When Verticillium dahliae enters the host plant, the first line of defense that it encounters is the cell wall. Plant pathogenic fungi (including V. dahliae) produce extracellular enzymes which degrade plant cell wall components in a coordinated action. Some of the genes that encode these cell wall degrading enzymes, i.e., xylanases, cellulases and non-specific-action genes, were the major focus of this Ph.D. programme. Gene inactivation bears a remarkable model for the determination of the functions of genes in any organism. In this research, this molecular genetic tool has been applied to examine the functional role of β-1,6-endoglucanase and β-1,4-endoxylanase genes in the pathogenicity of filamentous fungus Verticillium dahliae. The gene coding for β-1,6-endoglucanase is considered as one of the important genes that code for hydrolyzing enzymes released at the initial stages of infection by fungi for the depolymerization of the cell wall. To address the hypothesis that these enzymes are important in V. dahliae virulence, a gene encoding a β-1,6-endoglucanase (vdg6) was isolated from V. dahliae using genome walking technique. Nucleotide sequence analysis of the 3’ and 5’ ends of clones from a genomic library of the fungus showed the presence of part of the endo-1,6-β-glucanase gene in a 3.5kb genomic fragment. Using this clone as probe and by employing genome walking approaches the 3’ and 5’; of the gene were determined bringing the entire gene (vdg6) size to ~1800 bp. An internal fragment (1.2kb) of vdg6 was used to disrupt the wild-type gene of the tomato race 2 V. dahliae strain 123V and the knock-out mutant (VdB) strain was tested for pathogenicity on tomato plants. The result showed a 7.5% reduction in disease symptoms caused on tomato plants in comparison with the wild type. Growth on minimal medium supplemented with different carbon sources showed reduced ability of the mutant to breakdown cellulose, whereas growth on glucose, pectin and sucrose were similar to the wild type. Endo-β-1,4-xylanase catalyze the endohydrolysis of xylan, the major structural polysaccharide of the plant cell wall. In order to investigate the role of the β-1,4-endoxylanase gene(xylA) in virulence of V. dahliae, through the analysis of clones from a genomic library of V. dahliae strain 76 and shotgun ESTs from xylem sap growing fungus the xylA gene was isolated. Its nucleotide sequence was determined and the predicted amino acid sequence showed significant homology with family 11 xylanases. The gene was disrupted by targeted inactivation due to a single cross-over event in a V. dahliae race 2 tomato strain. The knock-out mutant (XA) was compared with the wild type strain for disease symptoms on tomato plants. The result showed a small (7%) reduction in disease severity in the mutant strain. Growth of the mutant strain on minimal medium containing cellulose as the sole carbon source was reduced compared to the wild type indicating for a role of xylA in the breakdown of complex components of the cell wall. Other cell wall degrading genes cloned were β-1,3-exoglucanase, β-1,4-endoglucanse and endoglucanase II. The major obstacle to the determination of the genes involved in the depolymerisation of cell wall and pathogenicity is function redundancy. In an attempt to overcome the hurdle created by this function redundancy in analyzing the functions of the above genes, first, the regulation of vdg6 by sucrose non-fermenting gene (VdSNF1) was checked. The results showed that vdg6 gene is under catabolite repression, it is expressed during pathogencity and is important for the virulence of V. dahliae. Secondly, double disruption mutants were constructed from the single VdB mutant and the signalling PKA (cAMP-mediated protein kinase A) gene, namely VP1, VP2, VP3 and VP4. Analysis of these double mutants showed an obvious link between vdg6 gene and cAMP-mediated PKA (VdPKAC1) and that the β-1,6-endoglucanase cell wall degrading gene contributes to the pathogenicity of the fungus. In conclusion, all experimental evidence from this study showed that cell wall degrading genes contribute to virulence and pathogenicity of the fungus, however, since most of them belong to families of genes with similar functions the system is very complex to unravel and fully understand the genetic basis of pathogenicity.
Characterization of Race-Specific Interactions Among Isolates of Verticillium dahliae Pathogenic on Lettuce