Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli

Wulff, Nelson Arno; Carrer, Helaine; Pascholati, Sérgio Florentino

doi:10.1590/s0103-90162003000400016

Cited by 7 publications

(7 citation statements)

References 35 publications

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“…This contention is supported by the following results: i) presence of bacterial cells in the interior of pits ( Figures 1C-D, Figure 2C, and Figure 3B); ii) degradation of the primary cell wall at the pit membranes by X. fastidiosa cells (Figures 3B-C), and iii) absence of colloidal gold immunolabeling against primary cell wall components. These observations of radial bacterial migration are supported by Fry et al (1994), who verified the production of proteases by X. fastidiosa from grapevines; by Simpson et al (2000) that identified precursors for polygalacturonases and cellulases in the X. fastidiosa genome and by Wulff et al (2003) who expressed cellulase genes from X. fastidiosa in E. coli. These studies all support the hypothesis that X. fastidiosa utilizes pit to facilitate intercellular migration.…”

Section: Discussionmentioning

confidence: 84%

Citrus sinensis leaf petiole and blade colonization by Xylella fastidiosa: details of xylem vessel occlusion

Alves¹,

Leite²,

Pascholati³

et al. 2009

Sci. agric. (Piracicaba, Braz.)

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Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is an important disease of citrus in Brazil. X. fastidiosa is restricted to xylem vessels of plants and knowledge regarding xylem colonization is still limited. Our goal was to verify how this bacterium colonizes and spreads within xylem vessels of sweet orange Citrus sinensis cv. Pêra. Petioles and pieces of leaf blades from naturally infected plant exhibiting characteristic symptoms were prepared for light microscopy (LM), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and immunogold labeling (IGL). Petioles from healthy plants were used as control. IGL results, using an antibody against wall hemicelluloses, revealed that the pit membrane of vessels was altered. Bacterial cells were observed in the pit between adjacent vessels. Results support the contention that X. fastidiosa produces cellulases to reach adjacent vessels. SEM revealed that colonization of sweet orange started with X. fastidiosa cells attaching to the xylem wall, followed by an increase in the number of bacterial cells, the production of fibrous material, and finally vessel occlusion by biofilm composed of copious amounts of amorphous material, strands and cells. Phenolic materials, hyperplasia and hypertrophy were noticed in leaves with gummy material. Xylem vessels frequently contained an unknown needle-like, crystallized matter blocking the vessel. Key words: ultrastructure, scanning electron microscopy, transmission electron microscopy, immunolabel COLONIZAÇÃO DE PECÍOLO E FOLHA DE Citrus sinensis POR Xylella fastidiosa: DETALHES DA OBSTRUÇÃO DE VASOS DO XILEMARESUMO: A clorose variegada dos citrus (CVC), causada por uma bactéria restrita ao xilema (Xylella fastidiosa), é uma importante doença de citros no Brasil, entretanto, pouco se sabe sobre a colonização dos vasos do xilema pela bactéria. O objetivo deste trabalho foi estudar como X. fastidiosa invade os vasos adjacentes do xilema e algumas das alterações expressas por plantas de laranja Pêra. Foram coletadas 15 amostras de pecíolos e áreas das folhas de plantas com sintomas característicos da doença, as quais foram preparadas para microscopia de luz, microscopia eletrônica de varredura (MEV) e transmissão (MET) e imunomarcação de polissacarídeos da parede primária. Pecíolos de folhas sadias foram utilizados como controle. Os resultados da imunomarcação para hemicelulose mostraram alterações na integridade da membrana da pontuação. A bactéria foi localizada nas imediações e no interior das pontuações entre dois vasos. Estas observações suportam a hipótese de que X. fastidiosa produz celulases para alcançar os vasos adjacentes do xilema. Imagens de MEV revelaram que a colonização dos vasos do xilema de laranja doce inicia-se com as células da bactéria aderidas às paredes do xilema, seguida pelo aumento no número de células bacterianas, produção de material extracelular e finalmente a obstrução do vaso. Também, nos tecidos das folhas, verificou-se a deposição de goma, compostos fenólic...

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Section: Discussionmentioning

confidence: 84%

Citrus sinensis leaf petiole and blade colonization by Xylella fastidiosa: details of xylem vessel occlusion

Alves¹,

Leite²,

Pascholati³

et al. 2009

Sci. agric. (Piracicaba, Braz.)

