp. carotovora.

Amino Acid Sequence↗

PubMed1997

Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp. carotovora subsp. carotovora that encodes a two-component sensor-regulator protein.

A mutant of Erwinia carotovora subsp. carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase. A cosmid clone, pEC462, was isolated from a wild-type E. carotovora subsp. carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans. The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors. Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P. tolaasii, RepA from P. viridiflava, LemA from P. syringae pv. syringae, and RpfC from Xanthomonas campestris pv. campestris. The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E. carotovora subsp. carotovora on potato tubers.

Aconitate Hydratase↗

PubMed2000

Cloning and disruption of pgx4 encoding an in planta expressed exopolygalacturonase from Fusarium oxysporum.

Fusarium oxysporum f. sp. lycopersici, the causal agent of tomato vascular wilt, produces an array of pectinolytic enzymes, including at least two exo-alpha1,4-polygalacturonases (exoPGs). A gene encoding an exoPG, pgx4, was isolated with degenerate polymerase chain reaction primers derived from amino acid sequences conserved in two fungal exoPGs. pgx4 encodes a 454 amino acid polypeptide with nine potential N-glycosylation sites and a putative 21 amino acid N-terminal signal peptide. The deduced mature protein has a calculated molecular mass of 47.9 kDa, a pI of 8.0, and 51 and 49% identity with the exoPGs of Cochliobolus carbonum and Aspergillus tubingensis, respectively. The gene is present in a single copy in different formae speciales of F. oxysporum. Expression of pgx4 was detected during in vitro growth on pectin, polygalacturonic acid, and tomato vascular tissue and in roots and stems of tomato plants infected by F. oxysporum f. sp. lycopersici. Two mutants of F. oxysporum f. sp. lycopersici with a copy of pgx4 inactivated by gene replacement were as virulent on tomato plants as the wild-type strain.

Amino Acid Sequence↗

PubMed2000

Transgenic expression of pear PGIP in tomato limits fungal colonization.

Transgenic tomato plants expressing the pear fruit polygalacturonase inhibitor protein (pPGIP) were used to demonstrate that this inhibitor of fungal pathogen endopolygalacturonases (endo-PGs) influences disease development. Transgenic expression of pPGIP resulted in abundant accumulation of the heterologous protein in all tissues and did not alter the expression of an endogenous tomato fruit PGIP (tPGIP). The pPGIP protein was detected, as expected, in the cell wall protein fraction in all transgenic tissues. Despite differential glycosylation in vegetative and fruit tissues, the expressed pPGIP was active in both tissues as an inhibitor of endo-PGs from Botrytis cinerea. The growth of B. cinerea on ripe tomato fruit expressing pPGIP was reduced, and tissue breakdown was diminished by as much as 15%, compared with nontransgenic fruit In transgenic leaves, the expression of pPGIP reduced lesions of macerated tissue approximately 25%, a reduction of symptoms of fungal growth similar to that observed with a B. cinerea strain in which a single endo-PG gene, Bcpg1, had been deleted (A. ten Have, W. Mulder, J. Visser, and J. A. L. van Kan, Mol. Plant-Microbe Interact. 11:1009-1016, 1998). Heterologous expression of pPGIP has demonstrated that PGIP inhibition of fungal PGs slows the expansion of disease lesions and the associated tissue maceration.

Base Sequence↗

PubMed2001

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Colletotrichum↗

PubMed2003

New NodW- or NifA-regulated Bradyrhizobium japonicum genes.

A cluster of genes coding for putative plant cell-wall degrading enzymes (i.e., genes for two endoglucanases [gunA and gunA2], one pectinmethylesterase [pme], and one polygalacturonase [pgl]) was identified by sequence similarities in the symbiotic region of the Bradyrhizobium japonicum chromosome. In addition, a systematic screen of the region revealed several genes potentially transcribed by the sigma(54)-RNA polymerase and activated by the transcriptional regulator NifA (i.e., genes for proteins with similarity to outer membrane proteins [id117 and id525] and a citrate carrier [id331 or citA] and one open reading frame without similarity to known proteins [id747]). Expression studies using transcriptional lacZ fusions showed that gunA2 and pgl were strongly induced by the isoflavone genistein in a NodW-dependent manner, suggesting a role of the gene products in early events of the nodulation process; by contrast, gunA and pme expression was very weak in the conditions tested. The gunA2 gene product was purified and was shown to have cellulase activity. beta-Galactosidase activity expressed from transcriptional lacZ fusions to id117, id525, and id747 in the wild type and in nifA and rpoN mutant backgrounds confirmed that their transcription was dependent on NifA and sigma(54). Despite the presence of a -24/-12-type promoter and a NifA binding site upstream of citA, no regulation could be demonstrated in this case. Null mutations introduced in gunA, gunA2, pgl, pme, citA, id117, id525, and id747 did not impair the symbiosis with the host plants.

Amino Acid Sequence↗

PubMed2003

Characterization of a Ralstonia solanacearum operon required for polygalacturonate degradation and uptake of galacturonic acid.

