PLANT BIOLOGY
ANIMAL BIOLOGY
SUBSCRIPTION
E-SUBSCRIPTION
 
MAP
MAIN PAGE

 

 

 

 

Sel’skokhozyaistvennaya Biologiya [Agricultural Biology], 2019, Vol. 54, № 3, p. 566-574.
(how to cite this article)

doi: 10.15389/agrobiology.2019.3.566eng

UDC: 632.3.01/.08:577.15

Acknowledgements:
Supported financially in part by Russian Science Foundation (project No. 15-14-10022)

 

ENZYMES FOR THE DEGRADATION OF RHAMNOGALACTURONAN I AS VIRULENCE FACTORS OF PHYTOPATHOGENIC BACTERIUM Pectobacterium atrosepticum

E.A. Kovtunov1, V.Yu. Gorshkov2, 3, N.E. Gogoleva2, 3, O.E. Petrova2,
E.V. Osipova2, Ch.B. Nuriakhmetova3, S.V. Tatarkin2, Yu.V. Gogolev2, 3

1ITMO University, SCAMT laboratory, 9, ul. Lomonosova, St. Petersburg, 191002 Russia, e-mail kovtunovea@mail.ru;
2Kazan Institute of Biochemistry and Biophysics, Federal Research Center Kazan Scientific Center RAS, PO box 30, Kazan, Republic of Tatarstan, 420111 Russia, e-mail gogolev.yuri@gmail.com (✉ corresponding author), gvy84@mail.ru, negogoleva@gmail.com, poe60@mail.ru, eva-0@mail.ru, tatarkins@gmail.com;
3Kazan (Volga region) Federal University, 18, ul. Kremlyovskaya,Kazan, Republic of Tatarstan,420008 Russia, e-mail ch_nuriakhmetova@mail.ru

ORCID:
Kovtunov E.A. orcid.org/0000-0002-3506-080X
Osipova E.V. orcid.org/0000-0002-8203-9798
Gorshkov V.Yu. orcid.org/0000-0002-9577-2032
Nuriakhmetova Ch.B. orcid.org/0000-0002-8626-0780
Gogoleva N.E. orcid.org/0000-0003-2404-1539
Tatarkin S.V. orcid.org/0000-0002-2422-0476
Petrova O.E. orcid.org/0000-0003-1618-5479
Gogolev Yu.V. orcid.org/0000-0002-2391-2980

Received November 6, 2017

 

Plant pathogenic pectobacteria (Pectobacterium genus) are well-known all over the world as the causal agents of the cultural plant diseases called soft rots. Rot symptoms are related to the extensive plant tissue maceration due to the production by microorganisms of the plant cell wall degrading enzymes. Most of the pectobacteria-secreted enzymes catalyze the cleavage of homogalacturonan. This polysaccharide, that is a linear homopolymer, consists of galacturonic acid residues and is the most abundant (by mass) pectic polysaccharide of plant cell walls. The knockout of genes of homogalacturonan-degrading enzymes is known to lead to reduced virulence of pectobacteria. In addition, the modification of another pectic compound — rhamnogalacturonan I also occurs in the course of infection process caused by pectobacteria. This compound is a ramified heteropolymer, the backbone of which consists of alternate rhamnose and galacturonic acid residues, and side chains are represented by galactose or arabinose residues. However, the role of pectobacterial enzymes for rhamnogalacturonan I degradation in the development of soft rots has not been previously ascertained. The present study is dedicated to the investigation of the necessity of P. atrosepticum SCRI1043 enzymes degrading rhamnogalacturonan I for a full development of soft rots in the plants infected by pectobacteria. By directed mutagenesis, we have obtained mutant forms of P. atrosepticum SCRI1043 deficient in genes encoding rhamnogalacturonyl hydrolase (genome locus eca3749) that cleaves the backbone of rhamnogalacturonan I, and galactanase (genome locus eca0852) that breaks side chains of this polymer. For the target gene knockout, mutant loci were constructed by overlap-extension PCR. Most of the original gene was replaced by kanamicin-resistance cassette. The obtained construction was ligated into a mobilized suicide vector and the resulting plasmid was transferred into donor E. coli СС118 strain cells. The recombinant plasmid with the mutant locus was introduced into P. atrosepticum SCRI1043 cells by three-parental mating. The P. atrosepticum SCRI1043 clones, in which the original locus was replaced by the mutant one, and the donor plasmid was eliminated, were selected on the selective media. The mutant strains P. atrosepticum SCRI1043∆3749 and P. atrosepticum SCRI1043∆0852 caused significantly less damage to the plant tissues of Brassica rapa spp. pekinensis Cha Cha cv. compared to parental wild-type strain. Herewith, the strain mutant in eca0852 locus encoding galactanase, the enzyme that cleaves side chains of rhamnogalacturonan I, was least virulent. The reduction in virulence, in this case, was not related to the suppression of homogalacturonan-degrading enzyme activity or less motility of bacteria. Thus, we have demonstrated that, first, rhamnogalacturonan I-degrading enzymes may be attributed to virulence factors of phytopathogenic pectobacteria, and second, the hydrolysis of the sides chains of rhamnogalacturonan I contributes more to the process of tissue maceration than the decay of the polymer backbone.

