It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidios... more 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.
Background and Aims: Tyloses and embolisms have been reported to impair water transport during th... more Background and Aims: Tyloses and embolisms have been reported to impair water transport during the development of Pierce's disease (PD), caused by the xylem-dwelling pathogen Xylella fastidiosa. This work investigates the relative importance of these xylem conduit obstructions in stems of inoculated Vitis vinifera plants. Methods and Results: After 18 weeks in a greenhouse, internodes from control and needle-inoculated Chardonnay vines were assessed for embolisms using non-destructive magnetic resonance imaging (MRI). Tyloses were identified by subsequent destructive histology and light microscopy examination. Embolism of individual or small sectors of vessels was detected by MRI analysis of a control shoot, usually in the proximity of the pith; virtually no tyloses were observed in the corresponding histological sections. From a symptomatic (inoculated) shoot, MRIs revealed large areas of embolised vessels extending radially towards the epidermis in most of the internodes; several individual or groups of two to three vessels appeared to be occluded with tyloses. Conclusions: In all assessed internodes, MRI-detected embolisms were found more frequently than tyloses, suggesting that over the course of PD embolism formation precedes tylose development. Significance of the Study: Embolisms appear to be the first and predominant type of occlusion in stems during early PD progression; nevertheless, their importance for explaining water transport impairment has been overlooked in most studies.
The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits... more 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.
Industrial processes to produce ethanol from lignocellulosic materials are available, but improve... more Industrial processes to produce ethanol from lignocellulosic materials are available, but improved efficiency is necessary to make them economically viable. One of the limitations for lignocellulosic conversion to ethanol is the inaccessibility of the cellulose and hemicelluloses within the tight cell wall matrix. Ferulates (FA) can crosslink different arabinoxylan molecules in the cell wall of grasses via diferulate and oligoferulate bridges. This complex cross-linking is thought to be a key factor in limiting the biodegradability of grass cell walls and, therefore, the reduction in FA is an attractive target to improve enzyme accessibility to cellulose and hemicelluloses. Unfortunately, our knowledge of the genes responsible for the incorporation of FA to the cell wall is limited. A bioinformatics prediction based on the gene similarities and higher transcript abundance in grasses relative to dicot species suggested that genes from the pfam family PF02458 may act as arabinoxylan feruloyl transferases. We show here that the FA content in the cell walls and the transcript levels of rice genes Os05g08640, Os06g39470, Os01g09010 and Os06g39390, are both higher in the stems than in the leaves. In addition, an RNA interference (RNAi) construct that simultaneously down-regulates transcript levels of these four genes is associated with a significant reduction in FA of the cell walls from the leaves of the transgenic plants relative to the control (19% reduction, P \ 0.0001). Therefore, our experimental results in rice support the bioinformatics prediction that members of family PF02458 are involved in the incorporation of FA into the cell wall in grasses.
Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtu... more Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR...
Polygalacturonase inhibitory protein (PGIP) was extracted from 'Shinli' pear tissue, purified and... more Polygalacturonase inhibitory protein (PGIP) was extracted from 'Shinli' pear tissue, purified and partially characterized. Extraction was carried out at 4°C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration, the extract was chromatographed on size-exclusion chromatography (S-100), and its active fractions were applied on concanavalin A-Sepharose. The PGIP activity was bound by the lectin, and then eluted using 1 M -methyl mannopyranoside, resulting in an 18-fold purification of the PGIP and demonstrating its glycoprotein nature. The following ion-exchange chromatography gave a PGIP that was 360-fold purified relative to the initial tissue extract, and having a 45 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibitory activity was tested against A. niger, C. acutatum and B. cinerea. The radial diffusion and reducing sugar assays showed that PGIP inhibition of three PGs was affected by pH. In vivo tests revealed that PGIP inhibited polygalacturonase from all three fungi. Heated for 20 min at 85°C, the inhibitory activity of PGIP was reduced by 85-90%, and it was completely suppressed after being heated at 100°C for 20 min.
It is conventionally thought that multiplication of the xylem-limited bacterium Xylella fastidios... more 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.
