Functional characterization of a cellulose binding xylanase from Fusarium oxysporum

Summary An extracellular endoxylanase from Fusarium oxysporum binds onto crystalline cellulose. A small peptide (~ 2kDa) could be isolated after partial proteolysis of the native protein. It consists of 18 amino acids, is located in the C-terminal region of the protein and corresponds functionally to a cellulose binding domain (CBD), the first one to be reported in a fungal xylanase. The amino acid sequence of this peptide shows no homology with any known CBD.     

Two xylanase genes of the vascular wilt pathogen Fusarium oxysporum are differentially expressed during infection of tomato plants

Two genes encoding putative family F xylanases from the tomato vascular wilt pathogen Fusarium oxysporum f.sp. lycopersici have been cloned and sequenced. The two genes, designated xyl2 and xyl3, encode proteins with calculated molecular masses of 33 and 39.3?kDa and isoelectric points of 8.9 and 6.7, respectively. The predicted amino acid sequences show significant homology to other family F xylanases. XYL3 contains a cellulose-binding domain in its N-terminal region. Southern analysis suggested that xyl2 and xyl3 homologs are also present in other formae speciales of F. oxysporum. Both genes were expressed during growth on oat spelt xylan and tomato vascular tissue in vitro. RT-PCR revealed that xyl3 is expressed in roots and in the lower stems of tomato plants infected by F. oxysporum f.sp. lycopersici throughout the whole disease cycle, whereas xyl2 is only expressed during the final stages of disease.     

Bacterial ectosymbionts and virulence silencing in a Fusarium oxysporum strain

In the present article we have ascertained the presence of a consortium of ectosymbiotic bacteria belonging to Serratia, Achromobacter, Bacillus and Stenotrophomonas genera associated to the mycelium of the antagonistic Fusarium oxysporum MSA 35 [wild-type (WT) strain]. Morphological characterization carried out on the WT strain, on the F. oxysporum MSA 35 without ectosymbionts [cured (CU) strain] and on the pathogenic F. oxysporum f.sp. lactucae (Fuslat 10) showed that the ectosymbionts, present only in the WT strain, caused a depleted production of micro conidia and aerial hyphae, and a change in shape and dimension of the latter. Virulence tests showed that the cured Fusarium was a pathogenic strain and, as shown by polymerase chain reaction and microscope analysis, pathogenicity was correlated with the capability of the cured hyphae of penetrating lettuce roots. Accordingly, the hyphae of the WT strain were impaired in entering the plant roots. Typing experiments provided evidence that both CU and WT strains belong to F. oxysporum f.sp. lactucae. This implies that the antagonistic effect of WT Fusarium is not a fungal trait, but it is due to the interaction with the ectosymbiotic bacteria. Expression analysis showed that fmk1, chsV and pl1 genes involved in F. oxysporum pathogenicity are not expressed in the WT strain whereas they are expressed in the cured fungus. These results, together with the hyphal characteristics, suggest that the inability of WT strain to penetrate the plant roots could be due to alterations in the expression profile of cell wall-degrading enzymes. In conclusion, we demonstrated a modulation of F. oxysporum gene expression in response to the interaction with the ectosymbiotic bacteria. Preliminary researches indicated that the presence of bacteria attached to the hyphae of antagonistic F. oxysporum is not an isolated phenomenon. Further investigations are necessary to better understand the rule and the diffusion of ectosymbiotic bacteria among antagonistic Fusarium.     

Identification of virulence genes in Fusarium oxysporum f. sp. lycopersici by large-scale transposon tagging

Forward genetic screens are efficient tools for the dissection of complex biological processes, such as fungal pathogenicity. A transposon tagging system was developed in the vascular wilt fungus Fusarium oxysporum f. sp. lycopersici by inserting the novel modified impala element imp160::gfp upstream of the Aspergillus nidulans niaD gene, followed by transactivation with a constitutively expressed transposase. A collection of 2072 Nia⁺ revertants was obtained from reporter strain T12 and screened for alterations in virulence, using a rapid assay for invasive growth on apple slices. Seven strains exhibited reduced virulence on both apple slices and intact tomato plants. Five of these were true revertants showing the re-insertion of imp160::gfp within or upstream of predicted coding regions, whereas the other two showed either excision without re-insertion or no excision. Linkage between imp160::gfp insertion and virulence phenotype was determined in four transposon-tagged loci using targeted deletion in the wild-type strain. Knockout mutants in one of the genes, FOXG_00016 , displayed significantly reduced virulence, and complementation of the original revertant with the wild-type FOXG_00016 allele fully restored virulence. FOXG_00016 has homology to the velvet gene family of A. nidulans . The high rate of untagged virulence mutations in the T12 reporter strain appears to be associated with increased genetic instability, possibly as a result of the transactivation of endogenous transposable elements by the constitutively expressed transposase.     

Cloning and optimized expression of a GH-11 xylanase from Fusarium oxysporum in Pichia pastoris

The endo-1,4-β-xylanase gene xyn11a from Fusarium oxysporum, member of the fungal glycosyl hydrolase (GH) family 11, was cloned and expressed in Pichia pastoris. The mature xylanase gene, which generates after the excision of one intron and the secreting signal peptide, was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The final construction was integrated into the genome of the methylotrophic yeast P. pastoris X33 and the ability to produce xylanase activity was evaluated in flask cultures. Recombinant P. pastoris efficiently secreted xylanase into the medium and produced high level of enzymatic activity (110 U/ml) after 216 hours of growth, under methanol induction. To achieve higher enzyme production, the influence of initial pH, methanol concentration, agitation and flask design was evaluated. Under optimum culture conditions, production of the recombinant xylanase increased by 50%, reaching a final yield of 170 U/ml, underpinning aeration as the most important factor in improving enzyme production.     

Role of cell wall-degrading enzymes in pathogenicity of Fusarium oxysporum

Fusarium oxysporum invades its host plants through the roots and colonizes the vascular system. It produces a great variety of cell-wall degrading enzymes (CWDE), such as cellulases, xylanases, pectinases and proteases. Our group has purified and characterized an endopolygalacturonase (PG1), two exopolygalacturonases (PG2 and PG3), an endoxylanase (XYL1) and an endo pectatelyase (PL1). We have isolated the following CWDE-encoding genes: pg1, pgx4, pg5, xyl2, xyl3, prt1 and pl1. Gene expression in different culture conditions has been determined by Northern analysis. The occurrence of these genes in different formae speciales has been analyzed by Southern analysis and PCR. All these genes are expressed during different stages of the interaction with the host plant indicating a possible role in pathogenesis. At present, targeted gene disruption is being carried out, in order to determine the role of each gene in the pathogenicity process.