Helwan University, Ain Helwan, Cairo, Egypt

ACCUMULATION OF PROTEINS IN CUCUMBERSEEDLINGS RESISTANT TO PECTOBACTERIUM

CAROTOVORUM SUBSP. CAROTOVORUM

H.H. El-Hendawy

Abstract

DNA, RNA and protein contents as well as protein banding pattern onSDS-PAGE of melon (Cucumis melo) and cucumber (C. sativus) seedlings inoculatedwith Pectobacterium carotovorum subsp. carotovorum strain 119A, which infects me-lon but not cucumber, and P. carotovorum subsp. carotovorum strain pep2A, whichinfects either plant species, were estimated. Seedlings inoculated with sterile dis-tilled water (SDW) and noninoculated were used as controls. No significantchange was detected in DNA levels of melon and cucumber seedlings inoculatedwith SDW and Pcc strain pep2A and melon seedlings inoculated with Pcc strain119A. However, increased RNA levels were observed in these treatments. Cucum-ber seedlings inoculated with Pcc 119A contained the highest level of DNA andRNA relative to all other treatments as well as the control. Although melon seed-lings inoculated with Pcc 119A and Pcc pep2A contained more protein bands thanthe noninoculated control, it contained reduced band percentage and reduced pro-tein contents. In contrast, melon seedlings inoculated with SDW and cucumberseedlings inoculated with the three types of inoculations contained higher numberof protein bands, higher band percentage as well as higher protein contents rela-tive to the noninoculated control. The highest level of protein was obtained in cu-cumber tissues inoculated with Pcc 119A which might suggest that these proteinscould play a role in the resistance of cucumber seedlings to this strain.

Key words: Erwinia carotovora, soft rot, melon, cucumber, pathogenesis-relatedproteins

Phytopathol. Pol. 37: 11–21© The Polish Phytopathological Society, Poznań 2005ISSN 1230-0462

Introduction

Soft rot Erwinia produce different types of cell wall degrading enzymes such aspectinases and cellulases (Collmer and Keen 1986, Perombelon and Salmond1995). These enzymes facilitate penetration of plant tissues by the pathogen andalso cause the release of degradation products of cell wall components. Theseproducts are used by plants as elicitors for the production of pathogenesis-relatedproteins (PRs), such proteins could be effective in inhibition of pathogen growth,multiplication and/or spread and can be responsible for systemic acquired resis-tance (SAR) (Benhamou 1996, Agrios 1997).

The soft rotting Pectobacterium carotovorum subsp. carotovorum (Erwinia carotovorasubsp. carotovora) strain 119A is pathogenic to melon but not cucumber. The fail-ure of this strain to induce soft rot disease symptoms on cucumber seedlings wasnot due to its failure to grow or to secrete pectate lyase enzyme in cucumber tissue(El-Hendawy et al. 1997). This study was undertaken to compare DNA, RNA andprotein concentration as well as protein profile of melon and cucumber seedlingsinoculated with strain 119A with those inoculated with P. carotovorum subsp.carotovorum (E. carotovora subsp. carotovora) strain pep2A, which is pathogenic to ei-ther melon and cucumber (El-Hendawy et al. 2002) to know whether cucumber re-sistance to strain 119A is associated with protein accumulation or not.

Materials and methods

Two strains of P. carotovorum subsp. carotovorum were used throughout thisstudy: strain 119A which had been isolated from infected melon plants(El-Hendawy 1983) and was capable to induce soft rot disease symptoms on melonseedlings, however, not on seedlings of the related species cucumber (El-Hendawyet al. 1997), and strain pep2A which had been isolated from rotten pepper fruitsand was capable to infect either melon and cucumber seedlings (El-Hendawy et al.2002). These strains were maintained on nutrient agar slopes at 4°C andsubcultured monthly.

Seeds of melon (Cucumis melo L.), cultivar group Musk or Netted melon, and cu-cumber (C. sativus L.), cultivar group Madina 106, were purchased from the Minis-try of Agriculture, Egypt. Seeds were sown in a clay soil. Three weeks after sowing,seedlings of each plant species were grouped into four batches, containing 30 seed-lings each, then one batch from each species was subjected to one of the followingfour treatments: 1) control seedlings noninoculated, 2) seedlings inoculated withsterile distilled water (SDW), 3) seedlings inoculated with strain 119A, 4) seed-lings inoculated with strain pep2A.

Inoculations and inoculum preparation were carried out as described byEl-Hendawy et al. (1997). Seedlings were inoculated through cotyledons. Eachcotyledon received 3.5 × 106 cfu of either strains.