Background and Aims: Tyloses and embolisms have been reported to impair water transport during th... more Background and Aims: Tyloses and embolisms have been reported to impair water transport during the development of Pierce's disease (PD), caused by the xylem-dwelling pathogen Xylella fastidiosa. This work investigates the relative importance of these xylem conduit obstructions in stems of inoculated Vitis vinifera plants. Methods and Results: After 18 weeks in a greenhouse, internodes from control and needle-inoculated Chardonnay vines were assessed for embolisms using non-destructive magnetic resonance imaging (MRI). Tyloses were identified by subsequent destructive histology and light microscopy examination. Embolism of individual or small sectors of vessels was detected by MRI analysis of a control shoot, usually in the proximity of the pith; virtually no tyloses were observed in the corresponding histological sections. From a symptomatic (inoculated) shoot, MRIs revealed large areas of embolised vessels extending radially towards the epidermis in most of the internodes; several individual or groups of two to three vessels appeared to be occluded with tyloses. Conclusions: In all assessed internodes, MRI-detected embolisms were found more frequently than tyloses, suggesting that over the course of PD embolism formation precedes tylose development. Significance of the Study: Embolisms appear to be the first and predominant type of occlusion in stems during early PD progression; nevertheless, their importance for explaining water transport impairment has been overlooked in most studies.
The pit membrane (PM) is a primary cell wall barrier that separates adjacent xylem water conduits... more 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.
Industrial processes to produce ethanol from lignocellulosic materials are available, but improve... more Industrial processes to produce ethanol from lignocellulosic materials are available, but improved efficiency is necessary to make them economically viable. One of the limitations for lignocellulosic conversion to ethanol is the inaccessibility of the cellulose and hemicelluloses within the tight cell wall matrix. Ferulates (FA) can crosslink different arabinoxylan molecules in the cell wall of grasses via diferulate and oligoferulate bridges. This complex cross-linking is thought to be a key factor in limiting the biodegradability of grass cell walls and, therefore, the reduction in FA is an attractive target to improve enzyme accessibility to cellulose and hemicelluloses. Unfortunately, our knowledge of the genes responsible for the incorporation of FA to the cell wall is limited. A bioinformatics prediction based on the gene similarities and higher transcript abundance in grasses relative to dicot species suggested that genes from the pfam family PF02458 may act as arabinoxylan feruloyl transferases. We show here that the FA content in the cell walls and the transcript levels of rice genes Os05g08640, Os06g39470, Os01g09010 and Os06g39390, are both higher in the stems than in the leaves. In addition, an RNA interference (RNAi) construct that simultaneously down-regulates transcript levels of these four genes is associated with a significant reduction in FA of the cell walls from the leaves of the transgenic plants relative to the control (19% reduction, P \ 0.0001). Therefore, our experimental results in rice support the bioinformatics prediction that members of family PF02458 are involved in the incorporation of FA into the cell wall in grasses.
Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtu... more Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR...
Polygalacturonase inhibitory protein (PGIP) was extracted from 'Shinli' pear tissue, purified and... more Polygalacturonase inhibitory protein (PGIP) was extracted from 'Shinli' pear tissue, purified and partially characterized. Extraction was carried out at 4°C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration, the extract was chromatographed on size-exclusion chromatography (S-100), and its active fractions were applied on concanavalin A-Sepharose. The PGIP activity was bound by the lectin, and then eluted using 1 M -methyl mannopyranoside, resulting in an 18-fold purification of the PGIP and demonstrating its glycoprotein nature. The following ion-exchange chromatography gave a PGIP that was 360-fold purified relative to the initial tissue extract, and having a 45 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibitory activity was tested against A. niger, C. acutatum and B. cinerea. The radial diffusion and reducing sugar assays showed that PGIP inhibition of three PGs was affected by pH. In vivo tests revealed that PGIP inhibited polygalacturonase from all three fungi. Heated for 20 min at 85°C, the inhibitory activity of PGIP was reduced by 85-90%, and it was completely suppressed after being heated at 100°C for 20 min.
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Papers by John Labavitch