Abstract
The endo-β-1,4-xylanase BcXyn11A is one of several plant cell-wall degrading enzymes that the phytopathogenic fungus Botrytis cinerea secretes during interaction with its hosts. In addition to its enzymatic activity, this protein also acts as an elicitor of the defense response in plants and has been identified as a virulence factor. In the present work, other four endoxylanase coding genes (Bcxyn11B, Bcxyn11C, Bcxyn10A, and Bcxyn10B) were identified in the B. cinerea genome and the expression of all five genes was analyzed by Q-RT- PCR in vitro and in planta. A cross-regulation between xylanase genes was identified analyzing their expression pattern in the ΔBcxyn11A mutant strain and a putative BcXyn11A-dependt induction of Bcxyn10B gene was found. In addition, multiple knockdown strains were obtained for the five endoxylanase genes by transformation of B. cinerea with a chimeric DNA construct composed of 50-nt sequences from the target genes. The silencing of each xylanase gene was analyzed in axenic cultures and during infection and the results showed that the efficiency of the multiple silencing depends on the growth conditions and on the cross-regulation between them. Although the simultaneous silencing of the five genes was observed by Q-RT-PCR when the silenced strains were grown on medium supplemented with tomato extract, the endoxylanase activity measured in the supernatants was reduced only by 40%. Unexpectedly, the silenced strains overexpressed the Bcxyn11A and Bcxyn11C genes during the infection of tomato leaves, making difficult the analysis of the role of the endo-β-1,4-xylanases in the virulence of the fungus.
Highlights
Endo-β-1,4-xylanases (E.C. 3.2.1.8) hydrolyze the β-(1,4)-xylosidic linkages between two xylosyl residues breaking the linear backbone of xylan (Pollet et al, 2010), the main hemicellulosic component of the plant cell wall
According to the Carbohydrate-Active Enzymes (CAZy) database, most xylanases belong to the glycosyl hydrolase families (GH10) and (GH11), some enzymes have been classified into families 5, 8, 16, 26, 30, 43, and 62 (Paës et al, 2012)
A Blast-P search of the B. cinerea B05.10 genome was carried out using as queries either BcXyn11A or well-characterized xylanase sequences from other fungi (Supplementary Table S2), with known 3D structure. This search allowed the identification of two xylanases of family glycosyl hydrolase families 10 (GH10) (Bcin03g03480 and Bcin05g06020) and two additional members of family GH11 (Bcin15g01600 and Bcin12g00090), which were named BcXyn10A, BcXyn10B, BcXyn11B and BcXyn11C, respectively
Summary
Endo-β-1,4-xylanases (E.C. 3.2.1.8) hydrolyze the β-(1,4)-xylosidic linkages between two xylosyl residues breaking the linear backbone of xylan (Pollet et al, 2010), the main hemicellulosic component of the plant cell wall. According to the Carbohydrate-Active Enzymes (CAZy) database, most xylanases belong to the glycosyl hydrolase families (GH10) and (GH11), some enzymes have been classified into families 5, 8, 16, 26, 30, 43, and 62 (Paës et al, 2012). In addition to the catalytic domain, these proteins frequently contain a carbohydrate-binding module (CBM) involved in binding to polysaccharides of the plant cell wall (Hervé et al, 2010). CBMs are classified into more than 60 families in the CAZy database, based on their amino acid sequence similarity (Cantarel et al, 2009)
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