The bacterial wilt pathogen Ralstonia solanacearum produces three extracellular polygalacturonases (PGs): PehA, PehB, and PehC. All three PGs hydrolyze pectin's polygalacturonic acid backbone, but each releases different reaction products. PehA and PehB contribute significantly to pathogen virulence, probably by facilitating root invasion and colonization. To determine the collective contribution of PGs to virulence and saprophytic survival, we cloned, characterized, and mutated the R. solanacearum pehC gene, which encodes a distinctive monogalacturonate-releasing exo-PG. The virulence of a pehC mutant on tomato was indistinguishable from that of its wild-type parent; thus, this exo-PG alone does not contribute significantly to wilt pathogenesis. Unexpectedly, a completely PG-deficient triple pehA/B/C mutant was slightly more virulent than a pehA/B mutant. PehC may degrade galacturonide elicitors of host defense, thereby protecting the pathogen from plant antimicrobial responses. A galacturonate transporter gene, exuT, is immediately downstream of pehC and the two genes are co-transcribed. It has been hypothesized that galacturonic acid released by PGs from plant cell walls nourishes bacteria during pathogenesis. To separate the pectolytic and nutrient-generating roles of the PGs, we made an exuT mutant, which still produces all three isozymes of PG but cannot uptake PG degradation products. This exuT mutant had wild-type virulence on tomato, demonstrating that metabolism of galacturonic acid does not contribute significantly to bacterial success inside the plant.

Amino Acid Sequence↗

PubMed2004

Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana.

The phytopathogenic bacterium Ralstonia solanacearum requires motility for full virulence, and its flagellin is a candidate pathogen-associated molecular pattern that may elicit plant defenses. Boiled extracts from R. solanacearum contained a strong elicitor of defense-associated responses. However, R. solanacearum flagellin is not this elicitor, because extracts from wild-type bacteria and fliC or flhDC mutants defective in flagellin production all elicited similar plant responses. Equally important, live R. solanacearum caused similar disease on Arabidopsis ecotype Col-0, regardless of the presence of flagellin in the bacterium or the FLS2-mediated flagellin recognition system in the plant. Unlike the previously studied flg22 flagellin peptide, a peptide based on the corresponding conserved N-terminal segment of R. solanacearum, flagellin did not elicit any response from Arabidopsis seedlings. Thus recognition of flagellin plays no readily apparent role in this pathosystem. Flagellin also was not the primary elicitor of responses in tobacco. The primary eliciting activity in boiled R. solanacearum extracts applied to Arabidopsis was attributable to one or more proteins other than flagellin, including species purifying at approximately 5 to 10 kDa and also at larger molecular masses, possibly due to aggregation. Production of this eliciting activity did not require hrpB (positive regulator of type III secretion), pehR (positive regulator of polygalacturonase production and motility), gspM (general secretion pathway), or phcA (LysR-type global virulence regulator). Wild-type R. solanacearum was virulent on Arabidopsis despite the presence of this elicitor in pathogen extracts.

Amino Acid Sequence↗

PubMed1982

An exopectate lyase of Butyrivibrio fibrisolvens from the bovine rumen.

An extracellular pectinolytic enzyme produced by Butyrivibrio fibrisolvens isolated from the bovine rumen was studied. The enzyme had a pH optimum of 8.0 to 8.5 and was stimulated by Ca2+ and inhibited by EDTA. The products of pectinolysis had an absorption peak at 235 nm and reacted with thiobarbituric acid, indicating a lyase type of action. The enzyme cleaved the substrates terminally from the reducing end; action on poly- and oligogalacturonates resulted in the formation of an unsaturated trigalacturonate. The enzyme was classified as an exopectate lyase (EC 4.2.2.9). A pectinesterase was also produced by B. fibrisolvens but polygalacturonase was not detected.

Animals↗

PubMed1975

Correlation of virulence with secretion in vitro of three wall-degrading enzymes in isolates of Sclerotinia fructigena obtained after mutagen treatment.

Correlation and regression analyses were carried out between the virulence (expressed as growth rate in apple fruits) and the secretion in vitro of three host wall-degrading enzymes by 119 isolates of Sclerotinia fructigena, most of which had been obtained following exposure of conidia to N-methyl-N'-nitro-N-nitroso-guanidine. Virulence was found to be significantly correlated (P less than 0-01) with alpha-L-arabinofuranosidase, but not with pectin esterase or, where enzyme interdependence had been statistically eliminated, with polygalacturonase. Approximately 35% of the total variability in virulence could be accounted for in terms of the three enzymes.

Arabinose↗

PubMed1999

Isolation of an extracellular protease gene of Erwinia carotovora subsp. carotovora strain SCC3193 by transposon mutagenesis and the role of protease in phytopathogenicity.

Using mini-Tn5CmR::gusA, a transposon that allows transcriptional fusions to a promoterless beta-glucuronidase gene, a mutant of Erwinia carotovora subsp. carotovora SCC3193 deficient in extracellular protease production and soft-rot pathogenicity in plants was isolated. The mutant, designated SCC6004, produced normal levels of pectate lyase, polygalacturonase and cellulase. The region of the transposon insertion was partially sequenced to permit the design of specific oligonucleotide primers to amplify a 2.7 kb Clal fragment from E. carotovora subsp. carotovora SCC3193. The DNA sequence of the cloned fragment contained two complete and one partial ORFs. One of the complete ORFs (ORF1) was designated prtW and encodes a secreted protease. The deduced amino acid sequence of PrtW showed a high overall identify of 60-66% to the previously described Erwinia chrysanthemi proteases, but no homology to other proteases isolated from different E. carotovora strains. Downstream from ORF1, a further complete ORF (ORF2) and a partial ORF (ORF3) were found, with deduced peptide sequences that have significant similarity to the Inh and PrtD proteins, respectively, from E. chrysanthemi, which are involved in protease secretion. Gene fusion to the gusA reporter was employed to charaterize the regulation of prtW. The prtW gene was found to be strongly induced in the presence of plant extracts. The mutant exhibited reduced virulence, suggesting that PrtW enhances the ability of strain SCC3193 to macerate plant tissue.

Artificial Gene Fusion↗