12 H.H. El-Hendawy

Seedlings of both melon and cucumber were harvested 24 h after inoculationfor protein and nucleic acid extraction and estimation. Nucleic acid isolationmethod based on that of Shibko et al. (1967) was used. Total DNA content was es-timated by diphenylamine (DPA) reaction according to Burton (1968), while RNAcontent was estimated by orcinol reaction described by Ashwall (1957). Theamount of DNA and RNA in each sample were quantitatively calculated from stan-dard curves, prepared from different concentrations of pure RNA or DNA.

Quantitative analysis of protein was carried out according to Thorne and Koller(1974). Seedlings protein was extracted with 0.1 N NaOH and determined by theBio-Rad Assay using freshly prepared bovine serum albumen as a standard.

SDS-PAGE was carried out according to Laemmli (1970). Total proteins wereextracted overnight using 0.2 M Tris-HCl buffer adjusted to pH 3.8 and containingsodium dodecyl sulphate (SDS). After centrifugation at 9000 rpm for 6 min, thesupernatant was collected. SDS slab gel of 12.5% acrylamide was used and molecu-lar mass markers were applied in each run. Molecular weight of protein bandingpattern of each sample was identified using gel-documentation system “GelAnalyser V 3.0 Computer Program”.

Statistical analysis of the results was carried out by variance analysis and thesignificance were determined using LSD values at levels of 1 and 5%. All data aremeans of three replicates.

Results

Inoculation of two weeks old melon seedlings with 1 × 107 cfu of Pcc 119A orPcc pep2A resulted in water soaked areas around the site of inoculation either oncotyledons or stems, 24 h after inoculation (Phot. 1). The whole seedling wastransformed into completely rotted mass within three–four days. Inoculation oftwo weeks old cucumber seedlings with the same number of Pcc 119A and Pccpep2A cells produced soft-rot disease symptoms only when cotyledons were inoc-ulated with strain pep2A.

No significant change in the level of DNA was observed in melon and cucumberseedlings inoculated with either SDW and Pcc pep2A and in melon seedlings inoc-ulated with Pcc 119A, relative to noninoculated control seedlings (Table 1). In con-trast, highly significant increase of DNA level was observed in cucumber seedlingsinoculated with Pcc 119A.

The results presented in Table 2 show that increased RNA level, of either plantspecies, was obtained in all types of inoculations. The highest RNA level was de-tected in cucumber seedlings inoculated with Pcc 119A. It reached up to 1.6 times,approximately, as that of the control.

Estimation of protein concentration in melon and cucumber seedlings revealedthat protein content of either plant species inoculated with SDW was significantlyincreased relative to the noninoculated seedlings. On the other hand, non-signifi-cant reduction in protein concentration was observed in melon seedlings inocu-

Accumulation of proteins in cucumber seedlings... 13

lated with either bacterial strains in comparison with the control. In contrast,increased protein level was detected in cucumber seedlings inoculated with eitherbacterial strains. In cucumber seedlings treated with Pcc 119A the protein contentwas found to be 2.3 times as that of the control (Table 3).

Analysis of proteins obtained from melon and cucumber seedlings withSDS-PAGE, indicated accumulation of certain proteins in the inoculated seedlings

14 H.H. El-Hendawy

Phot. 1. Pathogenicity of Pectobacterium carotovorum subsp. carotovorum strain 119A on melonseedlings. Two-week-old seedlings were inoculated with 1 × 107 cfu prepared from 24 h liquidculture (photograph taken 48 h after inoculation). 1– seedlings inoculated through stem, 2 –seedlings inoculated through cotyledons, 3 – seedlings inoculated with sterile distilled water

(photo by N. Tait)

Table 1DNA content of inoculated and noninoculated melon and cucumber

seedlings tissues ( g/g F.W.)

Treatment Melon Cucumber

Control 4.49 4.35

SDW 4.67 4.50

Pcc pep2A 4.43 4.30

Pcc 119A 4.40 5.65

LSD 1% 1.65 0.98

LSD 5% 0.99 0.59

in comparison with the noninoculated ones (Phot. 2, Table 4). For example, inocu-lation of either plant species with SDW resulted in the appearance of five new pro-tein bands in melon and other seven in cucumber with molecular weights of 53.15,49.82, 38.87, 29.30, 28.06 kDa and 75.0, 73.0, 51.61, 49.46, 35.17, 27.23, 26.26kDa, respectively (Table 4). On the other hand, four protein bands with molecularweights of 51.36, 48.29, 42.54 and 27.44 kDa, originally present in the control,were not detected in melon seedlings inoculated with SDW.