Keywords: Pectobacterium atrosepticum, pectic polysaccharides, rhamnogalacturonan I, glycosyl hydrolases.

 

REFERENCES

  1. Mansfield J., Genin S., Magori S., Citovsky V., Sriariyanum M., Ronald P., Dow M., Verdier V., Beer S.V.,. Machado M.A., Toth I., Salmond G., Foster G.D. Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol., 2012, 13(6): 614-629 CrossRef
  2. Perombelon M.C.M. Potato diseases caused by soft rot erwinias: an overview of pathogenesis. Plant Pathol., 2002, 51(1): 1-12 CrossRef
  3. Charkowski A., Blanco C., Condemine G., Expert D., Franza T., Hayes C., Hugouvieux-Cotte-Pattat N., Solanilla E.L., Low D., Moleleki L., Pirhonen M., Pitman A., Perna N., Reverchon S., Rodríguez Palenzuela P., Francisco M.S., Toth I., Tsuyumu S., van der Waals J., Van der Wolf J., Gijsegem F.V., Yang C.-H., Yedidia I. The role of secretion systems and small molecules in soft-rot Enterobacteriaceae pathogenicity. Annu. Rev. Phytopathol., 2012, 50: 21.1-21.25 CrossRef
  4. Gorshkova T.A. Rastitel'naya kletochnaya stenka kak dinamichnaya Sistema [Plant cell wall as a dynamic system]. Moscow, 2007 (ISBN 978-5-02-035598-9) (in Russ.).
  5. Tarasova N., Gorshkov V., Petrova O., Gogolev Y. Potato signal molecules that activate pectate lyase synthesis in Pectobacterium atrosepticum SCRI1043. World J. Microbl. Biot., 2013, 29(7): 1189-1196 CrossRef
  6. Walker D.S., Reeves P.J., Salmond G.P.C. The major secreted cellulase, CelV, of Erwinia carotovora subsp. carotovora is an important soft rot virulence factor. Mol. Plant Microbe In., 1994, 7(3): 425-431 CrossRef
  7. Mäe A., Heikinheimo R., Tapio Palva E. Structure and regulation of the Erwinia carotovora subspecies carotovora SCC3193 cellulase gene celV1 and the role of cellulase in phytophatogenicity. Mol. Gen. Genet., 1995, 247(1): 17-26 CrossRef
  8. Moleleki L.N., Pretorius R.G., Tanui C.K., Mosina G., Theron J. A quorum sensing-defective mutant of Pectobacterium atrosepticum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems. Mol. Plant Pathol., 2017, 18(1): 32-44 CrossRef
  9. Gorshkov V., Daminova A., Ageeva M., Petrova O., Gogoleva N., Tarasova N., Gogolev Y. Dissociation of a population of Pectobacterium atrosepticum SCRI1043 in tobacco plants: formation of bacterial emboli and dormant cells. Protoplasma, 2014, 251(3): 499-510 CrossRef
  10. Donlan R.M. Biofilms: microbial life on surfaces. Emerg. Infect. Dis., 2002, 8(9): 881-890 CrossRef
  11. Izano E.A., Amarante M.A., Kher W.B., Kaplan J.B. Differential roles of poly-N-glucosamine surface polysaccharide and extracellular DNA in Staphylococcus aureus and Staphylococcus epidermidis biofilms. Appl. Environ. Microb., 2008, 74(2): 470-476 CrossRef
  12. Flemming H.C., Wingender J. The biofilm matrix. Nat. Rev. Microbiol., 2010, 8: 623-633 CrossRef
  13. Gorshkov V.Y., Daminova A.G., Mikshina P.V., Petrova O.E., Ageeva M.V., Salnikov V.V., Gorshkova T.A., Gogolev Y. V. Pathogen-induced conditioning of the primary xylem vessels — a prerequisite for the formation of bacterial emboli by Pectobacterium atrosepticum. Plant Biology, 2016, 18(4): 609-617 CrossRef
  14. Gorshkov V., Islamov B., Mikshina P., Petrova O., Burygin G., Sigida E., Shashkov A., Daminova A., Ageeva M., Idiyatullin B., Salnikov V., Zuev Y., Gorshkova T., Gogolev Y. Pectobacterium atrosepticum exopolysaccharides: identification, molecular structure, formation under stress and in planta conditions. Glycobiology, 2017, 27(11): 1016-1026 CrossRef
