The study of pathogenicity genes in the ascomycete

Doctoral Dissertation uoadl:1309764 492 Read counter

ΠΜΣ Μικροβιακή Βιοτεχνολογία
Library of the School of Science
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Eboigbe Lugard
Dissertation committee:
Καθηγητής Μιλτιάδης Τύπας (επιβλέπων)
Original Title:
The study of pathogenicity genes in the ascomycete
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.
Verticillium dahliae, Verticillium dahliae, Cell wall degrading genes , Gene inactivation, Phytopathogenic assays on plants
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