Inoculation of Pcc pep2A into either plant species resulted in production of sixprotein bands in cucumber and four bands in melon with M.W. of 71.00, 50.35,48.73, 35.25, 27.22, 26.30 kDa and 75.00, 71.00, 39.18, 29.74 kDa, respectively(Table 4). These bands were not detected in the control plants. Also, seven moreprotein bands with M.W. of 75.00, 71.59, 51.40, 49.38, 35.14, 27.19, 26.26 kDaand four more bands with M.W. of 75.00, 68.30, 50.02, 28.39 kDa were observedin cucumber and melon seedlings inoculated with Pcc 119A, respectively (Table 4).

Accumulation of proteins in cucumber seedlings... 15

Table 2RNA content of inoculated and noninoculated melon and cucumber

seedlings tissues ( g/g F.W.)

Treatment Melon Cucumber

Control 1.673 1.785

SDW 1.833 2.018

Pcc pep2A 2.117 2.540

Pcc 119A 2.467 2.845

LSD 1% 0.300 0.727

LSD 5% 0.180 0.438

Table 3Protein concentration of melon and cucumber seedlings inoculatedwith sterile distilled water, Pcc pep2A and Pcc 119A (mg/g F.W.)

Treatment Melon Cucumber

Control 24.30 20.50

SDW 27.00 33.00

Pcc pep2A 22.00 24.50

Pcc 119A 23.00 48.00

LSD 1% 3.76 7.63

LSD 5% 2.27 4.60

Discussion

Inoculation of two weeks old melon seedlings with Pcc 119A or Pcc pep2A in-duced soft-rot disease symptoms either from stem or cotyledon inoculations. Incontrast, inoculation of two weeks old cucumber seedlings with either bacterialstrains produced soft-rot disease symptoms only when cotyledons were inoculatedwith strain pep2A. However, the majority of soft rotting Erwinia strains have beenfound to have a wide host range (Perombelon and Kelman 1980) but the failure ofsome strains to infect some plant species was reported elsewhere (Arsenijevic andObradovic 1996, El-Hendawy et al. 2002).

16 H.H. El-Hendawy

Phot. 2. SDS-polyacrylamide gel electrophoresis of proteins extracted from cucumber (lanes 1–4)and melon (lanes 5–8). M – molecular weight marker, 1 – seedlings inoculated with strain pep2A,2 – seedlings inoculated with strain 119A, 3 – noninoculated seedlings, 4 – seedlings inoculated

with SDW (photo by M. Monir)

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The inability of Pcc pep2A to infect cucumber seedlings from stem inoculationcould be attributed to certain defense mechanisms which were developed againstthis pathogen in stem but not cotyledons. However, Sasaki et al. (2002) found thatthe wound-induced tobacco peroxidase gene was induced preferentially in stemsand petioles but was negligible in leaf blades even 8 h after wounding.

Although no significant change was detected in DNA content of melon seed-lings with all types of inoculations as well as in cucumber seedlings inoculatedwith SDW and Pcc pep2A, highly significant increase of DNA content was observedin cucumber seedlings inoculated with Pcc 119A. Other studies on the induction ofDNA synthesis in plants infected with phytopathogenic bacteria showed that nei-ther nuclear nor cytoplasmic synthesis was inhibited until the end of the latent pe-riod (Sigee 1984). However, the increased level of DNA in cucumber seedlingsinoculated with Pcc 119A could be due to induction of DNA synthesis as a defenseresponse against the pathogen.

The highest RNA level was detected in cucumber seedlings inoculated with Pcc119A. However, in several plant diseases, infected plants, particularly resistantones, seem to contain higher levels of RNA than healthy plants (Agrios 1997). Theincreased level of RNA means subsequent increased transcription and synthesis ofsubstances involved in the defense mechanisms of plant cells.