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“…Xf has several genes whose sequences are similar to genes in other bacteria that encode cell wall-degrading polyglacturonase and b-1,4-glucanase (Wulff et al, 2003), but data for the presence of these enzymes in culture and in infected vines have been developed only recently (Agü ero et al, 2005; M.C. Roper, L.C.…”

Section: Discussionmentioning

confidence: 99%

Xylella fastidiosa Infection and Ethylene Exposure Result in Xylem and Water Movement Disruption in Grapevine Shoots

et al. 2006

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It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidiosa (Xf) within xylem vessels is the sole factor responsible for the blockage of water movement in grapevines (Vitis vinifera) affected by Pierce's disease. However, results from our studies have provided substantial support for the idea that vessel obstructions, and likely other aspects of the Pierce's disease syndrome, result from the grapevine's active responses to the presence of Xf, rather than to the direct action of the bacterium. The use of magnetic resonance imaging (MRI) to observe the distribution of water within the xylem has allowed us to follow nondestructively the development of vascular system obstructions subsequent to inoculation of grapevines with Xf. Because we have hypothesized a role for ethylene produced in vines following infection, the impact of vine ethylene exposure on obstruction development was also followed using MRI. In both infected and ethylene-exposed plants, MRI shows that an important proportion of the xylem vessels become progressively air embolized after the treatments. The loss of xylem water-transporting function, assessed by MRI, has been also correlated with a decrease in stem-specific hydraulic conductivity (K S) and the presence of tyloses in the lumens of obstructed water conduits. We have observed that the ethylene production of leaves from infected grapevines is greater than that from healthy vines and, therefore, propose that ethylene may be involved in a series of cellular events that coordinates the vine's response to the pathogen.

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“…The EGase was obtained from inclusion bodies purified from Escherichia coli expressing a recombinant X. fastidiosa EGase encoded by the gene (PD1851) showing homology to known EGase genes (Simpson et al, 2000;Wulff et al, 2003;Xia et al, 2004). The PG was cloned and purified from Aspergillus niger (Armand et al, 2000).…”

Section: Water Flow and Stem Infiltration Apparatusmentioning

confidence: 99%

“…The involvement of cell wall-degrading enzymes during PD first had been proposed based on indirect evidence from the development of internal symptoms and the location of bacteria in X. fastidiosainfected shoots, but the absence of evidence for bacterial enzyme production limited wide acceptance of this idea (Hopkins, 1989;Fry and Milholland, 1990a;Purcell and Hopkins, 1996). However, the observation of intervessel X. fastidiosa movement described above and reports that the X. fastidiosa genome contains several genes similar to those encoding cell walldegrading polygalacturonase (PG) and endo-1,4-bglucanase (EGase) in other bacteria (Simpson et al, 2000;Wulff et al, 2003) suggested the contrary. Indeed, a X. fastidiosa mutant disrupted in the pglA gene, which encodes an endo-PG, was restricted to the point of inoculation and unable to move systemically in grapevine, indicating that PG plays a major role in vessel-to-vessel movement (Roper et al, 2007).…”

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confidence: 99%

Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy andXylella fastidiosa-Infected Grapevines

Pérez-Donoso

Sun²,

Roper³

et al. 2010

Plant Physiology

109

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The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits, limiting the spread of xylem-localized pathogens and air embolisms from one conduit to the next. This paper provides a characterization of the size of the pores in the PMs of grapevine (Vitis vinifera). The PM porosity (PMP) of stems infected with the bacterium Xylella fastidiosa was compared with the PMP of healthy stems. Stems were infused with pressurized water and flow rates were determined; gold particles of known size were introduced with the water to assist in determining the size of PM pores. The effect of introducing trans-1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid (CDTA), oligogalacturonides, and polygalacturonic acid into stems on water flux via the xylem was also measured. The possibility that cell wall-degrading enzymes could alter the pore sizes, thus facilitating the ability of X. fastidiosa to cross the PMs, was tested. Two cell wall-degrading enzymes likely to be produced by X. fastidiosa (polygalactuoronase and endo-1,4-b-glucanase) were infused into stems, and particle passage tests were performed to check for changes in PMP. Scanning electron microscopy of control and enzyme-infused stem segments revealed that the combination of enzymes opened holes in PMs, probably explaining enzyme impacts on PMP and how a small X. fastidiosa population, introduced into grapevines by insect vectors, can multiply and spread throughout the vine and cause Pierce's disease.

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Cloning and expression of cellulase XF-818 of Xylella fastidiosa in Escherichia Coli

Cited by 7 publications

References 35 publications

Citrus sinensis leaf petiole and blade colonization by Xylella fastidiosa: details of xylem vessel occlusion

Citrus sinensis leaf petiole and blade colonization by Xylella fastidiosa: details of xylem vessel occlusion

Xylella fastidiosa Infection and Ethylene Exposure Result in Xylem and Water Movement Disruption in Grapevine Shoots

Cell Wall-Degrading Enzymes Enlarge the Pore Size of Intervessel Pit Membranes in Healthy andXylella fastidiosa-Infected Grapevines

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