A novel glycoside hydrolase 74 xyloglucanase CvGH74A is a virulence factor in Coniella vitis

Grape white rot is a destructive fungal disease occurring worldwide.Recently,Coniella vitis was identified as the predominant pathogen causing this disease in China.As the periderms of grape shoots are severely degraded by C.vitis,it was speculated that cell wall-degrading enzymes(CWDEs)might play a key role in the pathogenesis of this disease.Therefore,this study aimed to examine the hydrolytic activity of the CWDEs of C.vitis.The results showed that xylanase(Xy)and xyloglucanase(XEG)had high levels of hydrolytic activity both in vitro and in vivo.Furthermore,a high-virulence fungal strain exhibited higher levels of Xy and XEG activities compared with a low-virulence strain.The genome of the fungus was found to harbor two XEG-coding genes CvGH74A and CvGH74B,which belonged to the glycoside hydrolase(GH)74 family.The expression level of CvGH74A was found to be high during pathogen infection.CvGH74A gene deletion mutants were generated using the split-marker method.The deletion of CvGH74A decreased both the hydrolytic activities of XEG and Xy and also the ability of the fungus to infect the grape leaves.No differences in the hyphal growth,morphology of colonies,or conidiation were found between theΔCvGH74A mutant strains and the wild-type strain.Together,these results suggested that CvGH74A acted as an important virulence factor,and its enzymatic activity might regulate the virulence of the pathogen.This study was novel in reporting that GH74 XEG acted as a virulence factor in C.vitis.

Studies on Xyloglucanase during the Germination of Seeds of Tamarindus indica

Germinating seeds of Tamarindus indica contain endo-β-1, 4-xyloglucanases which degrade tamarind xyloglucan, but not carboxymethylcellulose (CMC). The xyloglucanases are isolated from the germinating tamarind seeds using 50 mM acetate buffer, pH 5.5 containing 0.5 M NaCl. The Km value is 0.667 g/liter and the enzyme is optimally active at pH 5.5 and stable between pH 4 - 6.5. The optimum temperature is 45?C and is quite stable upto 50?C. The activity declined by 50% at 60?C and is completely inactivated at 70?C. Highest xyloglucanase activity and specific activity are observed on the 23rd day of germination. The polyacrylamide gel electrophoresis (PAGE) indicated the presence of five isozymes of xyloglucanases which are visualized by activity staining separately with congo red and grams iodine. Isozyme 2 is the major xyloglucanase present throughout the germination period.

Overexpression of the recombinant xyloglucanase <i>sp-Xeg</i>from <i>Penicillium canescens</i>accelerates growth and rooting of transgenic aspen plants

Analysis of the properties of transgenic aspen clones with recombinant gene xyloglucanase sp-Xeg from fungi Penicillium canescens showed the presence of complex modifications both in the wood and the phenotype of plants. Biometric analysis revealed an increase in the height of transgenic plants as compared to control plants. Increasing in the height of the shoot of 24.8%, 25% and 26% was observed for lines PtXIVXeg1a, PtXVXeg1a, PtXVXeg1b, respectively. Also there was an increase in the number of internodes in some transgenic clones. For the first time we showed the change in plants rhizogenesis with the recombinant gene xyloglucanase. In 10 of the 25 lines the rooting efficiency in vitro exceeded the control value. The maximum value of the rhizogenesis was fixed for line PtXVXeg1a (2.5 times higher than the control value). The mass of the root system for 6 of the 25 clones in the greenhouse was higher by 20% than the control value. The pentosan content decrease was also detected in all wood samples of transgenic plants. The obtained data of xyloglucanase activity and pentosan content generally correlated with phenotypic modifications.

Functions of Xyloglucan in Plant Cells

While an increase in the number of xyloglucan tethers between the cellulose microfibrils in plant cell walls increases the walls＇ rigidity, the degradation of these tethers causes the walls to loosen. Degradation can occur either through the integration of xyloglucan oligosaccharides due to the action of xyloglucan endotransglucosylase or through direct hydrolysis due to the action of xyloglucanase. This is why the addition of xyloglucan and its fragment oligosac- charides causes plant tissue tension to increase and decrease so dramatically. Experiments involving the overexpression of xyloglucanase and cellulase have revealed the roles of xyloglucans in the walls. The degradation of wall xyloglucan in poplar by the transgenic expression of xyloglucanase, for example, not only accelerated stem elongation in the primary wall, but also blocked upright-stem gravitropism in the secondary wall. Overexpression of cellulase also reduced xylo- glucan content in the walls as cellulose microfibrils were trimmed at their amorphous region, resulting in increased cell volume in Arabidopsis leaves and in sengon with disturbed leaf movements. The hemiceflulose xylogiucan, in its function as a tether, plays a key role in the loosening and tightening of cellulose microfibrils： it enables the cell to change its shape in growth and differentiation zones and to retain its final shape after cell maturation.

Loosening Xyloglucan Accelerates the Enzymatic Degradation of Cellulose in Wood

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrUs was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.