Estimation of protein concentration gave significantly higher level of proteinsin melon and cucumber seedlings inoculated with SDW than in noninoculatedplants. Also, the number and percentage of protein bands obtained from seedlingsinoculated with SDW was higher than in noninoculated seedlings. However, it hasbeen reported that plants respond to wounding by switch on several defense mech-anisms for healing wounded tissues and protecting them from the subsequent in-vasion of pathogens. These mechanisms include inosculation of the cell wall bysuberization (Dean and Kolattukudy 1976), enhancement of phenyl-propanoidsynthesis (Collinge and Slusarenko 1987), crosslinking of the cell wall compo-nents (Bradley et al. 1992) and local or systemic induction of a set of defense-re-lated proteins (Farmer and Ryan 1990, Niki et al. 1998). Thus, the increasedprotein level as well as protein banding of seedlings inoculated with SDW could beattributed to protein accumulation as a response to wounding during inoculation.Although inoculation of melon seedlings with Pcc pep2A or Pcc 119A increased thenumber of protein bands, it reduced protein contents as well as the total percent-age of bands relative to the noninoculated seedlings. The increased number of pro-tein bands could be due to the production of new proteins with different molecularweights or degradation of the host proteins by the action of the pathogens proteas-es. Perombelon and Salmond (1995) reported the production of proteases by softrot Erwinias.

In melon seedlings inoculated with the two bacterial strains, higher level ofRNA was observed in comparison with the control, in contrast reduced proteincontents was detected. This could be attributed to two possible reasons: 1) thedamage of proteins by compounds released during cell wall degradation by thepathogen (Cooper 1984, Sigee 1993), or 2) the failure of mRNA to be translatedinto proteins due to inhibition of this process by compounds produced by the

18 H.H. El-Hendawy

pathogens or released from the host (Sigee 1993). Increased protein content aswell as increased number of bands and bands percentage were obtained when cu-cumber seedlings were inoculated with both bacterial strains. Seven protein bandswhich were not observed in the control were detected in either treatments. The in-crease in protein level was nonsignificant in case of seedlings treated with strainpep2A whereas, in seedlings treated with strain 119A it was highly significant.Taking into consideration that cucumber seedlings were resistant to degradationby the latter strain, it is more likely that the obtained high level of protein was cor-related with cucumber resistance. It has been reported that phytopathogenic bac-teria harbor a gene cluster that controls pathogenicity in susceptible plants and theability to elicit the hypersensitive reaction in non-host plants or resistant cultivarsof host plants (Lindgren et al. 1986, Galan and Collmer 1999). Some of these genesencode elements by which effector proteins are exported and delivered into thecytosol of host plant cells (Galan and Collmer 1999, Kjemtrup et al. 2000). Some ofthese effector proteins were found to interact with plant intracellular proteins andactivate the plant defense system. Furthermore, pathogenesis-related proteins ofcucumber have been detected before and identified as chitinases (Metraux et al.1989, van Loon 1999) and peroxidases (Rasmussen et al. 1995). Cucumberchitinase was found to have molecular weight of 28 kDa and accumulated in theintercellular space of the virus-infected, as well as the uninfected parts of the plant.They also have strong homology with a lysozyme/chitinase from Parthenocissusquinquefolia (L.) Planch. (Metraux et al. 1989). On the other hand, peroxidase iso-zymes from intercellular wash fluids of induced cucumber plants had molecularweights of approximately 30, 31 and 33 kDa and were found to be associated withsystemic acquired resistance in cucumber (Smith and Hammerschmidt 1990, Ras-mussen et al. 1995). In this study, protein bands with M.W. of 33.04 to 33.23 kDaapproximately, were detected in cucumber seedlings with different treatments.The levels of these proteins were higher than that in the noninoculated control.Also a protein band of M.W. of 27.91 was detected in cucumber seedlings inocu-lated with either Pectobacterium strains at a percentage higher than that of the con-trol. Moreover, protein bands of M.W. of 27.2 and 26.3 kDa were detected in theinoculated cucumber but not in the noninoculated control. These protein bandsmight belong to peroxidases and chitinases which were accumulated in cucumbertissues as a response to wounding or bacterial infection or both. Similarly, a pro-tein band of M.W. of 56.1 kDa which was observed in all treatments of melon andcucumber was detected at its highest level in cucumber seedlings inoculated withPcc 119A. Thus, this protein as well as the previously mentioned proteins mightplay a role in the resistance of cucumber seedlings to Pcc 119A.

Accumulation of proteins in cucumber seedlings... 19

Acknowledgements

Deepest thanks and appreciation to Prof. Dr. M.S.A. Soliman, Botany and Mi-crobiology Department, Faculty of Science, Helwan University, who contributedgenerously with his time and effort to the preparation of this work.

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Author’s address: Dr. H.H. El-Hendawy,Helwan University,Botany and Microbiology Department,Ain Helwan, Cairo,Egypte-mail: el_hendawi@hotmail.com

Accepted for publication: 15.01.2005

Accumulation of proteins in cucumber seedlings... 21