  15. Bell K.S., Sebaihia M., Pritchard L., Holden M.T.G., Hyman L.J., Holeva M.C., Thomson N.R., Bentley S.D., Churcher L.J., Mungall K., Atkin R., Bason N., Brooks K., Chillingworth T., Clark K., Doggett J., Fraser A., Hance Z., Hauser H., Jagels K., Moule S., Norbertczak H., Ormond D., Price C., Quail M.A., Sanders M., Walker D., Whitehead S., Salmond G.P.C., Birch P.R.J., Parkhill J., Toth I.K. Genome sequence of the enterobacterial phytopathogen Erwinia carotovora subsp. atroseptica and characterization of virulence factors. PNAS USA, 2004, 101(30): 11105-11110 CrossRef
  16. Sambrook J., Fritsch E.F., Maniatis T. Molecular cloning: a Laboratory Manual. 2nd ed. Cold Spring Harbor, NY, 1989.
  17. Lee M.K., van Iersel M. W. Sodium chloride effects on growth, morphology, and physiology of Chrysanthemum (Chrysanthemum morifolium). HortScience, 2008, 43(6): 1888-1891.
  18. Shevchik V.E., Robert-Baudoy J., Hugouvieux-Cotte-Pattat N. Pectat lyase PelI of Erwinia chrysanthemi belongs to a new family. J. Bacteriol., 1997, 179(23): 7321-7330 CrossRef
  19. Herrero M., de Lorenzo V., Timmis K.N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J. Bacteriol., 1990, 172(11): 6557-6567 CrossRef
  20. Grinter N.J. A broad-host-range cloning vector transposable to various replicons. Gene, 1983, 21(1-2): 133-143 CrossRef
  21. Datsenko K.A., Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. PNAS USA, 2000, 97(12): 6640-6645 CrossRef
  22. Kaniga K., Delor I., Cornelis G.R. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene, 1991, 109(1): 137-141 CrossRef
  23. Gorshkov V., Gubaev R., Petrova O., Daminova A., Gogoleva N., Ageeva M., Parfirova O., Prokchorchik M., Nikolaichik Y., Gogolev Y. Transcriptome profiling helps to identify potential and true molecular switches of stealth to brute force behavior in Pectobacterium atrosepticum during systemic colonization of tobacco plants. European Journal of Plant Pathology, 2018, 152(4): 957-976 CrossRef
  24. Robert-Baudouy J., Nasser W., Condemine G., Reverchon S., Shevchik V.E., Hugouvieux-Cotte-Pattat N. Pectic enzymes of Erwinia chrysanthemi, regulation and role in pathogenesis. The American Phytopathological Society, St. Paul, 2000.
  25. Matsumoto H., Umehara M., Muroi H., Yoshitake Y., Tsuyumu S. Homolog of FlhDC, a master regulator for flagellum synthesis: required for pathogenicity in Erwinia carotovora subsp. carotovora. J. Gen. Plant Pathol., 2003, 69: 189-193 CrossRef
  26. Hossain M.M., Shibata S., Aizawa S.I., Tsuyumu S. Motility is an important determinant for pathogenesis of Erwinia carotovora subsp. carotovora. Physiol. Mol. Plant P., 2005, 66(4): 134-143 CrossRef
  27. Duan Q., Zhou M., Zhu L., Zhu G. Flagella and bacterial pathogenicity. J. Basic Microb., 2013, 53(1): 1-8 CrossRef
  28. Pfeilmeier S., Saur I. M, Rathjen J. P., Zipfel C., Malone J. G. High levels of cyclic-di-GMP in plant-associated Pseudomonas correlate with evasion of plant immunity. Mol. Plant Pathol., 2016, 17(4): 521-531 CrossRef
  29. Harholt J., Suttangkakul A., Vibe Scheller H. Biosynthesis of pectin. Plant Physiol., 2010, 153: 384-395 CrossRef

back

 


CONTENTS

 

 

Full article PDF (Rus)

Full article PDF (Eng)